<%BANNER%>

Effect of Supplementing Essential Fatty Acids to Prepartum Holstein Cows and Preweaned Calves on Calf Performance, Metab...

Permanent Link: http://ufdc.ufl.edu/UFE0045027/00001

Material Information

Title: Effect of Supplementing Essential Fatty Acids to Prepartum Holstein Cows and Preweaned Calves on Calf Performance, Metabolism, Immunity, Health and Hepatic Gene Expression
Physical Description: 1 online resource (467 p.)
Language: english
Creator: Garcia Orellana, Miriam
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: calves -- linoleic-acid -- milk-replacer
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A series of experiments were conducted to examine the effect of supplementing essential fatty acids (FA) to prepartum Holstein cattle and newborn calves. The overall objective was to evaluate the effect of increasing intake of linoleic acid (LA) during the preweaning period on overall calf performance. In the first study prepartum cattle were fed one of three supplements, namely control(no fat), hydrogenated FA (SFA) or Ca containing essential FA (EFA). Colostrum FA profile of dams fed EFA reflected the concentration of LA in the fat supplement. Colostrum from nulliparous heifers was a better source of n-3 FA.Calves born from dams fed SFA had greater serum concentrations of total Immunoglobulin G (IgG), but efficiency of IgG absorption did not differ. In same study, 96 Holstein cattle were fed prepartum the same supplements as in experiment 1 and newborn calves were fed milk replacer (MR) of low LA (LLA) or high LA (HLA). Feeding SFA prepartum increased grain intake and average daily gain (ADG) without improving feed efficiency (FE) of calves born from fat-fed dams. Feeding HLA increased ADG, FE, plasma glucose and IGF-I, LA and its derivatives in liver, blood lymphocytes, phagocytosis by neutrophils and interferon-? from mononuclear cells. Expression of liver genes was strongly affected by the combination of prepartum diets and MR. Upregulated pathways included the PPAR signaling pathway, glycolysis/gluconeogenesis and oxidative phosphorylation whereas downregulated pathways included genes involved in inflammatory processes and ubiquitin-mediated proteolysis.Cardiomyopathy and tight junction pathways were upregulated in calves fedHLA-MR, but were downregulated if calves born from SFA- or EFA-fed dams. Calves born from fat-fed dams prepartum produced more milk at first lactation,possibly mediated by fetal programming. The last study aimed to determine the requirement of LA for preweaned calves. Heifers gained more BW in the first 30d of life as intake of LA increased. Wither and hip growth was greater in calves consuming LA exceeding 0.206 g/kg of BW0.75 during the 60-d study.Several markers of immunity were increased when LA was fed between 0.206and 0.333 g/kg of BW0.75.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Miriam Garcia Orellana.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Staples, Charles R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045027:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045027/00001

Material Information

Title: Effect of Supplementing Essential Fatty Acids to Prepartum Holstein Cows and Preweaned Calves on Calf Performance, Metabolism, Immunity, Health and Hepatic Gene Expression
Physical Description: 1 online resource (467 p.)
Language: english
Creator: Garcia Orellana, Miriam
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: calves -- linoleic-acid -- milk-replacer
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A series of experiments were conducted to examine the effect of supplementing essential fatty acids (FA) to prepartum Holstein cattle and newborn calves. The overall objective was to evaluate the effect of increasing intake of linoleic acid (LA) during the preweaning period on overall calf performance. In the first study prepartum cattle were fed one of three supplements, namely control(no fat), hydrogenated FA (SFA) or Ca containing essential FA (EFA). Colostrum FA profile of dams fed EFA reflected the concentration of LA in the fat supplement. Colostrum from nulliparous heifers was a better source of n-3 FA.Calves born from dams fed SFA had greater serum concentrations of total Immunoglobulin G (IgG), but efficiency of IgG absorption did not differ. In same study, 96 Holstein cattle were fed prepartum the same supplements as in experiment 1 and newborn calves were fed milk replacer (MR) of low LA (LLA) or high LA (HLA). Feeding SFA prepartum increased grain intake and average daily gain (ADG) without improving feed efficiency (FE) of calves born from fat-fed dams. Feeding HLA increased ADG, FE, plasma glucose and IGF-I, LA and its derivatives in liver, blood lymphocytes, phagocytosis by neutrophils and interferon-? from mononuclear cells. Expression of liver genes was strongly affected by the combination of prepartum diets and MR. Upregulated pathways included the PPAR signaling pathway, glycolysis/gluconeogenesis and oxidative phosphorylation whereas downregulated pathways included genes involved in inflammatory processes and ubiquitin-mediated proteolysis.Cardiomyopathy and tight junction pathways were upregulated in calves fedHLA-MR, but were downregulated if calves born from SFA- or EFA-fed dams. Calves born from fat-fed dams prepartum produced more milk at first lactation,possibly mediated by fetal programming. The last study aimed to determine the requirement of LA for preweaned calves. Heifers gained more BW in the first 30d of life as intake of LA increased. Wither and hip growth was greater in calves consuming LA exceeding 0.206 g/kg of BW0.75 during the 60-d study.Several markers of immunity were increased when LA was fed between 0.206and 0.333 g/kg of BW0.75.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Miriam Garcia Orellana.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Staples, Charles R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045027:00001


This item has the following downloads:


Full Text

PAGE 1

1 EFFECT OF SUPPLEMENTING ESSENTIAL FATTY ACIDS T O PREPARTUM HOLSTEIN COWS AND PRE WEANED CALVES ON CALF PERFORMANCE, METABOLISM, IMMUNITY, HEALTH AND HEPATIC GENE EXPRESSION By MIRIAM GARCIA ORELLANA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

PAGE 2

2 2012 Miriam Garcia Orellana

PAGE 3

3 To my loved parents: Maxi and Pascual, for their endless love and for all they taught me, not only with words but by examples. All I am and all I have achieved, have their hallmark. A mis queridos padres: Maxi y Pascual, por su infinito amor y por todo lo que me ensearon, no solo con palabras sino con ejemplos. Todo lo que soy y todo lo que he logrado tienen su inconfundible sello.

PAGE 4

4 ACKNOWLEDGMENTS I deeply thank my advisor Dr. Charles Staples for all of his support throughout my Ph D program I am very grateful to him for giving me the opportuni ty to pursue a Ph D degree under his guidance. I deeply appreciate the time he devoted to help me not only with academic and research topics but also his willingness to be a good listener providing great advice and good examples for living I also thank my supervisory committee members for their support and for inspiring me with the passion that they have as scientists and professors Specifically I thank Dr. Lokenga Badinga for his continuous encouragement to keep going under any circumstance s, Dr. Carlo we used to meet at the calf unit Dr. Gbola Adesogan for being such a friendly professor in the first class I took after arriving in the United States and also for allowing me to be become an un official member of his lab; and Dr. Jose Santos for his direct involvement in all of my research projects H e contributed to the design of my pro jects helped me with on farm research shared his scientific knowledge and reviewed my scientific writings A very special thank s goes to Dr. William Thatcher for his example of passion for gaining new knowledge, for his valuable guidance in analyz ing the microarray data and for his contribution to the writing of the corresponding chapter, all done without being an official member of my committee. Sincere thanks go to all student interns for their efficient and enthusiastic work at different point s during my studies namely Mauricio Favoretto, Rafael Marsola, Leonardo Martins, Armando Schlaefli, Pedro Bueno, Seth Jenkins and Yeong J. Jang. I also thank all of the dairy farm crew for their valuable help and for efficien tly solving m any miscellaneous issues. Special thanks go to the men and women at the university

PAGE 5

5 calf uni t, namely and more than in the experiments A s pecial t hank you goes to Dr. Fiona Maunsell for car ing for the health of the calves during my second study as if they were her own Thanks also go to Dr. Al an Ealy, Dr. Joel Yelich, Dr. Jeff Dahl, Dr. Klibs Galvo, and Dr. Jorge Hernandez for allowing me to work in their laborator ies T hank s also to Dr. Sergei Sennikov for his help with some chemical analyses to Joyce Hayen for her valuable help with the ins ulin assay to Jan Kivipe l to for her friendly answer to every question I had about equipment operation and for teaching me about fatty acid analysis. Thanks also go to Dr. Joel Brendemuhl and Joan n Fisher for all of their help completing the paper work req uired for gaining admi ssion to the Animal Sciences Ph D program and to the University of Florida for financial assistance as a graduate assistant Thanks also to the nicest administrat ors that I could ever meet namely Glenda Tucker, Sabrina Robinson and Shirley Levy for all of their help with many different things. Great appreciation goes out to all Animal Science graduate students I met during the years of my program It was always nice to be cheered up by their presence Special thank s go to my fellow graduate students, Leandro Greco for working with me every day during the first experiment and for helping me at every turn and to Ms. Dan Wang for her enthusiastic willingness to help with lab work and for her kind personal care. Also thank s go to Dr. J ae Shin for his involvement during the on farm animal work of my second study. Additional thanks go to Eduardo Ribeiro, Fbio Lima, Rafael Bisinotto, Suzgo Chapa, Dr. Sha Tao, Dr. Oscar Queiroz and Dr. Belen Rabaglino for their varied assistance with the animal studies, lab work, and data processing.

PAGE 6

6 I ow e a deep thank you to my Peruvian girl Dr. Kathy Arriola I am proud to have be en her friend for almost 20 years, for always being there to support me and for iola, who played the role of my parents while here. Thanks also go to my Peruvian boys Juan J. Romero and Miguel Zarate for their friendship and for being there when they were needed. Special thank s go to Dr. Milerky Perdomo for gifting me with her fri endship and for being such a good example of a brave women and mother. Thanks also go to all of my friends in Gainesville, e special ly Chaevien Clendinen, Tara Shakir, Ana Cabrera, Eduardo Alava, Erin Alava, Micheal Morgan and Emma Zapata They were key s ources of refreshment during my spare time these four years. Thanks also go to my aunts, uncles, and cousins for always keeping m e in their thoughts and prayers. Thanks to all of my friends that I left in Peru for their supportive friendship s never hinder ed by the distance. Almost last, but not less important, I deeply thank my parents for their immense and unconditional love, for support ing me at a ll circumstance s I am very proud of them T hey are my heroes. Deep thanks also go to my siblings Enrique, Ma rilu, Ramon and Efren for their love, friendship, complicity and emotional support. Thanks also go to my three nephews and five nieces, thinking of them was a balsamic cure during my times of homesick ness Most of all, I thank my Lord and Savior Jesus Christ for His incomparable love, for support ing me in at a ll time s even when I was walking far from him and for making me a better person little by little. Thanks also go to all my brothers and sisters from the coming me to this amazing Christian family and

PAGE 7

7 for their supportive prayers. Special thanks go to my pastor Aldo Mesa and his wife, for their spiritual support, care, and friendship.

PAGE 8

8 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 13 LIST OF FIGURES ................................ ................................ ................................ ........ 16 LIST OF ABBREVIATIONS ................................ ................................ ........................... 20 ABSTRACT ................................ ................................ ................................ ................... 24 C H A P T E R 1 INTRODUCTION ................................ ................................ ................................ .... 26 2 LITERATURE REVIEW ................................ ................................ .......................... 29 Overview of Fatty Acids ................................ ................................ .......................... 29 Nomenclature and Classification ................................ ................................ ...... 29 Sources ................................ ................................ ................................ ............ 31 Metabolism ................................ ................................ ................................ ....... 32 Essentiality ................................ ................................ ................................ ....... 36 Overview of Newborn Calf Immunity ................................ ................................ ....... 42 Innate Immunity ................................ ................................ ................................ 43 Passive Acquired Immunity ................................ ................................ .............. 46 Active Acquired Immunity ................................ ................................ ................. 50 Insulin and Growth Factors in Colostrum ................................ ................................ 53 Effect of Supplemental Fatty Acids on Passive Transfer ................................ ........ 55 Effect of Supplemental Fatty Acids on Total Fat and Fatty Acid Profile .................. 56 Colostrum ................................ ................................ ................................ ......... 56 Plasma ................................ ................................ ................................ ............. 58 Liver ................................ ................................ ................................ ................. 60 Effect of Supplemental Fatt y Acids on Preweaned Calves Performance ................ 62 Effect of Supplemental Fatty Acids during Pregnancy on Growth Performance and Hormonal and Metabolic Profile of Preweaned Calves ..... 63 Effect of Feeding Supplemental Fatty Acids to Preweaned Calves on their Growth Performance and Metabolic Profile ................................ ................... 67 Effect of Supplemental Fatty Acids Fed During Pregnancy on Offspring Health and Immunity ................................ ................................ ............................ 71 Effect of Supplemental Fatty Acids Fed to Preweaned Calves on Their Health and Immunity ................................ ................................ ............................ 73 Effect of Supplemental Fatty Acids on Hepatic Gene Expression ........................... 83 Regulation of Hepatic Peroxisome Proliferator Receptor .............................. 84 Regulation of Hepatic Sterol Regulatory Element Binding Protein ................... 86 Regulation of Hepatic liver X Receptor ................................ ............................. 88

PAGE 9

9 Regulation of Other Hepatic Receptors ................................ ............................ 90 Regulation of Hepatic Uptake and Binding of Fatty Acids ................................ 94 Regulation of Hepatic Fatty Acid Oxidation ................................ ...................... 95 Peroxisomal oxidation ................................ ................................ ............ 96 Oxidation ................................ ................................ .......... 97 Microsomal hydroxylation ................................ ................................ ....... 98 Regulation of Lipogenesis a nd Hepatic Steatosis ................................ ............ 99 Regulation of Glucose and Carbohydrate Metabolism ................................ ... 101 Regulation of Bile and Hepatic Cholesterol ................................ .................... 103 Regulation of Inflammation and Immune Response ................................ ....... 105 Effect on Oxidative Phosphorylation ................................ ............................... 106 Summary ................................ ................................ ................................ .............. 106 3 EFECT OF SUPPLEMENTAL ESSENTIAL FATTY ACIDS TO PREGNANT HOLSTEIN COWS ON COLOSTRUM FATTY ACID PROFILE AND CALF PASSIVE IMMUNITY ................................ ................................ ............................ 111 Background ................................ ................................ ................................ ........... 111 Materials and Methods ................................ ................................ .......................... 113 Experimental Design and Dietary Treatments ................................ ................ 113 Prepartum Body Weight, Feed Intake and Analyses ................................ ...... 114 Prepartum Ovalbumin Challenge and Assay for Bovine Anti OVA IgG .......... 115 Calving Management ................................ ................................ ..................... 116 Colostrum Feeding and Analyses ................................ ................................ ... 116 Blood Collection for Measures of Immunoglobulin and Protein Concentration ................................ ................................ .............................. 117 Estimation of Appropriate Passive Transfer and Efficiency of IgG Absorption 119 Statistical Analysis ................................ ................................ .......................... 119 Results ................................ ................................ ................................ .................. 121 Prepartum Cow Performance ................................ ................................ ......... 121 Immunoglobulin G Concentration and Fatty Acid Profile of Colostrum ........... 123 Transfer of IgG and Hormones by Feeding of Colostrum ............................... 124 Discussion ................................ ................................ ................................ ............ 127 Summary ................................ ................................ ................................ .............. 136 4 EFFECT OF SUPPLEMENTING ESSENTIAL FATTY ACID TO PREGNANT HOLSTEIN COWS AND THEIR PREWEANED CALVES ON CALF PERFORMANCE, IMMUNE RESPONSE AND HEALTH ................................ ..... 152 Background ................................ ................................ ................................ ........... 152 Materials and Methods ................................ ................................ .......................... 154 Prepartum Management ................................ ................................ ................. 154 Calves Dietary Treatments, Feeding Management and Analyses .................. 154 Housing, Body Weight and Immunizations ................................ ..................... 156 Calves Scoring for Health Assessment and Incidence of Health Disorders ... 157 Hormone and Metabolite Analyses ................................ ................................ 157 Markers of Immunity Analyses ................................ ................................ ....... 160

PAGE 10

10 Statistical Analyses ................................ ................................ ........................ 165 Results ................................ ................................ ................................ .................. 167 Plasma Fatty Acid Concentration and Profile ................................ ................. 1 67 Measures of Growth and Feed Efficiency ................................ ....................... 171 Metabolic and Hormonal Profile ................................ ................................ ..... 172 Incidence of Diarrhea and Poor Attitude ................................ ......................... 175 Blood Cell Population ................................ ................................ ..................... 176 Expression of Adhesion Molecule s and Phagocytic Activity of Neutrophils .... 177 Concentration of Acute Phase Proteins ................................ .......................... 178 Humoral and Cell Mediated Immune Responses ................................ ........... 178 Discussion ................................ ................................ ................................ ............ 179 Prepartum Supplementation of Fatty Acids Affects FA Profile and Immunity Measures of Calves ................................ ................................ .................... 179 Feeding Milk Replacer Enriched with Linoleic Acid Improved Growth, Feed Efficiency, and Immune Responses ................................ ............................ 187 Prepartum Supplementation of Fatty Acids Affects Calf Responses to a Linoleic Acid Enriched Milk Replacer ................................ .......................... 198 Summary ................................ ................................ ................................ .............. 199 5 EFFECT OF SUPPLEMENTAL ESSENTIAL FATTY ACIDS TO PREGNANT HOLSTEIN COWS AND THEIR PREWEANED CALVES ON CALF HEPATIC FATTY ACID PROFILE AND GENE EXPRESSION ................................ ............. 236 Background ................................ ................................ ................................ ........... 236 Materials and Met hods ................................ ................................ .......................... 238 Prepartum Management ................................ ................................ ................. 238 Calves Dietary Treatments, Feeding Management and Analyses .................. 238 Liver Biopsy ................................ ................................ ................................ .... 239 Calves Liver Fatty Acid Profile ................................ ................................ ........ 239 Total RNA isolation ................................ ................................ ......................... 240 Affymetrix Array Hybridization, washing, staining and scanning ..................... 241 Affymetrix Data Analysis ................................ ................................ ................. 241 Statistical Analysis ................................ ................................ .......................... 242 Results ................................ ................................ ................................ .................. 244 Liver Fatty Acid Content and Profile ................................ ............................... 244 Differential Expression of Genes in Liver ................................ ....................... 246 Enriched Ge ne Ontology Terms ................................ ................................ ..... 248 Enriched KEGG Pathways ................................ ................................ ............. 250 Heifers Productive and Reproductive Performance ................................ ........ 253 Discussion ................................ ................................ ................................ ............ 254 Regulation of Hepatic Total and Individual Fatty Acid Concentration ............. 254 Feeding of High Linoleic Acid in Milk Replacer Up regulated PPAR and its Target Genes ................................ ................................ .............................. 257 Feeding Fat Prepartum and High Linoleic Acid in Milk Replacer Upregulated PPAR Target Genes ................................ ................................ ................. 259 Feeding of Essential Fatty Acids Prepartum and High Linoleic Acid in Milk Replacer Enhanced Catabolic Processes and ATP Generation .................. 261

PAGE 11

11 Regulation of Carbohydrate Metabolism ................................ ........................ 264 Regulation of Protein Turnover ................................ ................................ ....... 265 Regulation of Inflammation and other immune processes .............................. 266 Feeding of Essential Fatty Acids Prepartum and High Linoleic Acid in Milk Replacer Improved Insulin Sensitivity ................................ ......................... 269 Fat and Fatty Acid Supplementation and its Risk and Prevention of Cardiomyopathic Diseases ................................ ................................ .......... 271 Prepartum Fat Feeding Influenced Future Adult Performance ....................... 273 Sum mary ................................ ................................ ................................ .............. 275 6 EFFECT OF FEEDING MILK REPLACER ENRICHED WITH INCREASING LINOLEIC ACID ON HOLSTEIN CALF PERFORMANCE, IMMUNE RESPONSE AND HEALTH ................................ ................................ ... 299 Background ................................ ................................ ................................ ........... 299 Materials and Methods ................................ ................................ .......................... 301 Enrollment and Management of Pregnant Cows ................................ ............ 301 Calving Management at Birth and Colostrum Feeding ................................ ... 302 Appropriate Passive Immune Transfer Identification ................................ ...... 302 Dietary Treatments, Feeding Management and Analyses .............................. 304 Body Weight and Immunizations ................................ ................................ .... 306 Calf Scoring for Health Assessment and Incidence of Health Disorders ........ 307 Hormone and Productive Metabolite Analyses ................................ ............... 308 Markers of Immunity Analyses ................................ ................................ ....... 310 Statistical Analysis ................................ ................................ .......................... 315 Results ................................ ................................ ................................ .................. 316 Measures of Growth and Feed Efficiency ................................ ....................... 318 Metabolic and Hormonal Profile in Plasma ................................ ..................... 318 Incidence of Diarrhea and Other Diseases ................................ ..................... 320 Blood Cell Populations ................................ ................................ ................... 321 Neutrophil Phagocytosis and Oxidative Burst ................................ ................. 323 Concentration of Acute Phase Proteins ................................ .......................... 323 Humoral and Cell Mediated Immune Responses ................................ ........... 324 Discussion ................................ ................................ ................................ ............ 326 Summary ................................ ................................ ................................ .............. 340 7 GENERAL DISCUSION AND CONCLUSIONS ................................ .................... 376 A P PE ND I X A LIST OF DIFFERENTIALY EXPRESSED GENES ................................ ............... 385 B DIFFERENTIALY E XPRESSED GENES FOR THE CONTRAST OF FAT ........... 408 C DIFFERENTIALY EXPRESSED GENES FOR THE CONTRAST OF FATTY ACIDS ................................ ................................ ................................ ................... 411

PAGE 12

12 D DIFFERENTIALY EXPRESSED GENES FOR THE CONTRAST OF MILK REPLACER ................................ ................................ ................................ .......... 414 E DIFFERENTIALY EXPRESSED FOR THE INTERACTION FAT BY MILK REPLACER ................................ ................................ ................................ .......... 416 F DIFFERENTIALY EXPRESSED FOR THE INTERACTION FATTY ACID BY MILK REPLACER ................................ ................................ ................................ 424 LIST OF REFERENCES ................................ ................................ ............................. 431 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 467

PAGE 13

13 LIST OF TABLES Table page 2 1 Common fatty acids terminology ................................ ................................ ...... 108 2 2 Fatty acid compositionof major sources of fatty acids in dairy cattle ................ 109 3 1 Ingredient c omposition of experimental diets fed to pregnant Holstein cattle starting at 8 weeks from expected calving date. ................................ ............... 138 3 2 Fatty acid profile of fat supplements fed to pregnant Holstein cattle sta rting at 8 weeks from expected calving date. ................................ ................................ 139 3 4 Mean concentrations of total, individual and group of fatty acids in colostrum of Holstein cattle ................................ ................................ ............................... 141 3 5 Passive immunity related parameters in calves born from Holstein cattle ........ 143 3 6 Concentrations of insulin and insulin like growth factor I in serum of calves. ... 144 3 7 Correlation coefficients among several variables in calves born from Holstein cattle. ................................ ................................ ................................ ................ 145 4 1 Ingredient and chemical com position of milk replacers and grain mix. ............. 201 4 2 Fatty acid profile of milk replacers and grain mix. ................................ ............. 202 4 3 Mean concentration of total plasm a fatty acids individual and group of FA before colostrum feeding in calves. ................................ ................................ .. 203 4 4 Mean concentrat ion of total plasma fatty acids, individual and group of FA expressed of calves fed milk re placer containing linoleic acid ......................... 205 4 5 Dry matter intake, body weight gain and feed efficiency of Holstein calves fed milk replacer containing linoleic acid. ................................ .............................. 20 7 4 6 Plasma concentrations of metabolites and hormones in Holstein calves fed milk replacer containing linoleic acid ................................ ................................ 209 4 7 Attitude and fecal scores and percent age of days with poor attitude and diarrhea in Holstein calves fed milk replacer containing linoleic acid. ............... 210 4 8 Mean co ncentration of blood cells and percentage of individual white blood cell s in Holstein calves fed milk replacer containing linoleic acid ...................... 211 4 9 Expression of adh esion molecules on surface of blood neutrophils and phagocytic activity of blood neutrophils as in Holste in calves ........................... 212

PAGE 14

14 4 10 Mean concentration of serum total protein, acute phase proteins, serum anti OVA IgG and interferon gamma produced in Holstein calves .......................... 213 5 1 Mean concentration fatty acid s in liver of Holstein ma le calves fed milk replacer containing linoleic acid ................................ ................................ ........ 279 5 2 Functional annotation clusters for main effects of upregulated enriched GO terms in liver of Holstein male calves ................................ ............................... 281 5 3 Functional annotation clusters for the interaction fat by milk replacer of upregulated enriched GO terms in liver of Holste in male calves ...................... 282 5 4 Functional annotation clusters for the interaction fatty acid by milk replacer of upregulated enriched GO terms in in liver of Holstein male calves ................... 283 5 5 Functional annotation clusters for main effects of downregulated enriched GO terms in liver of Holstein male calves ................................ ......................... 284 5 6 Functional annotation c lusters for the interaction fat by milk replacer of downregulated enriched GO terms in liver of Holstein male calves .................. 285 5 7 Functional annotation clusters for the interaction fatty acid by milk replacer of downregulated enriched GO terms in liver of Holstein male calves .................. 286 5 8 Functional annotation chart for enriched upregulated KEGG pathways for main factors and interactions in live r of Holstein male calves ........................... 287 5 9 Functional annotation chart for enriched downregulated KEGG pathways for main factors and interactions in liver of Holstein male calves ........................... 288 5 10 Productive and reproductive parameter of Holstein heifers .............................. 289 5 11 Incidence and m ain causes of culling of Holstein heifer s ................................ 290 6 1 Ingredient and chemical composition of diet fed to nonlactating, pregnant Holstein animals. ................................ ................................ .............................. 342 6 2 Fatty acid profile of sources o f fat t y a ci ds emulsifi er and basal milk replacer .. 343 6 3 Ingredient and chemical composition of milk replacers and grain mix fed to preweaned Holstein calves. ................................ ................................ .............. 344 6 4 Passive immunity related measures of newborn male and female Holstein calves assigned to treatments with increasing amounts of linoleic acid .......... 345 6 5 Dry matter intake, body weight gain, and feed efficiency of preweaned male and female Holstein calves fed increasi ng amounts of linoleic acid ................ 346

PAGE 15

15 6 6 Wither and hip height and growth of preweaned male and female Holstein cal ves fed increasi ng amounts of linoleic acid ................................ ................. 347 6 7 Plasma concentrations of gl ucose, plasma urea nitrogen, B hydroxybutyrate total cholesterol, insulin, and insu lin like growth factor I of pr eweaned calves 348 6 8 Health scores and percentage of days with poor attitude, fever, diarrhea and nasal discharge of preweaned male and female Holstein calves ..................... 349 6 9 Incidence of diseases in preweaned Holstein calves fed increasi ng amounts of linoleic acid ................................ ................................ ................................ .. 351 6 10 Mean co ncentrati on of blood cel ls and perce ntage of individua l white blood cells in preweaned male and female Holstein calves. ................................ ...... 352 6 11 Phagocytosis, oxidative burst, and me an fluorescence intensity of neutrophils in periph eral blood of preweaned male and f emale Holstein calves ................. 353 6 12 Mean concentration of plasma acute phase proteins, serum anti OVA IgG, cytokines, and proliferation of whole blood cells in preweaned calves ............. 354 6 13 Skin fold change measured after 6, 24, and 48 h of intradermal injection of 150 ug of phytohaem agglutinin in preweaned male and female calves ............ 355

PAGE 16

16 LIST OF FIGURES Figure pa ge 2 1 Structural fo rmula of linoleic and li nolenic acid s ................................ ............ 110 3 1 Dry m atter intake by nulliparous an d parous Holstein cattle su pplemented with no fat, saturated fatty acids, or essential fatty acids ................................ 147 3 2 Bovine anti OVA IgG concentration in serum of Holstein nulliparous and parous Holste in cattle ................................ ................................ ....................... 148 3 3 Body weight at birth of calves born from Holstein cattle su pplemented with no fat, saturated fatty acids or essential fatty acids ................................ .............. 149 3 4 Concentrations of total IgG before feeding and aft er 24 to 30 h of colostrum feeding in serum of calves. ................................ ................................ ............... 150 3 5 Concentrations of i nsulin and IGF I in serum of calves born from Holstein cattle. ................................ ................................ ................................ ................ 151 4 1 Plasma concentrations of fatty acids in calves at 30 to 60 d of age ................. 214 4 2 Plasmatic concentrations of gluc ose in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age ............................ 215 4 3 Plasmatic concentrations of urea N in Holstein calves fed milk replacer containing l ow or high linolei c acid from 0 to 60 days of age ............................ 216 4 4 Plasmatic concentrations of and hydroxybutyric acid and none sterified fatty acids in Holstein calves fed milk replacer containing low or high linoleic acid. 217 4 5 Plasmatic concentrations of total cholesterol in Holstein ca lves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age. ............. 218 4 6 Plasmatic concentrations of insulin in Holstein calves fed m ilk replacer containing low or high linol eic acid from 0 to 60 days of ag e ............................ 219 4 7 Plasmatic concentrations of insu lin like growth factor I in Holstein calves fed milk replacer containing l ow or high linoleic acid from 0 to 60 days of a ge ....... 220 4 8 Serum tot al protein concentrations in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age. ........................... 221 4 9 Attitude score of Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age ................................ ............................... 222 4 10 Fecal score of Holstein calves fed m ilk replacer con taining low or high linoleic acid from 0 to 60 days of age. ................................ ................................ .......... 223

PAGE 17

17 4 11 Blood concentrat ions of red and white blood cells in Holstein calves fed milk replacer containing low or high linoleic acid fr om 0 to 60 days of age.. ............ 224 4 12 Blood c oncentrations of neutrophils and lymphocyte in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age.. ..... 225 4 13 Blood concentrations of monocytes and eosinophils in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age ....... 226 4 14 Blood concentrations of eosinophils in Holstein calves fed m ilk replacer containing low or high linoleic acid from 0 to 60 days of age ............................ 227 4 15 Blood concentrations of bas ophils in Holstein calves fed mi lk replacer containing low or high linoleic acid from 0 to 60 days of age ........................... 228 4 16 Blood concentrations of platelets in Holstein ca lves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age. ........................... 229 4 17 Hematocrit concentrations in Holstein calves fed mil k replacer containing low or high l inoleic acid from 0 to 60 days of age. ................................ .................. 230 4 18 Me and f luorescence intensity of n eutrophils positive to CD62L in Holstein calves fed mil k replacer containing low or high linoleic acid from 0 to 60 days. 231 4 19 Me an f luorescence intensity of pha gocytic neutrophils positive and concentration of phagocytic blood neutrophils in Holstein calves ..................... 232 4 20 Percentage of blood neutrophils undergoing phagoyctiosis in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age. 233 4 21 Plasmatic concent ration of acid soluble protein in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age .............. 234 4 22 Plasmatic concentration of haptoglobin and serum anti OV A IgG in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days 235 5 1 Concentrations of C12:0, C14:0 and C16:0 in liver of Holste in calves fed milk replacer co ntainin g low or high LA from 1 to 30 days of age. ........................... 291 5 2 Conc entrations of linoleic and linolenic acid, and their derivatives in liver of Holste in calves fed milk replacer containing low or hig h linoleic acid .............. 292 5 3 Venn d iagram of the upregulated differential expressed genes in liver of mal e calves fed milk replacer containing low or high linoleic acid ............................. 293 5 4 Venn d iagram of the downregulated differential expressed genes in liver of mal e calves fed milk replacer containing low or high linoleic acid ................... 294 5 5 Upreg ulated genes in the PPARA KEGG pathway ................................ ........... 295

PAGE 18

18 5 6 Upregulated genes in the adipocyto kine KEGG pathway ................................ 296 5 7 Upregulated and downregulat ed genes in the t ight junction KEGG pathway. .. 297 5 8 Downregulated genes in the l eukocyte transendothelial migration KEGG pathway. ................................ ................................ ................................ ........... 298 6 1 Body weight gain and milk replacer intake during the first 30 d of life of preweaned Holstein calves fed increased intake of linoleic acid ...................... 356 6 2 Averages daily wither and hip growth during first 60 d of life of preweaned Holstein calves fed increased intake of linoleic acid. ................................ ........ 357 6 3 Plasm a concentrations of glucose and urea N of preweaned Holstein calves fed increased intake of li noleic acid. ................................ ................................ 358 6 4 Pl asma concentrations of BHBA and total cho lesterol in preweaned Holstein calves fed increased intake of linoleic acid. ................................ ...................... 359 6 5 Plasm a concentrations of insulin an d IGF I in preweaned Holstein calves fed increased intake of linoleic acid. ................................ ................................ ....... 360 6 6 Total serum protein in preweaned Holstein calves fed increas ed intake of linoleic acid. ................................ ................................ ................................ ...... 361 6 7 Attitude and fecal average weekly scores of preweaned Holstein calves fed increased intake of linoleic acid ................................ ................................ ........ 362 6 8 Rectal temperature first 14 days of life of preweaned Holstein calves fed increased intake of linoleic acid. ................................ ................................ ....... 363 6 9 Red blood cells a nd hematocrit concentration in Holstein cal ves fed increased intake of linoleic acid. ................................ ................................ ....... 364 6 10 Concentrations of w hite blood cells in Holstein calves fed increased intake of linoleic acid.. ................................ ................................ ................................ ..... 365 6 11 Concentrations of n eutrophi l s and lymphocyte s in blood of Holstein calves fed increased intake of linoleic acid. ................................ ................................ 366 6 12 Concentrations of m onocyte s and eosinophi ls in blood of Holstein calves fed increased intake of linoleic acid ................................ ................................ ........ 367 6 13 Concentrations of b asophils and platelet s in blood of Holstein calves fed increased intake of linoleic acid. ................................ ................................ ....... 368 6 14 N eutrophil phagocytosis and m e an f uore sce nce int ens it y o f ne ut ro phi ls in Holstein calves fed increased intake of linoleic acid. ................................ ........ 369

PAGE 19

19 6 15 Acid Soluble protein and Haptoglobin concentration in preweaned Holstein calves fed i ncreased intake of linoleic acid ................................ ....................... 370 6 16 Serum Anti OVA IgG conc entrations in male and female preweaned Holstein calves fed increased intake of linoleic acid ................................ ....................... 371 6 17 Lymphocyte proliferation in whole blood cells of Holstein calves fed increased intake of linoleic acid. ................................ ................................ ....... 372 6 18 T umor necrosis factor and interferon gamma produced by stimulated whole blood cells of preweaned Holstein calves ................................ .............. 373 6 19 Interfe ron produced by stimulated whole blood cells of preweaned Holstein male and female calves fed increased intake of linoleic acid. .............. 374 6 20 Skin fold change after phytohaemagglutinin injection as percentage of the baseline measure at 30 and 60 days of life of Holstein calves ......................... 375

PAGE 20

20 LIST OF ABBREVIATION S AA Arachidonic acid ACC Acetyl CoA carboxylase ADF Acid detergent fiber ADG Average daily gain ALA linolenic acid APO Apolipoproteins APT appropriate passive transfer ASP Acid soluble protein BCS Body condition sco re BP Biological process BVD Bovine viral diarrhea BW Body weight CCO Coconut oil CD18 integrin, adhesion molecule CD62L L selectin, adhesion molecule ChREBP C arbohydrate regulatory element binding protein CLA Conjugated linoleic acid CO Corn oil CYP Cytochrome P450 CYP7A1 Cholesterol 7 DEG Differentially expressed genes DHA Docosahexaenoic acid DM Dry matter DMI Dry matter intake

PAGE 21

21 DPA Docosapentaenoic acid E S E n r i c h m en t s c o re EFA Essential fatty acids EPA Eicosapentaenoic acid FA Fatty acid FABP Fatty acid binding protein FAME Fatty acid methyl esters FASN Fatty acid synthase FcRn Neonatal Fc receptors for IgG FE Feed efficiency (gain/intake) FO Fish oil FXR Farsenoid X receptor GK Glucokinase GLA linolenic acid GO Gene ontology HNF Hepatonuclear facto Hp Haptoglobin IFN Interferon Ig Immunoglobulin IGF Insulin like growth factor IGFBP IGF binding protein IL Interleukin KEGG Kyoto encyclopedia of genes and genomes LCFA Long chain fatty acids LDL Low density lipoprotein

PAGE 22

22 LPS lipopolysaccharide LXR Liver X receptor MCFA Medium chain fatty acids MDH Malate dehydrogenase MF Molecular function MHC Major h istocompatibility complex MLX Max like protein X MR Milk replacer MUFA Monounsaturated fatty acids n 3 3 fatty acids n 6 Family of 6 fatty acids NDF Neutral detergent fiber NEFA Nonsterified fatty acids NFkB Nuclear factor kB NRC The National Research Council OA Oleic acid OVA Ovalbumin PBMC Peripheral blood mononuclear cells PHA Phytohaemagglutinin PI3 Parainfluenza 3 PK Piruvate k inase PPAR Peroxisome proliferator receptor PUFA Polyunsaturated fatty acids rBST recombinant bovine somatotropin RXR Retinol X receptor

PAGE 23

23 SAO Safflower oil SCFA Short chain fatty acids SFA Saturated fatty acids SO Soybean oil SREBP Sterol regulatory elemen t binding protein STP Serum total protein TCR T cell receptor Th T helper cell TNF Tumor necrosis factor VLDL Very low density lipoprotein

PAGE 24

24 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Pa rtial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF SUPPLEMENTING ESSENTIAL FATTY ACIDS TO PREPARTU M HOLSTEIN COWS AND PRE WEANED CALVES ON CALF PERFORMANCE, METABOLISM, IMMUNITY, HEALTH AND HEPATIC GENE EXPRESSION By Mi riam Garcia Orellana December 2012 Chair: Charles R. Staples Major: Animal Sciences A series of experiments were conducted to examine the effect of supplementing essential fatty acids (FA) to prepartum Holstein cattle and newborn calves. The overall obj ective was to evaluate the effect of increasing intake of linoleic acid (LA) during the preweaning period on overall calf performance. In the first study prepartum cattle were fed one of three supplements, namely control (no fat), hydrogenated FA (SFA) or Ca containing essential FA (EFA). Colostrum FA profile of dams fed EFA reflected the concentration of LA in the fat supplement. Colostrum from nulliparous heifers was a better source of n 3 FA. Calves born from dams fed SFA had greater serum concentrations of total Immunoglobulin G (IgG), but efficiency of IgG absorption did not differ. In same study 96 Holstein cattle were fed prepartum the same supplements as in experiment 1 and newborn calves were fed milk replacer (MR) of low LA (LLA) or high LA (HLA). Feeding SFA prepartum increased grain intake and average daily gain (ADG) without improving feed efficiency (FE) of calves born from fat fed dams. Feeding HLA increased ADG, FE, plasma glucose and IGF I, LA and its derivatives in liver, blood lymphocytes, phagocytosis by neutrophils and interferon

PAGE 25

25 Expression of liver genes was strongly affected by the combinatio n of prepartum diets and MR. Up regulated pathways included the PPAR signaling pathway, glycolysis/gluconeogenesis and oxi dative phosphorylation whereas down regulated pathways included genes involved in inflammatory processes and ubiquitin mediated proteolysis. Cardiomyopathy and tight junction pathways were up regulated in calves fed HLA MR, bu t were down regulated if calves b orn from SFA or EFA fed dams. Calves born from fat fed dams prepartum produced more milk at first lactation, possibly mediated by fetal programming. The last study aimed to determine the requirement of LA for preweaned calves. Heifers gained more BW in th e first 30 d of life as intake of LA increased Wither and hip growth was greater in calves consuming LA exceeding 0.206 g/kg of BW 0.75 during the 60 d study. Several markers of immunity were increased when LA was fed between 0.206 and 0.333 g/kg of BW 0.75

PAGE 26

26 CHAPTER 1 INTRODUCTION Reaching an appropriate growth rate and health performance of dairy calves before weaning that would allow to double the birth weight by weaning period and minimize the incidence of diseases is one of the primary goals of dai ry herd management After birth d airy calves are immediately removed from their dams and transferred to a different unit to initiate the preweaning period in which they spend six to eight weeks consuming milk or milk replacer. The preweaning period, which requires APT of immunity, is often considered as the most critical period of early life (Beam et al., 2009; Furman Fratczac, 2011). The newborn calf is completely dependent of immunoglobulin (Ig) supplied by colostrum consumption because the epithelio chor ial placenta of cows prevents transfer of Ig during the fetal period (Kehoe and Heinrichs, 2007). Establishment of APT is crucial to reduce neonatal morbidity, mortality, and strengthen calf immunity (Quigley and Drewry, 1998; Donovan et al., 1998). Moreov er APT has been associated with improved weaning and postweaning body weight (BW; Robison et al., 1988) and with greater milk production ( DeNise et al., 1989 ). Several studies have evaluated different nutritional strategies to improve calf performance. Fe eding high energy diets for rapid growth during the pre weaning period have reduced both the age to reach the target breeding weight and costs associated with raising replacement heifers (Radcliff et al., 2000; Raeth Knight et al., 2009). In addition, the optimized feeding management of heifers during the preweaning period can have a positive impact on future milk production. One kg increase in average daily gain increased milk at first lactation by 850 kg (Soberon et al., 2012).

PAGE 27

27 The role of essential fatt y acids (EFA) in the growth and health of preweaned dairy calves is poorly understood. Pioneer studies (Jenkins et al., 1985; Jenkins et al., 1986; Jenkins and Kramer, 1986) supplemented the milk replacer (MR) of newborn calves with different sources of fa t and reported that concentration of EFA in liver and plasma reflected the composition of FA in the MR but classical symptoms of EFA could not be reproduced. Other studies used preruminant calf hepatocytes, cultured with different FA to evaluate oxidative and gluconeogenic activity of the liver (Mashek et al., 2002; Mashek et al., 2003, Mashek and Grummer, 2004). The type of FA used to incubate liver of preaweaned calves did not affect propionic acid metabolism to produce glucose and cellular glycogen. Howe ver, regardless the type of FA, the formation of both glucose and glycogen were decreased when FA concentrations increased from 0.1 to 1.0 mM. Limited information has been generated regar ding the role that dietary EFA might have in modifying the expression of genes in liver. Strategic feeding of pregnant cows during late gestation has been documented as having a tremendous impact on the future life of their offspring ( Osgerby et al., 2002; me of calves during the fetal period could be due to epigenetic regulation as a consequence of maternal nutrition during fetal development or nutrition during the first year of life (Funston et al., 2010; Singh et al., 2010). Few studies have evaluated the strategic supplementation of prepartum diets with EFA on the future life of their offspring. The present dissertation begins with an overview of the roles of fatty acids ( FA ) in calf metabolism and the calf immune system, including information from the mo st relevant and/or available studies evaluating the effect of fat supplementation on calf

PAGE 28

28 immunity and liver metabolism. Chapter 3 describes an experiment that was aimed to evaluate the effect of supplementing calcium salts of FA enriched in EFA on colostr um FA profile and production of total IgG and how that colostrum affected APT of calves born from those dams. The objective of Chapter 4 was to evaluate the effect of feeding EFA to dams during late gestation and to calves in their preweaning diets on calf growth, health, and immune responses. In Chapter 5, the liver FA profile and global gene expression of calves from Chapter 4 was evaluated. Chapter 6 is a second in vivo study that aimed to determine the requirement of linoleic acid (LA) of Holstein calv es during the preweaning period. Calves were fed a milk replacer with increasing concentrations of linoleic acid and the potential LA requirement was evaluated in terms of growth, health, and immune responses. The final chapter is a general conclusion and discussion of the major findings of the aforementioned studies.

PAGE 29

29 CHAPTER 2 LITERATURE REVIEW Overview of Fatty Acids Lipids are more than just high energy provider molecules. Their composition is as complex as proteins which are building blocks of amino acids The building blocks of the most common structure of lipids, triglycerides, are individually different fatty acids (FA) that are a ttached to a glycerol backbone. The lipid FA composition varies according to different sources such as animal or vegetab le origin. Although lipids of animal origin tend to have greater proportions of saturated FA (SFA) and those from vegetable origin tend to have a greater proportion of unsaturated FA, there are some fat that break this rule. For many years, lipids were con sidered simple inert molecules, with the single function of being a source of energy. However, classical pioneer studies found that specific FA were actually essential for animal health, reproduction and survival (Burr and Burr, 1929, 1930). Recent studies are focusing on identifying the different functions of the essential FA (EFA) and the mechanisms by which those FA perform. Some classical and new knowledge related to chemistry, sources, metabolism, and essentiality of FA will be detailed in the followin g sections. Nomenclature and Classification This section will introduce basic concepts of the most common nomenclature systems (IUPAC nomenclature, common or trivial names, and short terminology) used to classify FA according their chain length, u nsaturation number, and as insolubility in water but solubility in nonpolar solvents However eefe (2002) argued that even this definition is not an exact one beca use very short chain FA (SCFA,

PAGE 30

30 C1 C4) are soluble in water. The a uthor concluded that a more precise working definition is difficult given the complexity and heterogeneity of lipids. The IUPAC nomenclature is one of the systematic nomenclatures regulated by internationally accepted rules agreed on by chemists and biochemists (Gunstone, 1996). Under this nomenclature, the FA is named after the parent hydrocarbon, for example an 18 carbon FA is named as octadecanoic (Figure 2.1). Double bonds are described u considered as carbon number 1. A FA of 18 carbons with one double bond is named octadecenoic acid and one with 2 double bonds as octadecadienoic acid and so on. The double bond posit octadecenoic acid or simply 9 octadecenoic acid). The cis/trans terms are used to describe the geometric positions of double bonds. In the cis configuration adjacent hydrogen atoms are located on the sam e side of the double bond whereas in the trans configuration they are located on opposite sides (Gurr et al., 2002). Common (trivial) names were originally given before the chemical structure of the FA were elucidated and often were chosen to indicate the source of FA and are still used widely (Gunstone, 1996). Some examples of those n ames are palmitic acid (from linolenic acid (ALA, from linseed oil) and arachidonic acid (AA, from groundnut oil, Arachis hypogea ). Trivial names are not indicative of structure and can result in confusion when a name is assigned to a particular FA such as bovidic acid. The mor e carbons and double bonds a FA possesses, the more difficult a trivial name becomes, and more preferred are the IUPAC names. Good examples are eicosapentaenoic aci d (EPA) and docosahexaenoic

PAGE 31

31 acid (DHA) since they can have different isomers. However for convenience, EPA refers to the c 5,c 8,c 11,c14,c 17 isomer whereas DHA refers to the all cis 4,7,10,13,16,19 Systematic names for FA are too cumbersome for general use and shorter alternatives are used widely (Scrimgeour, 2005). One way to shorten the names are by using numbers in an abbreviated form such as 18:2 for octadienoic acid. But to better describe the isomeric form, other descriptor s have to be added such as 18:2 (9, 12), 18:2 (9c, 12c), and 18:2 (n 6). All of these refer to the same FA. The first number indicates the position of the double bonds in the C18 chain with reference to the carboxyl end counted as C1. The second formula confirms the cis configuration of the double bonds and the third one describes the FA in Greek terminology (Figure 2.1), which starts counting the carbon from the methyl group and describing this carbon as carbon (or n carbon) thus n 6 means that the first double bond is at carbon six counting from the methyl group (Gunstone, 1996). The (n) abbreviation or symbol is the most popular because of its simplicity and because most of the FA of nutritional importance can be named, but holds some limitations such as: cannot be used for FA with trans configuration, all double bonds can only be in the methylene interrupted position, and FA cannot have additional functional groups or have double bond systems 1). Sources A vast variety of v egetable and animal fat sources are available (table 2.2) for feeding ruminants such as oilseeds, rendered fats, purified vegetable oils, marine oils, and ruminally protected fats (e.g. hydrogenated FA, calcium salts of FA), with the latter being modified to prevent ruminal microbe metabolism. In preweaned calves, fat is an

PAGE 32

32 important source of energy. For economic reasons, milk fat is rarely used in commercial milk replacers (MR). Alternatively vegetable and animal fats are used commonly. Animal fat source s are tallow lard, and white grease. Vegetable oils such as coconut and palm also are used but vegetable and marine oils that pro vide long chain polyunsaturated FA (PUFA) are of minimal inclusion. The most common form of lipid in fats and oils is glycero lipids, which are essentially triglycerides (TG), accompanied by small amounts of phospholipids, mono glycerides, di glycerides, and sterols or sterol esters. The FA commonly found in TG are SFA (of varied length chain), monounsaturated FA (MUFA, mostly > 12 carbons), or PUFA (> 17 carbons) (FAO, 2010). All naturally occurring PUFA are in the cis 3 and n 6 being the most important families in terms of commonality of occurr ence and animal health and nutrition. Specifically LA (n 6) and ALA (n 3) are the only 2 recognized EFA whose functions will be discussed in detail later. Those 2 FA are parents of other FA which can be synthesized by elongation and desaturation enzymatic processes to generate family members of the same n group (FAO, 2010; Eastridge, 2002). The richest sources of LA are oil containing seeds such as safflowers, sunflowers, cotton, corn, and soybeans whereas the richest sources of ALA are canola and linseed oil ; marine fats are rich sources of very long chain PUFA such as EPA and DHA. Metabolism For dietary fats to be used by the body they must be metabolized in the lumen of the small intestine (or in prior compartments in ruminants). The digestion products s hould pass through the gut wall and be resynthesized in the intestinal epithelial cells

PAGE 33

33 and packaged for transport in the blood stream (Gurr et al., 2002). The composition of lipids entering the duodenum in ruminant cattle differs from the composition in n onruminant calves. This difference is due to the initiation of lipid metabolism in the forestomach of ruminant cattle whereas in nonruminant calves very little metabolism occurs before lipids enter the small intestine. Salivary lipase can begin to act on dietary TG upon ingestion (Bauchart, 1993). Lipid metabolism in ruminants is unique because the rumen compartment holds feeds for extended periods of time for microbial digestion prior to delivery of feeds further down the digestive tract where mammalian d iges tion occurs. However in newborn calves, lipid metabolism takes place just as it happens in nonruminants because their rumen has not developed a microbial population nor anatomical maturity. Metabolism of TG by lipases of ruminal anaerobic microbes inv olves an extensive hydrol i zation leading to the formation of free FA (FFA) which are subjected to partial hydrogenation by microbial hydrogenases. Stearic acid is the final product of a complete hydrogenation of 18 carbon FA. However what most commonly occ urs is an incomplete hydrogenation resulting in the formation of intermediate products of hydrogenation such as cis and trans isomers of monoenoic FFA (ie. C18:1 n 9 and C18:1 n 7) and isomers of PUFA such as conjugated LA (CLA) (Hocquette and Bauchart, 19 99). Hence the final product of microbial hydrolysis and biohydrogenation is a pool of FFA that are far more saturated than that of the dietary FA. In addition to long chain FFA arriving at the small intestine, fats may also include dietary TG escaping mic robial hydrolysis, microbial phospholipids (containing odd and branched chain FA), and phospholipids from bile and sloughed intestinal endothelial cells flowing toward the duodenum (Hocquette and

PAGE 34

34 Bauchart, 1999; Drackley and Andersen, 2006). Feeding large quantities of fat can have detrimental effects on microbial activity and animal productivity due to an inhibitory effect on cellulolytic microorganism that can depress fiber digestion (Eastridge, 2002). Various techniques of lipid protection such as lipid encapsulation and saponification of long chain FA have been developed to limit the extent of ruminal lipid hydrogenation and possible disturbances in fermentation (Hocquette and Bauchart, 1999). In preruminant calves, lipid digestion starts with pregastric lipases secreted by the specific for the 3 position of the glycerol carbon chain holding the FA which then releases only 1 FA per TG whereas Villeneuve and coworkers (1996) r eported a nearly similar pregastric lipase activity at positions 1 and 3 with preferential release of SCFA and medium chain FA (MCFA) from ingested TG. All dietary fat, including products of pregastric lipase digestion, arrive to the abomasum where they ar e emulsified by physical agitation and mixture with HCl (Drackley, 2008). The coagulation of milk casein in the abomasum is critical to slow down the movement of milk from the abomasum and increase the efficiency of the digestive process in the small intes tine of suckling calves. This results in a greater retention time of dietary TG which delays the postprandial increase of lipids in circulation (Hocquette and Baunchart, 1999; Guilloteau et al., 2009). Milk fat delivered to the abomasum undergoes some dig estion by pregastric lipase which remains active in the acid conditions of the abomasum (Drackley, 2008). After leaving the abomasum, the end products of gastric digestion pass into the duodenum, coming in contact with gall bladder and pancreatic secretion s.

PAGE 35

35 During the first month of life, pancreatic enzyme activities increase by 50 to 160% for most enzymes. Pancreatic lipase activity increases with age but this enzyme cannot express its full activity in older preruminants since colipase is a limiting fact or. This explains why lipids are sometimes poorly utilized in older preruminant calves (Guilloteau et al., 2009). Pancreatic lipase, in the presence of colipase and bile salts, hydrolyzes diglycerides and the remaining TG to 2 monoglycerides and FFA. Bile salts and 2 monoglycerols aid in the emulsifications of lipids and micelle formation. Micelles migrate to the brush border of the small intestine and facilitate absorption of FFA and monoglycerides through specific FA binding proteins (FABP) located in t he membrane of the enterocytes (Drackley, 2008; Hayashi et al., 2012). The transportation routes that a FFA can take will depend on its chain length. The FFA bound to albumin, specifically in the portal vein, by which MCFA arrive to liver and are oxidati on Their products are transferred to the Krebs cycle or utilized for synthesis of ketone bodies (Sato, 1994). On the other hand, long chain FA (LCFA) are reconverted to TG and packaged into lipoproteins, primarily chylomicrons, which are secreted from the cells into the extracellular space, where they are picked up by the lymphatic system and delivered to the vena cava. In this way, dietary FA are delivered to peripheral tissues for their use (Drackley, 2008). Apolipoproteins of chylomicrons are characteri zed by the prevalence of apolipoprotein B48 (APO B 48 ) with minor peptides of the APO C family and with variable amounts of APO A (Bauchart, 1993). Small amounts of very low density lipoproteins (VLDL) are synthesized in the small intestine but rather synt hesized by the liver The VLDL account for ~5% of total lipoproteins in preruminant calves, with

PAGE 36

36 APO B100 as the main APO constituent ( Wang et al. 2012 ). Lipoprotein lipase present on the surface of endothelial cells of capillary vessels hydrolyze TG in th e chylomicron and VLDL, facilitating the release of FFA and monoglycerides. Hence each of these compounds can enter the peripheral tissues. Tissues with high lipoprotein lipase activity in growing ruminants include adipose tissue, skeletal muscle, and hea rt whereas the remnant is preferentially taken by hepatocytes (Drackley, 2005). The liver secretes metabolized lipids in various forms such as acetate, ketone bodies, and lipoproteins containing TG, but the secretion of TG in VLDL is limited. As a conseque nce, young or adult ruminants are more prone to develop steatosis. This condition is primarily due to the processing and storage of TG rather than to de novo synthesis of TG which are actually lower in ruminant hepatocytes (Hocquette and Bauchart, 1999). Metabolism of FA in liver is finely regulated by transcription factors such as peroxisome proliferator activator receptor (RXR), liver X receptor (LXR), and sterol regulatory element binding protein (SREBP). Their role in syn oxidation of lipids will be discussed in detail in coming sections. Essentiality Using rats, Burr and Burr (1929) aimed to identify the effect of fat free diets on rats. Rats fed fat free diets developed a condition characterized by skin and tail lesions accompanied by hair loss, impaired reproduction, increased trans epidermal water loss, and weight loss. All these conditions were reversed when rats were fed diets of 2% lard (~10% LA). In a follow up study, Burr and Burr (1930) p urified the source of FA provided

PAGE 37

37 of fat deficiency. Better recovery was documented when rats were fed oils rich in LA rather than when fed purified LA. At that time, authors postulated that LA was a dietary essential, however the better responses obtained when linseed oil was fed already was shedding light on the essentiality of ALA pr e sent in the aforementioned oil Two years later Burr and coworkers (1932) reevaluate the supplementation of other individual FA and concluded that LA and ALA were similarly effective in enhancing recovery of rats fed free fat diets and that the mixture of LA and ALA was more effective than each individual FA per se. A study from Cunningham and Loosli (1954) did not support the essentiality of LA in the early life of calves fed fat free synthetic milk as observed in rats by Burr and Burr (1929, 1930) Calves developed leg weakness and muscular twitches within 1 to 5 wk of age and died if a source of fat was not supplied. Lard or coconut oil fed at 2% (wet basis) of milk prevented the appearance of symptoms. Hence authors concluded that in the first 5 wk of li fe, body reserves for EFA were enough to prevent deficiency of LA and ALA but that a source of fat was still needed during the first wk of life. On the other hand, Lambert et al. (1954), aiming to replicate studies by Burr and Burr (1929, 1930), fed fat fr ee MR to dairy calves. Authors reported that calves fed fat free milk had a marked retardation in BW gain, signs of scaly dandruff, dry hair, excessive loss of hair on the back, shoulders and tail, and diarrhea. Among the lipids that prevent development of or promoted prompt recovery from all the mentioned signs of EFA deficiency were butter oil, hydrogenated soybean oil plus lecithin, and mixed methyl esters of OA and LA. Differences among the various dietary groups in the plasma concentrations of LA and A A were small and the values for LA were low in all instances.

PAGE 38

38 Early studies reported that rats fed diets lacking ALA apparently grew and to ALA (Sander, 1988). Tinoco et al. (1978) fed diets free of ALA to rats and did not observed any signs of fat deficiency or disease but concentrations of ALA derivatives in certain tissues were decreased markedly. One year later in a review paper, Tinoco et al. (1979) conclu ded that if ALA was essential. Its role was focalized at the retina and brain level where ALA was found in greater concentrations. Later studies reviewed by Innis (1991) clearly demonstrated that ALA derivatives have a critical role in brain development during the fetal period and in early life. In an attempt to determine the requirement of LA in rats, using a requirement criteria for organisms to maintain constant concentrations of AA in different organs, Bourre et al. (1990) fed female rats increasing amounts of LA (from 0.15 to 3.2 g of LA/100 g of diet). They reported that dietary requirements for LA varied from 0.15 to 1.2 g of LA/100 g of diet, depending on the organ and the nature of the tissue FA (ie. constant concentrations of AA were found in nerve structu res when at least 0.15 g of LA/100 g of diet was fed, in testes and muscle with LA fed at 0.3 g/100 g of diet, in kidney with LA fed at 0.8 g/100 g of diet, and in liver, lung, and heart with LA fed at 1.2 g/100 g of diet). Authors concluded that the minim um dietary requirement ensuring constant concentrations of AA in all tissues and organs was estimated to be about 1.2 g of LA/100 g of diet. The essentiality of LA and ALA is clearly accepted currently. Although several studies have tried to determine po tential requirements, so far, only laboratory animals such mice and rats have determined requirements for LA but not for ALA (NRC, 1995)

PAGE 39

39 For other species, particularly humans, some recommendations have been released, but these recommendations most of the time do not focus on the actual role of individual FA but consider them as a group (e.g. n 6 FA) or even consider the EFA as a propor tion of one to another (e.g. LA:ALA or n 6 :n 3), a generalization that has led to a vast debate and controversy in recent years. Czernichow et al. (2010) reviewed studies reporting different effects of n 6 FA, particularly those reducing the risk of cardiac diseases. Authors considered that intakes of n 6 ideally above 10% of the total energy appear justified. However Ramsden et al. (2010) evaluated the same studies reviewed by Czernichow et al. (2010) but analyzed them in a different manner. They concluded that recommendations of Czernichow et al. (2010) for a beneficial effect of increasing LA was done considering the feedin g of diets rich in n 6 FA but that also included n 3 FA. He nce it is unlikely that increased intake of n 6 per se will reduce cardiac disease but may actually increase the risk by exacerbating the inflammatory process during cardiac and heart disease Ca lder and Deckelbaum (2011) discussed the two previous contradictory reviews and concluded that Ramsden et al. (2010) made a better assessment of assigning FA to their parent n group. However authors criticized both studies saying that they used FA termino logy too loosely, such as considering PUFA, n 6 PUFA and LA as common interchangeable terms. The lack of clear distinction among terms could lead to inaccurate statements and wrong advice. Authors recommended that advice should be given in terms of specifi c FA, based on the consideration that every FA has its own role and functionality. In an attempt to evaluate the ratio n 6:n 3 as a valid measurement of EFA requirement, Harris (2006) reviewed available literature and concluded that there is no

PAGE 40

40 evidence t hat lowering intake of n 6 FA (which will reduce the ratio) will result in a reduced risk of cardiac diseases, suggesting rather to focus on increasing the intake of n 3 without considering the n 6:n 3 ratio. Similarly a report by Stanley et al. (2007) sum marizing the conclusions of a workshop gathered by the United Kingdom Food Standards Agency concluded that the n 6:n 3 ratio is not a useful concept. Calder and Deckelbaum (2011) recommended to avoid discussing requirements or biological function of FA in terms of the ratio n6:n3 under the consideration that every individual FA within the n 6 and n 3 groups is not biologically equivalent to the others, and also that both groups of FA do not have complete opposite functions. Regarding a differential requir ement of LA due to gender, Greenberg et al. (1950) concluded that male rats had a daily requirement of LA between 50 to100 mg whereas that for females was 10 to 20 mg/d. Authors based their conclusion on the growth rate of rats fed different amounts of LA as compared to the growth of rats fed a fat free diet. Authors also pointed out that LA intake over 50 mg/d in female rats led to a decrease in BW gain. Some year s later, Pudelkewicz et al. (1968) evaluated the potential requirement of LA in rats by measur ing BW gain, skin health, and the triene:tetraene ratio (< 4) to determine EFA status The main factor leading to the recommended LA requirement for male or female rats (1.3 and 0.5% of dietary calories, respectively) was the C20:3 n 9 to AA ratio. In all evaluated tissues (liver, hearth and erythrocytes) and in plasma, the intake of LA that allowed a C20:3 n 9 to AA ratio of 0.4 or less was about 2.5 greater in males than females, with liver having the greater requirement Authors also reported that feedin g female rats with LA over 1.2% of calories increased the incidence of skin lesions and BW gain was reduced. For male rats, all amounts tested

PAGE 41

41 (up to 4.7% of calories) did not have negative impacts. The findings of Greenberg et al. (1950) and Pudelkewicz e t al. (1968) were the basis for the current recommendation of LA intake in growing rats by the NRC (1995). Similarly Nikkari et al. (1995) reported greater proportions of LA in cholesterol ester but a lower proportion in its derivatives in cholesterol este r and phospholipids fractions. On the other hand, other authors working with humans have not found an effect of gender when evaluating the proportion of FA in different fractions of serum phospholipids (Antar et al., 1967); in fractions of serum cholestero l ester, phospholipids and TG (Holman et al. 1979); or in plasma TG, FFA, different phospholipids fractions, or total phospholipids fraction in red blood cells (Manku et al., 1983). Moreno et al. (2006) r e ported that the FA profile of fat depots in pre a nd postweaning beef heifers had greater proportions of MUFA (primarily OA) but that proportions of SFA and PUFA did not differ when compared to males. Recently Dervishi et al. (2012) aimed to evaluate the FA profile of intramuscular fat in suckling lambs f rom ewes fed one of two diets with different FA profiles. Authors reported a differential effect of diet on some FA but gender did not affect the proportion of any FA in intramuscular fat. The question of essentiality of LA and ALA has been answered in di fferent studies in the last 80 years. H owever specific requirements of EFA of all livestock species still need to be determined. Some studies performed in rats have pointed out a differential requirement due to gender but more studies are needed to confir m this statement particularly if the observed differential effect of gender seen in rats held true for preruminant calves.

PAGE 42

42 Overview of Newborn Calf Immunity The immune system is a versatile defense system that has evolved to protect multicellular organism s from invading foreign pathogens. It is composed of different cells and molecules capable of recognizing and eliminating an unlimited variety of foreign invaders acting together in a dynamic network. The immune molecules and cells are grou ped into two sys tems, namely, innate and adaptive (acquired) immunity, which interact and collaborate to protect the body (Kind et al., 2007). Invader pathogens must first overcome numerous surface barriers, such as skin, enzymes and mucus that can have direct antimicrob ial activity or physical barriers to prevent attachment of the microbe. The keratinized surface s of the skin or the mucus lined body cavities are ideal habitats for most organisms; hence microbes must breach the ectoderm. Any organism that breaks through t his first barrier encounters molecules and cells of the two immune defense systems, the innate and acquired immune responses (Delves and Roitt, 2000). The newborn calf is born into an environment populated by a vast amount of pathogens such as bacteria, vi ruses, and parasites with the capacity to overtake the pathogenic invasion, because it has the ability to recognize and eliminate different pathogens. However, the immune through interaction with infectious agents. Different factors such as antibodies obtained from intake of colostrum provide assistance during the nai ve phase of calf immunity, as a calf ages its interactio n with pathogens through vaccination or natural encounters lead to a mature response (Woolums, 2010).

PAGE 43

43 Innate Immunity The innate immune system developed early in evolution consists of all immune defenses that lack memory. Thus a common feature of innate re sponses is that they remain unchanged however often the antigen is encountered (Delves and Roitt, 2000). Given the limited exposure to antigens in utero and the nai ve adaptive system, newborns must rely on their innate immune system for protection to a s ignificant extent. The tasks of this system are to shield the body from microbial invasion, reducing the number and virulence of microorganisms, and to coordinate and instruct the acquired immune responses (Levy, 2007). Responses to pathogen invasion initi ates within minutes to hours with the activation of the innate immunity system. Most components of this system are present before the onset of infection and constitute a set of disease resistance mechanisms that are not specific to a given pathogen. Innate immunity is composed of cellular and molecular components (soluble factors) that recognize classes of molecules peculiar to frequently encountered pathogens (Kindt et al., 2007). The main innate immune response is an acute inflammation as a response to in fection with pathogens in which cells and molecules of the immune system move into the affected tissue (Delves and Roitt, 2000). The cellular components of the innate immune system originate in the bone marrow. These include granulocytes (neutrophils, eosi nophils, and basophils), macrophages, dent ritic cells, natural killer cells, and T lymphocytes. Macrophages possess receptors for carbohydrates that are not normally exposed on the cells of vertebrates, such as mannose, and therefore can discriminate between foreign and self molecules (Delves and Roitt, 2000). Macrophages and neutrop hils have receptors for

PAGE 44

44 antibodies and complement, which coat the microorganism and lead to enhanced engulfment (phagocytosis) of these microorganisms. Dentritic cells are localized in different tissues and constantly sample the environment to identify inf ectious agents when they first encounter the host (Woolums, 2010). Dentritic cells have surface receptors capable of distinguishing pathogen associated molecular patterns on the surface of microorganisms that activate them, and once activated, they become antigen presenting cells migrating to the local draining lymph node, where they present antigens to T cells (Delves and Roitt, 2000). A principal role of eosinophils is to fight against parasites. Eosinophilia has been related to an increased response aga inst parasites (Ganheim et al., 2007). Recent studies indicate that basophils, in addition to releasing histamine as an allergic response, also can produce a vast array of effector molecules such as cytokines and might aid the maintenance of a Th2 cytokine dependent immunity (Siracusa et al., 2010). Natural killer cells are especially important in fighting viral infection and cancer; T lymphocytes can secrete cytokines that modify the function of oth er immune cells and can kill host cells infected with vi ruses, bacteria, or parasites (Woolums, 2000). Soluble factors of the innate immunity include the many proteins of the complement system, enzymes such as lysozyme and proteins such as lectins and defensins, which bind non specifically to various classes of molecules typical of infectious agents. Once bound, the soluble factors may impair or kill the infectious agent (Woolums, 2000). Moreover, activation of the complement cascade generates highly reactive and powerful activation products with chemotactic, i nflammatory, and cytotoxic activities which are key features for initiation of the inflammatory process

PAGE 45

45 (Zipfel, 2009). Acute phase proteins are other soluble factors of the innate immunity system. Their concentrations in plasma increase rapidly in respon se to infection, inflammation, and tissue injury and are commonly used as markers or inflammation (Delves and Roitt, 2000). Cytokines constitute another group of soluble mediators, acting as messengers both within the immune system and between the immune s ystem and other systems of the body, forming and integrated networks highly involved in regulation of immune responses (Delves and Roitt, 2000). The main feature of innate immunity after pathogen invasion is to trigger an acute inflammatory response. Compl ement proteins coat the surface of pathogens and also serve as chemoattractants of neutrophils. Vascular permeability is increased due to release of histamine and complement proteins. In parallel, substances released by p athogens and damaged tissues up regu late expression of adhesion molecules on inflamed endothelium, alerting cells, such as neutrophils, of the presence of infection. Circulating neutrophils recognize adhesion molecules expressed on the endothelium surface through its receptor L selectin (CD6 2L) initiating the process of rolling along the vessel wall and becoming activated. Once activated by chemoattractants and chemokine, they rapidly shed CD62L from their surface and replace them with another adhesion molecule named integrin (CD18). This one primarily binds to intracellular adhesion molecule 1 expressed on inflamed endothelium under the influence of inflammatory mediators. The activated neutrophils pass through the vessel walls, moving up the chemotactic gradient t o accumulate at the site of infection, where they are well placed to phagocyte pathogens and kill them by production of toxic intracellular molecules including reactive oxygen species hydroxyl radicals, hypochlorous acid, nitric

PAGE 46

46 oxide, antimicrobial catio nic proteins and peptides, and lyzosyme (Delves and Roitt, 2000; Ley et al., 2007). Chase et al. (2008) reviewing the competence of cells and soluble factors of innate immunity in newborn calves, reported the following: a) complement activity at birth is a pproximately 50% of that in adult cows, increasing gradually and rising to ~50% of adult levels by 4 wk of age; b) the number of circulating neutrophils around birth is approximately 4 times higher than in 3 wk old calves; c) neonatal neutrophils and macro phages have reduced phagocytic ability but their capacity is increased after the ingestion of colostrum; d) by 1 wk of age, neutrophils are functional and able to mount an effective response, improving gradually to adult levels by 5 mon of age; and e) the number of circulating natural killer cells is also lower at 1 wk of age (3% of total lymphocytes), increasing to 10% by 6 wk of age. Passive Acquired Immunity The transfer of Ig from the dam to the neonate is termed passive transfer in the majority of spe cies and the transfer of Ig starts occurring during the fetal period (Weaver et al., 2000). The exception is ruminant animals which deliver no Ig to the neonate prepartum. Therefore the newborn calf is completely dependent on the Ig supply from colostrum b ecause the epithelio chorial placenta of cows prevents transfer of Ig during the fetal period (Kehoe and Heinrichs, 2007). Colostrum is the first secretion of the mammary gland and the first feed offered to newborn calves. In addition to providing the neede d amount of Ig to e nsure APT it also provides other essential nutrients that have passed across the placenta in minimal proportions, as well as other nutrients that are needed to satisfy the nutritional requirement of calves during first hours of life (Ke hoe and Heinrichs, 2007).

PAGE 47

47 The most important component of colostrum for the life of the calf are the Ig. Colostrum contains 5 types of Ig (IgG, IgA, IgM, and IgE IgD ). However IgG accounts for 85 to 90% of the total Ig. Specifically IgG is divided in two subclasses, IgG1 and 1976). However in colostrum, the proportion of IgG1 was about 5 times greater than that of IgG2 (Sasaki et al., 1976). Concentrations of IgG subc lasses in serum of calves fed colostrum reflect the greater proportion of IgG1 found in colostrum. Studies have reported values of 7 to 9 times greater concentrations of IgG1 compared to IgG2 (Hidiroglou et al., 1995; Godden et al., 2009). The pool of Ig r eaching the intestine and able to be transported across the intestinal epithelium was initially assumed to occur by non selective pinocytosis (Klaus et al., 1969; Jones and Waltman, 1972). However later studies discovered the existence of specific Ig recep tors known as neonatal Fc receptors (FcRn) present in intestinal ep it h elium, initially identified in human epithelial cells of the intestine, suggesting its involvement in IgG binding and transfer of passive immunity (Israel et al., 1997). A potential prot ective mechanism of FcRn in favor of ci rculating IgG that prevents premature degradation and clearance from circulation has been recently hypothesized (Goebl et al., 2008). Establishment of APT is crucial to reduce neonatal morbidity and mortality, streng then calf immunity, and increase calf life spam (Robison et al. 1988, Quigley and Drewry, 1998; Donovan et al., 1998). Calves are considered to have an APT if they Weaver et al., 2000). Other authors consider serum total protein (STP) as a good

PAGE 48

48 APT (Donovan et al., 1998, Calloway et al., 2002). Appropriate passive transfer has been associate d with improved weaning and postweaning weight. A correlation analysis including ~900 heifers from birth to 180 d of age indicated a positive relationship of serum IgG concentrations at 24 to 48 h of age with average daily gain (ADG) and weaning weight. Au thors also reported an increased mortality in heifers having < 1.2 g/dL of serum IgG at 24 to 48 h of age (6.8 vs. 3.3%) when compared with heifers with greater concentrations of IgG (Robison et al. 1988). Concentrations of serum IgG at 24 to 48 h also wer e positively associated with mature equivalent milk and fat production during the first lactation, although no effect was found for age at first calving. Regression analyses of mature equivalent milk on IgG concentration was 8.5 kg of milk per 1 g/L of IgG The regression of mature equivalent fat on serum IgG concentrations was 0.28 kg per 1 g/L of IgG ( DeNise et al., 1989 ). Different factors could prevent dairy calves from reaching APT. The time of colostrum feeding, the quality (in terms of IgG concentra tion of colostrum), and the quantity of colostrum fed are reported as the most critical factors determining an APT. Neonatal calves have an ability to absorb complete proteins such as Ig, however this capacity is lost with in a few hours after birth before gut closure occurs. It is recommend feeding calves as soon as they are born. Stott et al. (1979, a, b, c) in a series of studies demonstrated that the rate of Ig absorption depended primarily on the amount of colostrum fed and how soon after birth the inge stion occurred. Final gut closure was not dependent on the amount of colostrum provided but it depended on the timing of colostral feeding; in non fed calves, their ability to absorb entire Ig through their intestine

PAGE 49

49 will last until 24 h of life, with a ma rked decrease after 12 h of age. However in calves fed right after birth, the gut closure occurs earlier (Stott et al., 1979a). At a fixed time of feeding, amount of colostrum fed is the main factor affecting rate of Ig absorption. However if first feeding is delayed, it would negatively affect the rate of Ig absorption (Stott et al., 1979b). When feeding colostrum in ranging amounts from 0.5 to 2 L, Stott et al. (1979c) reported a positive linear correlation of amount of colostrum fed and time of feeding o n serum total IgG concentration. Neither BW (33 to 52 kg) at birth nor colostrum IgG concentrations (28.4 to 46 g/L) were correlated with the maximum absorption observed when feeding at an early age and with greater amounts. Concentration of IgG in colostr um, in spite of Stott et al. (1979c) reporting no effect of colostrum IgG concentrations on serum IgG, has been reported to have a positive correlation with serum IgG concentrations after colostrum feeding. Morin et al. (1997) found that in fact, as Stott and coworkers confirmed, the most important factors associated with serum IgG concentrations were volume of colostrum fed and timing of administration when colostrum IgG1 was low (23.9 g/L). However when colostrum had of greater IgG1 concentration (60 g/ L) and fed at the same volume and time, Holstein calves had more IgG1 in serum. In addition they also reported that feeding 4 L of high quality colostrum at 0 h resulted in greater IgG in serum than those of calves fed only 2 L of the same high quality co lostrum, concluding that feeding this amount did not saturate the absorptive mechanisms. On contrary, Jaster (2005) evaluated the best option to feed colostrum to Jersey calves. Author reported that provision of high quality colostrum (IgG1, 84 vs. 31 g/L) in 2 feedings (2 L at 0 and 12 h) resulted in greater serum IgG concentrations than calves fed same 4 L in a single feeding.

PAGE 50

50 Active Acquired Immunity A general concept is that it is not until the innate immune system is overcome by pathogens or infectio n that the adaptive immune system is activated. However, in recent years, close interactions between components of both i nnate and adaptive immunity indicate that both function as a highly interactive and cooperative system, producing a combined response m ore effective than either system could produce by itself. The advantages of the acquired immune response are the following: 1) specificity, the ability to maximize the efficacy of the immune response while minimizing unnecessary collateral damage and 2) me mory, which provides protection from future infection with the same pathogen (Palm and Medzhitov, 2009). The main soluble factor in the acquired immune response is the antibody, also defined as Ig, produced by B lymphocytes and found in different parts of the body including fluids. Antibodies bind to molecules on pathogens and prevent them from infecting the host or target them for destruction by immune cells. Different types of antibodies are produced by B cells, including IgM, IgG, IgA, IgE, and IgD. Thes e Ig have different functional characteristics and exist in variable concentrations in different parts of the body. Levels of antibodies increase slowly the first time a pathogen is encountered, but in subsequent encounters with the same or similar pathoge ns, antibody levels can increase very rapidly (Woolums, 2010). Like B lymphocytes, T lymphocytes also arise in the bone marrow but migrate to the thymus glands to mature. Maturing T cells express a unique antigen binding molecule called T cell receptor (TC R) on their membranes. Two types of T cells are differentiated based on the membrane glycoproteins present on their surfaces, namely T helper (Th) exp ressing CD4 and T cytotoxic expressing CD8. Most TCR can

PAGE 51

51 recognize only antigens that are bound to cell me mbrane proteins called major histocompatibility complex (MHC) molecules. The MHC molecules are grouped into 2 classes, namely MHC I, expressed by nearly all nucleated cells and class MHC II, expressed onl y by antigen presenting cells After encountering na i ve T cells with antigen presenting cells the T cell proliferates and differentiates into memory T cells and various effector T cells (Kindt et al. 2007). Helper T cells assist a wide variety of other cells to respond optimally to infection. They do this through production of cytokines and expression of surface molecules that can stimulate other cells to improve their activity. They are further subdivided into groups called Th1, Th2, Th0, and Th17 which are based on the combination of cytokines expressed b y a given Th cell. The Th1 type cytokines [interferon (IFN interleukin (IL 2)] play a key role in initiating early resistance to pathogens and induction of cell mediated immunity, enhancing macrophage stimulation and phagocytic activity of viruses and other pathogens that live inside host cells (Mar odi, 2002). The Th2 cytokines (IL 4 and IL 5) are particularly important in the induction of antibody production, especially on mucosal surfaces (Woolums, 2010). A polarization of Th cells is measured as the ratio of IFN 4, a lower ratio indicates a b ias for a Th2 response, which has been associated with impaired cell meditated activity (Mizota et al., 2009). Cytotoxic T cells may kill target cells by one of at least three distinct pathways. Two involve direct cell to cell contacts between effector and target cells. The third is mediated by cytokines, such as IFN which are produced and secreted as long as TCR stimulation continues. These cytokines affect the opposed target cell. The TNF engages its receptor on the t arget cell and triggers

PAGE 52

52 the caspase cascade leading to target cell apoptosis, whereas IFN transcriptional activation of the MHC I antigen presentation pathway, leading to enhanced presentation of endogenous peptides by MHC I (Andersen et al., 200 6). Chase et al. (2008), reviewing the particularities of B and T lymphocyte function in newborn calves, reported that the following: a) the number of circulating B cells is greatly reduced in the first wk of age (~4% of the total lymphocytes) compared to ~20% to 30% in adults, which is reached by 6 to 8 wk of age; b) the low number of B cells result in a prolonged lack of endogenous antibody response, even in the face of an apparent Th2 cytokine bias in neonates; c) circulating IgA, IgG1, and IgG2 do not reach appreciable concentrations until 16 to 32 d after birth; d) T cell subsets have an adult like ratio (CD4:CD8) in neonates with ~ 20% for Th cells and ~10% for T cytotoxi c cells; and e) mitogen activation of T lymphocytes is slightly depressed at birth and remains constant through 28 d after birth. In preruminant calves, the FA profile of the phospholipids fraction of cells, including immune cells, is expected to reflect t he FA profile of the diet due to the lack of rumen functionality. Thus modifying the dietary FA profile is expected to also modify that of the immune cells. Chapkin et al (1988) fed mice four different diets, CCO (0% LA and ALA); safflower oil ( SAO, 78.2% LA), borage oil (BO, 36.6% LA and 25.2% GLA), fish oil ( FO, 1.2% of LA, 16.9% EPA and 12% DHA). The FA profile of PBMC phospholipids fractions differed with the diet fed to mice. Mice fed CCO or BO had PBMC phospholipids fractions with the lowest proport ion of LA. Concentration of mead acid, an indicator of EFA deficiency was higher in mice fed CCO. Interestingly, concentration of

PAGE 53

53 AA was quite constant except in the PBMC phospholipids fractions of mice fed FO which was lower compared to other diets (6.3 vs. 14.7% of total fat). Regarding n 3 FA, ALA was not detected in either PBMC phospholipids fractions whereas EPA, DPA, and DHA were only detected in mice fed FO diet. Insulin and Growth Factors in Colostrum enriched in different growth factors; however they were not fully investigated (Georgiev, whereas in humans, it is epidermal growth. Epidermal g rowth concentrations remain high during the lactation period whereas IGF are high only i n colostrum (Georgiev, 2008b; Blum and Baumrucker, 2008). In general, regardless of the type of diet, concentrations of IGF and insulin are higher in colostrum than in blood (Oda et al., 1989). Among the postulated physiological characteristics of IGF and insulin, the most critical in newborn calves is enhancing the growth and development of the gastrointestinal tract by affecting cellular proliferation and differentiati on (Roffler et al., 2003; Georgiev et al., 2003). Calves fed colostrum compared to those deprived of colostrum exhibited an enhanced epithelial cell proliferation as evidenced by greater circumference, area, and height of the villus. Authors assumed this r esponse to be due to the presence of growth promoting factors in colostrum (Buhler et al., 1998). Later studies verified the positive effect on development of the intestinal tract if IGF I was present in colostrum but not if IGF I was provided orally or by parenteral administration (Roffler et al., 2003; Georgiev et al., 2003). However, studies evaluating the effect of

PAGE 54

54 diet manipulation on concentration of growth factors in colostrum and their transfer to the newborn are scarce. Sparks et al. (2003) arbitr arily grouped newborn calves as having low (< 10 ng/mL) or high (> 10 ng/mL) concentrations of IGF I in serum before colostrum feeding. After 48 h of colostrum feeding, no differences were reported between groups for IGF I and IGF binding protein ( IGF BP) t ypes 2, 3, 4, and 5. Authors found a negative correlation of IGF I at 0 h to the difference between serum IGF I at 48 and 0 h (r = 0.82) because calves born with greater concentrations of IGF I had a significant decrease at 48 h after colostrum feedin g, whereas a positive correlation of concentrations of IGF I in colostrum and IGF I in serum at 48 h was detected (r = 0.45). In an attempt to evaluate the effect of bovine somatotropin (rBST) on IGF I concentrations in colostrum and calves, Pauletti et al (2007) found that prepartum supplementation with rBST increased concentrations of IGF I in colostrum but concentrations of IGF I in serum were lower at the first or second day of colostrum feeding regardless of supplementation with rBST. Hammon et al. ( 2000) delayed the intake of colostrum in calves and reported that calves fe d colostrum within the first 2 to 3 h of birth, their IGF I concentrations were greater and were maintained during the first 36 h after colostrum feeding compared to delays of more than 12 h, however feeding colostrum did not increase IGF I concentrations. On the other hand, insulin concentrations increased after first colostrum feeding but not if the delay in colostrum feeding was over 24 h. All previous studies have hypothesized th at reduced serum concentrations of IGF I, although in greater concentrations in colostrum, might be due to colostrum IGF having a local effect rather than being absorbed into circulation.

PAGE 55

55 Effect of Supplemental Fatty Acids on Passive Transfer Among the ma in factors contributing to ensure an APT are timing, quality (in terms of Ig concentration), and quantity of colostrum f ed. These factors have been extensively evaluated. However a limited number of studies have involved the effect of supplementing E FA pre partum. Rajaraman et al. (1997) fed newborn calves with skim colostrum and CCO as a replacer of the milk fat or normal colostrum. Authors did not find differences in AP (IgG1 > 15 g/L) or in the activity of PBMC during the first week of life. However conce ntrations of fat soluble vitamins were lower in the treatment group deprived of milk fat. Dietz et al. (2003) supplemented pregnant beef cows with no oil source, safflower seeds (6.4% of dietary DM), or whole cottonseeds (14.3% of dietary DM). Both seeds a re rich in LA. Authors reported no difference in colostral concentrations of IgG (85, 96, and 83 g/L, respectively) and calf birth weight. Only serum of calves born from cows fed control or whole cottonseed diets were measured for IgG concentration after 3 6 h of birth and no difference was reported (38.6 and 37.1 g/L of IgG, respectively) between calves born at ambient temperatures > 6 C. Later, Lake et al. (2006c) classified late gestation beef cows as in BCS 4 or 6 and measured the serum IgG concentration in their calves after 18 h of birth finding no difference in calves born from dams have low vs. high BCS (15.6 vs. 13.4 g/L of IgG). Novak et al. (2012 b ) restricted the intake of energy by 13% (by increasing intake of NDF) in late gestation dairy cows and reported no difference in DM intake. Colostrum IgA was greater from cows of lower energy intake; however total Ig, IgG (17.3 vs. 16.2 g/L, high and low energy respectively), IgA, and IgM did not differ after colostrum feeding at 3 d of age. Similarly birt h weight did not differ. Serum IGF I was not affected

PAGE 56

56 by the prepartum diets when measured at 3 d of age (130.6 vs. 99.3 ng/mL for high and low energy intake treatments, respectively). Limited studies have evaluated the effect of feeding fat supplements to cows on fatty acid (FA) composition of colostrum and most of them did not include the effect of parity. However, few studies using dairy cows and ewes supplemented with CLA have reported not effect of parity in total CLA ( Kelsey et al., 2003; Tsiplakou et al., 2006). primiparous ewes produced greater proportion of LA, GLA, ALA, EPA and total CLA. Moreover the few studies performed with cows, regardless parity considerati on have focused on supplementation of n 3 or CLA FA instead of n 6 FA. Effect of Supplemental Fatty Acids on Total Fat and Fatty Acid Profile Colostrum A limited number of studies have evaluated the effect of supplementing EFA prepartum on colostrum FA pr ofile of ruminants. Most were focused on supplementation of n 3 FA. An early study conducted by Noble and coworkers (1978) supplemented pregnant ewes with a protected PUFA supplement (70% sunflower oil + 30% SO). Intake of fat was increased from 8 to 1 wk prior to calving from 3.4 to 37.6 g/d and 9.4 to 113 g/d for the control and supplemented group, respectively but caloric intake remained the same. The major FA present in colostrum was OA. Linoleic acid accounted for less than 1% in colostrum of ewes fe d control diets but was 8% in colostrum of ewes supplemented with PUFA. The presence of elongated FA derived from LA or ALA were not detected. Capper et al. (2006) fed pregnant ewes Ca salt of palm oil ( Megalac, 4.1% of C16:0 and 2.0% of LA, % of concentr ate DM basis) or FO ( 1.5% of LA, 0.4% of EPA

PAGE 57

57 and 0.04% of DHA, % of concentrate, DM basis). Ewes fed Megalac during the prepartum period produced colostrum with greater proportions of LA, C18:0, C18:1 trans and OA but proportions of C16:0 and AA did not d iffer with fat supplement. Supplementing ewes with FO increased the proportions of CLA c 9 t 11, ALA, EPA, and DHA in colostrum. In addition, supplementation of FO reduced colostrum yield and total fat concentration. Aiming to evaluate the effect of prepartu m supplementation of FA on colostrum FA profile, Santschi et al. (2009) fed prepartum cows a control diet (90.6% C16:0, 4.7% LA, and 0.5% ALA, % of total FA) or a linseed supplement (6.6% C16:0, 19.3% LA, and 53.6% ALA, % of total FA). Authors reported tha t colostrum from linseed supplemented cows had lower proportions of C16:0 but greater proportions of C18:0, CLA c9 t11, and ALA. Proportions of OA, LA, AA, EPA, and DPA did not differ due to fat supplement. Leiber et al. (2011) supplemented prepartum cows (n = 6) with seeds rich in LA (safflower) or ALA (linseed) and reported that only prepartum cows fed seeds rich in ALA increased the proportion of this FA in colostrum but did not influence the proportions of EPA and DHA. Authors concluded that those physi ologically necessary FA were maintained to avoid deficiency regardless of the type of FA fed prepartum. Studies have reported that supplementation of FA during the prepartum period modifies the FA profile of colostrum. A common feature among studies is th at the dietary FA profile tended to be reflected in colostrum FA. However efficiency of transfer is poor particularly for PUFA with LA and ALA derivatives appearing to have a preferential synthesis in mammary gland to ensure proper proportions of AA, EPA a nd DHA regardless of dietary FA.

PAGE 58

58 Plasma Early studies using pregnant ewes focused on assessing the dietary transfer of LA and AA to the offspring in utero (Noble et al.1978, Soares, 1986). However those studies did not report concentrations of any of the n 3 FA. In order to test the effect of differences in maternal intake of LA in late gestation, Elmes et al. (2004) fed late gestation ewes diets differing in LA concentration, namely a control diet (3.8% total fat, DM basis; 29% LA and 22% ALA, % of total FA) or high LA diet (4.5% total fat, DM basis; 41% LA and 16% ALA, % of total FA). They reported that plasma phosphatidylcholine fraction of fetus (138 gestational d), had increased proportions of LA, GLA, AA, DPA and DHA were higher in fetus from high LA supplemented ewes; ALA was undetectable, authors hypothesized that the high availability of LA in dams tissue might increased the enzymatic activity of desaturases and elongases increasing the proportion of LA and ALA derived FA. Lake et al. (2006b) suppl emented lactating beef cows with no fat or safflower seeds rich in LA or OA. Diets did not affect the total fat concentration in plasma, but calves suckling dams fed safflower seeds rich in LA or OA had greater plasma concentrations of those respective FA. Concentration of C18:0 was greater in plasma of calves suckling cows supplemented with safflower seed regardless the type of FA enriched in the seed, whereas EPA was greater in calves suckling no fat supplemented cows and fewer in calves suckling cows su pplemented with safflower seed rich in LA. Moallem and Zachut (2012) supplemented cows during the last 22 d of gestation with encapsulated fat containing 240 g/d of SFA, 300 g/d of linseed oil (15 g of LA and 56 g of ALA daily), or 300 g/d of FO (5.8 g of EPA and 4.3 g of DHA daily). No differences in plasma concentrations of LA, ALA, AA, EPA and DPA in calves were

PAGE 59

59 detected before colostrum feeding. However, plasma concentrations of GLA and DHA were 1.3 and 1.7 times greater in the FO group than the other g roups, respectively. Authors concluded that DHA supplementation, and not its precursor ALA, was needed in the diet to increase concentration of this critical FA in fetal development. In an attempt to evaluate the rise in plasma cholesterol concentrations, Wrenn et al. (1973) fed preweaned calves with an increase proportion of LA (14.1 vs. 2.5% of total fat) in milk. Growth was not affected by differential intake of LA, similarly cholesterol did not differ. Jenkins et al. (1985) reported that using tallow ( 3.8% LA), CCO (3.2% LA) or CO (52.7% LA) as sources of fat in MR resulted in calves fed CCO having greater concentrations of plasma free cholesterol but not cholesterol ester ( 50 to 57% of lipid fraction), which was the main lipid f raction in plasma follow ed by phosphatidylcholine (25 to 30% of lipid fraction) Interestingly, LA in plasma cholesterol ester and phosphatidylcholine fractions was only greater when calves were fed CO. Concentration of AA did not differ in the cholesterol ester fr action, but was lowest in the phosphatidylcholine fraction of calves fed CO. In a follow up study, Jenkins and coworkers (1986) evaluated the use of tallow (2.0% LA), CAO (20.4% LA) or 1:1 mixture of tallow + CAO on plasma lipid fractions of calves. The cholesterol ester (41 to 46% of total fat) and phosphatidylcholine (35 40% of total fat) fractions were in greater concentrations. However concentration of LA in both lipid fractions was greater when tallow + CAO were fed, whereas concentration of AA was greater when feed ing tallow In a following study, Jenkins and Kramer (1986) fed MR with 4 different FA sources: CCO (0.1% LA), CCO + CO (95% C CO + 5% CO, 2.8% LA), CCO + CAO (92.5% C CO + 7.5% CAO, 1.6% LA), or tallow (5% LA). Authors reported that fat source did not

PAGE 60

60 chang e the total concentration of fat in cholesterol ester or phosphatidylcholine fractions. However cholesterol ester reflected better the dietary FA profile with fewer concentrations of C12:0 and C14:0 and greater concentrations of LA in calves fed tallow In calves fed CCO + CO, concentrations of C12:0 and C14:0 were greater and LA was similar as in calves fed tallow Concentrations of AA in cholesterol ester were greater in calves fed CCO + CAO, followed by similar concentrations when CCO + CO or tallow was fed. Jenkins and Kramer (1990) evaluated the inclusion of FO in MR containing primarily tallow and vegetable fats. Tallow and CCO served as fat sources in the control MR resulting in a n 6:n 3 of 7.1. In the other MR, half of the control was replaced with either CO alone (n 6:n 3 = 36.5) or with a mix of CO and FO in the two following ratios: 2/3 CO and 1/3 FO (n 6:n 3 = 3.1) and 1/3 CO and 2/3 FO (n 6:n 3 = 1.0). No difference was detected in ADG and FE due to MR. Total fat in plasma was lowest w hen FO was added to the MR. C holesterol ester and phosphatidylcholine were the most abundant lipid fractions in plasma. Regardless the lipid fraction, concentration of LA was greater in calves fed C + CO but that of AA was greater in calves fed the highest proportio n of FO. Feeding any proportion of FO increased EPA concentration in cholesterol ester and phosphatidylcholine fractions, but only that of DHA in phosphatidylcholine whereas the highest proportion of FO was needed to increase the concentration of DHA in cholesterol ester fraction. Liver Fatty liver is a critical condition that leads to impaired liver function. Several studies in humans (Reddy and Rao, 2006; Cave et al. 2007; Semple et al. 2009; Thomson and Knolle, 2010) have documented very well the effe ct of hepatic steatosis in

PAGE 61

61 liver function and the multiple etiologies of this disorder. Dairy cows in the transition period face a high risk for fatty liver, due to the high demand of nutrients for milk production, accompanied by a limited intake that forc es the cow to mobilize corporal tissue and generate intermediates of en ergy such as FFA. When these F FA are taken up by the liver in high quantities, the oxidative and secretive capacity of lipids by liver is exceeded. Hence, the arriving FFA are only part ially oxidized forming ketone bodies or reesterified to TG, which end up accumulating in liver decreasing the metabolic function of liver Bobe et al. (2004) wrote a comprehensive review of the pathology and etiology of fatty liver in dairy cows. The auth ors concluded that fatty liver is a multifactorial, multifaceted disease with nutritional factors as the main drivers of this condition. Jenkins and Kramer (1986) fed MR with 4 different FA sources : CCO (0.1% LA), CCO + CO (95% C CO + 5% CO, 2.8% LA), CCO + CAO (92.5% C CO + 7.5% CAO, 1.6% LA), or tallow (5% LA). Feeding tallow reduced the total fat in liver and this was due to a lower proportion of TG (5%), which was the greatest lipid fraction in the CCO MR (48.55), whereas, proportion of phosphatidylcholin e was the highest in the other diets (23.3% CCO, 34.8% CO, 32.4% CAO, and 43.% tallow ). The FA profile of TG was the only one containing significant proportions of C12:0 and C14:0 and they were in greater proportions in calves fed CCO MR. Proportions of LA and ALA in the TG fraction also better reflected the dietary FA profile. On the other hand AA was not present in the TG fraction but in the phosphatidylcholine fraction and was greater in liver of calves fed CO or CAO. Jenkins and Kramer (1990) evaluated the inclusion of FO in MR containing primarily tallow and vegetable fats. Tallow and CCO served as fat sources in the control

PAGE 62

62 MR resulting in a n 6:n 3 of 7.1. In the other MR, half of the control was replaced with either CO alone (n 6:n 3 = 36.5) or with a mix of CO and FO in the two following ratios: 2:3 CO and 1:3 FO (n 6:n 3 = 3.1) and 1:3 CO and 2:3 FO (n 6:n 3 = 1.0). Phospholipids were the greater fraction in calf liver. Individual phospholipid fractions [ phosphatidylcholine (52%), sphingomyelin (1.2 %), and phosphatidyl ethanolamine(21%)]; as well as total fat did not differ due to MR. Concentration of LA was greater in calves fed CO, whereas AA concentration was greater in calves fed the control and high FO MR. Jambrenghi et al. (2007) supplemented l ambs with a control diet (3.3% fat, 39.8% of LA as % of total fat) or a high fat diet enriched with LA (7.9% fat, 45.5% of LA as % of total fat) for a 45 d finishing period. Feed intake and final BW were not changed. However, the FA profile of the liver wa s influenced by the diet. Concentrations of C16:0, C16:1, and C18:0, ALA, and EPA were greater for control calves, whereas OA, LA, AA, and DHA were greater for the group fed more fat and LA. Effect of Supplemental Fatty Acids on Preweaned Calves Performanc e Obtaining good growth and health performance of dairy calves before weaning is one of the primary goals of a dairy herd management. Dairy herd managers have to deal with challenging circumstances once the calf is born, such as to ensure appropriate passi ve transfer of IgG from colostrum (Beam et al., 2009) and prevention and treatment of diseases such as diarrhea, omphalitis, septicemia, and pneumonia which are among the most commonly diagnosed diseases leading to morbidity and mortality in calves (Donova n et al., 1998). To prevent a high incidence of calf diseases and profitability of the herd, care should be taken not only during the preweaning period but

PAGE 63

63 also during the gestation period, particularly during the last trimester of gestation, during which time the fetus has its greatest development. Effect of Supplemental Fatty Acids during Pregnancy on Growth Performance and Hormonal and Metabolic Profile of Preweaned Calves Early studies using human subjects have reported a direct effect of nutritional st atus in late pregnancy on fetal growth and birth weight. Naeye et al. (1973) evaluated 467 gestating women and reported that low calorie intake during late gestation was highly and negative correlated with fetal growth and weight. Kramer (1987) reviewed 89 5 publications related to potential causal reasons of intrauterine growth retardation in human subjects and reported that regardless of racial origin and economic status, poor gestational nutrition was a common cause of lighter birth weight. One of the mos t evaluated nutrients to produce adverse effects on the offspring was protein. Anthony et al. (1986) and Carstens et al. (1987) fed protein levels below the requirement for maintenance of cows during late gestation and although BW and BCS of cows at calvin g was lower for undernourished cows, the birth weight of their calves did not differ. More recent studies using ruminants found contradictory effect of undernutrition during late gestation. Osgerby et al. (2002) fed pregnant lambs a diet meeting only 70% o f total nutrient requirements and reported that undernourished fetuses at 135 d had lighter heart, pancreas, thymus, gut, and kidney weights; bone growth also was affected; Dwyer et al. (2003) reduced the nutritional intake of pregnant lambs by 35% and rep orted a 9.3% reduction in birth weight and a reduced ability of offspring to suckle their dams. On the other hand, Hess (2003) evaluated 18 studies that supplemented late gestation beef cows with fat and results were not consistent; that is, calf birth wei ght was decreased (n=2), increased (n=3), or unchanged (n=12). Hess (2003) therefore

PAGE 64

64 concluded that fat supplementation of dams in late gestation did not affect birth weight. Similarly, Banta et al. (2006) aimed to evaluate the effect of LA supplementation in middle and late gestation cows by feeding 0.68 kg of soybean meal, 3.01 kg of soybean hull or 1.66 kg of sunflower seed rich in LA. All diets provided same intakes of CP and RDP but soybean hull and sunflower supplements provided 2.34 more Mcal/d. Auth ors rep orted no effect of supplements o n birth and weaning weight s of calves. Later, the same authors (Banta et al., 2011) adjusted the supplements to provide same intakes of N and energy by feeding 0.23 kg of soybean hull, 0.68 kg of sunflower seed rich i n LA plus 0.23 kg of soybean hull, or 0.64 kg of mid oleic sunflower seed plus 0.23 kg of soybean hull and reported similar response as in their previous study. In a review article by Barker (1997) adapt to a limi ted supply of nutrients and in doing so they permanently change their programming event is diab etes. Pettitt et al. (1987) reported that offspring of diabetic women had twice the risk of developing diabetes than offspring of non diabetic women; even though when the incidence of this condition was adjusted using maternal weight and birth weight as co variate. Fowden et al. (20 06), based in previous studies, identified the most probable period s in which fetal programming occur The se potential periods start pre conceptual and pre implantation, in which either under or overnutrition can affect birth wei ght and incidence of disease later in the offspring. The majority of fetal maturation occurs during late gestation, where many tissues undergo structural and functional changes in

PAGE 65

65 preparation for extrauterine life (Funston et al., 2010). In fact, several s tudies have evaluated the effect of undernourishment in late gestation on the metabolism of offspring. Although most studies have reported a low birth weight with concomitant effects on metabolic response of offspring (Barker et al., 1993; Barker, 1997; Oz anne and Hales, 2002; Drake and Walker, 2004 2005 ; Gicquel et al., 2008) some studies have reported that metabolic response of offspring can be programmed in uterus without a change in birth weight (Pettitt et al., 1987; Ferezou Viala et al., 2007). Funst on et al. (2010) reviewed how maternal nutrition affects conceptus growth and postnatal responses in beef cattle T he most common negative effects reported when pregnant cows were undernourished were on birth weight, health, growth, reproduction, carcass w eight and carcass quality. Singh et al. (2010) reviewed the factors that account for phenotypic variation in milk production by dairy cows. They concluded that a substantial proportion of the unexplained phenotypical variations were due to epigenetic regul ation (change in gene expression without modifying DNA sequence) as a consequence of maternal nutrition during fetal life or nutrition during the first year of life. Recently Soberon et al. (2012) reported a positive correlation of preweaning ADG with firs t lactation yield; for every 1 kg of preweaning ADG, heifers, on average, produced 850 kg more milk during first lactation. They concluded that increased growth before weaning results in some form of epigenetic programming resulting in a positive effect on milk yield. Few studies have evaluated the effect of supplementing FA during late gestation or early lactation on their effect on overall calf performance. Early studies reported better growth rate of calves by providing extra calories using fat through a concentrate

PAGE 66

66 source (Espinoza et al., 1995). Bottger et al. (2002) supplemented beef cows from 3 d through 90 d post partum with isonitrogenous and isocaloric supplements, a control, a safflower seed rich in LA (76% LA) or rich in OA (72% OA). They reporte d that calf BW gain during the supplementation period was not influenced by supplement fed; neither did 205 d adjusted weaning weights. Encinias and coworkers (2001, 2004) supplementing pregnant beef cows or ewes with fat rich in LA versus a control diet o f low fat, did not find any effect of additional prepartum fat in birth and weaning weight of their offspring. Lake et al. (2005) fed lactating beef cows with isocaloric and isonitrogenous diets of low (1.2% of DM) or high (5% of DM) fat, by providing a s upplement rich in LA or in OA. They reported no effect of diets in BW gain of suckling calves. In a companion paper Lake and coworkers (2006a) reported increased concentrations of plasma glucose in calves suckling cows supplemented with LA compared to thos e fed control diets, but no change in insulin, IGF I or NEFA was reported due to dam diets. Greater plasma glucose accompanied with no change in insulin concentrations might indicate reduced sensitivity of peripheral tissue for uptake of glucose. Chechi an d Chema (2006) fed pregnant rats and their pups with diets of 20% fat rich in SFA (15% LA) or PUFA (70% LA). They reported that pups fed SFA pre and postweaning had the highest concentration of plasma total cholesterol, whereas the PUFA/PUFA fed group had the lowest, but plasma triglyceride concentration did not change among groups. The cholesterolemic effect of SFA/SFA diets might be due to increased proportions of LDL cholesterol.

PAGE 67

67 Undernutrition during late gestation in women results in reduced birth wei ght of the offspring. However, in beef cows, supplementation of fat during prepartum has yielded contradictory results, with most of the studies reporting no effect of fat supplementation on calf birth weight and preweaning BW. At the best of our knowledge no study has evaluated the metabolic and immune response of preweaned dairy calves born from EFA supplemented cows, this topic warrants further investigation, considering the recent discover of potential fetal programming effect of nutrition during early life in future offspring productivity. Effect of Feeding Supplemental Fatty Acids to Preweaned Calves on their Growth Performance and Metabolic Profile Few studies are available in which preruminant dairy calves were fed increased amounts of LA. The firs t studies were done in an attempt to replace milk fat in skim milk with vegetable sources of fat in order to reduce the cost of raising calves. Later, studies have focus in the supplementation of specific sources of FA. Early work of Jacobson et al. (1949) intended the evaluated the use of different types of SO in total replacement of milk fat (3% wet basis) in calf performance. They reported that crude expeller SO produced poor growth, severe scours and high mortality, whereas performance of calves fed hyd rogenated SO equaled that of calves fed whole milk. In a second companion study (Murley et al., 1949) totally replaced milk fat with hydrogenated, refined or crude SO (3% wet basis) and reported, similar results but that feeding refined SO resulted in fewe r incidences of scours than crude SO. Calves fed either refined or crude SO had the poorest growth. From the same lab, Richard et al. (1980) replaced milk fat with 2% (web basis) of SO, CO or tallow and did not find any effect on ADG but feeding vegetable oils increased plasma cholesterol

PAGE 68

68 concentrations. Authors indicated that fat globule size after reconstitution of milk by Some years later, the laboratory of K.J. Jenkins in Ontario, Canada, evaluated th e supplementation of specific FA by reconstituting skim milk and sweet whey with different fat sources as the only feed of calves. In one study, Jenkins and coworkers (1985) reported the use of CCO (3.2% LA), tallow (3.8% LA) or CO (52.7% LA) as total sour ces of fat in MR (~20% fat DM basis). Calves fed CCO or tallow had greater ADG and feed efficiency (FE) than calves fed CO. This likely occurred as a result of severe scouring by calves fed CO. In a follow up study, Jenkins and coworkers (1986) evaluated t allow (2.0% LA), CAO (canola oil, 20.4% LA), CO (53.2% LA), and a 1:1 mix of tallow + CAO or tallow + CO as only fat sources of reconstituted skim milk (~20% fat DM basis) for calves. Again authors reported that reconstituted milk with CO promoted scours and poor calf gains, which was not reversed when tallow + CO. Feeding tallow + CAO or tallow AL did not produce scours and resulted in calves with better ADG and FE. In other study, Jenkins and Kramer (1986) replaced fat in skim milk with 4 different fat sources: CCO, CCO + CO (95% CCO + 5% CO), CCO + CAO (92.5% CCO + 7.5% CAO), or tallow All calves, regardless of the fat source fed, were free of diarrhea. Increasing intake of EFA by including CO or CAO did not affect BW gain and FE; however feeding tallo w increased ADG, DMI and FE when compared to calves fed milk containing just CCO but not when CCO was combined with CO or CAO. Leplaix Charlat et al. (1996) fed 5 wk old calves for 17 d, with a 23% fat MR (DM basis) containing either tallow (3.7% LA) or SO (51.2% LA) with or without additional dietary cholesterol (1% of MR, DM basis) aiming to evaluate the plasmatic distribution of

PAGE 69

69 lipoproteins. Calves fed SO had greater concentrations of total fat in liver and total cholesterol in plasma compared with cal ves fed tallow The effect of high LA diets was due to increased concentrations of high density lipoprotein without modification of LDL or VLDL. Plasmatic concentrations of NEFA were reduced dramatically when SO was fed. The inclusion of dietary cholestero l had no effect on NEFA when SO was fed but reduced NEFA when tallow was fed. Plasmatic levels of APO B were similar in calves fed either source of fat but increased about 3 fold when cholesterol was included in the diet. Piot et al. (1999) fed 2 wk old ca lves for 19 d, a MR formulated with either CCO (42% C12; 3% LA) or tallow (22% C16:0; 38% C18:1; 2.4% LA) and reported no difference in ADG, MR intake and plasma concentrations of B HBA. However, calves fed CCO had decreased plasma concentrations of glucose and insulin. Whether these decreased plasma concentrations were due to reduced secretion of insulin by MCFA or an enhanced ability of peripheral tissue for glucose uptake could not be defined since calves had no difference in ADG. In an attempt to evaluat e the effects of feeding isocaloric, isonitrogenous MR that varied in the amount and type of FA, Mills et al. (2010) fed calves MR with 23% fat and varied proportions of MCFA as the only feed. MR contained 2% MCFA (control) or 32% MCFA supplied by either C CO (23% C8:0) or caprilate (23.6% C12:0). After insulin challenge calves fed caprilate had a greater decrease in plasma glucose concentration. Empty BW gain was better for control calves. Liver of calves consuming CCO was heavier and contained 15% more fat (as is basis) than the other two groups. Authors stated that it was unclear why CCO induced lipid accumulation in the liver, but

PAGE 70

70 increased capacity of initial FA oxidation and subsequent preferential de novo synthesis or chain elongation of MCFA may have occurred. In order to prove whether vegetable fat mixtur es could be used instead of lard (15.2 % DM basis), Huuskonen et al. (2005) fed calves with MR containing 3 different fat sources, namely mixture 1 [palm oil (75%) + CCO (25%); 7% LA, 0.1% ALA], mixt ure 2 [palm oil (75%) + CCO (20%) + rapeseed oil (5%); 8.0% LA, 0.7% ALA], or lard (12.1% LA, 1.2% A LA). During the preweaning period, ADG or number of days with diarrhea did not differ, but calves fed lard had a reduced and poor FE. Post weaning ADG also was not affected by the type of fat fed preweaning. Calves fed MR with mixture 2 tended to have lower starter intake during the preweaning period, but total DMI did not differ among treatments. Berr et al. (1993) aimed to study excretion of cholesterol by n 3 and n 6 PUFA. Authors fed rats with 3 different sources of fat (~9 % wet basis). Feeding FO reduced plasma concentrations of total cholesterol but not when CCO or SAO were fed. This decrease was due primarily to the decrease in high density lipoprotein cholesterol concentrations which is one of the main mechanism by which feeding of n 6 FA reduced the concentration of circulating cholesterol Recently, the few studies evaluating dietary inclusion of EFA or its derivatives to newborn calves have focus ed in the supplementation of n 3 FA from vegetable (ALA) or animal (EPA and DHA) origin. Ballou and DePeters (2008) evaluated the inclusion of FO in calf MR to replace 1 or 2% of the fat of a control MR (20% fat, DM basis).They reported no effect on growth ADG, FE or serum concentrations of glucose, insulin, urea nitrogen, NEFA, and TG. However at day 20, calves fed FO only had lower NEFA and

PAGE 71

71 TG concentrations, no clear pattern in metabolite concentrations was evident and interpretation of these temporal r esults was difficult. Hill et al. (2009) fed calves a grain mix containing 0, 0.125 or 0.25 5% Ca salt of linseed oil or 0.25% Ca salt of FO (DM basis). No effect of oils was detected during the first 28 d of life. When the first 56 d of life was evaluate d, ADG and hip width increased linearly as flax oil increased in the grain mix whereas serum concentrations of urea N and glucose decreased. The better ADG and lower serum concentrations of glucose in calves supplemented with flax oil might indicate a bett er sensitivity of tissue for glucose to be utilized for protein synthesis. Hill et al. (2011) fed newborn calves with MR (16% fat DM basis) containing only fat sources to which NeoTec 4 (blend of butyric acid, CCO and flax oil) replaced 0 or 1% of the anim al fat. The NeoTec 4 contained 7 times more butyrate and MCFA and 2 times more ALA. Intake of MR as percentage of BW and grain mix intake did not differ, but calves fed NeoTec 4 had 10% better ADG and FE tended to be greater, possible as a result of improv ed immune response, as suggested by reduction of diarrhea incidence in calves fed NeoTec 4. Early studies reported that vegetable oils rich in long chain FA in replace of fat in MR have resulted in detrimental effects in calf performance, whereas CCO had resulted in improved performance similar to that of tallow Recently, studies have not revealed clear effect of n 3 or n 6 supplementation on calf performance, making this area in need for more research. Effect of Supplemental Fatty Acids Fed During Pregn ancy on Offspring Health and Immunity A very limited number of studies have evaluated the effects of supplementing FA during late gestation on immune response of cattle offspring. Most of the studies

PAGE 72

72 supplementing fat prepartum were c onducted using beef cattle. Since calves stay with their dams after birth, during the preweaning period, more variables are encountered when assessing on calf performance. Das (2003) proposed that the negative correlation between breast feeding and insulin resistance and diabetes mellitus can be related to the presence of significant amount of PUFA in human breast milk, and that the provision of PUFA during late pregnancy and lactation can prevent diabetes mellitus from developing. In a review article, Enke et al. (2008) indicated that dietary PUFA and their derivatives consumed during mid to late gestation had a programming effect on early immune development and immune maturation by regulating numerous metabolic processes as well as by modifying gene expres sion. Recently, Klemens et al. (2011) evaluated the odds ratio of incidence of allergic diseases and production of inflammatory cytokines due to fat supplementation using 5 randomized controlled trials. They concluded that supplementation during pregnancy but not during lactation reduced the risk of allergic diseases and production of inflammatory cytokines. Petit and Berthiaume (2006) fed beef cows isonitrogenous and isocaloric diets starting in late gestation. Diets contained either Megalac (14% of conc entrate, DM basis), linseed (33.2% of concentrate, DM basis), or no fat supplement. Calves born from dams fed fat had lower rate of mortality both at birth and at weaning although birth weight and ADG preweaning were not different. During the last 55 d of gestation, Lammoglia et al. (1999) fed beef cows isocaloric and isonitrogenous diets of 1.7 or 4.7% fat (safflower seeds fed at 0 and 6.7% of dietary DM). Calves were fed standard colostrum and challenged to cold stress conditions (0 C

PAGE 73

73 for 140 min). Calves born from cows fed safflower seeds kept their body temperature throughout the stress period, whereas control calves decreased their body temperature after 70 min. Calves fed safflower seed also had lower cholesterol concentrations in plasma after 60 min o f cold exposure, whereas glucose concentration in plasma was ~40% greater the whole 140 min of cold stress, suggesting that increased glucose availability resulted in better heat production. In contrast, Dietz et al. (2003) reported that the body temperatu re and plasma concentrations of glucose were not affected in calves born from cows fed no supplemented dat, safflower seeds, or whole cottonseed. Encinias et al. (2004) reported that lambs had lower incidence of mortality and a greater number of lambs were weaned per ewe fed isocaloric diets of 10 vs. 0% safflower seeds. However, neither birth weight nor weaning weight were affect by feeding of safflower seeds. Similarly, plasmatic concentrations of NEFA and glucose did not differ within 48 h post birth. Si milar intake of energy by pregnant ewes was proposed as a cause for lack of diet effect. Effect of Supplemental Fatty Acids Fed to Preweaned Calves on Their Health and Immunity As indicated earlier, initial studies of supplementat ion of LA to calves were done by partially replacing milk fat. Those initial studies had in common a greater incidence of diarrhea by calves fed additional LA. Authors concluded that the likely causes of increased diarrhea were type of oil and poor quality process during homogenization or dispersion of fat supplements into dry skim milk. (Jacobson et al., 1949; Murley et al., 1949). Jenkins et al. (1985, 1986) reported that calves fed CO alone or a 1:1 mixture of CO + tallow had greater incidence of diarrh ea than calves fed tallow CAO or a mixture

PAGE 74

74 of tallow + CAO. However when Jenkins and Kramer (1986) replaced 5% of CCO with CO, calves did not suffer from diarrhea. Later, Jenkins (1988) fed calves with MR containing tallow CO, or CO with aspirin (to inhi bit potential role of prostaglandin promoting scours). Feeding CO, regardless the inclusion of aspirin, produced more, less ADG, and worse FE than when tallow was fed. However, compared with Jenkins et al. (1985), the incidence of diarrhea was the lesser i n later study although the same diets were used in both studies. Low pressure dispersion of CO was used in the 1985 study whereas a homogenizer was used in 1988 study, which resulted in smaller globules of fat (<1 um vs. 1 to 20 um). A vast amount of in vi tro and in vivo studies have evaluated the potential of FA to modify different markers of immune response. However, a limited number of them have evaluated the effect of feeding LA specifically. Moreover, most of those studies have been performed using hum ans or rodents. Kelley et al. (1989, 1990) fed adult human subjects diets of low or high LA concentrations by reducing or increasing the proportion of fat respectively. In both studies they were unable to detect any dietary effect on number of circulating T and B lymphocytes and on in vitro proliferation of PBMC to different mitogens and production of complement proteins. Total number of circulating leukocytes also was unchanged. Actual concentrations of LA in tissues or blood were not measured. A potential reason of lack of LA effect might be low differential concentrations of LA in tissues between subjects on test diets. Barone et al. (1989) evaluated the reduction of LA intake on immunity of young men by reducing total fat (< 30% of dietary calories) inta ke. They reported that activity of b lood isolated natural killer cells was increased. However,

PAGE 75

75 subjects were not directly controlled so actual intake of LA could not be determined. In a later study, Heber et al. (1990) fed young men with a low fat diet (< 20% of calories) supplemented or no with CCO or SAO. Activity of natural killer cells was increased when men were fed low fat diet compared to the baseline measure but oil supplementation did not affect the activity of natural killer cells. These results i ndicate that amount of fat unluckily source of FA modify the activity of natural killer cells. Yaqoob et al. (2000) supplemented the diet of adult human subjects with 9 g/d of SAO for 12 wk and was unable to effect in lymphocyte proliferation, natural kill er cells activity, or production of cytokines (TNF IL 1 IL 2 or IFN ) by PBMC. However, a potential reason for lack of effect was that the FA profile of plasma phospholipids or PBMC were not altered by the feeding of SAO, thus reducing the chance for LA to modify activity of immune cells. In vitro and animal controlled studies (better experimental control) have more marked effects of supplemental LA than studies using humans supplemented with LA. Calder et al. (1990) cultured murine macrophages in pr esence of a variety of FA. Those FA were rapidly taken up by the cells enriching the neutral and phospholipid fractions with the FA from the medium. Macrophages enriched with C14:0 or C16:0 showed a decreased ability to phagocyte unopsonized zymosan partic les whereas those enriched with LA, ALA, AA, EPA or DHA had an enhanced phagocytic ability with AA having the greatest effect on rate of uptake. A change in FA profile of phospholipid fraction of lymphocytes affecting the membrane fluidity was proposed as the mechanism of improved phagocytic activity in calves supplemented with PUFA. Thanasak et al. (2005) cultured bovine PBMC with 2 doses (125 and 250uM) of LA or ALA and reported that

PAGE 76

76 the greater concentration of LA inhibited proliferative response of PBCM to mitogens whereas ALA had no effect on proliferation. Increased concentrations of ALA decreased the co ncentrations of leukotriene B4 whereas LA had no effect However prostaglandin E2 a prostaglandin with immunomodulatory effect, was increased in ALA m edia. A potential contrasting effect of leukotriene B4 and prostaglandin E2 functions might be the reason for the inhibitory effect of LA on lymphocyte proliferation. Later Gorjao et al. (2007) evaluated the proliferative response of human lymphocytes to I L 2 stimulation when cells were incubated with different doses of various FA. Oleic acid and LA stimulated proliferation at non toxic concentrations (<75uM) that could induce apoptosis and necrosis whereas other FA decreased proliferation by causing cell d eath (C16:0 and C18:0) or cell cycle arrest and apoptosis (EPA, DHA) if concentration were >25uM. Wallace et al. (2001) fed mice a low fat control diet or diets supplemented with CCO, SAO of FO; the FA profile of phospholipids fraction of spleen lymphocyt es reflected the diet. Thymidine incorporation into Concanavalin A stimulated lymphocytes and IL 2 production were greater after CCO feeding whereas IFN production was decreased when feeding SAO or FO. The ratio of IFN :IL 4 was used as the ratio of pro duction of Th1:Th2 type cytokines. This ratio was lower for mice fed SAO or FO; whereas, mRNA expression of cytokines at 4 and 8 h indicated that the production of cytokines affected by the feeding of specific FA was regulated at the level of gene expressi on. Rodrigues et al. (2010) fed rats doses (0, 0.11, 0.22, 0.44 g/kg BW) of OA or LA. Neutrophil migration was greater in mice fed the 2 greater doses of OA but only the lower dose (0.11 g/d) of LA was needed to enhance neutrophil migration in response to

PAGE 77

77 intraperitoneal injection of glycogen. Enhanced migration may be possible due to an increase in expression of CD62L, production of the chemoattractant CINC 2 and enhanced rolling of neutrophils, all were enhanced with both FA but no FA was found to incr ease expression of CD18, an important integrin in the process of neutrophil extravasation. In the presence of lypopoliscaccharide (LPS), only LA reduced the production of CINC 2 The ratio of Th1:Th2 cell s or of their derived cytokines (IFN :IL 4) are measured to evaluate the polarization of the immune system toward antibody mediated (Th1 < Th2) or cell mediated immunity (Th1 > Th2). For 4 wk Mizota et al. (2009) fed liquid sources of fat to mice subj ects to change the dietary ratio of n 6:n 3 (0.25, 2.27 or 42.9) due changes in LA and ALA. Production of IFN by mononuclear cells from splenocytes declined when LA r ich diets were fed relative to greater ALA diets Whereas i nterleukin 4 was reduced when either lower or greater LA rich diets were fed T hus the ratio of IFN :IL 4 was greater in mice fed the high ALA diet, indicating, contrary to the common a nt inflammatory definition of n 3 FA, that n 3 enriched diets at the level evaluated here had infla mmatory properties. In a later in vivo study, Diwakar et al. (2011) evaluated the impact of feeding rats with different proportions of LA and ALA The 4 experi mental diets were: diet 1:53.6% LA and 0.45% ALA, diet 2: 40% LA and 8.8% ALA diet 3: 32.2% LA and 16.7% ALA, and diet 4 : 9.9% LA and 32.2% ALA Supplementation of diets rich in ALA (greater than D1), increased the proportions of ALA, EPA, and DHA in the membrane of splenocytes and peritoneal macrophages. Proliferation of splenocytes stimulated with concanavalin A and phytohaemaglutynin (PHA) decreased when any of the 4 diets was fed. A similar

PAGE 78

78 effect happened with the production of nitrite at 12 h post stimulation of peritoneal macrophages. Production of leukotriene B4 by peritoneal macrophages was only decreased by diet 3 and 4 but TNF 2 concentrations did not differ. These responses a gree with others reporting ant inflammatory effect of diets rich in ALA. In this study this effect was executed by the decreased proliferation of lymphocytes and potential reduction of the phagocyti c activity of immune cells. In an attempt to evaluate the effect of n 6 FA to alter immune function Thanasak et al. (2004) fed castrated goats either olive oil (10% LA) or CO (55% LA) for 3 wk. Goats in the CO group had greater LA concentrations in both p lasma and erythrocyte at 21 d after supplementation. Goats fed CO experienced a reduction in the percentage of blood lymphocytes expressing 4 integrin (CD49d) at day 21. However no change were observed in lymphocyte proliferation after concanavalin A or PHA stimulation, in total white blood cell count, or in lymphocytes expressing CD2, CD4, CD8, CD21 or MHC II. Authors could not give a concl usive mechanism for these immune responses but stressed that a combination of all mechanism by which FA perform their action such as changes in membrane fluidity might affect intracellular interaction, receptor expression, nutrient transport, signal transd uction, regulation of gene expression, protein acylation or calcium rele ase might be potential factors. Beef calves undergo stress during long distance shipping and arrival in new environment which induce inflammatory response. Feeding of soybean seed with high LA might exacerbate that inflammatory response which might lead to undesirable animal performance. Farran et al. (2008) transported crossbreed heifer calves (~200 kg initial BW) from Kansans to use in 35 d receiving diet experiments. Heifers were fed

PAGE 79

79 diets containing tallow (2.3% LA and 0.3% ALA), linseed (15.9 LA and 54.2% ALA) or soybean seed (54% LA and 8% ALA). Changes in plasma FA profile were in parallel to the diets. Calves fed soybean seed had the lower ADG and FE, but percentage of calves tre ated for bovine respiratory disease did not differ due to fat supplements. A group of calves were challenged with LPS and resulting rectal temperatures were lower for soybean seed and linseed fed groups whereas concentrations of plasma TNF were greater f or heifers fed soybean seeds when compared to those fed tallow Diets did not affect plasma haptoglobin, fibrinogen, or total white blood cell count after LPS challenge. In a study performed at the University of Florida, Araujo and coworkers (2010) in a f irst trial, evaluated the effect of supplementing rumen inert SFA ( 2.1% of dietary DM, 1.7% LA; Energy Booster 100) or PUFA ( 2.5% dietary DM, 28.5% LA, Megalac R) and a control non fat supplemented diet for 30 d after transportation and feedyard entry of B radford steers (218 kg BW). Steers fed rumen inert SFA had decreased DMI and tended to gain less BW compared with control steers but no effect was detected for plasma concentrations of fibrinogen and ceruloplasmin. In a second trial, Brahman crossbreed hei fers (276 kg BW) were fed diets of 0 or 5.7% of Megalac R starting 30d before transportation to ensure adaptation to diets. No effect of diets was detected on DMI and ADG post transport. Also no difference in plasma concentrations of ceruloplasmin was dete cted; however, plasma concentrations of haptoglobin were lower during the first week post transportat ion for heifers fed Megalac R. A raise in circulating concentrations of haptoglobin is a liver response to proinflammatory cytokines Megalac R primarily contains LA but also minimal concentrations of ALA. I t could be

PAGE 80

80 that the production of cytokines might change in response to a specific FA (LA or ALA) or a combination of both FA (LA + ALA) that in turn modified the synthesis of haptoglobin. H owever a redu ced inflammatory response that could detrimentally affect performance could not be ruled out, in fact DMI, ADG and /or FE post transport was not improved by the feeding of Megalac R. Other University of Florida study, Silvestre and coworkers (2011) fed t ransition dair y cows Ca salts of palm oil or SAO N eutrophils of cows fed SAO from 30 d prepartum to 35 d postpartum had greater concentrations of vaccenic acid (0.45 vs. 0.96%), LA did not differ (20.6 vs. 23.2%), lower concentrations of c 9, t 11 CLA (1.69 vs. 0.85%) and ALA (1.43 vs. 1.02%. Cows fed SAO had increased plasma concentrations of haptoglobin and fibrinogen. The percentage of blood neutrophils with phagocy tic and oxidative burst activities were not affected by diets but mean number of E. coli ph agocyt iz ed per neutrophil and mean intensity of H 2 O 2 produced per neutrophil were increased in cows fed SAO at 4, and 4 and 7 d postpartum respectively. Percentage of neutrophils positive to CD62L and CD18 were greater in cows supplemented with SAO at 4 a nd 7 d postpartum. Throughout the evaluation period, mean number of CD62L expressed per neutrophil was greater in cows fed SAO but number of circulating neutrophils expressing CD62L was lower whereas mean number of CD18 did not change with diets. Concentra tion of TNF in incubated isolated neutrophils at 35 d postpartum was greater in cows fed SAO diets both with and without LPS stimulation, but total mass increase did not differ with diets. Concentration of IL 1 was greater in neutrophils when cells of cows fed SAO were stimulated with LPS and mass change was also greater. Supplementation of LA during the transition period enhanced the

PAGE 81

81 inflammatory and acute responses of dairy cows to better cope during immunesuppresed period prone to exacerbate incidence of diseases Lake et al. (2006c) conducted two experiments to determine the effect of maternal lipid supplementation on calf response to an antigenic challenge, in trial 1 a control low fat diet and a diet containing high LA safflower seeds (9.5% of diet DM), both is o caloric and isonitrogenous, were fed to primiparous beef cows from 1 to 40 d of lactation. Calves born from cows fed safflower seeds had a lower and delayed response of antibody production in response to an ovalbumin (OVA) challenge. In a second trial cow s were blocked by BCS at birth and were supplemented with no oils seeds, high linoleate safflower seeds (8.1% of dietary DM), or high oleate safflower seeds (7.6% of dietary DM). Calves born from cows supplemented with LA or OA had lower serum concentratio ns of anti OVA IgG but cell mediated immune response were not affected. Potential change in FA profile of lymphocyte affecting membrane fluidity and/or lymphocyte proliferation was proposed as the potential cause of impaired production of antibodies in cal ves suckling from LA supplemented cows. To the best of our knowledge there is no study evaluating the inclusion of LA in MR to modify activity of different markers of immune response in newborn calves, however, some work were recently developed to evalua te the effect of n 3 FA from animal or vegetal origin. Ballou and DePeters (2008) evaluated the inclusion of FO in calf MR to replace 1 or 2% of the fat of the control MR (20% fat, DM basis. Authors reported no differences in fecal score, concentrations of white blood cells, hematocrit, total protein, and phagocytic activity of polymorphonuclear leukocytes in blood. However, production of anti OVA IgG was attenuated after the second OVA injection in

PAGE 82

82 calves fed 1% but not 2% of FO. Differential effect on ger minal center affinity maturation of Ig class switching might be the mechanism by which a quadratic response in IgG production was observed when supplementing FO. Hill et al. (2011) fed newborn dairy calves with a control MR containing 15% of animal fat or a 15% fat MR containing 1% of NeoTec 4 (blend of butyric acid, CCO and LSO). Calves fed NeoTec 4 had fewer numbers of days with an abnormal fecal score and also the average fecal score tended to be lower. After pasteurella vaccination, the relative mRNA abundance (respect to non vaccinated and non NeoTec 4 supplemented calves) of TNF IL 4, IL 6, and IL 10 did not differ pre or post vaccination, but the change in relative mRNA abundance from pre to post vaccination them was negative for TNF ended to be positive for IL 4 These effects coincided with lower rectal temperatures and less refusal of MR after vaccinations in calves fed NeoTec 4 Additionally, calves fed NeoTec 4 had greater antibody titer post vaccination with parainfluenza 3 (PI3) and bovine virus diarrhea type I Results indicate that butyric acid and ALA can cause a reduced inflammatory response by potentially reducing the synthesis of proinflammatory cytokines and changing the immune response over an antibody type instead to a cell mediate response. A very limited amount of in vivo studies have focused in the effect of supplementing LA to evaluate the modification of immune response in dairy calves From the available studies, including other species or adult cattle, LA seems t o modify the activity of diffe rent cells of the immune system. The primary effect of LA by itself or its derivatives appears to be the induction of inflammatory response by increasing the production of proinflammatory eicosanoids, synthesis of proinflammat ory cytokines, and

PAGE 83

83 enhancing the migration and activity of leukocytes on injured tissues. Contrary, ALA could have an opposite effect on inflammation through increasing the production of antinflammatory eicosanoids and cytokines and by reducing the migrati on and activity of leukocytes. However, some invitro studies have demonstrated that LA could reduce the activity of immune cells and production of proinflammatory cytokines when cell s were cultured with greater concentrations of LA. All these effects might indicate that under in vivo circumstances, the physiological status of preweaned calf could modify the need of LA and their effect on immune cells. Future research should focus in the effect of increased intakes of LA modifying different parameters of imm une response. Effect of Supplemental Fatty Acids on Hepatic Gene Expression The liver plays a critical role in the systemic circulation; its strategic position in blood circulation ( connected to systemic circulation by vena cava and hepatic artery and to i ntestines trough portal vein and bile duct) allows the liver to carry out all its different metabolic functions. Among the metabolic functions are lipid, carbohydrate, and protein metabolism, including protein generation and metabolism of toxic or waste pr oducts (Thomson and Knolle, 2010). Liver disorders or diseases, primarily fatty liver, can lead to impairment of liver function. Hence it is of high importance to provide a balanced diet to avoid excess ive accumulation of FA in liver primarily because of excessive weight loss in cattle. Different dietary strategies have been evaluated in humans to reduce the risk of hepatic steatosis and many of these studies have been replicated recently in cattle, primarily in transition cows, which are the group with hi gher risk of fatty liver. Grummer (2008) divided the nutritional strategies to prevent fatty liver in cows into two groups: a) diet formulation to increase energy density and b) inclusion of feed additives with different

PAGE 84

84 modes of action such as reduction o f adipose lipolysis, enhanced hepatic VLDL secretion or increased hepatic FA oxidation. Supplementation of n 6 and n 3 PUFA have became an important dietary strategy in humans and rodents, similar to including supplementation of PPAR agonists (Clarke, 2001 ; Sekiya et al., 2003; Guo et al., 2006a,b; Rakhshandehroo, et al., 2009 ) with even more recent evaluation in cattle (Litherland et al., 2010; White et al., 2011a, b; Bionaz et al., 2012). Polyunsaturated FA elicit their effects by coordinating suppressi on of lipid synthesis and upregulating FA oxidation in liver. Clarke et al. (2001) and Sampath and Ntambi (2005) reviewed different studies supplementing n 3 and n 6 FA. Authors concluded that n 3 FA had a more potent activity than n 6 FA and that suppress ion of lipid synthesis in liver is a more sensitive pathway regulated by PUFA than the lipid oxidation pathway. Before the discovery of nuclear receptors capable of binding FA and establishment of a direct role of FA in gene regulation, it was established that FA can affect cell signaling and gene expression by affecting membrane phospholipid content or through the production of eicosanoids (Sampath and Ntambi, 2005). Regarding livestock animals, more studies on PUFA regulation of gene expression have bee n carried out using pigs and chickens but scarcely any using ruminants. Definitely more studies are needed to elucidate the important mechanisms by which FA can exert their regulatory function on gene expression in dairy cattle. Regulation of Hepatic Pe roxisome Proliferator Receptor A well described ligand activated nuclear transcription factor is PPAR which plays important roles in lipid and carbohydrate metabolism Upon activation PPAR forms a heterodimer with a FA and RXR his heterodimer b inds to PPAR responsive elements in the regulatory regions of target genes to influence gene expression

PAGE 85

85 (Schmitz and Ecker, 2008). Two isoforms, PPAR and PPAR are the most well understood isoforms. The PPAR primarily expressed in the liver where it is involved in promoting gluconeogenesis and stimulating the transcription of genes that are critical for peroxisomal and mitochondrial oxidation of FA, as well as a regulator of other transcription factors (Weickert and Pfeiffer, 2006; Calder, 2 012). Among all transcription factors, PPAR has a wide e ffect on expression of genes for different processes of lipid metabolism as well as on other pathways such as glucose and amino acid metabolism, and inflammation. For each of those processes PPAR aff ects the expression of several genes. A good proportion of these genes were already identified due to the presence of a PPAR response element in the promoter region (Rakhshandehroo et al., 2010). Effect of PUFA on PPAR Fatty acids and more sp ecifically PUFA are natural ligands of PPAR (CV 1 cells) and analyzed the binding ability of different FA to PPAR reported that MCFA (C12:0 to C16:0) were weak activators of PPAR reas the best activators were ALA, AA, and LA. They reported also that derivatives of lipoxygenase metabolism such as 8(s) hydroxyeicosatetraenoic acid was a potent ligand of PPAR whereas leukotriene B4 was a weak one. Hostetler et al. (2005) evaluate d the affinity of different forms of FA for PPAR using a direct fluorescence ligand binding assay in E. coli strains expressing the recombinant mouse PPAR protein. They concluded that saturated or unsaturated LCFA acyl CoA and certain PUFA ( cis and trans parinaric acid and C18:4) were able to bind PPAR with higher affinity whereas, regardless of chain length, SFA were not

PAGE 86

86 significant binders. Recently Bionaz et al. (2012) used bovine kidney cells cultured with a PPAR agonist and individual 12 carbon FA to evaluate the differential expression of 30 genes involved in lipid metabolism and inflammation. They reported that out of 15 genes, well known to be target genes of PPAR in nonruminants, 10 were upregulated by the PPAR agonist in bovine kidney cells Interestingly, the stronger activation effect was induced by C16:0, C18:0, and EPA followed by C20:0 and CLA c 9 t 1. Authors concluded that the preference for SFA in bovine may be due to adaptation of PPAR in ruminants to cope with greater availability of SFA in their diets. Regulation of Hepatic Sterol Regulatory Element Binding Protein Three isoforms of SREBP have thus far been identified; SREBP 1a and SREBP 1c. The SREBP 1 form is an important regulator of genes involved in lipid synthesis whereas SREB P 2 has been shown to control genes important to cholesterol homeostasis. The SREBP 1c is the major isoform in rodent and human liver (Sampath and Ntambi 2005). The SREBP are initially synthesized as large proteins and have to undergo a maturation process that includes reduction in size and further transit to the nucleus. Once in the nucleus, it binds to cis elements, termed sterol regulatory elements, in the promoters of target genes and induces the transcription of a variety of genes involved in choleste rol, TG, and FA synthesis (Sampath and Ntambi 2005). A fasting refeeding experiment in rodents (Horton et al., 1998) indicated that refeeding enhanced expression of FA biosynthetic enzymes compared to the prefasting condition but expression of cholesterol synthetic enzymes only returned to the prefasting level. The expression of SREBP 1 and SREBP 2 followed exactly the same pattern of expression after the refeeding state. These findings led to the hypothesis that the inhibitory effect of PUFA on lipogenic gene expression could occur via either

PAGE 87

87 repression of SREBP mRNA or inhibition of SREBP maturation (Horton et al., 1998; Sampath and Ntambi, 2005). Effect of PUFA on SREBP activity. Yahagi et al. (1999) supplemented wild and transgenic mice that over expres sed a mature form of SREBP 1 in liver, with different sources of FA. In wild mice, SFA and MUFA sources did not reduce the expression of mature SREBP 1 whereas EPA and DHA were more potent depressors of SREBP 1 expression followed by LA. The rate of decrea se in mature SREBP 1 paralleled those in mRNA for lipogenic enzymes such as FA synthase (FASN) and acetyl CoA carboxylase (ACC). In the transgenic mice, dietary PUFA did not reduce the amount of SREBP 1 protein. This result excluded the possibility that PU FA accelerated the degradation of mature SREBP 1. These results demonstrated that the suppressive effect of PUFA on lipogenic enzyme genes in the liver is caused by a decrease in the mature form of SREBP 1 protein, which is presumably due to the reduced cl eavage of SREBP 1 precursor protein. In an attempt to evaluate the effect of cholesterol supplementation on regulation of transcription factors, Kim et al. (2002) fed mice 3 sources of fat (5% diet) namely triolein, SO (rich on LA) and FO (rich in EPA and DHA) supplement with or without 2% of cholesterol (cholesterol is a potent inducer of stearoyl CoA desaturases expression) Authors reported that when a high cholesterol diet was combined with either SO or FO, maturation of SREBP 1 mRNA was repressed wh ereas levels of mRNA, protein synthesis, and enzymatic activity of stearoyl CoA desaturase 1 were increased. Interestingly, mice of the same dietary group had increased levels of SREBP 1 mRNA, however the mRNA levels of SREBP 1 target genes such as FASN a nd LDL receptor

PAGE 88

88 were decreased. Results indicated that the main control of PUFA mediated suppression of SREBP 1 target genes is through repressing SREBP 1 maturation and demonstrated that cholesterol regulates stearoyl CoA desaturase 1 gene expression thr ough a mechanism independent of SREBP 1 maturation. The mechanisms by which PUFA affect SREBP is still unclear. One recent study (Di Nunzio et al., 2010) evaluated the effect of different FA to suppress SREBP activity and regulate the flow of nonesterified cholesterol using hepatic hepG2 cells. The supplementation of FA reduced SREBP activity in the order of EPA = LA = AA > ALA = DHA = DPA > OA. Likewise, the incorporation of PUFA increased nonesterified cholesterol flow from the plasma membrane to intracel lular membranes. Suppression of SREBP activity by PUFA may depend on the degree of incorporation into cellular lipids, and it may be associated with increased flow of nonesterified cholesterol between the plasma membrane and intracellular membranes. Regul ation of Hepatic liver X Receptor The LXR are transcription factors belonging to the nuclear receptor super family. Two isoforms exist, LXR LXR These receptors are recognized as important regulators of cholesterol metabolism, lipid biosynthesis, and glucose homeostasis as well as regulators of the storage and oxidation of dietary fat (We ickert and Pfeiffer, 2006). T his receptor is activated by binding to oxysterols, which are derived from the cholesterol oxidative process. After binding, LXR needs to form an obligated heterodimer with RXR before binding the DNA on the LXR responsive elem ent (Ducheix et al., 2011). This receptor plays a crucial role in regulation of FA metabolism by activating the expression of SREBP 1c (Yoshikawa et al., 2003) and carbohydrate regulatory element binding protein (ChREBP) (Cha and

PAGE 89

89 Repa, 2007) when binding t o its promoter region in each of these two transcription factors. Independently, LXR can also bind LXR response elements on the promoter region of FASN, ACC, and stearoyl CoA desaturase 1 (Ducheix et al., 2011). Effect of PUFA on LXR activity. In an attemp t to investigate the molecular mechanism by which dietary PUFA decrease hepatic SREBP 1c expression, Yoshikawa et al., (2002) established mouse SREBP 1c promoter luciferase reporter assays in HepG2 cells and HEK293 cells. Supplementation of EPA in the medi um withHepG2 or HEK293 cells, both co transfected with LXR 1c promoter activity when the heterodimer LXR/RXR was activated. Deletion of the two liver LXR responsive elements present in the SREBP 1c promoter region eliminated the suppressive effect of PUFA. Authors evaluated the effect of different FA on their ability to decrease SREBP 1c promoter activity resulting in the order: AA > EPA > DHA > LA, whereas SFA had no effect and oleic acid had minimal effect. These results ind icate that both LXR responsive elements are important PUFA suppressive elements suggesting that PUFA could be deeply involved in nutritional regulation of cellular FA concentrations by inhibiting the LXR SREBP 1c system, which enhances lipogenesis The sam e group, Yoshikawa et al. (2003), using similar methodology demonstrated that PPAR 1c promoter activity induced by LXR, concluding that deletion of the two LXR response elements in the SREBP 1c promoter region were responsible for this in hibitory effect of PPAR In contrast to the regulation of LXR activity by PUFA reported by Yoshikawa et al. (2002, 2003), Pawar et al. (2003) concluded that PUFA suppressed SREBP 1 and its target genes by other mechanisms than by LXR. They used primary hepatocytes and

PAGE 90

90 FTO 2B hepatoma cells supplemented with different PUFA. Authors reported a similar response in both cells when rats were fed diets of 10% FO. They reported that EPA in primary hepatocytes or FO in in vivo conditions suppressed hepati c SREBP 1c regulated genes ( FASN, S14, glycerol 3 phosphate acyltransferase and l iver pyruvate kinase) and induced PPAR 4A, mitochondrial HMG CoA synthase, acyl CoA synthetase 1, and acyl CoA oxidase] but SREBP 1c for their activat ion (CYP7Aq, ATP binding cassette subfamily G5 and G8), concluding that the PUFA suppression of SREBP 1 and its target lipogenic genes is A more recent study (Howell et al, 2009) indicated that hepatic cells transfected with LXR resp onsive element had an increased activity of full length SREBP 1c when treated with an LXR agonist. However this activity was reduced when cells were treated chimeric protein. Thes e results did not favor the idea of competitive antagonism of ligand binding, but they demonstrated that n 3 PUFA effectively mitigated the induction of SREBP 1 via reduced trans activation capacity of LXR. Regulation of Other Hepatic Receptors Hepatocyte nuclear factor 4 It is a highly conserved nuclear receptor th at binds to direct repeated elements as a homodimer. This receptor seems to be indispensable for hepatocyte differentiation and hepatic functions, such as cholesterol and lipoprotein secretion. It is expressed mainly in liver, kidney, intestine, and pancreas and is capable of activating target genes even in the absence of a ligand (Sampath and Ntambi 2005).

PAGE 91

91 Hertz et al. (1998) studied the binding of recombinant HNF 4 dimer to its cognate C3P promot er as a function of the degree of unsaturation and chain length of fatty acyl CoA. Binding was activated by different C14 to C16 saturated fatty acyl CoA but was inhibited by (C18:0) CoA and (C18:3, n 3) CoA. When amounts of HNF 4 were limited, C3P bindin g was dependent on concentrations of C14:0 CoA within the range of concentrations required for ligand binding to HNF 4 Both activation of C3P binding by C14:0 CoA and inhibition by C18:3 CoA were observed using mammalian HNF 4 Inhibitor kB and necro sis factor kB (NF k B). These factors are present in the cytoplasm of cells, in their inactive form, as a h eterodimer. Phosphorylation of inhibitor translocate to the nucleus modifies the transcription of a variety of genes involved in inflammation, including cytokines, adhesion molecules, acyl CoA oxidase 2, and inducible NO synthase (Calder, 2012). vely regulated by a positive effect on the transcription of its target genes, EPA more potently inhibits et genes (Camandola et al., 1996). Products of the AA metabolism through the activity of P450 epoxygenases, such as different AA derived One derivate group is epoxyeicosatrienoic acids, which have vasodilatory properties and can prevent the nuclear accum the prevention of inhibitor (Node et al., 1999).

PAGE 92

92 Retinol x Receptor ( RXR ) This receptor has already been mentioned as the heterodimer that PPAR needs for its nuclear tr anslocation. However, RXR first needs to be activated by its natural ligand 9 cis retinoic acid. Once bound to its ligand, RXR is indirectly involved in different cellular processes such as transduction of the retinoid signaling pathway and lipid anabolism and catabolism (Schmitz and Ecker, 2008). Lengqvist et al. (2004) evaluated the activation of RXR by different FA in transfected cells with an RXR expression vector by direct addition of the tested FA. Whereas DHA, EPA, and AA were robust activators of RXR C16:0 and C18:0 were not. It was also demonstrated that the activation of RXR was not due to presence of PPAR or any other ligand from other r eceptor factors. Farnexoid X receptor (FXR). Farnexoid X receptor is a nuclear receptor controlling the expression of genes whose products are critically important in bile acid and cholesterol homeostasis. Stimulation of FXR enhances the expression of a short heterodimer protein, which has a negative feedback effect on LXR activity (Schmitz and Ecker, 2008). Zhao et al. (2004) evaluated transfected HepG2 with FXR fusion protein for its ability to bind FA and reported a positive binding affinity of FA for FXR in the order ALA > AA > DHA, whereas C16:0 and C18:0 had no binding activity on FXR. The expression of the FXR target genes, bile salt export pump and kininogen, were differentially regulated by PUFA supplementation. Bile salt export pump was induced with PUFA supplementation but kinonegin expression was depressed. Through this selective mechanism of regulation of target FXR genes, PUFA may contribute to the beneficial effect on lipid metabolism by preventing the accumulation of cholesterol in liver an d circulation, enhancing its transport as part of bile acids

PAGE 93

93 ChREBP and max like protein X (MLX). The ChREBP is a transcription factor involved in mediating glucose responsive gene activation. It is most abundantly expressed in tissues in which lipogenes is is highly active, such as the liver and its activity is enhanced in diets rich in carbohydrates. ChREBP was recognized initially by its ability to bind the carbohydrate response element within the promoter region of the PK gene (Yamashita et al., 2001). Later other studies determined that ChREBP additionally induces positive transcriptional effects on lipogenic enzymes such as ACC and FASN (Dentin et al., 2004). Additionally, Stoeckman et al. (2004) utilized human embryonic kidney 293 cells to identify i f MLX was a heterodimer partner of ChREBP regulating the expression of glucose responsive genes. The cotransfection of plasmids expressing either ChREBP or MLX with a carbohydrate response element containing reporter plasmid into human embryonic kidney 2 93 cells did not activate the promoter containing ChRE on target lipogenic genes; however the expression of both ChREBP and MLX significantly enhanced promoter activity for reporters containing carbohydrate response element from several lipogenic enzymes. The role of ChREBP in li pogenesis has led researchers to evaluate its potential role in the physiopathology of hepatic steatosis, which in humans has been highly correlated with further diseases such as obesity, insulin resistance, and type 2 diabetes (Pos tic et al., 2007). To prevent the occurrence of these diseases, the feeding of PUFA has been evaluated to prevent the negative impact of high carbohydrate and high SFA diets through PUFA capacity to reduce the activation of ChREBP. Dentin et al. (2005) fed mice a 10% fat diet containing either C18:0, C18:1, or a mix of PUFA containing 45% LA, 5% EPA, and 3.5% DHA. Mice supplemented with PUFA but not with C18:0 or

PAGE 94

94 C18:1 suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translo cation from the cytosol to the nucleus, independently of an activation of the AMP activated protein kinase. Inhibition of translocation was accompanied by an inhibition of liver piruvate kinase and FASN, key lipogenic genes. Regulation of Hepatic Uptake a nd Binding of Fatty Acids Dietary FA esterified in chylomicron TG or in VLDL TG are derived both dietary and endogenous biosynthesis. Tryglicerides are hydrolyzed into FA by the action of lipoprotein lipase Upon hydrolysis, dietary NEFA enter into the c ell, similar to albumin bound NEFA mobilized from storage depots. The mechanism by which NEFA enter the cell are still unclear (Bordoni et al, 2006). Pownall and Hamilton (2003) discussed the controversies regarding the contribution of passive diffusion of FA versus protein mediated FA transport and concluded that both models have their validity and would lead to a common rationalized model. Some studies evaluated the expressi on of FA transport protein genes (also known as SCL27 family and composed by 6 s ubfamilies). Motojima et al. (1998) discovered a genes coding for FA transport proteins being up regulated by PPAR A direct effect of PPAR in this upregulation was verified when PPAR null mice were used and no change in FA transport protein was detec ted. Rakhshandehroo et al. (2009) comparing the differential co regulation of genes by PPAR in human and mouse hepatocytes, reported that the solute carrier family 27 (fatty acid transporter), member 2, was co upregulated in both species. The mammalian f atty acid binding protein ( FABP ) family binds long chain FA with high affinity; this family comprises a group of high affinity intracellular FA binding proteins with both unique and overlapping functions. The FABP family modulates intracellular lipid homeo stasis by regulating FA transport in the nuclear

PAGE 95

95 and extra nuclear compartment of the cell, impacting systemic energy homeostasis and other unique functions depending on the cell type. Liver FABP have been hypothesized to be involved in lipid absorption by the enterocyte and in hepatocyte lipid transport and lipoprotein metabolism (Storch and McDermott, 2009). Liver FABP was postulated to be responsible to aid PPAR targets such as FA to reach the nuclear receptor. Wolfrum et al. (2001) reported that live r FABP and PPAR are co localized in nucleus of mouse primary hepatocytes and that liver FABP has the ability to directly interact with PPAR and PPAR but not with RXR or PPAR The interaction of liver FABP and PPAR was independent of the ligand binding, but activation of PPAR was in positively correlated with concentration of liver FABP for all ligands tested. Among the ligands tested to enhance activation of PPAR was found to have the shallowest slope, with the steepest slope in decre ased order of: ALA > OA > AA. In an attempt to evaluate the molecular mechanisms responsible for the pleiotropic effects of PPAR agonists, Guo et al. (2006a) treated mouse hepatocytes with 3 different PPAR agonists. Authors documented that all agonist s enhanced PPAR t ransactivation. Among the differentially expressed genes (DEG) the most prominent group was that of lipid metabolism with FABP1 increasing about 20 to 30 fold with all agonists. However, duck hepatocytes supplemented with LA or EPA caus ed an upregulation of PPAR and their target genes acyl CoA oxidase and lipoprotein lipase but no change was reported for liver FABP (Liu et al., 2011). Regulation of Hepatic Fatty Acid Oxidation Regulation of lipid metabolism is coordinated mainly by th e liver, which actively metabolizes FA as fuel and continuously produces VLDL particles to provide a constant

PAGE 96

96 supply of FA to peripheral tissues. Oxidation of FA in liver occurs through the 3 main following pathways: peroxisomal oxidation, mitochondrial oxidation, and hydroxylation, with most of the enzymes of these pathways being tightly regulated by PPAR sturbances in these pathways are the basis for hepatic steatosis and alterations in plasma lipoprotein concentrations (Rakhshandeh roo et al., 2010). Peroxisomal oxidation Peroxisomes are known to be involved in many aspects of lipid metabolism, including synthesis of bile acids and plasmalogens, synthesis of cholesterol and isoprenoids, alpha oxidation, glyoxylate and H 2 O 2 metabol ism, and oxidation of very long straight chain or branched chain acyl CoA (Rakhshandehroo et al., 2010). The role of PUFA in peroxisomal oxidation is through the activation of PPAR. The activation not only enhances the proliferation and size of peroxi s omes but also up regulates different key enzymes involved in the oxidative process. At present, three different types of FA are known to fully rely on peroxisomes for oxidation. These include the following: 1) very long chain FA such as C24:0 and C26:0; 2) the 2 methyl branched chain FA pristanic acid (2, 6, 10, 14 tetramethylpentadecanoic acid); and 3) the bile acid synthesis intermediates dihydroxycholestanoic acid and tr ihydroxycholestanoic acid In addition, LC FA can be oxidized in peroxisomes but are preferentially oxidized in mitochondria ( Wanders and Waterham, 2006; Wanders et al., 2010). Peroxisomes contain the full enzymatic machinery to oxidize FA, although oxidation does not go to completion. In general, the architecture of the peroxisoma l oxidation system is comparable to that of mitochondria and consists of subsequent

PAGE 97

97 steps of: dehydrogenation, hydration, dehydrogenation again, and thiolytic cleavage. Among the enzymes involved in peroxisomal oxidation and found to be up regulated by PPAR in liver of humans and/or rats are acyl CoA oxidase 1, enoyl CoA, and hydratase 3 hydroxyacyl CoA dehydrogenase that have PPAR response elements in their promoter regions (Rakhshandehroo et al., 2010). The end products of peroxisomal oxidation are shuttled to mitochondria, either as carnitine esters and/or as free FA for final oxidation (Wanders et al., 2010). Oxidation This process provides energy, as ATP yield for every oxidation cycle, to different cellular processes, with SC FA (< C8), MC FA (C8 to C12), and LC FA (C12 to C20) as principal targets. Mitochondrial oxidation results in progressive shortening of FA into acetyl CoA subunits, which either condenses into ketone bodies or enters into the tricarboxylic acid cycle for f urther oxidation to water and carbon dioxide (Reedy and Rao, 2006). Mitochondrial oxidation is primarily regulated by control of its key gene carnitine palmitoyltransferase 1 Among the regula tors of carnitine palmitoyltransferase 1 are: carnitine conc entrations, malonyl CoA, FA, fatty acyl CoA, and different peroxisome proliferators (Reddy and Rao, 2006). Genes that control the import of FA into the mitochondria are upregulated by PPAR activates the major enzymes within the oxi dation pathway including vario us acyl CoA dehydrogenases mitochond rial trifunctional enzyme and genes involved in oxidation of unsaturated FA. In addition PPAR the synthesis of ketone bodies via mitochondrial HMG CoA synthase and HMG CoA lyas e (Rakhshandehroo et al., 2010).

PAGE 98

98 Microsomal hydroxylation The mammalian CYP4 family of P450 enzymes catalyzes the preferential hydroxylation of FA (e.g., hydroxylases of the CYP4 family are known to convert AA to its metabolite 20 hydro xyeic osatetraenoic acid ). The enzymes of this family differ in their substrate specificities in terms of FA chain length and degree of unsaturation. In some instances, these enzymes exhibit preferential affinities for prostaglandins and leukotrienes, but almost invariably preferentially catalyze over 1 hydroxylation of their substrates (Johnston et al., 2011). Expression of CYP4A genes is extremely sensitive to PPAR indicating that CYP4A genes may serve as PPAR performed in human hepatocytes have revealed significant induction of CYP4A11 by the PPAR hydroxylation of SFA and unsaturated FA may lead to the generation of high affinity PPAR including 20 hydroxyeicosatetraenoic aci d or 20 OH EPA from EPA and 20 OH DHA from DHA, and 20 hydroxyeicosatetraenoic acid from AA, with a potential inhibition of synthesis of the former ligand by the n 3 derivate oxidases (Harmon et al., 2006). oxid oxidation in myeloid cells and hepatocytes. Degradation is accompanied by loss of biological activity. Interestingly, the degradative process of leukotriene B4 with subsequent loss of its biological proinflammatory function, takes place at microsomal oxidation in hepatocytes by the activity of degradative enzymes. The activity of these enzymes is increased by the proliferation of PPAR by binding to leukotriene B4 (Crooks a nd Stockley, 1998).

PAGE 99

99 Regulation of Lipogenesis and Hepatic Steatosis Whereas several factors contribute to enhance lipogenesis such as LXR, SREBP, and ChREBP, PPAR any of the 3 FA oxidati on systems discussed above has a key role in lipid homeostasis and prevention of hepatic steatosis (Reddy and Rao, 2006). Early studies supplemented different FA sources to rats fed fat free diets (Clarke et al., 1977). Rats supplemented with LA (3% of die t, as fed basis) for 7 d reported a decreased activity of FASN and ACC as well as a reduction in the deposit of total FA in liver. Toussant et al. (1981) fed rats a fat free diet or diets supplemented with SAO at 5 or 10% of diet (as fed basis). Authors di d not find a reduction in FASN activity when rats were fed diets of 5% SAO. However, when rats were fed diets of 10% of SAO, the enzymatic activity of ACC was reduced as was the synthesis of FA in liver. Berger et al. (2002) evaluated the effect of increas ing dietary concentration of PUFA relative to a control diet (10% fat, 0% AA, and DHA) on mice global hepatic gene expression. The diets were: 0% AA + 0% DHA, 0.5% AA, 0.5% DHA, or 0.5% AA + 0.5% DHA. Supplementation of 0.5% of DHA or a mixture of AA + DHA decreased the expression of SREBP with respect to mice fed the control diet whereas supplementation of AA did not. Regardless of the type of fat fed, expression of PPAR was not affected, although most of its target genes were, particularly those contain ing PPAR response elements. Among the PPAR target that were down regulated in hepatocytes of mice fed diets containing FA were: acetyl CoA synthetase 1 and ATP citrate ly ase, whereas only FASN was down regulated when AA or AA + DHA were supplemented. The r ate of down regulation was stronger with the combination of FA rather than with single FA which was unexpected.

PAGE 100

100 Piot et al. (1999) reported that calves fed CCO compared with tallow had a greater oxidation rate of C12:0 in liver, and the liver contained more fat. They concluded that the incomplete oxidation of C12:0 led to the synthesis and elongation of FA to be finally deposited in the liver. Gruffat Mouty et al. (1999), when comparing the rate of secretion of VLDL in rat and calf liver, reported no reducti on in the rate of synthesis of APO B100 between species. They concluded that there may be a defect in VLDL assembly and/or secretion which could affect the export of VLDL TG from calf liver. Later the same group (2001) reported that the feeding of CCO to c alves increased the infiltration of FA into liver tissue by reducing the synthesis of APOB. Sato et al. (2005) fed chickens with sources of fat with different lengths of FA and reported that C12:0 was the most potent FA in reducing the synthesis of mRNA AP OB at the transcriptional level. Jambrenghi et al. (2007) supplemented lambs with a control diet (3.3% fat, 39.8% LA as % of total fat) or a LA diet (7.9% fat, 45.5% LA as % of total fat) for a 45 d finishing period. The expression of cytosolic ACC and FA SN were reduced in the LA group even though the intake of total fat was more than twice that compared to lambs fed the control diet. However, microsomal and mictochond rial acyl chain elongation activity were increased in lambs fed LA, with a concomitant in crease in 9 desaturase activity in liver microsomes. One of the roles of PPAR is to reduce the plasmatic concentration of TG. The mechanism by which this happens is probably through reducing the synthesis of VLDL. Newly discovered roles of PPAR in in tracellular lipid trafficking and metabolism may be responsible to enhance reduction of plasma lipids. Nevertheless, the actual target

PAGE 101

101 genes underlying the suppressive effect of PPAR on hepatic VLDL production remain to be elucidated (Rakhshandehroo et a l., 2010). Activation of PPAR by an agonist can also increase the clearance of TG rich lipoproteins VLDL and chylomicrons by enhancing the activity of the lipoprotein lipase through activation of APOA5 which is a positive regulator of lipoprotein lipas e or through down regulation of APOC3 which is an inhibitor of lipoprotein lipase activity. On the other hand, PPAR activation can also down regulate the activity of lipoprotein lipase by up regulating the activity of ANGPTL4, which inhibits the clearance of TG rich proteins by stimulating the inactivation of lipoprotein lipase (Kersten, 2008). These different regulatory mechanisms of lipoprotein lipase indicate that PPAR can induce both pro and anti lipolytic pathways with predominately prolipolytic activi ty under continued PPAR activation. Regulation of Glucose and Carbohydrate Metabolism Important players in glycolysis are: transporters for glucose entry and the key glycolytic enzymes, phosphofructokinase and PK (Peeters and Baes, 2010). Among the tran scription factors having a direct role in carbohydrate metabolism are PPAR and ChREBP. Notable changes in carbohydrate gene expression due to PPAR activation are only observed in mouse hepatocytes rather than human hepatocytes (Peeters and Baes, 2010). Hence for the effect of PPAR regulation of expression of genes in carbohydrate metabolism, only studies with rodents will be presented. Yamada and Noguchi (1999) summarized the nutrient and hormonal regulation of PK gene expression and indicated that mos t in vitro studies done with rats reported that feeding PUFA (LA, EPA, and DHA) reduced the expression of PK in hepatocytes by up to 70%.

PAGE 102

102 Jump et al. (1994) evaluated the effect of 300 M of GLA, ALA, AA, or EPA on PK expression in rat hepatocytes. These FA inhibited the expression of PK gene to a similar extent as did triolein. In the same study, feeding FO (10% of diet) enhanced the rate of reduction of glycolytic enzymes GK, PK, and MDH in hepatocytes in the pre meal and post meal states compared to he patocytes from rats fed triolein. Enzymatic concentration of PK in rat hepatocytes decreased 25% when fed LA (3% of dietary DM) for 7 d compared to that from rats fed a fat free diet, while a non significant reduction of GK enzymatic activity was detected (Clarke et al., 1977). On the other hand, Toussant et al. (1981) fed rats a fat free diet or diets supplemented with SAO (5% as fed basis), tallow (5% as fed basis), or C18:0 (10% as fed basis). The feeding of SAO reduced GK activity, but the other treatm ents did not change in respect to the control diet. A further evaluation of the fat free diet and the LA supplemented diet (5% of LA, as fed basis) did not change the enzymatic activity of glycolytic enzymes GK, phosphofructokinase, and PK. Berger et al. (2002) evaluated the effect of increasing the dietary concentration of PUFA relative to a control diet (10% fat, 0% AA and DHA) on global hepatic gene expression. The diets were: 0% AA + DHA, 0.5% AA, 0.5% DHA, or 0.5% AA + 0.5% DHA. Supplementation of eit h er PUFA diet resulted in the up regulation of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase in rat liver. Although expression of PPAR and c AMP signaling were not modified by feeding PUFA, authors speculated that the higher expression o f phosphoenolpyruvate carboxykinase may be mediated with an overall effect on limiting fat accumulation and shunting metabolic flux to gluconeogenesis.

PAGE 103

103 Unlike the demonstrated effect of PUFA to enhance gluconeogenesis in rats, other studies have documente d PUFA to have a negative effect on gluconeogenesis in cultured bovine hepatocytes. Gluconeogenesis activity was measured through the synthesis of glucose using propionate as a precursor. Mashek et al. (2002) measured glucose production in hepatocytes from weaned ruminating calves treated first with 1mM of C16:0 and then additionally added either 1mM of C16:0, C18:1, C18:2, C18:3, C20:5, or C22:6. Hepatocytes treated with C18:1 produced more glucose from added propionate than those produced by adding C20:5 or C22:6, even though all three LCFA were reported as inducing greater oxidation. Later Mashek and Grummer (2003) tested the same set of FA but used hepat ocytes from preruminant calves. At this time, only C22:5 affected gluconeogenesis from propionate and that was to decrease it. Finally Mashek and Grummer (2004) used monolayer cultures of hepatocytes from preruminant calves treated with1 mM of C16:0 and supplemented them with 0.1 or 1 mM of LA, CLA c 9 t 11, or CLA t 10 c 12. Regardless of FA concentrations, the type of FA did not affect propionic acid metabolism to produce glucose, cellular glycogen or the combination of both. Regardless of the type of FA, the formation of both glucose and glycogen were decreased when FA concentrations increased from 0.1 to 1 .0 mM. Regulation of Bile and Hepatic Cholesterol Bile acids are amphipathic molecules derived from cholesterol in the liver. Its synthesis generates bile flow from the liver to the intestine. Bile acids facilitate biliary excretion of cholesterol, endoge nous metabolites, and xenobiotics in addition to their function in intestinal absorption of lipids and nutrients. The liver has a critical role in maintaining cholesterol homeostasis by balancing multiple pathways such as de novo cholesterol and bile acid synthesis, dietary cholesterol uptake, biliary cholesterol

PAGE 104

104 excretion, lipoprotein synthesis, and reverse cholesterol transport (Li and Chiang, 2009). Transcription factors closely related with bile and cholesterol metabolism in liver are SREBP, H N F 4 FXR and PPAR The latter is the most diversified target gene of PUFA. Since PPAR have a regulatory effect on the former transcription factors, so do the PUFA have a regulatory effect on bile and cholesterol metabolism. The HNF 4 is known for its activit y in stimulating cholesterol 7 hydroxylase (CYP7A1), which is a rate limiting enzyme in the conversion of cholesterol to bile acids in liver. PPAR agonists were evaluated for their potential to reduce the activation of CYP7A1 using HepG2 cells through lu ciferase reporter activities (Marrapodi and Chiang, 2000). The heterodimer PPAR /RXR did not prevent the binding of HNF 4 to CYP7A1. However, it significantly reduced the expression of HNF 4 by binding the HNF 4 to a conserved sequence in the PPAR r esponse element, which is the binding site for HNF 4 This prevented the transactivation of CYP7A1 by HNF 4 (Marrapodi and Chiang, 2000). Lower levels of sterols are sensed by the SREBP cleavage activating protein (SCAP). This protein aids to the matur ation of the SREBP, which upon translocation to the nucleus, bind to promoters of SREBP in target genes related to synthesis and metabolism of cholesterol. When levels of cholesterol are increased, the SREBP cleavage activating protein complex is retained in the endoplasm reticulum to stop the maturation/activation of SREBP (Bengoechea Alonso and Ericsson, 2007). Bile acids are physiological ligands for FXR as are PUFA. A study has revealed that the downregulation of CYP7A1 by FXR did not require binding to DNA, suggesting a potential indirect effect (Castillo Olivares and Gil, 2000). FXR also inhibits the entry of

PAGE 105

105 intestinal bile acids into hepatocytes by repressing the expression of hepatic bile acid uptake transporters (Niu et al., 2011). Regulation of In flammation and Immune Response The role of FA in regulation of gene expression within the immune cells can be done through different mechanisms that include effects on receptor activity, on intracellular signaling process, or on transcription factor activa tion (Calder, 2008). Changes in FA profile of membrane phospholipids might be expected to influence immune cell function in a variety of ways such as 1) alteration of the physical property of the membrane such as membrane fluidity and lipid raft conformati on, 2) effects on cell signaling pathways either through modifying the expression, activity, or avidity of membrane receptors, modifying intracellular signaling transduction mechanisms, modifying transcription factor activation and then gene expression, 3) alteration in the production pattern of lipid mediators that have different biological functions (Calder, 2008). Bouwens et al. (2009) evaluated the supplementation of FA to human subjects fed one of three diets: 1) 1.8 g of EPA + DHA, 2) 0.4 g of EPA + D HA, or 3) SAO (79% OA, % of total FA). The oils (900 mg of oil/d) were fed in capsules on a daily basis for 26 wk. Microarray data from PBMC RNA (pretreatment baseline was the reference for each treatment group) resulted in PBMC from subjects fed the highe st dose of EPA+DHA having significant decreases in the expression of genes involved in inflammatory pathways such as eicosanoid synthesis, interleukin signaling, mitogens activated protein kinase signaling, NFkB toll like receptor signaling, oxidative stre ss, cell adhesion, PPAR signaling, LXR/RXR activation and hypoxia signaling. Interestingly, the group fed SAO (rich in OA) also had downreg ulated genes involved in different

PAGE 106

106 pathways of inflammation (80% of overlapping pathways as in the high EPA + DHA die t) as well as all the same pathways related to cell adhesion. Unexpectedly, expression of PPAR downreg ulated in PBMC of humans fed the high EPA + DHA diet. Effect on Oxidative Phosphorylation Oxidative phosphoryla tion is the culmination of the energy yielding metabolism in aerobic organisms. All oxidative steps in the degradation of carbohydrates, fats and amino acids converge at this final stage of cellular respiration, in which the energy of oxidation drives the synthesis of ATP (Nelson and Cox, 2008). The major components of the mammalian system of oxidative phosphorylation are the four complexes of the respiratory chain, NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquino l:cytochrome c reductase (complex III), cytochrome c oxidase (complexIV), and F 1 F 0 ATP synthase (complex V) (Schagger and Pfeiffer, 2001). This mechanism is critical to provide of ATP for different metabolic processes. Summary The first strategic feeding o f FA was to increase the energetic density of diets. However, the studies of Burr and Burr (1929, 1930) determined the essentiality of LA and ALA. Strategic feeding during prepartum and preweaning period are the most influential periods affecting future an imal performance. The newborn calf is born deprived of Ig, with a nai ve immune system, hence ensuring APT is critical for the subsequent encounter with different pathogens. Future research should be oriented to optimize calf nutrition by strategic

PAGE 107

107 supplementation of critical nutrients to boost animal immune response, preventing risk of disease, hence optimizing growth and overal l efficiency.

PAGE 108

108 Table 2 1. Common fatty acids terminology [ Nomenclature and classification of lipids. Chemistry and properties. Chapter 1 in: Foods Lipids: Chemistry, Nutrition and Biotechnology Marcel Dekker (Pages 21 and 24, tables 4 and 5) Inc., New York, USA ] Systematic name a Common Name Shorthand b Saturated Fatty Acids Dodecanoic Lauric 12:0 Tridecanoic 13:0 Tetradecanoic Myristic 14:0 Pentadecanoic 15:0 Hexadecanoic Palmitic 16:0 Heptadecanoic Margaric 17:0 Octadecanoic Stearic 18:0 Nonadecanoic 19:0 Eicosanoic Arachidic 20:0 Docosanoic Behenic 22:0 Unsaturated Fatty Acids c 9 Hexadecenoic Palmitoleic 16:1 n 7 c 9 Octadecenoic Oleic 18:1 n 9 c 9,c 12 Octadecadienoic Linoleic 18:2 n 6 c 9,c 1 2,c 15 Octadecatrienoic Linolenic 18:3 n 3 c 6,c 9,c 12 Octadecatrienoic alpha Linolenic 18:3 n 6 c 8,c 11,c 14 Eicosatrienoic Dihomo gamma linolenic 20:3 n 6 c 5,c 8,c 11,c 14 Eicosatrienoic Arachidonic 20:4 n 6 c 5,c 8,c 11,c 14,c 17 Eicosapentaenoi c EPA 20:5 n 3 c 7,c 10,c 13,c 16,c 19 Docosapentaenoic DPA 22:5 n 3 c 4,c 7,c 10,c 13,c 16,c 19 Docosahexaenoic DHA 22:6 n 3 a c x is the double bounded carbon atom in cis configuration and x is the number of that carbon atom counting from the carboxyl end. b Number of carbon atoms : number of double bonds. For unsaturated fatty acids, n x indicates the first double bonded carbon counting from the methyl end.

PAGE 109

109 Table 2 2. Fatty acid composition (% of total fatty acids) of major sources of fatty acids i n dairy cattle sources Total FA 1 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:4 C20:5 C22:5 C22:6 Vegetable oils 2 Palm 88.4 0.4 1.1 43.8 4.4 39.1 10.2 0.3 Coconut 85.0 48.2 18.5 8.7 2.7 6.0 1.5 0.1 Safflower 88.9 6.1 2.3 13.4 76 0.3 0.5 0.5 Canola 88.9 0.1 5.1 1.7 60.1 21.5 9.9 Linseed oil 88.8 0.1 5.5 3.7 19.3 16.2 53.4 Cottonseed 88.7 0.8 24.2 2.3 17.4 53.2 0.2 Corn 88.8 12.3 1.9 27.7 56.1 1.0 Soybean 88.8 0.1 10.8 3.9 23. 9 52.1 7.8 Sunflower 88.9 0.5 0.1 6.4 4.5 22.1 65.6 0.5 Animal fats and blends Tallow 3 88.7 3.0 25.1 19.7 42.1 3.0 0.3 Yellow grease 4 88.6 0.2 1.0 21.3 6.1 41.5 21.4 1.4 Fish oil 5 90.5 8.3 16.9 3.2 10.3 1.5 2.1 0.9 13.2 2.4 12.5 Lard 6 1.7 30.2 22.6 26.1 12.1 1.2 Commercial fats Megalac 7 82.5 1.4 3.1 47.4 4.6 34.7 5.5 0.2 Me galac R 7 82.5 1.0 1.9 32.4 5.0 23.4 30.5 3.1 Energy booster 100 5 98.0 2.9 29.1 55.3 6.3 0.3 1 Calcu l a ted with the corresponding fatty acid composition e xcept for commercial fats (manufacturer claims). 2 Dubois et al., 2007 except for linseed oil (Sterk et al., 2010). 3 Onetti et al., 2002. 4 Avila et al., 2000. 5 Ballou et al., 2009. 6 Huuskonen et al ., 2005. 7 Theurer et al., 2009.

PAGE 110

110 Figure 2 1. Structural formula of linoleic acid (omega 6) and linolenic acid (omega 3)

PAGE 111

111 CHAPTER 3 EFECT OF SUPPLEMENTA L ESSENTIAL FATTY AC IDS TO PREGNANT HOLS TEIN COWS ON COLOSTRUM FA TTY ACID PROFILE AND CALF PASSIVE IMMUNIT Y Background Attaining an appropriate growth rate and health performance of dairy calves b efore weaning that would allow to double the birth weight by weaning period and minimize the incidence of diseases is one of the primary goals of dairy herd management Dairy farmers must manage health challenges once the calf is born (Beam et al., 2009; D onovan et al., 1998). Therefore to minimize the outbreak of calf diseases and not jeopardize the profitability of the herd, immediate and effective care of the newborn calf shoul d occur right after birth by effective feeding of colostrum of good concentrat ion of immunoglobulin G (IgG > 50 g/L) in order to ensure APT The transfer of immunoglobulins (Ig) from the dam to the neonate is termed passive transfer. With the exception of ruminants, transfer of Ig begin s in the fetal period (Weaver et al., 2000). Th erefore the newborn calf is completely dependent on the supply of Ig from colostrum because the epithelio chorial placenta of cows prevents transfer of Ig during the fetal period (Kehoe and Heinrichs, 2007). Establishment of APT is crucial to reduce neonata l morbidity and mortality, and strengthen calf immunity (Quigley and Drewry, 1998; Donovan et al., 1998). Moreover APT has been associated with improved weaning and postweaning body weight (BW; Robison et al., 1988) and with greater milk production ( DeNise et al., 1989 ). Colostrum is rich in Ig, particularly IgG which account s for 85 to 90% of total Ig. T ransportation of the pool of IgG reaching the intestine across intestinal epithelium initially was assumed to occur by non selective pinocytosis (Klaus et al., 1969; Jones and Waltman, 1972). However later studies discovered the existence of specific Ig

PAGE 112

112 receptors known as neonatal Fc receptor (FcRn) present in intestinal epithelium (Israel et al., 1997). The FcRn was initially identified in human epithelial cells of intestine, suggesting its involvement in IgG binding and transfer of passive immunity (Israel et al., 1997). A potential protective mechanism of FcRn in favor of circulating IgG that prevents its premature degradation and clearance from circulatio n has been recently hypothesized (Goebl et al., 2008). Fatty acid profile of enterocyte cell membrane tends to reflect that of the diet; hence greater supplementation of PUFA might change the fluidity of membrane and expression of receptors. In addition to Ig, colostrum has been documented to contain significant concentrations of different growth factors (Georgiev, 2008b; Blum and Baumrucker, 2008). Compared to colostrum deprived calves, calves fed colostrum exhibited an enhanced epithelial cell prolifera tion as evidenced by greater villous circumference, area, and height (Buhler et al., 1998). Later studies verified the positive benefits of insulin like growth factor I (IGF I) present in colostrum on development of the intestinal tract but the benefit was lacking when IGF I was administered orally or parenterally (Roffler et al., 2003; Georgiev et al., 2003). However, studies evaluating the effect of maternal diet manipulation on concentration of growth factors in colostrum and their transfer to the newbor n are scarce. Limited studies have evaluated the effect of feeding fat supplements to cows on fatty acid (FA) composition of colostrum and most of them did not include the effect of parity. However, few studies using dairy cows and ewes supplemented with CLA have reported not effect of parity in total CLA ( Kelsey et al., 2003; Tsiplakou et al., 2006). However, Mierlita et al. (2011) when comp reported that

PAGE 113

113 primiparous ewes produced greater proportion of LA, GLA, ALA, EPA and t otal CLA. Moreover the few studies performed with cows, regardless parity consideration have fo cused on supplementation of n 3 or CLA FA instead of n 6 FA. The hypothesis of this study was that supplementing dam diets with LA modifies the FA profile of col ostrum and really improves efficiency of IgG absorption. Therefore the objective was to evaluate the effect of supplementing Ca salts of FA enriched with LA and ALA to Holstein cattle in late gestation on colostrum FA profile and production and transfer of total and specific IgG. An additional goal was to evaluate the change in serum concentrations of insulin and IGF I in calves after colostrum feeding. Materials and Methods Experimental Design and Dietary Treatments The experiment was conducted at the U FL) from October 2008 to June 2009. All procedures for animal handle and care were nulliparous (n = 28) and previously parous (n = 50) Hol stein cattle were sorted according to calving date, parity, BW, and body condition score (BCS) and assigned to one of three treatments at 8 wk before their expected calving date. Dietary treatments were the following: no fat supplementation (Control), 1.7 % of Specialties, Dundee, IL), and 2.0% of dietary DM as Ca salts of FA enriched with EFA, to have low concentrations of total FA and EFA, whereas SFA and EFA diets were isoenergetic and all diets were isonitrogenous (Table 3 1). Proportions of unsaturated FA were minimal in the SFA supplement compared to the EFA supplement (Table 4 2).

PAGE 114

114 During t he first 4 wk of the experimental period ( 8 to 4 d relative to calving), cows were housed in a sod based pen and fed as groups according to the dietary treatments. At 4 wk before the expected calving date, cows were moved to a sod based pen equipped with Calan gates (American Calan Inc., Northwood, NH) and daily DM intake (DMI) was measured. Cows were wei ghed using a digital scale at 8 and 4 wk before the expected calving date and at calving. At the same time, BCS was determined using a 5 point scale (fro m 1 meaning extremely skinny to 5 meaning obese) divided into 0.25 points using the Elanco Animal Health BCS chart (Elanco, 1996). Prepartum Body Weight, Feed Intake and Analyses Prepartum diets were prepared as a total mixed ration and offered once daily (1000 h). Feed offered was adjusted daily to achieve 5 to 10% orts. Orts were collected and weighed daily. A bermudagrass silage sample was collected once a week and analyzed for DM by drying in a forced air convection oven (American Scientific, LLC, Model DN 41) at 55C for 48 h or until constant weight, in order to maintain the formulated DM ratio of forage to concentrate (56:44, DM basis) Dried silage and hay samples (collected once weekly) were ground to pass through a 1 mm screen using a Wiley Mill ( Arthur H. Thomas, Co, Philadelphia, PA). Samples of concentrate mixtures were collected once weekly and composited monthly. Forages and concentrates were analyzed for ash (600C for 2 h, AOAC, 2000), and neutral (NDF) and acid detergent fiber (ADF) accordi ng Van Soest (1991) using an ANKOM 200 Fiber Analyzer (ANKOM, Macedon, NY). amylase and sulfite were used in the NDF assay. Nitrogen concentration was determined using a Vario MAX CN Macro Elementar Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany) by the Dumas combustion method (AOAC, 2000) and protein conce ntration was calculated as N x 6.25.

PAGE 115

115 Concentrations of FA in prepartum diets were estimated based on available composition of FA in individual ingredients whereas estimated intake of LA per cow was estimated using the CPM dairy FA submodel. Energy intake d uring prepartum was calculated based on the DMI and estimation of the energy concentration of diets by the NRC (2001) model. The last 14 d before calving were used for calculation of DMI. Prepartum Ovalbumin Challenge and Assay for Bovine Anti OVA IgG Cows were injected subcutaneously (s.c.) with 1 mg of OVA ( Sigma Aldrich, Saint Louis, MO) diluted in Quil A adjuvant solution (0.5 mg of Quil A in 1 mL of PBS Accurate Chemical & Scientific Corp., Westbury, NY) using sterile procedures upon study enrollment ( 60 d relative to expected calving date), and again 30 d after the first injection. A blood sample (10 mL) was collected just prior to each vaccination with OVA and at calving. Blood samples were collected in a tube without anti coagulant (Vacutainer, Be cton Dickinson, Franklin Lakes, NJ) and serum was separated at room temperature, followed by 15 min of centrifugation ( 2095 x g Allegra X 15R centrifuge, Beckman Coulter, Inc). Serum concentration of bovine anti OVA IgG was measured by an enzyme linked immunosorbent assay (ELISA) as described by Mallard et al. (1997). Positive and negative control sera to bovine anti OVA IgG were obtained from a pool of sera of known high (sera of cows 1 wk after second OVA injection) and low (sera of cows never exposed to OVA) concentrations of OVA, respectively. All samples from the same cow or calf were analyzed in the same plate. All plates contained a balanced number of animals from each diet. Results were corrected by dividing the experimental sample by the positive control at the same specific dilution. Results of each dilution were averaged

PAGE 116

116 and the average of 2 dilutions was reported. Intra and inter assay coefficients of variation were 9.2 and 9.7%, respectively. Calving Management Calves were born from December 24 th 2008 through April 5 th 2009. Pregnant cattle gave birth to calves in a sod based pen. All cows were monitored for signs of calving initiation every 30 min between 0530 to 1530 h and then every 2 h between 1530 and 0530 h. Ease of calving was scored according to Sewallem et al. (2008) as unassisted (1), easy pull (2), hard pull (3), and surgery (4). Within 2 h of birth calves were weighed, ear tagged, and the umbilical cord was disinfected with 10% Betadine solution (Purdue Frederick Co., Norwalk, CT ). Calves were temporarily housed in individual hutches (1 x 1 m) equipped with a heat lamp and finally moved to individual wire hutches (1 x 1.5 m) when they were between 6 to 16 h of age. Colostrum Feeding and Analyses Within 2 h of birth, cows were milk ed with a cow side vacuum pump. Colostrum quality was recorded using a colostrometer. Immediately after weighing, calves were given 4 L of colostrum from their own dam regardless of IgG concentration using an esophageal feeding tube. When an animal did n ot produce sufficient colostrum for her calf, colostrum from another animal fed the same treatment was used to feed that calf. Remnant colostrum (> 1 L having IgG concentration > 50 g/L) after calf feeding was stored ( 4C). A sample of colostrum (10 mL) f rom each dam was collected to determine concentration of bovine total IgG by single radial immunodiffusion ( VMRD Inc., Pullman WA). Colostrum samples were diluted 1:5 with double distilled water. Diluted samples (3 radial immunodiffusion plates containing agarose gel with

PAGE 117

117 anti bovine IgG. Plates containing the samples were left undistu rbed for 23 h at room temperature. Resulting ring diameters were measured with a monocular comparator (VMRD Inc., Pullman WA). A standard curve was plotted with reference sera (4, 8, 16 and 32 g/L of IgG) supplied by the manufacturer. Concentrations of IgG in diluted samples were read from the standard curve and correction for the dilution factor was applied afterwards. Intra and inter assay variations were 3.0 and 3.3%, respectively. A colostrum sample from each dam (~100 mL) was freeze dried (Labconco Ka nsas City, MO) and delivered to Michigan State University for analysis of FA. Briefly total FA from freeze dried colostrum samples were extracted using the method of Hara and Radin (1978). Fatty acid methyl esters (FAME) were prepared by base catalyzed tra nsmethylation (Christie, 1989). The FAME were quantified using a GC 2110 Plus gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a split injector (1:100 split ratio) and a flame ionization detector using a CP Sil 88 WCOT fused silica column (100 m 0.25 mm i.d. 0.2 chromatographic conditions were described by Kramer et al. (2001). The FAME were identified by comparison of retention times with known FAME standards (Supelco 37 component FAME mix, cis/trans FAME mix, bacterial acid methyl ester mix, and polyunsaturated FA No. 3 mix from Supelco Inc., Bellefonte, PA; GLC reference standard 463 and conjugated LA (CLA) mixture #UC 59 M from Nu Chek Prep, Elysian, MN). Short chain FAME were corrected f or mass discrepancy using the correction factors published by Ulberth and Schrammel (1995). Blood Collection for Measures of Immunoglobulin and Protein Concentration Calf blood was collected via jugular venipuncture before colostrum feeding and again betwe en 24 to 30 h after colostrum feeding. Blood samples were collected in a

PAGE 118

118 tube without anti coagulant (Vacutainer, Becton Dickinson, Franklin Lakes, NJ), and serum was separated at room temperature followed by 15 min of centrifugation at 2095 x g (Allegra X 15R centrifuge, Beckman Coulter, Inc). Serum total protein (STP) concentrations were determined using an automatic temperature compensated hand refractometer (Reichert Jung; Cambridge Instruments Inc. Buffalo, NY). Serum total IgG concentrations were m easured in serum diluted 3:4 with distilled water. Final concentrations of IgG were obtained from the curve plotted with the standards provided by the manufacturer as described in the previous section for colostrum IgG analysis. In order to test the mater nal transference of a specific IgG by feeding of colostrum, serum of calves at 0 h (before feeding colostrum) and at 2 d of age were analyzed for bovine anti OVA IgG using an ELISA procedure as described by Mallard et al. (1997). Details of the procedure w ere described in a previous section for prepartum cattle Intra and inter assay coefficients of variation were 8.8 and 11.7%, respectively. Concentrations of insulin and IGF I were analyzed in sera samples at 0 and 24 to 30 h to verify their transfer from colostrum feeding. Concentration of IGF I was analyzed I ELISA, Diagnostic Systems Laboratory, Inc.) with some modifications in sample pre treatment. Releasing IGF I from their binding protei ns was done with half of the indicated volumes for sample pre treatment reagents to maintain the final suggested dilution of samples (1:30). A control sample was run in duplicate in each plate. The intra plate variation for IGF 1 of control samples was 2.4 %, whereas the inter plate variation was 3.2%. Insulin concentrations were analyzed using a double antibody radioimmunoassay (Badinga et

PAGE 119

119 al., 1991) in serum samples collected at 0 and 24 h of life. Intra and inter assay variations were 7.3 and 14.6%, resp ectively. Estimation of Appropriate Passive Transfer and Efficiency of IgG Absorption 1 g/dL after 24 h of colostrum feeding (Tyler et al., 1996; Weaver et al., 2000). The app arent efficiency of IgG absorption (AEA, %) was calculated according to Quigley and Drewry (1998) assuming that serum volume was 9.9% of calf BW (Quigley et al., 1998) using the following equation: (IgG concentration in serum at 24 h of life in g/L [0.09 9 was used as an indicator of APT (Donovan et al., 1998; Calloway et al., 2002). Statistical Analysis The experiment had a block randomized design. On a weekly basis, a cohort of cows at 8 wk before the expected calving date was blocked by parity (nulliparous and parous) and BCS and, within each block, randomly assigned to one of three treatments. Test of block in the model was not significant and thus was deleted. Depen dent variables with more than one observation within experimental unit were analyzed as repeated measures using the mixed procedure of SAS 9.2 (SAS Institute, 2009). Repeated measure data were tested to determine the structure of best fit, namely compound symmetry, compound symmetry heterogeneous, autoregressive 1, and autoregressive 1 heterogeneous as indicated by a Schwartz Bayesian information criteria value closest to zero (Littell et al., 1996). For the analysis of serum bovine anti OVA IgG in cows, th e measurement determined at 8 wk before expected calving day was used as a covariate. Cow nested within treatment and parity was used as a random term. The following model was used:

PAGE 120

120 Y ijkl = + T i + P j + (TP) ij + CL( ij ) + D l + (TD) il + (PD) jl + (TPD) ijl + + E ijkl Where: Y ijkl = dependant variable; = overall mean; T i = fixed effect of treatment i (control, SFA, and EFA); P j = effect of parity j (nulliparous and parous); (TP) ij = effect of treatment by parity interaction; CL( ik ) = random effect of cow nes ted within treatment l = effect of day relative to calving (l = 60, (TD) il = effect of treatment by day interaction; (PD) jl = effect of parity by day interaction; (TPD) ijl = effect of treatment by parity by day interaction; E ijkl = residual error. For nonrepeated measures regarding dams the preceding model was used after removing day and interactions with day. Calf variables were analyzed using nonrepeated measures analysis using the mixed procedure of SAS 9.2 (SAS Institute, 2009). Calf nested within treatment and parity was a random term. The statistical model for the analysis was the following: Y ijkl = + T i + P j + (TP) ij + CL( ij ) G k + (TG) ik + (PG) jk + (TPG) ijk + E ijkl Where: Y ijk = dependant variable; = overall mean; T i = fixed effect of treatment i (control, SFA, and EFA); P j = effect of parity j (nulliparous and parous); (TP) ij = effect of treatment by parity interaction; CL( ij ) = random effect of calf nested within treatment and parity (k = 1, 2, G k = effect of gender (male and female); (TG) ik = effect of treatment by gender interaction; (PG) jk = effect of parity by gender interaction; (TPG) ijk = effect of treatment by parity by gender interaction; and E ijkl = residual error. All variabl es were tested for normality of residuals using the Shapiro Wilk test (SAS version 9.2, SAS Inst. Inc., Cary, NC). Non normally distributed data were transformed as suggested using the guided data analysis of SAS and back transformed using the LINK and ILI NK function of GLIMMIX procedure respectively Temporal

PAGE 121

121 responses to treatments were further examined using the SLICE option of the MIXED or GLIMMIX procedure. Appropriate orthogonal contrasts were performed for dam variables [1) fat supplement = FAT (SFA + EFA) vs. no fat, 2) FA supplement = FA (EFA vs.SFA), 3) effect of parity, 4) contrast 1 by parity interaction, and 5) contrast 2 by parity interaction]. Additional contrasts for calf variables included gender interactions with each of the above contrast s. If any 3 way interaction or the interaction of gender by parity were not significant ( P > 0.25), the interaction was dropped from the model and the new model was rerun (Bancroft, 1968). Coefficients of correlation were estimated using the CORR procedure of SAS (SAS Institute 2009) to describe the relationships between and within and tended to be Results Prepartum Cow Performance Sevente en of the enrolled dams did not have sufficient days in Calan gates so intake data is provided for 61 cattle Intake was stable until the last 1 to 3 d at which time DMI decreased markedly (effect of day, P < 0.01, Figure 3 1). As expected both DMI (11.8 v s. 10.0 kg/d) and net energy of lactation intake (17.3 vs. 14.7 Mcal/d) were greater in parous cows compared to nulliparous heifers (effect of parity, P < 0.01, Table 3 3). Neither feeding fat prepartum nor the type of fat affected DMI. Intake of DM prepar tum was correlated positively with gestation length (r = 0.31, P = 0.01 Table 3 7 ) and with BW change during last 8 wk prepartum (r = 0.58, P < 0.01). Concentrations of serum anti OVA IgG increased with increased number of injections of OVA as expected (e ffect of day, P < 0.01, Figure 3 2). Parities responded

PAGE 122

122 in a like manner to OVA injections. Throughout the prepartum period, cattle fed SFA had greater mean concentration of serum anti OVA IgG than cattle fed EFA (0.65 vs. 0.45 OD, P = 0.02, Table 3 3). H olstein cattle (n = 78) consumed their assigned diets for a mean of 56 d and this did not differ among dietary treatments or parities (Table 3 3). Body weight and BCS at enrollment were similar for cattle on all diets with means of 616 kg and 3.41, 610 kg and 3.31, and 616 kg and 3.31 for BW and BCS for cattle fed control, SFA, and EFA, respectively (Table 3 3). As expected, at enrollment nulliparous heifers weighed less than parous cows (527 vs. 701 kg, P < 0.01) but BCS did not differ (3.36 vs. 3.35). How ever at calving, parous cows fed the control diet tended to have a greater mean BCS than parous cows fed fat (3.51 vs. 3.40) whereas BCS of nulliparous heifers fed fat tended to have a greater BCS compared to those not supplemented with fat (3.40 vs. 3.31, FAT by parity interaction, P = 0.10, Table 3 3). However BW gain between enrollment and calving was not affected by dietary treatment and did not differ between parities (mean of 54.3 kg). Length of gestation was shorter for nulliparous heifers compared to parous cows (275 vs. 278 d, P < 0.01) but was not affected by feeding fat. In general, mean value for calving score was low because cattle that had calving scores greater than 2 were not enrolled in order to avoid confounding effects of prepartum diets with stress at calving on calf measures. Nevertheless nulliparous heifers fed the control diet had a greater mean calving score compared to those fed fat (1.25 vs. 1.00) whereas calving score of parous cows did not differ due to fat feeding (1.06 vs. 1.09, FAT by parity interaction, P = 0.04).

PAGE 123

123 Immunoglobulin G Concentration and Fatty Acid Profile of Colostrum Of the 78 enrolled Holstein cattle only 70 cows produced colostrum. Volume of colostrum produced was not affected by diets but nulliparous heifers p roduced less colostrum (3.6 vs. 7.0 kg, P < 0.01, Table 3 3). Total IgG concentration in colostrum were greater in nulliparous heifers fed the control diet vs. fat supplemented diets (102 vs. 83 g/L) but the dietary effect was the opposite in colostrum fro m parous cows (96 vs. 115 g/L, FAT by parity interaction, P = 0.05 ). Total concentration of FA in colostrum was not affected by fat source or parity and averaged 6.9 g/100 g of DM (Table 3 4). Parity had a marked effect on proportion of individual and gro ups of FA in total colostrum FA Proportions of FA < or > C16:0 were greater in nulliparous heifers (20.3 vs. 17.7% and 43.6 vs. 39.4% of total FA for < and > C16:0, respectively, P 0.01). On the other hand, proportion of C16 (C16:0 and C16:1) was greater for parous cows compared to nulliparous heifers (42.6 vs. 35.8% of total FA, P < 0.01). The proportion of total SFA, monounsaturated FA (MUFA), and n 6 FA were not different betwee n parities. However total polyunsaturated FA (PUFA, 4.61 vs. 4.02% of total FA, P < 0.01), total CLA (0.32 vs. 0.19% of total FA, P < 0.01), total branched FA (1.36 vs. 0.97% of total FA, P < 0.01), total C18:1 trans FA (2.22 vs. 1.46% of total FA, P < 0.0 1), and total n 3 FA (1.00 vs. 0.54% of total FA, P < 0.01) were all greater in nulliparous heifers compared to parous cows Although many FA tested significant for the effect of FAT, the effect was mainly due to the feeding of EFA vs. SFA; hence feeding f at prepartum had minimal effects on proportions of FA in colostrum Proportions of C14:1 (0.54 vs. 0.41%, % of total FA, P = 0.01) and C16:1 (1.88 vs. 1.63%, % of total FA, P < 0.01) were de crea sed whereas that of C18:0 was in creased (8.4 vs. 9.6%, % of to tal FA, P < 0.01) by fat feeding.

PAGE 124

124 Both parities fed EFA as compared with those fed SFA produced colostrum with greater proportions of LA (3.35 vs. 2.31% of total FA, P < 0.01) and C20:2 n 6 (0.04 vs. 0.02% of total FA, P < 0.01). The other n 6 FA were inc reased by supplementing EFA only in colostrum from nulliparous heifers (0.61 vs. 0.54% for AA, 0.33 vs. 0.28% for C20:3 n 6, and 0.13 vs. 0.10% for C22:4) but not from parous cows (0.39 vs. 0.43% for AA, 0.24 vs. 0.27% for C20:3 n 6; 0.08 vs. 0.08% for C22 :4; FA by parity interaction, P 0.03). T otal proportions of n 6 FA were greater in colostrum from cattle fed EFA compared to those from cattle fed SFA (4.31 vs. 3.21% of total FA, P < 0.01) with LA accounting for approximately 75% of the total n 6 FA. Proportions of individual n 3 FA were affected minimally by diets. Specifically, ALA, C20:3 n 3, and DHA did not differ. Cattle fed EFA had lower proportions of eicosapentaenoic acid (EPA) than those fed SFA (0.08 vs. 0.10 % of total FA, P < 0.01). All seven identified C18:1 trans FA were greater or tended to be greater in colostrum from cattle fed EFA compared to those fed SFA. Hence, sum of all individual C18:1 trans FA were greater in colostrum from cattle fed EFA compared to those fed SFA (2.06 vs. 1.58% of total FA, P < 0.01). Similar ly, both of the identified CLA ( c 9, t 11 CLA and t 10 c 12 CLA) were also greater in EFA fed cattle (0.33 vs. 0.21% sum of CLA of total FA, P < 0.01). Transfer of IgG and Hormones by Feeding of Colostrum Calves born from parous cows were heavier than those b orn from nulliparous heifers (42.4 vs. 36.8 kg, P < 0.01 Table 3 5 ). Also, as expected, males were heavier than females at birth (41.0 vs. 38.2 kg, P = 0.02 data not shown ). Males born from cattle fed SFA tended to be heavier than males born from cattle fed EFA (43.2 vs. 39.6 kg) whereas birth weight of females did not differ (38.3 vs. 39.7 kg; FA by gender

PAGE 125

125 interaction, P = 0.06, Figure 3 3). Calves were fed the same amount of colostrum (4 L). Hence intake of IgG by calves reflects the concentration of Ig G in the colostrum they consumed. Calves born from nulliparous heifers fed the control diet consumed more IgG than calves born from nulliparous heifers fed fat (410 vs. 340 g of IgG) whereas calves born from parous cows fed fat consumed more IgG than calv es born from parous cows fed the control diet (459 vs. 383 g of IgG; FAT by parity interaction, P = 0.04 ). Serum total protein at birth (mean of 4.77 g/dL) and after colostrum feeding (mean of 5.81 g/dL) did not differ due to diet fed prepartum nor to pari ty. Concentration of IgG in colostrum was correlated positively with STP measured in serum of calves at 24 to 30 h after colostrum feeding (r = 0.50, P < 0.01). Serum concentration of total IgG at birth was low but tended to be greater in males born from dams fed the control diet than in males born from dams fed fat whereas females showed the opposite effect (Figure 3 4 A, FAT by gender interaction, P = 0.09). Contrary serum concentration of total IgG at 24 to 30 h after feeding of colostrum was greater i n males born from cows fed fat as compared to those males born from cattle fed control diet (2.78 vs. 2.03 g/dL) whereas that of females did not differ due to diet (FAT by gender interaction, P = 0.03, Figure 3 4 B). Concentration of IgG in colostrum was n ot correlated with calf serum concentration of IgG at birth (r = 0.02, P = 0.89 Table 3 7 ) but was positively correlated with serum IgG after colostrum feeding (r = 0.54, P <0.01). In addition, a strong positive correlation existed between serum concent rations of total IgG and STP measured in calves 24 to 30 d after feeding of colostrum (r = 0.81, P < 0.01).

PAGE 126

126 Regardless of gender, calves born from dams fed SFA tended to have greater concentrations of serum total IgG after 24 to 30 h of colostrum feeding, than those born from dams fed EFA (2.83 vs. 2.44 g/dL, P = 0.07, Table 3 5). This trend became significant when total serum IgG was expressed as a proportion of STP (43.5 vs. 38.2%, P = 0.05 ). Concentrations of a specific IgG (i.e. anti OVA IgG at 24 to 3 0 h after colostrum feeding) followed the same pattern; that is, calves born from dams fed SFA had greater serum concentrations of anti OVA IgG compared to dams fed EFA (1.13 vs. 0.90 OD, P = 0.01). The AEA of IgG consumed did not differ between calves bor n from dams fed SFA or EFA but these calves, as a group, had a better AEA than calves born from dams fed the control diet (27.9 vs. 23.4 %, P = 0.03, Table 3 5). Males were more efficient in absorbing IgG than females (28.6 vs. 24.1%, P = 0.02 data not sh own ). The AEA was correlated positively with serum concentrations of total IgG (r = 0.42, P < 0.01) and STP (r = 0.24, P = 0.03 Table 3 7 ) in calves at 24 to 30 h after colostrum feeding whereas AEA was correlated negatively with the concentration of IgG in colostrum (r = 0.39, P < 0.01). Serum concentrations of insulin and IGF I differed according to sampling day. Insulin increased from 1.01 ng/mL at birth to 1.69 ng/mL ( P = 0.01 Table 3 6 ) at 24 to 30 h after feeding of colostrum whereas IGF I concentr ations showed an opposite response with means of 90.7 and 69.8 ng/mL for birth and 24 to 30 h after colostrum feeding, respectively (Figure 3 5; effect of day, P < 0.01). Neither diet, parity, nor gender affected serum concentrations of insulin at birth ( Table 3 6). However at 24 to 30 h after feeding of colostrum female calves tended to have greater circulating concentrations of insulin than male calves ( 1.98 vs. 1.36 ng/mL, Figure 3 5 A, effect of gender, P = 0.10).

PAGE 127

127 Fat feeding during prepartum increase d serum IGF 1 concentrations of female calves at (104.7 vs. 83.7 ng/mL) but decreased of that of males (82.5 vs. 104.7 ng/mL, Figure 3 5 B, FAT by gender interaction, P = 0.04). After 24 to 30 h of colostrum feeding, fee ding fat prepartum continued to hav e a negative impact on serum IGF 1 of male calves (59.0 vs. 77.3 ng/mL) but prepartum diet did not a ffec t serum IGF 1 of female s (81.5 vs. 77.7 ng/mL, Figure 3 5 B, FAT by gender interaction, P = 0.09). Serum concentrations of insulin and IGF 1 at birth were correlated positively with birth weight (r = 0.24, P = 0.03 for insulin and r = 0.27, P = 0.01 for IGF 1 Table 3 7 ). At 24 to 30 h after feeding of clostrum serum insulin was correlated positively with AEA (r = 0.23, P = 0.04). Discussion Although not in this study, r eduction in DMI during the prepartum period of dairy cows supplemented with diets of similar density but different FA composition was reported by others (Douglas et al., 2004; Moallen et al., 2007; Duske et al., 2009). On the contrary, Petit et al. (2007) did not report a difference in DMI when isocaloric diets formulated with linseed or energy booster were fed (12.9 vs. 12.1 kg/d, respectively). Similarly, Caldari Torres et al. (2011) did not detect differences in DMI of prepartum cows Fairlawn, OH, 63.6% of LA 1.8% of dietary DM). Greater reduction of DMI by supplemental fats h as been associated with the feeding of more unsaturated fats (Allen, 2000). A possible mechanism by which unsaturated FA reduce DMI could be its function as a signal of satiety and energy status (Bradford et al., 2008). Recently, Allen and Bradford (2012) listed a series of observations from previous studies as evidences

PAGE 128

128 favoring oxidation of fuels in liver as the most likely mechanism in volved in regulation of intake in dairy cows fed energy dense diets. In a recent published meta analysis, Rabiee et al. ( 2012) evaluated the effect of fat supplements grouped as tallow, Megalac (rich in C16:0 and C18:1 FA), seed oils (rich in LA), hydrolyzed FA, or n 3 FA rich Ca salts, each compared to their respective control diets. Authors reported that all fat supplement s decreased DMI by an estimated mean of 0.88 kg/d per cow. However, ALA rich Ca salts induced the most dramatic reduction in DMI (2.1 kg/d per cow). Milk yield tended to improve in cows supplemented with Megalac and ALA rich Ca salts. The combined effect o f Ca salts of FA on DMI and milk production indicate that this supplement could improve efficiency of milk production. In the present study production of colostrum was not affected by fat supplementation nor source of FA The current finding contrasts to t hat of Banchero et al. (2004) and Hashemi et al. (2008) who reported greater production of colostrum by ewes supplemented with more energetic diets. The EFA supplement used in our current study is partially protected from hydrolysis and hydrogenation in t he rumen because it is in the Ca salt form, hence a greater proportion of LA and ALA in Megalac R can reach the intestine for further absorption and utilization. Consequently, greater concentrations of LA and ALA and their derivate FA might have been found in peripheral tissues and in fluids such as colostrum of cows. Studies have reported that when different ruminally protected sources of FA such as Megalac (rich in C16:0), Ca salts of FO (rich in EPA and DHA), safflower seed oil (rich in LA), or linseed o il (rich in ALA) were fed to pregnant cows, an

PAGE 129

129 increased proportion of the enriched FA was found in colostrum (Noble et al., 1978; Capper et al.; 2006; Santschi et al., 2009; Lei ber et al., 2011). Calculated intake of LA based upon actual DMI was 53.7, 58. 8, and 98.6 g/d for cattle fed control, SFA, and EFA diets, respectively. Linoleic acid accounted for 75% of total n 6 FA in colostrum and was in greater concentrations when cattle were fed EFA, as was C20:2 n 6 and C22:4 n 6. Additionally, greater proport ions of total and individual CLA as well as total C18:1 trans FA were detected in colostrum of cattle fed EFA which agree with others who measured FA profile of colostrum of cows supplemented with FO or linseed oil during the prepartum period (Capper et al .; 2006; Santschi et al., 2009). Plasma FA profile of prepartum cattle in the current study were not analyzed, but in agreement to the findings in colostrum FA profile, Lessard et al. (2004) found greater proportions of LA and C181 trans FA in plasma of tr ansition cows supplemented with micronized soybeans compared to those supplemented with linseed or only greater proportions of C181 trans when compared to those cows supplemented with Megalac. The fact that increased concentrations of trans isomers of mono and di unsaturated FA were detected in colostrum of dams fed EFA indicates that the Ca salt form was not fully protecting the LA. The metabolism of LA by ruminal microorganisms will result in the formation of CLA and C18:1 trans FA (Lundy et al., 2004). Based on these results, the enzymatic elongase/desaturase activity in the mammary gland was prioritizing the synthesis of LA derivatives to the detriment of the synthesis of ALA derivatives This is suggested because cattle fed EFA had lower proportions o f EPA in colostrum, although proportions of ALA, DHA, and total n 3 FA did not differ between cattle fed the two sources of FA. Studies using humans reported that

PAGE 130

130 increased supplementation of LA or ALA increased the proportions of their corresponding deriv atives in plasma (Chan et al., 1993; Goyens et al., 2006; Liou et al., 2007). In the current study, colostrum fat from nulliparous heifers had greater proportions of ALA, AA, EPA, DPA, and DHA whereas LA was greater in colostrum FA of parous cows Addition ally total C18:1 trans and CLA c 9, t 11 were greater in colostrum FA of nulliparous heifers In chapter 4 it is reported that calves born from parous cows had lower proportions of EPA, DPA and DHA in plasma before colostrum feeding than that of nulliparous heifers which matches with the proportions detected in colostrum in this study Previous studies using human subjects found a negative relationship between parity and DHA concentrations in dams and in their neonates (Al MD et al., 1997). In contrast, Van Gool et al. (2004) failed to match the parity effect detected in dam serum due to greater production of colostrum by multiparous cows can be ruled out since the total FA concentration in colostrum remained unchanged due to parity. Limited research exits on the colostrum FA profile and this makes it hard to hypothesize about preferential synthesis of EFA derivatives in nulliparous heifers However, considering that nulli parous heifers were raised in sod base pens, with some access to pasture whereas parous cows were kept in free stall barns, it can be possible that nulliparous heifers were mobilizing fat with greater proportions of PUFA obtained from previous access to pa sture than that of parous cows A recent study from Liu et al. (2011) reported that multiparous yak had greater proportions of total MUFA, total PUFA, CLA

PAGE 131

131 c 9 t 11, ALA, and DHA compared to primiparous yak fed the same diet These results contradict to find ings of the current study. Authors attribute d the greater proportions of these FA in multiparous yak to a greater growth and development of the mammary gland in the older animals; however total short chain and medium chain FA were not constantly greater in multiparous yak. Some studies have evaluated the parity effect on FA composition of milk. Mierlita et al. (2011) evaluated the effect of parity on milk FA from sheep and reported increased proportions of ALA, EPA, CLA c 9, C18:1 trans 11, and total C18:1 trans FA in nulliparous sheep which is in agreement with the findings of the current study. However parity effects on DPA and DHA were not detected as was found in the current study. The major individual CLA detected in the current study was CLA c 9, t 11, w hereas CLA t10, c12 was detected only in cows fed EFA but in limited proportions. Contrary to results in the current study studies that evaluated milk of ewes reported no effect of parity on total CLA (Kelsey et al., 2003; Tsiplakou et al., 2006). Mierlit a et al. (2011) also reported lower proportions of C18:0 in primiparous cows, hypothesizing that an incomplete biohydrogenation and/or a rapid passage of digesta was occurring in primiparous cows that prevented complete biohydrogenation, hence allowing th e increase in CLA c 9, t 11 and total C18:1 trans FA delivered to the lower tract. However, in the current study, C18:0 proportions were greater in nulliparous heifers disagrees with their hypothesis. Mallard et al. (1997) evaluated the responses of prepa rtum cows to OVA challenge and classified them as high or low responders. Cows with greater serum concentrations of anti OVA IgG had a lower incidence of diseases. Some researchers

PAGE 132

132 have hypothesized that reduction in serum Ig concentrations around calving could be due to greater sequestration by the mammary gland (Detilleux et al., 1995). In the current study we did not measure concentrations of anti OVA IgG in colostrum but total IgG in colostrum was greater in cattle fed SFA and the transfer of this spec ific antibody to the serum of calves also was greater if they were born from cattle fed SFA. In agreement with our findings, Mallard et al. (1997) and Watger et al. (2000) reported that cows with a greater response to prepartum OVA injections supplied grea ter concentrations of antibody to the mammary gland, therefore to the calf through feeding of the colostrum. Linoleic acid is commonly seen as an inducer of inflammatory responses. However some in vitro studies have reported that moderate amounts of LA cou ld partially inhibit lymphocyte proliferation (Karsten et al., 1994; Gorjao et al., 2007) which assumes an anti nflammatory effect of LA. In the current study cattle fed EFA had lower concentrations of anti OVA IgG in serum and total IgG in colostrum whic h might indicate an anti nflammatory property of LA. Nevertheless all calves fed 4 L of good quality colostrum within 2 h of birth had > 2.2 g of total IgG per L of serum which is about 100% more than the minimum needed to ensure APT. Only a few studies hav e evaluated the effect of additional fat with greater proportions of LA in isocaloric prepartum diets on measures of passive immunity and those were primarily done using beef cows. Dietz et al. (2003) fed cows isocaloric diets differing in concentrations o f LA. Authors did not report differences in concentrations of colostrum IgG or in serum IgG of calves after colostrum feeding. Lake et al. (2006c) aimed to evaluate the effect of prepartum energy balance on passive transfer of Ig. Prepartum beef cows were nutritionally managed to achieve

PAGE 133

133 different BCS at partition (4 vs. 6). Prepartum cows targeted to have greater body condition were fed a more energy dense diet. No differences in IgG concentration of serum collected 48 h after birth was detected due to BC S of dams (15.6 vs. 13.4 g/L of IgG). This result contrast s with the current study, in which neither intake of energy prepartum nor BCS at calving differed from dams fed SFA or EFA but concentrations of serum total IgG and anti OVA IgG were greater for cal ves born from dams fed SFA. Studies done with beef cows as those indicated above, are different from studies done with dairy cows. Beef calves are allowed to suckle their dams, whereas dairy calves are removed from their dams and normally force fed colostr um. Hence, concentration of serum IgG after feeding of colostrum in beef calves can be a combination of different factors including willingness of calf to drink colostrum and timing of intake whereas in dairy calves under the current experimental conditio ns, volume and timi ng of colostrum feeding we re standardized along calves which prevent ed these variables from a ffect ing serum IgG and AEA. Considering studies done with dairy cows, our results are in contrast to those of Novak et al. (2012 b ) who did not f ind any effect of lower intake of energy (88 vs. 100% of required energy) by prepartum Holstein cows on total concentrations of Ig and IgG in colostrum and serum of calves at 3 days of age (1.62 vs. 1.73 g/L of serum IgG). Adequate management of time of c olostrum feeding and total intake of IgG are important factors influencing APT (Heinrichs and Elizondo Salazar, 2009). Calves in our current study were fed within 2 h of birth. Therefore, the only factor left to potentially affect APT is intake of IgG. Bec ause all calves were offered the same volume of colostrum, the concentr ation of IgG in the colostrum was of primary importance. Calves

PAGE 134

134 born from nulliparous heifers fed the control diet had greater intake of IgG than calves born from nulliparous heifers fe d either source of fat. However this greater intake was not reflected in a greater AEA or a greater serum concentration of total IgG in this group of calves. The improved AEA in calves born from cattle fed either SFA or EFA, which was accompanied by a tren d for greater serum concentrations of IgG, included calves born from nulliparous heifers Hence, the improved AEA in c alves born from nulliparous heifers fed fat, that also consumed less IgG compared to calves born from nulliparous heifers fed the control diet, might simply reflect the inverse relationship of IgG intake and AEA as reported by others (Quigley et al., 1994; Garry et al., 1996) and also identified in our present study (r = 0.40, P < 0.01, data not shown) However we hypothesize that reduced A EA was not only due to a simple effect of greater intake of IgG saturating the receptors for IgG in the enterocyte and therefore limiting the absorption of available IgG I n the current study, calves born from parous cows fed any source of fat had greater intake of IgG but also had a greater AEA as compared to calves born from parous cows fed the control diet. Hence the improved AEA of calves born from cattle fed fat might indicate that the feeding of fat to t he dam may allow the calf to more efficiently ab sorb IgG. Lessard et al. (2006) challenged prepartum dairy cows with an OVA injection at 6 and 3 wk prepartum and measured transfer of anti OVA IgG into the colostrum. Multiparous cows supplemented with micronized soybeans (20.3% of dietary DM) had a gre ater increase in concentration of anti OVA IgG in colostrum than cows fed either a low fat or a high ALA diet. They concluded that dietary PUFA may influence the secretory function of mammary epithelial cells of multiparous

PAGE 135

135 cows by modifying the FA profile of those epithelial cells and therefore modulating the transfer of blood IgG to the mammary gland. The most recent mechanism discovered by which Ig are transported across the intestinal epithelium is with the assistance of FcRn, which in humans was identi fied in epithelial cells of the intestine, suggesting its involvement in binding of IgG and transfer of passive immunity (Israel et al., 1997). Later FcRn was not only associated with enhanced transport of IgG but also with protecting circulating IgG from degradation (Goebl et al., 2008). Composition of FA in cell membranes has been associated with a modified response of cells to expression of receptors such as those of the immune cell. Therefore it is valid to hypothesize that dams supplemented with fat (S FA or EFA) can pass those FA to the calf in utero through the placenta. Those FA become part of the enterocytes of the calf which influence the activity of FcRn resulting in improved efficiency of absorption of IgG. However, based on our results, we cann ot assign the benefit in AEA to a specific type of FA since no difference in AEA was identified between calves born from cattle fed SFA vs. EFA. Oda et al. (1989) reported that regardless of prepartum diet type, concentrations of IGF I, and insulin were g reater in colostrum than in plasma of prepartum cows In the current study, the concentrations of IGF I and insulin in colostrum and in serum of parturient cows were not measured. However, the lack of effect of diets on IGF I and insulin concentrations in serum of calves before and after colostrum feeding would not necessarily mean that concentration of these growth factors did not differ in colostrum due to prepartum diets. The beneficial effect of increased concentrations of IGF I found in colostrum has b een associated with an improved local effect on gastro intestinal tract

PAGE 136

136 development ( Hammon et al., 2000; Georgiev, 2008b; Blum and Baumrucker, 2008 ). However with the current findings we can not rul e out that calves born from dams fed dies with different F A profile might have different ial development of their gastrointestinal tract, in disregard of no difference s in serum IGF I after colostrum feeding. Sparks et al. (2003) re ported a negative correlation between IGF I at 0 h and the difference between ser um IGF I at 48 and 0 h (r = 0.82), which was confirmed in the present study for insulin (r = 0.54) and IGF I (r = 0.65). Sparks et al. (2003) also reported a positive correlation of IGF I in colostrum with IGF I in serum of calves after 48 h of colostru ms intake (r = 0.45). These results might suggest that colostrum with greater IGF I concentrations allow calves to maintain greater concentrations of serum IGF I after colostrum intake, even though actual mean values of serum IgG are decreased from birth t o that measured 1 to 2 d after colostrum feeding. Lack of effect of prepartum diets on serum IGF I after colostrum feeding might suggest that colostrum IGF I concentrations did not differ among prepartum diets. Summary The FA profile of colostrum of cattle fed EFA reflected the concentration of LA in the fat supplement and its metabolism in the rumen of the pregnant cattle Increased 6 derivatives indicate that elongase/ desaturase activities in the mammary gland were active Ho wever, increased proportions of total and individual CLA as well as total C18:1 trans FA in colostrum of cattle fed EFA indicate that the Ca salt of EFA was not completely effective in preventing the processes of biohydrogenation by ruminal microbes. Inter estingly, colostrum of nulliparous heifers appeared to be a better source of n 3 FA (ALA, EPA, DPA, and DHA) than that of parous cows

PAGE 137

137 Intake of IgG did not differ due to dietary treatments but serum concentrations of total IgG and anti OVA IgG after colo strum feeding were greater in calves born from cattle supplemented with SFA vs. EFA Hence feeding of newborn calves with colostrum of prepartum Holstein cattle fed SFA instead of EFA would enhance APT. Feeding of fat prepartum improved AEA across parities from 23.3 to 27.9% regardless of type of fat supplemented. It is possible that cattle fed fat gave birth to calves that had a more efficient mechanism to transfer IgG into circulation, possibly by modifying the activity of FcRn receptors in the intestinal tract due to the likely differential composition of FA in the cell membrane. Concentrations of serum IGF I in calves did not increase but were reduced with the feeding of colostrum and were not affected by the type of diet. This might indicate that IGF I is poorly absorbed into circulation or that IGF 1 is used to enhance proliferat ion and differentiat ion epithelial intestinal cells.

PAGE 138

138 Table 3 1. Ingredient composition of experimental diets fed to pregnant Holstein cattle starting at 8 weeks from expecte d calving date. Prepartum diets 1 Control SFA EFA Ingredient, % of DM Bermuda silage 56.0 56.0 56.0 Ground barley 8.0 8.0 8.0 Peanut meal 10.0 10.0 10.0 Citrus pulp 21.9 20.2 19.9 Saturated fatty acids 2 1.7 Ca salts of fatty acids 3 2.0 Mineral mix 4 4.1 4.1 4.1 Nutrient composition, (DM basis) NE L 5 Mcal/kg 1.42 1.49 1.5 CP, % 14.0 14.0 14.0 NDF, % 47.4 47.4 47.4 ADF, % 25.3 25.3 25.3 Fatty acids, % 1.68 3.37 3.35 Linoleic acid 6 g/d 57 62 116 1 Control = no fat suppleme nt ed; SFA = saturated fatty acids; EFA =essential fatty acids. 2 Energy Booster 100 (Milk Specialties, Dundee, IL). 3 Megalac R (Church & Dwight, Princeton, NJ). 4 Contains (DM basis) 34.5% corn meal, 5.0% dicalcium phosphate, 16.0 calcium carbonate, 10% calcium sulfate, 5% magnesium oxide, 10% magnesium sulfate, 4% sodium chloride, 1.7% Zinpro 4 plex (Zinpro, Minneapolis, MN), 0.4% Rumensin 80 (Elanco Animal Health, IN), 0.35% Sel Plex 2000 (Alltech Biotechnology, Nicholasville, KY), 0.002% Ca iodate, and a vitamin premix. Each kg contains 24.5% CP, 9.8% Ca, 1.5% P, 4.2% Mg, 3.2% S, 1.7% Na, 10.7 % Cl, 475 mg of Zn, 160 mg of Cu, 456 mg of Mn, 7.4 mg of Se, 37.4 mg of Co, 13.2 mg of I, 118,000 IU of vitamin A, 27,500 IU of vitamin D, 2,600 IU of vitamin E, and 770 mg of monensin. 5 Calculated from the estimation of energetic values of individual ingredients using the NRC software (2001) and considering intake at 3X of maintenance. 6 Considering 12 kg of DMI (CPM dairy fatty acid submodel).

PAGE 139

139 Table 3 2. F atty acid (FA) profile of fat supplements fed to pregnant Holstein cattle starting at 8 weeks from expected calving date. SFA 1 EFA 2 FA % of identified FA C14:0 3.3 1.0 C14:1 ND 3 ND C15:0 0.4 ND C16:0 35.1 34.3 C16:1 0.4 0.1 C17:0 1.5 0.1 C18:0 51.6 4.5 C18:1 3.1 27.1 C18:2 ND 27.4 0.7 2.3 Other FA 3.8 3.2 1 SFA = Energy Booster (Milk Specialties, Dundee, IL). 2 EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 ND = Not detected.

PAGE 140

140 Table 3 3. Performance of nulliparous and parous Holstein cattle fed diets supplemented without fat (control), with saturated fatty acids (SFA), or with essential fatty acids (EFA) the last 8 weeks of pregnancy. Dam Diet 1 P values 3 Measure Control SFA EFA SEM FAT FA P FAT x P FA x P Parity 2 Prim Mult Prim Mult Prim Mult N o of cows 4 4 16 8 13 6 14 DMI 5 kg 10.6 11.6 10.2 12.4 9.3 11.5 0.7 0.67 0.16 <0.01 0.41 0.98 NE L Intake 6 Mcal/d 15.0 16.5 15.2 18.4 13.8 17.0 1.0 0.73 0.16 <0.01 0.38 0.93 Serum anti OVA IgG 7 OD 0.34 0.56 0.68 0.63 0.43 0.48 0.09 0.22 0. 02 0.37 0.19 0.56 N o of cows 8 8 17 11 16 9 17 Days in diets 54.6 54.4 54.8 56.7 53.7 57.2 1.59 0.44 0.85 0.20 0.30 0.59 BW enrollment, kg 538 694 511 709 532 701 23.7 0.91 0.80 <0.01 0.52 0.54 BCS enrollment 3.34 3.47 3.36 3.27 3.31 3.32 0.09 0.26 1.00 0.84 0.31 0.52 BW calving, kg 587 752 569 777 583 743 21.9 0.93 0.65 <0.01 0.62 0.28 BCS calving 3.31 3.51 3.36 3.41 3.44 3.38 0.07 0.82 0.68 0.30 0.10 0.45 BW change, kg 49.4 58.4 57.2 67.6 50.7 42.4 12.7 0.96 0.21 0.72 0.73 0.46 Gestation length, d 275 276 275 278 273 279 1.35 0.25 0.62 <0.01 0.19 0.28 Calving ease Score 9 1.25 1.06 1.00 1.13 1.00 1.06 0.08 0.12 0.66 0.97 0.04 0.66 Colostrum 10 Kg 4.13 7.71 3.14 6.59 3.44 6.65 1.09 0.33 0.87 <0.01 0.90 0.91 IgG colostrum 10 g/L 102 96 83 122 83 109 11.1 0.99 0.59 0.04 0.05 0.56 1 Control = no fat supplemented; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 Null = nulliparous. 3 P values for orthogonal contrasts and in teractions. FAT= (SFA + EFA) vs. Control, FA = EFA vs. SFA, P = parity. 4 Total of 61 cattle that were allocated to the Calan gate system. 5 Day effect, P < 0.01. 6 Day effect, P < 0.01. 7 Day effect, P < 0.01; parity by day interaction effect, P = 0.03. 8 Scoring system: unassisted (1), easy pull (2), hard pull (3), and surgery (4). 9 Total of 70 cows after removing 8 cows that did not produce colostrum collected.

PAGE 141

141 Table 3 4. Mean concentratio ns of total fatty acids (FA, % of colostrum DM ) individual and group of FA (g of FA/100 g of total FA) in colostrum of Holstein cattle fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Dam diets 1 P values 2 Measure Control SFA EFA SEM FAT FA Parity (P) FAT x P FA x P Parity 3 FA Null Parous Null Parous Null Parous Total FA, % 7.82 7.05 5.65 6.33 7.84 6.58 1.08 0.37 0.28 0.62 0.80 0.39 C4:0 2.11 1.72 2.25 1.80 2.21 1.79 0.11 0.31 0.80 <0.01 0 .84 0.89 C6:0 1.17 0.93 1.24 0.91 1.18 0.93 0.05 0.76 0.71 <0.01 0.58 0.52 C8:0 0.59 0.47 0.61 0.44 0.59 0.45 0.03 0.77 0.93 <0.01 0.62 0.64 C10:0 1.24 1.07 1.20 0.93 1.12 0.97 0.08 0.18 0.79 0.01 0.82 0.51 C12:0 2.28 1.86 2.15 1.70 2.05 1.72 0.14 0.19 0.78 <0.01 0.89 0.70 C14:0 11.4 10.6 10.8 9.7 10.1 9.5 0.66 0.09 0.47 0.13 0.98 0.71 C14:1 c9 0.48 0.60 0.36 0.50 0.34 0.46 0.05 0.01 0.54 <0.01 0.92 0.80 C16:0 35.2 40.9 34.0 40.7 33.7 40.4 1.20 0.41 0.81 <0.01 0.66 0.98 C16:1 c9 1.62 2.15 1.45 1.95 1.38 1.76 0.07 <0.01 0.07 <0.01 0.43 0.43 C18:0 9.70 7.15 11.14 8.14 10.63 8.57 0.47 <0.01 0.94 <0.01 0.98 0.33 C18:1 t 4 0.02 0.01 0.01 0.01 0.02 0.02 0.002 0.02 <0.01 <0.01 0.71 0.50 C18:1 t 5 0.01 0.01 0.01 0.01 0.02 0.01 0.001 0.01 <0.01 <0.01 0.87 0. 92 C18:1 t 6 8 0.22 0.15 0.21 0.15 0.25 0.19 0.01 0.12 <0.01 <0.01 0.91 0.84 C18:1 t 9 0.18 0.13 0.19 0.14 0.21 0.15 0.01 0.01 0.07 <0.01 0.43 0.84 C18:1 t 10 0.21 0.16 0.21 0.19 0.28 0.30 0.03 0.02 0.01 0.51 0.40 0.58 C18:1 t 11 1.08 0.59 1.04 0.57 1.33 0 .77 0.06 0.07 <0.01 <0.01 0.83 0.40 C18:1 t 12 0.26 0.18 0.25 0.19 0.33 0.25 0.01 <0.01 <0.01 <0.01 0.65 0.47 C18:1 c 9 21.7 22.7 22.0 23.3 22.3 21.9 1.32 0.89 0.70 0.55 0.78 0.56 C18:1 c 11 0.99 0.89 0.97 0.89 1.04 0.87 0.06 1.00 0.68 0.02 0.84 0.49 C18: 2 n 6 2.16 2.34 2.33 2.28 3.20 3.50 0.10 <0.01 <0.01 0.08 0.77 0.10 C18:3 n 6 0.03 0.03 0.03 0.04 0.03 0.03 0.003 0.68 0.29 0.01 0.65 0.90 C18:3 n 3 0.44 0.31 0.46 0.32 0.45 0.35 0.02 0.23 0.41 <0.01 0.80 0.31 CLA c 9 t 11 0.25 0.15 0.22 0.13 0.33 0.20 0. 02 0.12 <0.01 <0.01 0.61 0.19 CLA t 10 c 12 0.000 0.000 0.000 0.000 0.001 0.002 0.001 0.26 0.06 0.75 0.82 0.71 C20:2 n 6 0.03 0.02 0.03 0.02 0.04 0.03 0.002 0.00 <0.01 <0.01 0.37 0.69 C20:3 n 9 0.03 0.01 0.03 0.01 0.03 0.01 0.002 0.09 0.05 <0.01 0.62 0.96 C22:0 0.09 0.06 0.09 0.06 0.09 0.06 0.005 0.88 0.82 <0.01 0.38 0.98 C20:3 n 6 0.29 0.21 0.28 0.27 0.33 0.24 0.02 0.04 0.65 <0.01 0.31 0.02 C20:3 n 3 0.01 0.00 0.01 0.01 0.01 0.01 0.001 0.71 0.27 0.01 0.10 0.53 C20:4 n 6 0.49 0.37 0.54 0.43 0.61 0.39 0.02 <0.01 0.42 <0.01 0.23 0.03 C20:5 n 3 0.11 0.05 0.13 0.07 0.11 0.05 0.01 0.16 <0.01 <0.01 0.59 0.99

PAGE 142

142 Table 3 4. Continued. Dam diets 1 P values 2 Measure Control SFA EFA SEM FAT FA Parity (P) FAT x P FA x P Parity 3 Null Parous Null Paro us Null Parous C24:0 0.06 0.04 0.06 0.04 0.07 0.04 0.003 0.99 0.35 <0.01 0.18 0.93 C22:4 n 6 0.09 0.07 0.10 0.08 0.13 0.08 0.01 <0.01 0.01 <0.01 0.09 0.01 C22:5 n 3 0.35 0.13 0.38 0.16 0.41 0.14 0.02 0.04 0.79 <0.01 0.26 0.10 C22:6 n 3 0.05 0.01 0.06 0.01 0.06 0.004 0.003 0.64 0.52 <0.01 0.83 0.52 Unknown FA 0.35 0.33 0.35 0.31 0.35 0.32 0.01 0.53 0.84 0.03 0.94 0.84 Other FA 4.77 3.59 4.79 3.62 4.73 3.57 0.13 0.99 0.68 <0.01 0.95 0.99 Total C16 41.7 38.0 43.7 39.7 45.3 40.5 1.8 0.13 0.54 0.01 0.82 0.83 65.8 66.1 65.6 65.8 63.6 65.8 1.5 0.55 0.49 0.46 0.74 0.52 26.0 27.5 26.0 27.7 26.4 26.1 1.4 0.87 0.69 0.41 0.76 0.51 4.07 3.56 4.35 3.69 5.40 4.83 0.14 <0.01 <0.01 <0.01 0.68 0.77 Total CLA 0.30 0.18 0.26 0.15 0.41 0.25 0.02 0.05 <0.01 <0.01 0.67 0.21 Total BCFA 1.37 1.00 1.37 0.98 1.33 0.93 0.07 0.59 0.57 <0.01 0.78 0.98 trans 0.10 0.08 0.11 0.08 0.11 0.08 0.01 0.31 0.32 <0.01 0.99 0.72 trans 1.97 1.23 1.91 1.24 2.44 1.68 0.09 0.01 <0.01 <0.01 0.85 0.68 3 0.96 0.51 1.02 0.56 1.03 0.55 0.04 0.08 0.98 <0.01 0.78 0.69 6 3.08 3.04 3.30 3.12 4.34 4.27 0.12 <0.01 <0.01 0.36 0.68 0.65 n 6 : n 3 3.24 6.14 3.26 5.88 4.25 7.92 0.36 0.04 <0.01 <0.01 0.69 0.17 1 Control = no fat supplemented; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 Null = nulliparous. 3 P values for orthogonal contrasts and interactions. FAT = (SFA + EFA) vs. Control, FA = EFA vs. SFA, P = parity.

PAGE 143

143 Table 3 5. Passive imm unity related parameter s in calves born from Holstein cattle fed diets supplemented with no fat (control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before calculated calving date. Dam Diet 1 P values 3 Measure Cont rol SFA EFA SEM FAT FA P FAT x P FA x P G FAT x G FA x G Parity 2 Null Parous Null Parous Null Parous N calves 8 17 11 16 9 17 Birth BW 4 kg 37.2 39.8 37.8 43.7 35.5 43.8 1.32 0.13 0.40 <0. 01 0.06 0.38 0.02 0.69 0.06 STP 5 g/dL 4.83 4.82 4.78 4.62 4.79 4.80 0.11 0.44 0.39 0.57 0.75 0.42 0.85 0.19 0.57 IgG intake 6 g 410 383 344 487 336 431 37.0 0.94 0.42 0.04 0.04 0.54 ST IgG 7 g/dL 0.02 0.02 0.03 0.02 0.01 0.02 0.01 0.77 0.34 0.95 0.66 0.44 0.29 0.09 0.37 24 h after birth STP, g/dL 6.35 6.16 6.21 6.58 6.33 6.23 0.21 0.67 0.59 0.90 0.39 0.25 0.75 0.11 0.71 ST IgG, g/dL 2.40 2.21 2.69 2.97 2.51 2.36 0.22 0.09 0.07 0.90 0.52 0.32 0.92 0.03 0.89 ST I gG, % of STP 37.5 35.2 42.3 44.6 39.1 37.3 2.57 0.05 0.05 0.79 0.59 0.42 0.82 0.03 0.94 Anti OVA IgG, OD 1.03 1.04 1.16 1.10 0.91 0.89 0.08 0.80 0.01 0.76 0.71 0.85 0.47 0.74 0.99 AEA 8 % 23.7 23.0 30.5 28.6 27.3 25.1 2.27 0.03 0.14 0.41 0.73 0.95 0.02 0 .20 0.33 1 Control = no fat supplemented; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 Null = nulliparous. 3 P values for orthogonal contrasts and interactions. FAT = (SFA + EFA) vs. Control, FA = EFA vs. SFA, P = parity, G = gender. Three way interactions were not significant. 4 Parity by gender, P = 0.07. 5 Serum total protein. 6 Gender not included in the model. 7 Serum total IgG. 8 Apparent eff iciency of IgG absorption, % = [IgG concentrat ion in serum at 24 h of life (0.099 x BW at birth)] IgG intake] x100.

PAGE 144

144 Table 3 6. Concentrations of insulin and insulin like growth factor I in serum of calves born from Holstein cattle fed diets supplemented with no fat (control), saturated fatty aci ds (SFA), or essential fatty acids (EFA) starting at 8 wk before calculated calving date. Dam diet 1 P values 3 Measure Control SFA EFA SEM FAT FA P FAT x P FA x P G FAT x G FA x G Parity 2 Null Parous Null Parous Null Parous N calves 8 17 11 16 9 17 Birth Insulin, ng/mL 1.31 0.70 1.24 1.30 0.99 0.73 0.24 0.72 0.12 0.11 0.20 0.45 0.13 0.25 0.24 IGF I, ng/mL 97.3 87.8 100.5 89.3 81.4 102.0 10.7 0.96 0.74 0.99 0.46 0.12 0.33 0.04 0.90 24 h after birth Insulin, ng/mL 1.27 1.77 1.73 1.82 1.47 1.84 0.43 0.63 0.79 0.33 0.67 0.70 0.10 0.87 0.58 IGF I, ng/mL 71.4 84.2 72.7 77.7 57.4 70.4 8.08 0.26 0.19 0.12 0.88 0.55 0.02 0.09 0.87 1 Control = no fat supplem ented; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 Null = nulliparous. 3 P values for orthogonal contrasts and interactions. FAT = (SFA + EFA) vs. Control, FA = EFA vs. SFA, P = parity, G = g ender. Three way interactions were not significant.

PAGE 145

145 Table 3 7. Correlation coefficients among several variables 1 in calves born from Holstein cattle fed diets supplemented with no fat (control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before calculated calving date. First row within each measure corr espond s to r values and second row i correspond s to P values. PP BWC PP DMI Colost IgG BW birth IgG 0 h IgG 24 h TSP 0 h TSP 24 h TSP diff AEA IGF I 0 h IGF I 24 h Insulin 0 h Insulin 24 h IGF I diff Insulin diff Gest. Length 0.22 0.31 0.11 0.59 0.00 0.11 0.20 0.09 0.01 0.16 0.10 0.05 0.20 0.02 0.17 0.17 0.07 0.01 0.38 <0.01 0.97 0.30 0.07 0.43 0.93 0 .17 0.33 0.65 0.05 0.86 0.11 0.10 PP BW change 0.58 0.01 0.03 0.18 0.06 0.17 0.20 0.12 0.00 0.15 0.17 0.28 0.31 0.02 0.13 <0.01 0.96 0.83 0.15 0.64 0.18 0.10 0.33 0.97 0.24 0.18 0.02 0.01 0.88 0.30 PP DMI 0.09 0.21 0.28 0.09 0.24 0.18 0.08 0.07 0.07 0.03 0.12 0.22 0.10 0.14 0.51 0.10 0.03 0.47 0.06 0.14 0.54 0.58 0.60 0.80 0.34 0.07 0.44 0.26 Colost. IgG 0.10 0.02 0.54 0.10 0.50 0.54 0.39 0.03 0.19 0.11 0.07 0.14 0.04 0.42 0.89 <0.01 0.42 <0.01 <0.01 <0.01 0.80 0.12 0. 38 0.57 0.26 0.77 BW birth 0.06 0.13 0.14 0.11 0.02 0.27 0.27 0.13 0.24 0.13 0.21 0.25 0.59 0.24 0.18 0.31 0.82 0.02 0.01 0.22 0.03 0.22 0.05 0.02 IgG 0 h 0.08 0.02 0.02 0.05 0.01 0.02 0.12 0.08 0.04 0.07 0.4 7 0.86 0.84 0.68 0.90 0.83 0.27 0.47 0.72 0.53 IgG 24 h 0.11 0.81 0.76 0.42 0.02 0.01 0.07 0.18 0.02 0.17 0.32 <0.01 <0.01 <0.01 0.85 0.92 0.51 0.10 0.85 0.12 TSP 0 h 0.25 0.15 0.02 0.17 0.04 0.25 0.18 0.21 0.02 0.20 0.85 0.12 0.73 0.02 0.09 0.06 TSP 24 h 0.87 0.24 0.10 0.05 0.10 0.19 0.08 0.20 <0.01 0.03 0.35 0.66 0.36 0.07 0.46 0.07 TSP diff 0.16 0.09 0.13 0.08 0.05 0.01 0.10 0.17 0.40 0.23 0.4 6 0.64 0.89 0.38 AEA 0.16 0.05 0.12 0.23 0.25 0.14 0.16 0.66 0.31 0.04 0.03 0.25 IGF I 0 h 0.59 0.27 0.04 0.65 0.12 <0.01 0.01 0.69 <0.01 0.26 IGF I 24 h 0.19 0.23 0.06 0.07 0.03 0.56

PAGE 146

146 Table 3 7 Continued. PP BWC PP DMI Colost IgG BW birth IgG 0 h IgG 24 h TSP 0 h TSP 24 h TSP diff AEA IGF I 0 h IGF I 24 h Insulin 0 h Insulin 24 h IGF I diff Insuli n diff Insulin 0 h 0.12 0.13 0.54 0.25 0.24 <0.01 Insulin 24 h 0.15 0.77 0.16 <0.01 IGF I diff 0.20 0.06 1 Gest. Length= gestation length; PP BW change = Body weight change during the last 60 d of gestation; PP DMI= prepartum dry matter intake; Colost. IgG= Concentration of IgG in colostrum; BW birth= body weight at birth; 0 h = corresponding variable measured in seru m of calves before colostrum feeding; 24 = corresponding variable measured in serum of calves after 24 30 h of colostrum feeding; diff= difference of measures after and before colostrum feeding; TSP= total serum protein; AEA= apparent efficiency of IgG absorption.

PAGE 147

147 A B Figure 3 1. D ry matter intake of Holstein cattle supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. A) N ulliparous heifers B) P arous cows. Effect of parity P = 0.03. Effect of days relative to calving, P < 0.01. 4 6 8 10 12 14 16 DMI, kg/d Days relative to calving Ctl = 10.8 SFA = 10.7 EFA = 9.30 4 6 8 10 12 14 16 DMI, kg/d Days relative to calving Ctl = 11.8 SFA = 12.1 EFA = 11.1

PAGE 148

148 Figure 3 2. Bovine anti OVA IgG concentration in serum of Holstein cattle supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. A) N ulliparous heifers (Null) B) P arous cows. weeks 8 and 4 relative to calving. Effect of day was P < 0.01. Effect of feeding SFA vs. EFA was P = 0.01. Effect of interaction of parity by day was P = 0.03. 0.2 0.0 0.2 0.4 0.6 0.8 1.0 Null Parous Null Parous Null Parous 8 (COV) 4 Calving Serum anti OVA IgG, OD Control SFA EFA Weeks relative to calving

PAGE 149

149 Figure 3 3. Body weight at birth of calves born from Holstein cattle supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Gender, P = 0.02, interaction fatty acid by gender, P = 0.06. 30 35 40 45 50 Male Female Male Female Male Female Control SFA EFA Body Weight (kg)

PAGE 150

150 A B Figure 3 4. Concentrations of total IgG in serum of calves born from Holstein cattle supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving da te. A) B efore colostrum feeding effect of fat (SFA + EFA) by gender, P = 0.09. B ) A fter 24 to 30 h of colostrum feeding effect of feeding SFA vs. EFA, P = 0.07 and effect of fat (SFA + EFA) by gender, P = 0.03. 0.01 0.00 0.01 0.02 0.03 0.04 0.05 Male Female Male Female Male Female Control SFA EFA Serum IgG (g/dL) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Male Female Male Female Male Female Control SFA EFA Serum IgG (g/L) Minimum

PAGE 151

151 A B Figure 3 5. Serum concentrations o f hormones of calves born from Holstein cattle supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date A) Insulin concentration, Effect of gender at day 1, P = 0.10. B) IGF I concentration, Effect of fat by gender at day 0, P = 0.04, at day 1, P = 0.09, effect of age, P = 0.02. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Male Female Male Female Day 0 Day 1 Insulin, ng/mL Control SFA EFA 0 20 40 60 80 100 120 Male Female Male Female Day 0 Day 1 IGF I, ng/mL Control SFA EFA

PAGE 152

152 CHAPTER 4 EFFECT OF SUPPLEMENT ING ESSENTIAL FATTY ACID TO PRE GNANT HOLSTEIN COWS AND TH EIR PRE WEANED CALVES ON CAL F PERFORMANCE, IMMUNE RESPON SE AND HEALTH Background Doubling the birth weight at weaning and minimize the incidence of diseases is the primary goals of dairy herd management. Dairy farmers have to deal with critical circumstances and health challenges once the calf is born (Beam et al. 2009, Donovan et al., 1998). Therefore, to prevent high incidence of calf diseases and to avoid jeopardizing the profitability of the herd, effective care should be taken not only during the preweaning period but also during the gestation period, part icularly during the last trimester of gestation, during which time the fetus has its greatest development. Early studies in human subjects have reported a direct effect of the nutritional status of pregnant women during late pregnancy on fetal growth and b irth weight. Kramer (1987) reviewed 895 publications related to potential causes of intrauterine growth retardation in human subjects and reported that poor gestational nutrition was a common cause of lighter birth weight. More recent studies in ruminants found contradictory effects of undernutrition during late gestation on birth weight (Osgerby et al., 2002; Dwyer et al. 2003). Hess (2003) evaluated 18 studies that supplemented fat to late gestation beef cows and concluded that fat supplementation did not affect birth weight. Funston et al. (2010) reviewed the effects of maternal nutrition on future performance of beef cows, whereas Singh et al. (2010) reviewed the factors accounting for phenotypic variation in milk production by dairy cows. Both authors c oncluded that a substantial proportion of the unexplained phenotypical variations were due to epigenetic regulation (change in gene expression without modifying DNA sequence) as a

PAGE 153

153 consequence of maternal nutrition during fetal life or nutrition during the first year of life. Recently Soberon et al. (2012) reported an increase of 850 kg of milk in the first lactation per 1 kg increase of ADG during the preweaning period. They concluded that increased growth rate before weaning resulted in some form of epigen etic programming with a positive effect on milk yield. Few studies have evaluated the effect of supplementing diets with different FA sources during late gestation on overall calf performance. The few available studies were done in beef cattle and resulted in no effect of supplemental fat on birth and weaning weight (Bottger et al., 2002; Encinias et al., 2001, 2004). Beef calves suckling cows supplemented with LA affected metabolic profile and antibody production but growth was not affected (Lake et al., 2 005; 2006a, 2006b, 2006c). A limited number of studies have evaluated the supplementation of increased intakes of LA to preweaned dairy calves. The laboratory of J.K. Jenkins in Ontario, Canada was among the first ones to evaluate the replacement of milk f at with other sources of less expensive fat such as vegetable oils. These studies (Jenkins et al., 1985, 1986; Jenkins and Kramer, 1986) are the foundation to better understand the effects of feeding vegetable and animal oils, aiming to increase the intake of EFA on calf growth, diarrhea incidence, and FA profile of important tissues involved in lipid metabolism such as liver heart and plasma S ome work was recently published to evaluate the effect of omega 3 (n 3) FA from animal or vegetable origin (Ballo u and DePeters., 2008; Hill et al., 2011). However, there is no study evaluating the inclusion of LA in MR to modify activity of different markers of immune responses in newborn calves. The hypothesis of the current study

PAGE 154

154 was that supplementing prepartum a nd preweaning diets with LA would improve overall performance of calves. In addition, it was hypothesized that calves born to dams not supplemented with LA would have a greater response to LA feeding in MR than calves born to dams fed diets sup plemented wi th LA The objective was to evaluate the effect of supplementing diets with fat enriched with LA during late gestation and feeding LA enriched MR during the first two months of life on calf growth, health, and immune responses. Materials and Methods Prepa rtum Management FL) from October 2008 to June 2009. All procedures for animal handling and care were nulliparous (n = 35) and previously parous (n = 61) Holstein cattle were sorted according to calving date, parity, body weight (BW), and body condition score (BCS) and assigned to one of the three treatments at 8 wk before their expected calving date. Prep artum treatments: supplementation (Control), 1.7% of dietary dry matter (DM) of Churc h and Dwight, Princeton, NJ) as well as cattle general management were the same as those indicated in chapter 3. Calves Dietary Treatments, Feeding Management and Analyses All procedures regarding calving management at birth and colostrum feeding were don e according details presented in Chapter 3. Calves were blocked by gender (n = 56 females and 40 males) and dam diet and randomly assigned to receive a MR containing

PAGE 155

155 low (LLA, 0.56% LA, DM basis) or high concentrations of LA (HLA, 1.78% LA, DM basis) for 6 0 d starting at birth. Milk replacer (Tables 4 1 and 4 2 Webster City, IA) was fed at 0600 and 1230 h daily at a constant rate of 0.149 g of LA/kg of BW 0.75 for the LLA treatment group and 0.487 g of LA/kg of BW 0.75 for the HLA treatment gr oup, respectively. Milk replacer was fed exclusively the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 Warm water (~38 to 42C) was added to the powdered MR at the time of each feeding in order to prepare an 11% DM MR. The amount of fat intake per kg of BW 0.75 remained constant throughout the experiment. Calves were weighed weekly and amounts of MR fed were adjusted weekly based upon BW. Refusal of MR was recorded daily. Coconut oil was the sole fat source in the LLA MR whereas a mixture of CCO and porcine lard were the fat sources in the HLA MR. The LA intake from the LLA MR was below the minimum recommend for laboratory rats (NRC, 1995) for optimum growth performance. For comparison, typical on farm practice for calves weighing 40 kg to be f ed 4 L of milk daily (10% of BW) containing 3.5% triglycerides of which 3.13% is LA. Intake of LA would be ~3.8 g of LA daily. Calves weighing 40 kg in the current study consumed 2.5 and 7.8 g of LA daily when fed LLA and HLA MR, respectively. A single g rain mix (1.17% LA, DM basis) was offered in ad libitum amounts from 31 to 60 d of age (Tables 4 1 and 4 2 ). Barley and peanut meal were chosen to formul at e the grain mix because they contain the lowest concentration of LA among traditional grain and prot ein meal supplements, respectively. Peanut meal contained 2 ppb of a flatoxin (Quamta Lab. Selma, TX). Amounts of grain mix offered and refused were measured daily. Clean water was available at all times. Powdered MR and grain

PAGE 156

156 mix were sampled weekly and c omposited monthly. Monthly composites were analyzed (Dairy One, Ithaca, NY) for minerals (Ca, P, Mg, K, Na, I, Zn, Cu, Mn, Mo, Co, and S) and CP. Additional analyses for the grain mix were ether extract, ADF, and NDF. Housing, Body Weight and Immunizations During the 60 d of the experimental period, calves were housed outside in individual wire hutches (1 m 1.5 m) bedded with sand. Body weights for measures of growth were taken at birth, before colostrum feeding, and at 30 and 60 d before the morning feed ing. At birth, calves were administered intranasal TVS 2 (Pfizer Co., New York, NY) to prevent infectious bovine rhinotracheitis (IBR) and parainfluenza 3 (PI3) and oral calf guard (Pfizer Co. New York, NY) to control infection for rotavirus and corona vi rus. At 3 wk of age Bovishield Gold 5 (Pfizer Co., New York, NY) was administered by s.c. injection for prevention of IBR, bovine virus diarrhea [types I and II], PI3, and bovine respiratory syncytial virus. The same dose of Bovishield Gold 5 was repeated at wk 5 plus an injection of Ultrabac 7 (Pfizer Co., New York, NY) to protect calves from diseases caused by Clostridium. A dose of Ultrabac 7 was repeated at wk 7 including an injection of Pinkeye Shield XT4 (Novartis, Inc., Larchwood, IA). Starting at 6 wk of age, a 5 day oral treatment with Corid (Merial Limited, Duluth, GA) to treat and prevent coccidiosis was followed by 5 d with an antihelmintic (Safeguard; Merck & Co., Inc., Whitehouse Station, NJ). Calves experiencing diarrhea were given electrolyt es (Gener Lyte, Bio Vet Inc., Blue Mound, WI), bismuth subsalicylate (Bismusol; First Priority, Inc., Elgin, IL), and sulfadimethoxine (Albon boluses, Pfizer Co., New York, NY) for 5 d. If the diarrheic condition reoccurred in a given calf, the same treatm ent was re administered.

PAGE 157

157 Calves Scoring for Health Assessment and Incidence of Health Disorders Attitude and fecal scores were recorded daily according to the scoring system of Magalhes et al. (2008). Attitude [1) responsive, 2) non active, 3) depressed, or 4) moribund] and fecal consistency scores [1) feces of firm consistency, no diarrhea, 2) feces of moderate consistency, soft, no diarrhea, 3) runny feces, mild diarrhea, or 4) watery feces, diarrhea] were recorded for each calf after the first MR feedin g between 0800 to 1000 h. Incidence of health disorders were recorded daily for each individual calf. Rectal temperature of calves displaying signs of any disease was measured. Fever o C. Diarrhea was diagnosed by p resence of watery feces (fecal score > 2). One calf was diagnosed with chronic pneumonia starting at 38 d of age, consequently only its measures before 30 d of age were considered for all statistical analyses. Hormone and Metabolite Analyses Before colostr um was fed, a jugular blood sample was collected from each calf and again within 24 to 30 h after colostrum feeding. Blood samples were collected into a clot activated tube (Vacutainer, Becton Dickinson, Franklin Lakes, NJ), and serum was separated at room temperature. Tubes were centrifuged for 15 min at 2095 x g (Allegra X 15R centrifuge, Beckman Coulter, Inc). Blood was collected from the jugular vein twice a week for the first 30 d of age and once a week thereafter, into clot activated and K 2 EDTA tubes for serum and plasma collection, respectively. Plasma and serum were separated by centrifugation and then stored at 20 o C for later analyses. Plasma metabolites such as glucose, plasma urea N (PUN), and cholesterol were analyzed 2 times weekly the first 30 d and 1 time per week from 31 to 60 d of age. hydroxybutyric acid (BHBA) were

PAGE 158

158 measured once a week from 1 to 60 d of age. Serum samples at 0 and 1 d of age and plasma samples at 14, 28, 42 and 56 d of age were used to analyze insulin and insulin like growth factor I (IGF I). A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to measure plasma glucose (Bran and Luebbe Industrial Method 339 19; Gochman and Schmitz, 1972) and PUN (B ran and Luebbe Industrial Method 339 01; Marsh et al., 1965). Samples were run in singlet, including in each run a control sample which was run in duplicate. Inter assay variations were 2.6 and 4.4% for PUN and glucose, respectively. Plasma concentrations of NEFA were determined using a commercial kit (NEFA C kit; Wako Diagnostics, Inc., Richmond, VA) with a method modified by Johnson (1993). Plasma concentrations of BHBA also were determined using a commercial kit (Wako Autokit 3 HB; Wako Diagnostics, Inc. Richmond, VA). Samples were run in duplicate for NEFA (intra assay variation of 2.2%), whereas samples for BHBA were run in singlet, including a control sample which was run in duplicate. Intra and inter assay variations for BHBA were 3.5 and 5.9% respe ctively. Total cholesterol concentrations (Cholesterol E kit, Wako Diagnostics Inc., Richmond, VA) were analyzed in serum at 0 h and in plasma twice a week the first 30 d of life and once a week thereafter. Each sample was analyzed in triplicate and one sa mple was removed if the coefficient of variation was greater than 5%. Intra and inter assay variations were 2.5 and 4.8%, respectively. Concentrations of IGF (Active non extraction IGF I ELISA, Diagno stic Systems Laboratory, Inc., Webster TX) with some modifications in sample pre treatment similar to those indicated in chapter 3

PAGE 159

159 The intra plate variation for IGF I of control samples was 2.4%, whereas the inter plate variation was 3.2%. Insulin concent rations were analyzed using a double antibody radioimmunoassay (Badinga et al., 1991 ). Intra and inter assay variations were 7.3 and 14.6%, respectively. The FA extraction and methylation procedures were the same for feed and plasma samples. It was perfo rmed by the 2 step methylation procedure according to Kramer et al. (1997) with some modifications. Briefly, feed ingredients (500 mg) and freeze dried plasma samples (1.5 ml of fresh plasma of calves at 0 d before colostrum feeding, and at 30 and 60 d of age) were weighed or transferred respectively to a screw capped (Teflon TM lined caps) culture tubes. One mL of internal standard (C19:0, 1mg/mL of benzene) was added in order to calculate total FA concentration. Lipid was extracted by adding 2 mL of sodiu m methoxide (Acros, New Jersey, USA), vortexing, and incubating in a 50 o C water bath for 10 min. After cooling for 7 min, 3 mL of 5% methanolic HCl (Fisher Scientific, Hampton, NH, USA) was added and the tubes were vortexed. The tubes were incubated in an 80 o C water bath for 10 min, removed from water bath, and allowed to cool for 10 min. One mL of hexane and 6.5 mL of 6% K 2 CO 3 were added. The tubes were vortexed and centrifuged at 1455 x g for 10 min. The upper layer was carefully transferred into crimp to p vials and stored at 20 o C for further analysis. Fatty acid methyl esters were determined using a Varian CP 3800 gas chromatograph (Varian Inc., Palo Alto, CA) equipped with auto sampler (Varian CP 8400), flame ionization detector, and a Varian capillary column (CP SIL 88 FS, 100 m x and detector temperatures were maintained at 250 o

PAGE 160

160 was injected via the auto sampler into the column. The ov en temperature was set initially at 120C for 1 min, increased by 5C/min up to 190C, held at 190C for 30 min, increased by 2C/min up to 220C, and held at 220C for 15 min. The peak was identified and calculated based on the retention time and peak are a of known standards. Markers of Immunity Analyses Blood for hematologic analysis and for markers of immunity in fresh blood, were collected from puncture of the jugular vein into heparinized vacutainer tubes at 2, 7, 14, 21, 30, 40, and 60 1 d of age. S amples were kept at ambient temperature with constant inversion. A Bayer Advia 120 cell counter (Fisher Diagnostic, Middletown, VA) was used to quantify the population of blood cells. Analysis was performed within 2 h of collection. Phagocytic activity of blood neutrophils was evaluated the same days as blood cells population was quantified. Whole blood samples were collected in duplicate for quantification of blood cells. Samples were kept under constant rotation on a Clay Adams nutator (BD, San Jose, CA) until the neutrophil concentration was obtained from sample of the heparinized blood (100 10 3 Conjugate. For samples with greater concentrations of neutrophils, proportional amounts of reconstituted product were adde d. Samples were incubated for 2 h at 37 o C with continuous rotation (Clay Adams nutator; BD, San Jose, CA). A control sample for each animal was included, following the same process as described above but without

PAGE 161

161 using Conjugate E. coli. After incubation, p hagocytosis initiated by the presence of E.coli was stopped by placing the samples on crushed ice. Samples were lysed for red blood cells using 2.5 mL of lysing buffer (44.94 g of NH 4 Cl, 5.0 g of KHCO 3 and 0.185 g of K 2 EDTA in 10 L of double distilled wa ter). Tubes were vortexed and left at room temperature for 15 min followed by a 5 min centrifugation at 931 x g (Allegra X 15R centrifuge, Beckman Coulter, Inc). The supernatant was removed and the pellet was broken a part by gently shaking. To each tube 2. 5 mL of FACS buffer (2% of fetal bovine serum, 0.1% of sodium azide in PBS) was added and immediately centrifuged for 5 min at 931 x g Tubes were then placed on crushed ice and transported to the University of Florida Flow Cytometry Core Lab. FACSFlow she Jose, CA) was added to each tube. For each sample the optical features of 50,000 neutrophils were acquired using a Facsort flow cytometer equipped with a 488 nm argon ion laser for excitation at 15 mW (BD Biosciences, San Jose, CA) and CellQuest software (Becton Dickinson, San Jose, CA). Forward (roughly proportional to the diameter of the cell) and side (proportional to membrane irregularity) scatters were used for preliminary identification of neutrophil cells on dot plots (Jain et al., 1991). Density cytograms were generated by linear amplification of the signals in the forward and side scatters. Percentage fluorescence of positive events was correlated with the proportion of neutrophils able to phagocytize E. coli whereas geometric mean fluorescence intensity (MFI) was interpreted as mean number of bacteria ingested per neutrophil. Expression of adhesion molecules on neutrophil surface was performed according to Silvestre et al. (2011) with some modifications. Bri efly, monoclonal mouse antibovine L selectin (CD62L, IgG1 isotype, Serotec Raleigh, NC ) and a mouse anti

PAGE 162

162 integrin (CD18, IgG1 isotype, Serotec Raleigh, NC ) that cross reacts with bovine CD18 were used. Additionally, an isotype mouse control antibody (IgG1 isotype, Serotec) was used to correct for non specific binding of CD62L and CD18 antibodies to the cells. Blood from each sample (3 mL) was placed in a 50 mL polypropylene tube and lysis buffer (44.94 g of NH 4 Cl, 5.0 g of KHCO 3 and 0.185 g of K 2 EDTA in 10 L of double distilled water) was added up to a final volume of 50 mL, left at room temperature for 15 min, and then centrifuged for 10 min at 931 x g Supernatant was decanted and pellet re suspended in 15 mL of lysis buffer and left for 10 min at room temperature, then centrifuged for10 min at 931 x g Supernatant was decanted and reconstituted with 15 mL of FACS buffer (2% of fetal bovine serum, 0.1% of sodium azide in PBS) and centrifuged for 10 min at 931 x g Supernantant was decanted and the pellet cells were re suspended in 1 mL of FACS buffer and kept on crushed ice until staining. The cell immunostaining of a negative control with and without any antibody and for each and control antibody in FACS buffer) were added to each individual tube and incubated at room temperature for 25 min. FACS buffer was added into each tube (2.5 mL) and centrifuged for 5 min at 233 x g Supernatants were decanted and each tube received 5 of antimouse IgG (polyclonal IgG isotype, Serotec) and then incubated for another 25 min. Cells were washed with FACS buffer (2.5 mL) and centrifuged for 5 min at 233 x g Supernatants were decanted and 0.4 mL of the FACS fixative solution (2% of fetal bo vine serum and 0.1% of sodium azide in 0.5% formalin) was added to each tube to re suspend the cell pellet. Flow cytometer settings were similar to that for neutrophil

PAGE 163

163 phagocytic activ ity. Percentage of neutrophil cells positive for CD62L and CD18 were obt ained based upon gated cells. Also, the geometric MFI of the labeling kit, an indicator of the number of receptors on the surface of each neutrophil cell, was obtained in the histogram for the gated cell populations. Blood was collected from the jugular v ein twice a wk the first 30 d of age and once a week thereafter into clot activated and K 2 EDTA tubes. Before obtaining the plasma from each sample, hematocrit concentration was measured using heparinized micro hematocrit capillary tubes (Fisherbrand, Therm o Fisher Scientific Inc.) centrifuged (Microspin 24 tube micro hematocrit centrifuge, Vulcon Technologies, Grandview, Mo) for 3 min and read in a micro hematocrit tube reader (Model CR, Damon/IEC, Needham Heights, MA). Plasma and serum were separated by ce ntrifugation for 15 min at 2095 x g (Allegra X 15R centrifuge, Beckman Coulter, Inc) and then stored at 20 o C for later analyses. Serum before storing was analyzed for serum total protein (STP) using an automatic temperature compensated hand refractometer Concentrations of haptoglobin (Hp) and acid soluble protein (ASP) were measured in all collected samples. Calves were injected subcutaneously (s.c.) with 0.5 mg of OVA (Sigma Aldrich, Saint Louis, MO) diluted in Quil A adjuvant solution (0.5 mg of Quil A in 1 mL of PBS, Accurate Chemical & Scientific Corp., Westbury, NY) at 2, 20, and 40 d of age. Concentrations of bovine anti OVA IgG were measured in serum on the same days of injection and at 60 d of age. Serum concentrations of bovine anti OVA IgG were measured by enzyme linked immunosorbent assay (ELISA) as described by Mallard et al. (1997) and detailed in chapter 3 Intra and inter assay coefficients of variation were 9.2 and 9.7%, respectively.

PAGE 164

164 Concentrations of plasma Hp were determined by measuri ng the differences of H 2 O 2 with Hp hemoglobin (Hb) as described previously (Makimura and Suzuki, 1982) Concentrations of Hp are reported as arbitrary units (optical density x 100) resulting from the absorption reading at 450 nm Intra and inter assay co efficients of variation were 6.0 and 10.9%, respectively. Concentrations of ASP were determined according to Nakajima et al. (1982) with some modifications Plasma samples (50 L) were incubated with PCA solution (1 mL 6 M perchloric acid, Fisher Scientif ic, Hampton, NH, USA). The intra and inter assay coefficients of variations were 2.6 and 5.9%, respectively. Isolation of peripheral blood mononuclear cells (PBMC) was done at 15 2 and 30 1 d of age according to Caldari (2009) with some modificatio ns. Briefly, 5 tubes of blood (10 mL each) were collected from each calf from the jugular vein into heparinized tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ). Blood samples were transported to the laboratory at ambient temperature and the isolat ion was initiated within 3 h of blood collection. Tubes were centrifuged for 15 min at 931 x g at room temperature (Allegra X 15R centrifuge, Beckman Coulter, Inc). The buffy coat containing most of the white blood cells, was transferred using sterile tr ansfer pipettes to a 13 mL tube (Sarstedt Inc., Newton, NC) containing 2 mL of medium 199 (M 199, Sigma Aldrich, Saint Louis, MO). The buffy coat and M 199 medium were mixed by pipetting up and down several times. This cell suspension was transferred slow ly on top of 2 mL of Fico/Lite LymphoH (Atlanta Biologicals, Lawrenceville, GA). The cell suspension/Fico/Lite LymphoH solution was centrifuged for 30 min at 524 x g at room temperature. Mononuclear cells were collected from the Fico/Lite interface and

PAGE 165

165 tra nsferred to pre labeled 13 mL culture tubes containing 2 mL of red blood cell lysing buffer (Sigma Aldrich, Saint Louis, MO). Exactly 20 sec after transferring, the solution was neutralized with 8 mL of 1X DPBS (Sigma Aldrich, Saint Louis, MO). The soluti on was centrifuged at 524 x g for 15 min at room temperature. The supernatant was removed by aspiration with a sterile glass pipette attached to a vacuum pump and the pellet containing the PBMC was resuspended in 4 mL of M 199 media by pipetting up and dow n 10 times with a sterile transfer pipette. The supernatant was resuspended in modified M 199 (M 199 media supplemented with 5% horse serum, 500 U/mL of penicillin, 0.2 mg/mL of streptomycin, 2 mM of glutamine, 10 5 mercaptoethanol; all reagents from S igma Aldrich, Saint Louis, MO). The PBMC were counted using the Trypan blue dye (Sigma Aldrich, Saint Louis, MO) by exclusion method. The cell suspension was adjusted to 2 x 10 6 cells/mL. Cell suspension in a total volume of 2 mL was plated in triplicate with modified M 199 media Aldrich, Saint Louis, MO) on a 6 well plate (Corning Inc., Corning, NY). Plates were incubated for 48 h at 37 o C at 5% CO 2 After incubation, plates were centr ifuged for 10 min at 524 x g and the supernatant was stored at 80C for analysis of cytokine production. Quantification of IFN development kit (R&D systems, Minneapolis, MN). Stimulated and non stimulated samples were run in triplicate and the most variable replicate was not considered. The intra assay coefficient of variation was 9.2%. Statistical Analyses Dam diets (n = 3) and MR (n = 2) were arranged in a 3 x 2 factorial randomized bloc k design. On a weekly basis, a cohort of Holstein cows at 8 wk before the expected

PAGE 166

166 calving date was blocked by parity (nulliparous and parous) and BCS. Within each block, cattle were assigned randomly to one of the three dietary treatments. Calves after bi rth were blocked by dam diet and gender and randomly assigned to one of the two MR. A total of 40 male and 56 female calves were enrolled. Repeated measurement analysis was conducted on nearly all variables using the PROC MIXED procedure of SAS (Release 9 .2) according to the following model: Y ijklm i j ij k ik jk ijk + Cl( ijk ) + W m im + jm ijm km ikm jkm ijkm jklm Where Y ijklm i is the fixed effect of dam diet (control, SF j ij is the k ik jk is the interaction of MR and gender, ij k is the interaction of dam diet, MR, and gender, Cl( ijk ) is the random effect of calf im jm is the in teraction ijm km is the ikm is the interaction of dam diet, gender, and age, jkm ijkm is the inte raction of dam ijklm is the residual error. For nonrepeated measures, the same model was used after removing the age effect and their interactions. All variables were tested for normality of residuals using the Shapiro Wil k test (SAS version 9.2, SAS Inst. Inc., Cary, NC). Non normally distributed data were transformed as suggested using the guided data analysis of SAS and back transformed

PAGE 167

167 using the LINK and ILINK function of GLIMMIX respectively Data were tested to determ ine the structure of best fit, namely compound symmetry, compound symmetry heterogeneous, autoregressive 1, and autoregressive 1 heterogeneous as indicated by a Schwartz Bayesian information criteria value closest to zero (Littell et al., 1996). If repeate d measures were taken on unequally spaced intervals, the sp(pow) covariance structure was used. Different temporal responses to treatments were further examined using the SLICE option of the MIXED or GLIMMIX procedure. The following orthogonal contrasts we re performed [1) dam diet of no fat vs. fat (SFA + EFA), 2) dam diet of SFA vs. EFA, 3) HLA vs. LLA MR, 4) interaction of contrasts 1 and 3, and 5) interaction of contrasts 2 and 3]. If any 3 or 4 way interaction including the effect of time were not sign ificant ( P > 0.25), the interactions were dropped from the model and the model was rerun (Bancroft, 1968). Differences discussed in the text were significant at P P Results Plasma Fatty Acid Concentrat ion and Profile Mean plasma concentrations of total FA at birth ranged from 1.14 to 1.34 mg/mL (Table 4 3 ). Regardless of the diet fed prepartum, palmitic acid and OA made up ~60% (~30% each) of the total FA in plasma of calves at birth followed by stearic acid at approximately 13.5%, palmitoleic at 5.2%, AA at 4.7%, and LA at 3.7% Docosahexaenoic acid was the n 3 FA with the greatest concentration with a mean of approximately 0.7%. Total FA concentration in plasma was not affecte d by parity (1.23 vs. 1.31 % for calves born from nulliparous heifers and parous cows respectively P = 0.23). However, calves from nulliparous heifers had lower concentrations ( P < 0.01) of n 6 FA, namely LA (2.9 vs. 4.5% of total FA) and AA (4.0 vs. 5.4% of total FA) but

PAGE 168

168 greater concentrations ( P < 0.01) of n 3 FA, namely EPA ( 0.39 vs. 0.08% of total FA) and DHA (0.87 vs. 0.51% of total FA). Although all cattle consumed the same basic TMR the last 8 wk before calving, these specific FA differences may have been because nulliparous heifers consumed more fresh pasture than parous cows in previous months as fresh grass usually contains more n 3 FA than stored forages. In summary, calves born from nulliparous heifers had lower concentrations of total n 6 FA (8.7 vs. 12.8, P < 0.01) bu t greater concentrations of total n 3 FA (1.82 vs. 1.13, P < 0.01). Compared to cattle fed control diet feeding fat prepartum did not appreciably change the total FA concentration or the profile of FA in plasma of the calves at birt h (Table 4 3 ). Total pr oportions of SFA, MUFA, and PUFA were not affected by dam diets. Plasmatic concentrations of total FA at birth tended to be greater for calves born from dams fed EFA instead of SFA (1.33 vs. 1.21 mg/mL, P = 0.09 ). Cattle supplemented with EFA prepartum gav e birth to calves having or tending to have greater proportions ( P = 0.03) of LA (4.4 vs. 3.3%) and total n 6 FA (11.8 vs. 10.3%; P = 0.06) in plasma compared to calves born from cattle fed SFA. The effect of fat type was the opposite for some n 3 FA. Catt le supplemented with EFA prepartum gave birth to calves with lower plasmatic proportions of total n 3 FA (1.30 vs. 1.67%; P < 0.02), specifically EPA (0.19 vs. 0.29%; P = 0.03) and DHA (0.60 vs. 0.80%; P < 0.01) compared to calves born from cattle fed SFA. Although calves born from cattle fed EFA tended to have more circulating FA ( P = 0.09 ), the increase was only 10%, hence when correcting the proportions of FA by this increased total FA, calves born from cattle fed EFA still had lower circulating amounts of DHA ( P = 0.05) but circulating amounts of EPA ( P = 0.52) were not different. Plasma concentrations of some FA found in greater

PAGE 169

169 concentrations in SFA (C1 6:0, C18:0, and C18:1 ) instead of in EFA supplement were not increased in calf plasma by feeding SFA prepartum. Mean daily intake of LA during the first 30 d, when MR was the only feed, was 2.6 and 8.6 g/d by calves fed LLA and HLA MR, respectively, whereas for the second 30 d of life, intake of LA from MR and grain mix was 9.4 and 16.4 g/d for calves fed LLA and HLA MR, respectively. Intake of ALA was minimal since the LLA MR did not contain ALA and the HLA MR only contained ALA at 0.15% of DM. Average intake of ALA during the first 30 d was 0 and 0.5 g/d by calves fed LLA and HLA MR, respectively and 0.5 and 1.3 g/d for calves fed LLA and HLA MR for the second 30 d, respectively. The FA profile of plasma changed dr amatically from birth (Table 4 3 ) to th at when calves were 30 to 60 d old (Table 4 4 ). The main changes were in proportions of C16:0, C18:1 cis, LA, and ALA. Mean concentrations at birth and at the 30 to 60 d of age period were approximately 30 and 16% for C16:0, 29 and 11% for C18:1cis, 4 and 44% for LA, and 0.06 and 0.70% for ALA. Fat concentration in plasma increased from 1.27 at birth to 2.02 mg/100 mL of plasma for older calves, an increase of ~60%. The feeding of fat or different FA during the prepartum period had no or little effect on the FA profile of plasma of calves at 30 to 60 d of age (Table 4 4 ). Proportion of total saturated FA in plasma of calves born from cattle fed SFA was greater ( P = 0.01) than in those born from EFA fed cattle however only proportions of C12:0 (0.74 vs. 0.53%) and C14:0 (3.7 vs. 3.4%) were increased ( P LA and DHA were in greater and lower conc entrations in newborn calves born from cattle fed EFA instead of SFA, respectively and the same pattern tended to be evident ( P in plasma of calves at 30 to 60 d of age. I nteractions of dam diet and MR were not detected for any FA except

PAGE 170

170 2 minor FA C12:0 and C14:1 and some EFA derivatives Calves fed HLA MR instead of LLA MR and born from cattle fed control diets tended to have a decreased proportion of plasma AA (3.21 vs. 2.82%) and DHA (0.26 vs. 0.20%) whereas proportions of AA (3.15 vs. 3.10%) a nd DHA (0.22 vs. 0.22%) in plasma of calves fed LLA and HLA MR respectively, and born from dams fed fat did not differ (FAT by MR interaction, P 0.10). Likewise, calves fed HLA MR and born from cattle supplemented with EFA had a greater proportion of plasma DPA (0.36 vs. 0.28%) whereas DPA proportions in plasma of calves were not affected (0.30 vs. 0.30%) when fed HLA and born from SFA supplemen ted cattle (FA by MR interaction, P = 0.01). As expected, the main factor affecting plasma FA at d 30 to 60 was the type of MR fed. Plasma concentrations of LA and ALA at birth did not differ in calves assigned to receive LLA or HLA MR treatments. By repla cing a portion of the CCO in the LLA MR with porcine lard in the HLA MR, the proportions of MCFA were decreased ( P < 0.01, Table 4 4 ) in plasma, namely C12:0 from 0.82 to 0.48% and C14:0 from 4.8 to 2.3%. Likewise, feeding HLA MR decreased proportion ( P < 0.01) of C18:1 c 9 from 11.3 to 10.1%. Fatty acids found in greater concentrations in porcine lard compared to CCO were increased in plasma of cal ves fed porcine lard namely C16:1 (1.1 vs. 1.4%, P < 0.01), LA (40.9 vs. 46.3%, P < 0.01), and ALA (0.68 vs. 0.81%, P < 0.01). Calves fed HLA had an reduced proportion of intermediate FA perhaps reflecting attenuation of the enzymatic elongation and desaturation processes of LA, namely GLA(0 .19 vs. 0.35, P < 0.01), C20:3 (0.95 vs. 1.35%, P < 0.01), and AA (3.0 vs 3.2%, P = 0.05) but not of C22:4 (0.23 vs. 0.24, P = 0.84). Responses of the n 3 FA to feeding HLA MR were not consistent. Plasma proportions of EPA were decreased (0.12 vs. 0.07%, P < 0.01), of

PAGE 171

171 DPA were increased (0.29 vs. 0.33%, P < 0.01), and of DHA were unchanged (0.23 vs. 0.22%, P = 0.40). Despite similar intakes of MR, total FA concentration in plasma was about 8% less in calves fed HLA vs. LLA MR (1.94 vs. 2.09 mg/100mL of plasma, P = 0.01). The dietary FA profile changed when grain feeding star ted at 31 d of age and this resulted in a change in the FA profile of the plasma of calves at 30 compared to 60 d of age (Figure 4 1). Plasma proportions of C16:0, LA, AA, and DHA decreased ( P < 0.01) whereas plasma proportions of C14:0, C18:0, and ALA inc reased ( P in calves at 30 compared to 60 d of age. Interaction of age and MR were not significant ( P > 0.05) for any FA except LA was reduced to a greater extent due to age when LLA was fed (42.1 vs. 39.7% of total FA) instead of HLA MR (46.6 vs. 46.0% of t otal FA, MR by age interaction, P = 0.08). Measures of Growth and Feed Efficiency Body weight of calves at birth did not differ due to dam diet and averaged 40.2, 41.5, and 41.0 kg for calves born from dams fed Control, SFA, and EFA diets respectively (T able 4 5 ). Male calves enrolled in the HLA MR group were heavier than that of male calves enrolled in the LLA MR group (45.3 vs. 42.0 kg), whereas mean female birth weights did not differ (37.6 vs. 38.6 kg, MR by gender interaction, P = 0.04 data not show n ). Serum concentrations of IgG measured 24 to 30 h after feeding per 100 mL of serum which indicates an appropriate passive transfer (Tyler et al., 1996; Weaver et al ., 2000). The calf that failed to meet an appropriate passive transfer (0.65 g of IgG/dL) was born from a SFA cow and assigned to the HLA MR.

PAGE 172

172 Calves fed the HLA MR had consistently greater ADG than calves fed the LLA MR (an increase of 18, 9 and 15% for the first 30 d ( P = 0.02), the second 30 d ( P = 0.05), and the whole 60 d period ( P < 0.01), respectively Table 4 5 ) for both female and male calves. Total intake of grain mix (mean of 11.7 kg of DM across genders) during the last 30 d of the study was n ot affected by type of MR fed. However intake of MR was greater ( P = 0.03) for calves fed the HLA MR during the 31 to 60 d period because the heavier calves in the HLA group would have been offered more MR per the design of the feeding regimen. Nevertheles s total DMI (kg or kg as a % of BW) did not differ between MR groups over the 60 d study. This improved gain without changing DMI over the 60 d resulted in better efficiency ( P = 0. 01) of BW gain from feed intake during the 60 d study for calves fed the HL A MR (0.63 vs. 0.59). Therefore the improved ADG and FE was due to the superiority of the HLA MR formulation rather than to greater intake of the grain mix. The effect of the HLA MR was independent of the type of diet fed to the dams of the calv es (dam di et by MR interaction, P > 0.10 ). However the type of fat supplement prepartum did influence calf performance. Calves of both genders born from cattle fed SFA prepartum gained more BW over the 60 d period ( P = 0.04) compared with calves born from cattle fed EFA prepartum (30.0 vs. 27.4 kg). This greater gain was due to a tendency ( P = 0.07) for calves to consume more DM (48.8 vs. 45.6 kg) mainly as a result of a tendency for greater intake of grain mix during the last 30 d of the study (13.1 vs. 10.9 kg of DM, P = 0.06). However FE of calves was not improved by feeding SFA prepartum. Metabolic and Hormonal Profile Concentrations of plasma glucose were greatest at 2 d of age, exceeding 100 mg/dL, but decreased to between 85 and 95 mg/dL for the remainder of the study

PAGE 173

173 (effect of age, P < 0.01, Figure 4 2). Although mean concentrations of plasma glucose were not affected by dam diet, mean plasmatic concentration of glucose tended to be greater at 2 d of life but lower at 19 and 30 d of life for calves born from cattle fed fat compared to calves born from control cows (dam diet by age interaction, P = 0.07, Figure 4 2). Mean glucose concentration in plasma was 3.1 percentage units greater (92.7 vs. 89.9 mg/100 dL, P = 0.03) in calves fed HLA than in calves fed LL A (Table 4 6). This was true throughout the 60 d study as the MR by age interaction was not significant. Plasma concentrations of PUN were greater the firs t 30 d and began decreasing upon initiation of grain intake (effect of age, P < 0.01, Figure 4 3). Me an concentration of PUN was greater ( P = 0.05) in calves born from dams fed fat (8.27 vs. 7.61 mg/dL) than dams fed control diets Mean plasma concentrations of PUN tended to be lower ( P = 0.06) for calves fed HLA 7.75 vs. 8.35 mg/dL and this held true thr oughout the study (Figure 4 3). Plasma concentrations of BHBA peaked during the second week of life, gradually decreased until 30 d of age, then gradually increased once grain intake began (effect of age, P < 0.01, Table 4 6, Figure 4 4 A). Mean concentr ation of BHBA in plasma of calves born from cattle fed fat tended to be greater than that for calves born from control dams (1.21 vs. 0.94 mg/dL, P = 0.06 ). Calves fed LLA MR had greater mean concentrations of plasma BHBA than those fed HLA MR (1.36 vs. 0. 87 mg/dL, P < 0.01). Plasma concentrations of NEFA were greatest in the first wk of life (approximately 312 Eq/L), gradually decreasi ng for 3 wk before plateauing at less than half of initial values of approximately 150 Eq/L (Figure 4 4 B, effect of age, P < 0.01). Neither prepartum nor preweaning diets affected concentrations of plasma NEFA.

PAGE 174

174 Plasma concentrations of total cholesterol rose from approximately 30 mg/dL at P < 0.01, Figure 4 5). Both the type of dam diet and MR affected plasma cholesterol. Calves fed HLA MR, regardless of the di et fed to their dams, had lower plasma concentrations of total cholesterol starting at approximately d 19 compared to those fed LLA MR (MR by age interaction, P = 0.01, Figure 4 5). In addition, the dam diet tended to influence the effect of the MR. Plasm a cholesterol concentrations of calves born from control dams were not affected by MR (87.9 vs. 85.3 mg/dL) but concentrations tended to be greater when calves born from dams fed fat were fed LLA vs. HLA MR (96.1 vs. 82.1 mg/dL, FAT by MR interaction, P = 0.08). Plasma concentrations of insulin were low at birth as expected, but doubled once feeding commenced (Figure 4 6 A, B). Concentrations were relatively steady until grain intake began (after wk 4) after which concentrations increased as a mean of all d iets (effect of age, P < 0.01). Neither dam diet nor MR affected mean concentration of plasma insulin although feeding HLA MR resulted in a greater numerical mean concentration of plasma insulin compared to feeding LLA MR (1.44 vs. 1.28 ng/mL, P = 0.14, Ta ble 4 6 ). For IGF 1, plasma concentrations were greatest at birth, decreased to < half 2 wk later, then rising until reaching concentrations by 8 wk similar to those recorded at birth (effect of age, P < 0.01, Figure 4 7 A, B). In a similar pattern to tha t for insulin, calves fed HLA tended to have greater mean concentrations of plasma IGF 1 compared to those fed LLA (59.7 vs. 53.2, P = 0.08 ). Concentrations of STP were not affected by prepartum or preweaned diets, but greater concentrations were seen the first wk of life (Figure 4 8).

PAGE 175

175 Incidence of Diarrhea and Poor Attitude Calf attitude was generally responsive throughout the 60 d stud y with a mean of 1.04 (Table 4 7 ). Likewise fecal consistency across the study also was quite acceptable with a mean of 1 .18. Severity (greater mean score) of poor attitude was greater during the first 2 wk of age whereas severity of diarrhea increased at 2 wk of age (age, P < 0.01, Figures 4 9 and 4 10). Neither main effects of prepartum diet nor type of MR had any effect on scores. However, the mean score for attitude tended to be greater in calves fed HLA vs. LLA MR if they were born from control cattle (1.06 vs. 1.03) but were not changed if the dam was fed either fat source prepartum (1.04 vs. 1.03, FAT by MR interactio n, P = 0.06). This pattern also was true for mean fecal score. The mean score for feces was greater in calves fed HLA vs. LLA MR if they were born from control cattle (1.22 vs. 1.12) but were not changed if the dam was fed either fat source prepartum (1.21 vs. 1.17, FAT by MR interaction, P = 0.03. The treatment effects on the percentage of days with poor attitude and diarrhea followed the same pattern. During the first 30 d, feeding HLA rather than LLA MR to calves born from dams not fed fat increased the percentage of days with poor attitude (12.3 vs. 5.3%) whereas no effect of MR was detected on attitude if fat was fed to calves born from dams fed fat prepartum (5.8 vs. 8.0%, FAT by MR interaction, P = 0.01). This was a 2 d difference in poor attitude du ring the first 30 d of life. This same interaction was detected ( P = 0.02) for attitude when the first 60 d were evaluated. Feeding a HLA MR proved beneficial if dams were fed fat prepartum. During the first 30 d of life, percentage of days with diarrhea were reduced if calves born from dams fed fat were fed the HLA MR (9.2 vs. 15.4%) whereas diarrhea days were increased by feeding HLA MR to calves born from

PAGE 176

176 dams not fed fat (5.3 vs. 12.3%, FAT by MR interaction, P < 0.01). This same interaction was detec ted ( P < 0.01) for attitude when the first 60 d were evaluated. Blood Cell Population Concentrations of total red (mean of 8.4 10 3 /L) and white (mean of 8.65 10 3 /L) blood cells were not affected by diets but increased with age ( P < 0.01, Table 4 8 Figures 4 11 A and B). Similarly, blood concentrations of neutrophils (mean of 3090/L), monocytes (mean of 380/L), and basophils (mean of 110/L) were not affected by diets but by age ( P < 0.01, Figure 4 12 A, 4 13 A and 4 15 respectively). Concentration s of blood neutrophils were greater the first wk of life and decreased to the lowest starting at 2 wk of life which matches with the period of greatest health challenges. Lymphocyte concentrations were greater in calves fed HLA vs. LLA MR (4.61 vs. 4.20 10 3 P = 0.04) and increased with age ( P < 0.01, Figure 4 12 B) with the greatest increase occurring between birth and 2 wk of age. Blood concentrations of eosinophils of calves fed HLA MR tended to be greater at 7 and 14 d of age compared to those fed L LA MR (MR by age interaction, P = 0.07, Figure 4 13 B). This decrease in eosinophil concentration at d 7 of life of calves fed LLA MR occurred primarily in calves born from cattle fed the control or SFA diets prepartum (dam diet by MR by age interaction, P = 0.01, Figures 4 14 A and B). Platelet concentrations in calves increased 2 to 3 fold from birth to the second week of age and then gradually decreased (effect of age, P < 0.01, Figure 4 16 A). Feeding EFA prepartum resulted in calves having lower platel et concentrations at 7 d of age ( P = 0.06) but greater concentrations at 60 d of age ( P = 0.05) compared with other diets (dam diet by age interaction, P = 0.03, Figure 4 16). Mean platelet concentration was greater for calves fed LLA vs. HLA MR (801 vs. 7 15 10 3 P = 0.03, Figure 16 B).

PAGE 177

177 Proportion of individual classes of white blood cells (%) followed the same pattern 8 ) with two exceptions. Proportion of lymphocytes was not affected by the MR fed but that of m onocytes was greater for calves fed HLA vs. LLA MR and born from cattle fed the control diet (4.51 vs. 4.09%) whereas the opposite was true for those born from cattle fed fat prepartum (3.87 vs. 4.32%, FAT by MR interaction, P = 0.05). Calves fed HLA MR t ended to have greater hematocrit than those fed LLA MR (35.9 vs. 34.4%, P = 0.08). Concentrations increased after birth but started falling after 9 d of age until d 42 when they increased again (Figure 4 17). Expression of Adhesion Molecules and Phagocyti c Activity of Neutrophils Proportion of neutrophils expressing CD18 and CD62L was not affected by dam or calf diets and means were 94.4 and 98.2% across diets, respectively. Likewise the MFI of CD18, an indicator of mean number of CD18 expressed per neutro phil, was not affected by diets. However MFI of CD62L tended to be greater in calves born from dams fed the control diet than calves born from dams fed fat supplemented diets (382 vs. 338, P = 0.10, Table 4 9 Figure 4 18). Mean florescence intensity, an i ndicator of the number of E. coli phagocytized per neutrophil, was greater for calves born from dams fed EFA compared to those born from dams fed SFA (121 vs. 113, P = 0.04 Figure 4 19 A) however calves born from dams fed SFA tended to have greater concen trations of phagocytic neutrophils (3.40 vs. 2.89 10 3 blood, P = 0.08, Figure 4 19 B) in blood. Phagocytic activity of blood neutrophils tended to be greater for calves fed HLA vs. LLA MR (96.3 vs. 95.6%), with the difference observed after 7 d of age (Figure 4 20).

PAGE 178

178 Concentration of Acute Phase Protein s Plasma concentration of ASP was greatest right after calving (~230 mg/L) and decreased gradually until plateauing at ~60 mg/L around 30 d of age (effect of age, P < 0.01, Figure 4 21). Feeding HLA rather than LLA MR reduced ASP concentrations to a great er extent in calves born from control dams (94.1 vs. 72.3 mg/L) compared to the response in calves of dams fed fat prepartum (90.0 vs. 82.0 mg/L, FAT by MR interaction, P = 0.04, Table 4 10 ). Concentration of ASP was lower in calves fed HLA (78.8 vs. 91.4 mg/L, P < 0.01) but the difference tended to be accentuated after 12 d of age (Figure 4 21, MR by age, P = 0.09). Calves born from dams fed fat tended to have increased plasma concentrations of haptoglobin (1.04 vs. 0.95, P = 0.06) and the concentrations increased after 2 d of age reaching a peak at 9 d of age (Figure 4 22 A). Humoral and Cell Mediated Immune Responses Injection of OVA into calves at 2 and 20 d of age did not have any effect on the concentration of anti OVA IgG in serum, whereas the incre ase was minimal after the 3 rd injection at 40 d of age (Figure 4 22A). Production of bovine anti OVA IgG was greater in calves born from dams fed SFA than in calves born from dams fed EFA between 2 and 20 d of age (dam diet by age interaction, P < 0.01, Ta ble 4 10 Figure 4 22 B). Production of IFN A is presented as the difference in concentrations of stimulated minus nonstimulated cells. In general, values of IFN used At 15 d of age, calves born from cows fed SFA tended to have greater differential production of IFN P = 0.08, Table 4 10 ). At 30 d of age, stimulated PBMC from calves fed the HLA MR had a

PAGE 179

179 greater differential production of IFN pg/mL, P = 0.05). Discussion Prepartum Supplementation of Fatty Acids Affects FA Profile and Immunity Measures of Calves Calves have a high demand for EFA derivatives such as DHA for central nervous system development. However the epitheliochorial placenta of cows is less permeable to free FA, partially limiting their uptake (Moallem and Zachut, 2012). In sheep (Campbell et al., 1994) and humans (Koletzko et al., 2007) a preferent ial materno fetal transfer of DHA across the placenta has been demonstrated, which is aided by the presence of placental FA transport proteins. Mean plasma concentrations of total FA at birth were similar to those reported by Jenkins et al. (1988) for 3 d old calves but greater than that of Noble et al. (1975) for newborn calves. The FA profiles of calf plasma were quite similar to those reported by Moallen and Zachut (2012) for newborn calves from cows fed 240 g/d of saturated fat, 300 g of linseed oil, or 300 g of FO prepartum. In this study, only proportions of ALA and DHA differed due to diet. Dams supplemented with EFA had an expected daily intake of 116 g of LA compared to 57 and 62 g/d for dams fed no supplemental fat or SFA, respectively. Intake of ALA was influenced minimally by the type of fat supplemented. As previously reported for newborn lambs ( Noble et al.,1978; Soares, 1986), calves born from cows fed EFA (rich in LA) had increased concentrations of LA in plasma but AA concentration was unaff ected by type of diet. However FA such as GLA and C20:3 n 6, which are precursors of AA in the elongation desaturation steps, were greater in calves born from dams fed EFA, in agreement with the findings of Soares (1986) studying lambs born

PAGE 180

180 from LA supplem ented ewes. The increased proportions of these intermediate FA might indicate that the enzymatic activity of FA desaturases and elongases that are the same for both n 6 and n 3 groups of FA were preferentially metabolizing LA over ALA in dams supplemented with fat enriched in LA, although final end products of AA and C22:4 were not increased significantly. Interestingly, supplementing SFA prepartum increased the proportions of EPA and DHA in plasma of newborn calves. This result is opposite to that of Elmes et al. (2004) who reported that increased intake of LA in pregnant ewes not only increased the proportion of LA, GLA, C20:3 n 6, and AA but also of DPA and DHA but not of ALA. These authors concluded that the overall activity of desaturases and elongases were very active in ewes fed more LA, so that the synthesis of longer chain FA were enhanced in both n 6 and n 3 FA groups. Burdge and Calder (2005) reviewed 23 studies supplementing ALA and concluded that greater supplementation of ALA prioritized the syn thesis of its derivate LC FA, similarly greater supplementation of LA should increase the synthesis of its derivatives. However Moallem and Zachut (2012) did not find increased proportions of ALA derivatives (EPA and DHA) when feeding prepartum cows linseed oil as compared to cows supplemented with saturated FA. These results indicate that the enzymatic processes of desaturation/elongation were either not activated by the increased supply of ALA or that the extra supply of ALA was metabolized. On the other hand, the greater synthesis of n 6 derivatives ( GLA and C20:3) in plasma of calves from dams fed LA in the current study might have depressed the elongation and desaturation of ALA, hence calves born from cattle fed EFA had lower

PAGE 181

181 proportions of those ALA d erived FA (EPA and DHA). The current results are in agreement with most studies using humans where supplementation of high amounts of LA reduced the synthesis of long chain n 3 FA, thus favoring the elongation of n 6 FA because of competition for 6 desaturase, the first limiting enzyme in this process. Chan et al. (1993) reported increased concentrations of EPA in the plasma phospholipid fraction of men fed low LA whereas Liou et al. (2007), feeding healthy men a diet rich in LA, reported greater concentrations of LA but lower concentrations of EPA in the plasma phospholipid fraction Another important finding is the parity effect on proportion of EFA and their derivatives. Calves born from nulliparous heifers had increased plasma concentrations of n 3 FA such as EPA, DPA, and DHA but decreased LA and AA. Although the plasma of dams was not analyzed for FA, the FA profile of colostrum was analyzed. Nulliparous heifers produced colostrum with greater concentrations of ALA, AA, EPA, DPA, and DHA where as LA was greater in colostrum of parous cows (Chapter 3). A previous study in humans reported a negative relationship of parity with DHA concentrations in blood of mothers and their neonates (Al et al., 1997) but another study did not detect negative effe ct of increased parity on dam n 3 FA in the offspring (Van Gool et al., 2004). It is not known why mature cows might have a preferential synthesis of FA derivate from LA instead of those derived from ALA, w hich could increase the risk of deficiency of crit ical FA for brain development in offspring. However, nulliparous animals may have mobilized fat with greater proportions of PUFA and possibly transferred this to their calves because unluckily parous cows, they were raised in sod base pens, with some acces s to pasture.

PAGE 182

182 Several studies have reported that undernutrition during pregnancy can decrease birth weight in humans (Naeye et al., 1973; Kramer, 1987) and sheep (Osgerby et al., 2002; Dwyer et al., 2003). Yet, the fetal metabolic environment can have long term metabolic effects on the offspring without necessarily affecting birth weight (Pettitt et al., 1987; Ferezou Viala et al., 2007). However supplementation of different lipid sources to nutritionally adequate diets for pregnant beef cows have not affec ted calf birth weight (Hess, 2003; Banta et al., 2006; Banta et al., 2011) when isocaloric and isonitrogenous diets were fed. Dams fed SFA ate more DM than dams fed EFA (Greco et al., 2010) and calves born from dams fed SFA were numerically 0.5 kg heavie r than calves born from dams fed EFA. Whether these positively related responses of DMI and birth weight (Osgerby et al., 2002; Dwyer et al., 2003) were the drivers promoting increased grain intake (75 g/d average) during 31 to 60 d of age by calves born f rom cows fed SFA is unclear. This greater intake of grain helped contribute to calves gaining 2.7 kg more between birth and weaning than calves born from dams fed EFA. This increased intake of grain would not necessarily cause a change in plasma concentrat ions of energy and protein metabolites. Feeding a grain mix along with MR to dairy calves did not change plasma concentrations of glucose, insulin, BHBA, or IGF 1 compared to calves fed MR alone (Laarman et al., 2012). Similarly calves born from dams fed SFA and EFA did not differ in plasma concentrations of glucose, PUN, IGF I, insulin, and BHBA. Calves born from dams fed the control diet had lower concentrations of PUN than those fed fat. Elevation of circulating PUN could result from supply of more prot ein than the calf could utilize. Bascom et al. (2007) fed calves with MR of 29 or 20% CP, and

PAGE 183

183 found reduced concentrations of PUN in calves fed the 20% CP MR. However other studies feeding increased concentrations of CP in MR did not affect PUN concentrati ons (Daniels et al. 2008). Metabolism of dietary nutrients basically occurs after feeding, hence it would be improbable that dietary CP in prepartum diets could directly affect PUN concentrations of their offspring in early life than is the MR fed, even mo re considering that all three prepartum diets were isonitrogenous. However, it might be that a low fat diet prepartum modified the ability of calves to use energy and protein to meet their needs, although plasma concentrations of glucose were not affected by prepartum diets. In addition to aiding the clotting process, platelets, have been reported to be involved in recruitment of leukocytes to sites of vascular injury and inflammation and release of pro and antinflammatory factors, all mostly associated w ith incidence of atherosclerosis, sepsis, or hepatitis (Smyth et al, 2009). Lam et al. (2011) reported that platelets enhanced transendothelial migration of neutrophils. It is important to indicate that regardless of the diet, plasma concentrations of plat elets increased dramatically during the first 2 wk of life which is in agreement with Knowles et al. (2000) and Brun Hansen et al. (2006) and support the hypothesis that platelets have a clear role enhancing neutrophil migration to injured tissues in calve s undergoing an outbreak of diarrhea. Activity of immune cells, more than their concentration per se, could be influenced by the FA composition of their membrane. This may affect cell signaling, production of eicosanoids, and fluidity to modify activity of receptors or their expression by regulating activity of target genes (Jump, 2002; Yaqoob and Calder, 2007; Calder, 2012). The

PAGE 184

184 importance of CD62L and CD18 expression on the neutrophil surface is due to their role in the processes of rolling and tethering neutrophils on the endothelium to enhance its migration to the injured tissue (Simon et al., 2000; Ley et al. 2007). These receptors are said to be constitutively expressed, therefore they should not be influenced by diet as happened in the current study a nd other using calves (Pang et al. 2009; Corrigan et al., 2009). However, current findings contrast to that of Novak et al. (2012a) who reported a lower proportion of monocytes expressing CD62L in diarrheic calves and to Silvestre et al. (2011) who reporte d an increase in percentage of neutrophils positive to CD62L and CD18 in transition dairy cows fed Ca salts of SAO. Even though CD62L and CD18 are constitutively expressed, they could be down atus and dietary management. After calving, a lower number of CD62L expressed per neutrophil was associated with neutrophilia in cows, which might indicate the inability of neutrophils to migrate to the infection zone, hence increasing the risk of infectio ns (Weber et al. 2001). A similar association was reported in abruptly weaned calves 2 d postweaning when compared with preweaned calves (Lynch et al., 2010). In this study, circulating neutrophils from calves born from dams fed fat tended to have a decrea sed number of CD62L receptors (MFI) compared to calves born from cows not fed fat. Weber et al. (2001) and Lynch et al. (2010) indicated that adhesion molecule expression can be inversely related to neutrophil concentration because the greater the adhesion intensity, the greater the movement of neutrophils out of circulation. If this relationship holds true in the current study, calves born from fat supplemented dams would have experienced a reduced movement of neutrophils from the blood stream.

PAGE 185

185 Neutrophil function is incomplete if the neutrophils that are able to migrate to the infection zone are not able to phagocyte pathogens. Consequently an enhanced ability to phagocytize would potentially result in reduced incidence of diseases. However, such effects h ave been equivocal in studies using calves to evaluate the effect of different stressors on phagocytic activity of blood neutrophils (Pang et al., 2009; Hulbert et al., 2011). Circulating neutrophils in calves born from dams fed EFA phagocytized more bacte ria per neutrophil compared to those of calves born from dams fed SFA. Although the neutrophils in EFA calves were more efficient, number of circulating neutrophils with ability to phagocytize tended to be lower, likely resulting in similar number of bacte ria phagocytized by neutrophils in calves from dams fed the 2 fat sources. Actual concentrations of Hp increased before the attitude and fecal scores reached their highest point, which agree with studies reporting the validity of Hp as a predictor of the inflammatory process (Ganheim et al., 2007; Cray et al. 2009). Haptoglobin is absent or present in very low concentrations in healthy animals but under subclinical inflammatory disorders, its concentration increases (Ganheim et al., 2007; Cray et al., 200 9). When calves had respiratory and digestive tract infections, plasma concentrations of Hp were increased compared to healthy calves (Deignan et al., 2000; Heegaard et al., 2000; da Silva et al., 2011). In the current study calves born from dams fed SFA o r EFA had greater plasma concentrations of Hp compared to calves born from cows fed the control diet, which was due to a greater rise in concentration at the time Hp peaked in all calves (~9 d of age). This agrees with the finding of Bueno and coworkers (2 010) who reported that mice supplemented with lard (rich in long chain SFA) instead of SO increased the expression of genes coding for Hp in white adipose

PAGE 186

186 tissue. Authors hypothesized that lard could induce a proinflammatory condition, increasing the produ ction of proinflammatory cytokines which are inducers of acute phase protein production. In the current study, the time when Hp reached its highest concentration was between 5 and 9 d of age which coincided with the period of initiation of diarrheic events in calves. Interferon presentation, cell cycle growth and apoptosis, leukocyte trafficking, and B cell depletion (Arens et al., 2001; Chen and Liu, 2009). On the other hand, Ig, speci fically IgG, directly could kill or neutralize pathogens or indirectly serve as a cell surface receptor for antigens permitting cell signaling and activation through its presentation by professional antigen presenting cells (Schroeder and Cavacini, 2010; R ath et al, 2012). Stimulating the PBMC from 15 d old calves born from dams fed SFA resulted in increased production of IFN OVA IgG from 2 to 20 d of age when compared to calves born from dams fed EF A. Anti OVA IgG concentrations found in calves were primarily those derived from passive transfer with colostrum because all dams were vaccinated with OVA prepartum. Newborn calves have a biased preferential T helper 2 (Th2) response, which is responsibl e for a strong antibody production and a reduced Th1 response (Chase et al., 2008). The Th1 type cytokines play a key role initiating early resistance to pathogens and induction of cell mediated immunity (Marodi, 2002) In the current study, greater produc tion of IFN Th2 to a Th1 meditated immune response. This preferential Th pattern might be aided by the greater anti OVA IgG response. Total IgG concentrations in serum were also

PAGE 187

187 greater (Chapter 3) in calves born from dams fed SFA after 24 to 30 h of colostrum feeding. Other immune cells in colostrum were not measured but it is possible that colostrum from dams fed SFA in addition to having greater total IgG might have had a greater numbe r of CD4+, CD8+, and T cells, because the latter two cell types can produce IFN In summary, prepartum supplementation of EFA changed the FA status of calves as evidenced by changes in their FA profile. Feeding fat prepartum did not have a negative influence on health and immunity with the exception that plasma concentrations of haptoglobin were greater at 5 and 9 d after birth suggesting that inflammation was increased in these calves whereas lower expression of CD62L indicated a reduced proinflamm atory response. Specific source of FA differentially affected some markers of immune response such as concentrations of anti OVA IgG, and production of IFN this may have been due to greater intake of grain than calves born from EFA supplemented dams. However this increased intake did not affect the concentration of energetic metabolites and anabolic hormones Feeding Milk Replacer Enriched with Linoleic Acid Improved Growth, Feed Efficiency, and Imm une Responses Calves are born as preruminants with a preference for milk intake which delays the initiation of ruminal development or makes it very limited until grain intake increases. Consequently, metabolism of nutrients in the rumen, including fat, is very limited. Limited microbial activity in the underdeveloped rumen prevents or limits hydrolysis and biohydrogenation of dietary FA; thus the FA profile of plasma of preweaned calves is expected to reflect the diet.

PAGE 188

188 Newborn calves, assigned to either MR, at birth had similar plasma concentrations of LA, ALA and all their FA derivatives as well as total plasmatic concentrations of these FA. Plasma concentrations of LA increased markedly at 30 and 60 d of life from that of birth (~11.5 fold increase). This change occurred gradually starting right after the first day of life (~ 2.5 fold increase from that at birth). Concentrations of LA became relatively stable around 3 wk of age (Noble et al., 1975). Two potential mechanisms occurring in placenta might accou nt for the decreased plasma concentrations of LA in newborn calves, namely increased desaturation activity in the placenta and selective uptake of FA by placental FA binding proteins (Moallem and Zachut, 2012). Gradual increase of LA postpartum might be a combination of a release from the regulatory effects of the placenta in transferring FA and an enhancement by the increased dietary intake of fat from colostrum, milk, and grain. Concentration of total FA in plasma was less in calves fed the HLA MR which may have resulted from a greater digestibility of the FA in porcine lard compared to CCO. Murley et al. (1949) reported that plasma fat concentration was reduced in calves consuming a more vs. a less digestible SO. The effect of feeding MR of different FA profiles had a profound impact on the FA profile of calf plasma. Feeding a MR containing a highly saturated FA fat source (CCO containing a high concentration of medium chain FA, LLA) resulted in elevated plasma concentrations of C10:0, C12:0, and C14:0. T hese results are in agreement with previous studies supplementing short and medium chain FA in humans (Hill et al., 1990) and calves (Jenkins and Kramer, 1986). Reveneau et al. (2012) found increased proportions of medium chain FA in omasal digesta of CCO supplemented cows, resulting in milk with greater proportions of

PAGE 189

189 medium chain FA. Swift et al. (1990) reported that human enterocytes can incorporate medium chain FA as substrates for triglyceride synthesis when diets contain high proportions of those FA. Other studies also have documented transfer of dietary medium chain FA into milk fat of dairy cows supplemented with CCO, even though efficiency of transfer decreased with increased intakes of CCO (Vyas et al., 2012; Hollmann and Beede, 2012). Likewise, ca lves fed a MR containing a combination of CCO and a highly unsaturated FA fat source (porcine lard containing mainly C18:1 and LA, HLA) had increased plasma concentrations of LA and ALA. These results were similar to the findings of Wrenn et al. (1973), an d Jenkins and Kramer (1986, 1990) when diets rich in LA or ALA were fed to calves during the preweaning period. Plasma of calves fed HLA MR at 30 to 60 d of age had decreased proportions of LA derivatives (GLA, C20:3, and AA) compared to those fed LLA MR from 30 to 60 d of age. However, newborn calves born from dams fed EFA as compared to those fed SFA had similar proportions of all identified LA derivatives. The lack of effect in LA derivatives when feeding EFA prepartum, particularly in AA, also contras ts with several previous studies which reported an increased concentration of AA due to enhanced synthesis from its parent FA, LA (lambs, Soares, 1986; rats, Lands et al., 1990; and pigs, Novak et al., 2008) and also was recently reviewed by Gibson et al. (2011). It is important to point out that the liver of calves fed HLA MR tended to have greater proportions of AA (Chapter 5). Jenkins et al. (1985) reported a greater proportion of LA but decreased AA in plasma of calves fed CO compared to those fed CCO or beef tallow as the fat sources

PAGE 190

190 in MR. Later Jenkins and Kramer (1990) found that increased intake of LA from CO increased LA but reduced AA concentration in plasma compared to calves fed tallow plus CCO which agrees with our current findings. Authors su ggested a reduced activity EPA yet concentrations of DPA and DHA did not change. In the current study calves fed HLA MR had increased plasma concentrations of DPA. It is not clear why, in early life, calves fed HLA MR may not have enhanced desaturase/elongase activity favoring elongation of LA as that of pregnant dams fed greater amounts of LA. A potential reason might be that neonatal calves have a preferential synthes is of DHA to cope with the needs of brain tissue, enhancing DHA synthesis regardless of the balance of LA and ALA. This hypothesis is supported by the similar plasma concentrations of DHA in calves fed any of the MR and by the increased proportions of EPA and DPA, precursors of DHA synthesis, found in calves fed LLA and HLA MR, respectively. Improved ADG and FE in calves fed HLA MR were consistent during the periods of feeding MR alone (1 to 30 d), MR plus grain mix (31 to 60 d), and total experimental day s (1 to 60 d). Efficiency of gain was improved because calves fed HLA MR had greater ADG and no difference in MR intake and total DMI during the first and second 30 d, respectively. Studies regarding the feeding of increased amounts of LA on calf performan ce are limited. Beef calves born from cows supplemented with safflower seed rich in LA during the prepartum and lactation periods resulted in calves having similar BW at birth and at weaning, even though diets rich in LA were more energetically dense (Enci nias et al., 2001, 2004; Bottger et al., 2002;). When Lake and coworkers (2005, 2006a,b,c) fed isocaloric and isonitrogenous diets enriched in LA to lactating beef cows,

PAGE 191

191 BW at weaning was not affected. Intake of LA from the control diets and intake of LA f rom dry feed that calves were consuming may have been sufficient so that intake of LA above those values did not improve calf performance. The first studies using dairy calves that totally replaced milk fat with vegetable oils rich in LA resulted in poor ADG and FE. However the major reason for poor diet utilization (greater incidence of diarrhea and poor gain) was the inferior quality of high LA vegetable oil (crude expeller SO) plus poor preparation of CO (large size of oil droplets) (Jacobson et al., 19 49; Murley et al., 1949; Jenkins et al., 1985, 1986). However in current commercial practice, no MR is composed of 100% vegetable oils supplementing a basal MR to orphaned lambs wi th daily intakes of 1 g of SAO or FO did not produce any change in ADG or FE. Authors did not report whether lambs had diarrhea. Jenkins et al. (1985) reported that ADG and FE by calves were not different when CCO (3.2% of FA as LA) or tallow (3.8% of FA a s LA) were the sources of dietary fat. Hence both fat sources supported the same performance in calves even though mean carbon length of FA differed and LA supply was similar. The replacement of tallow by lard rich in LA in the current study could have an enhanced improvement of ADG and FE in calves fed CCO due to increased intake of LA. Pattern of metabolites in plasma of calves are in agreement with the difference in growth performance between calves fed LLA and HLA MR. Regardless of age, calves fed HLA M R had increased plasma concentrations of glucose, which was accompanied by a tendency for increased plasma concentrations of IGF I and only a numerical increase in plasma insulin ( P = 0.14). Plasma concentrations of PUN and BHBA were

PAGE 192

192 maintained lower in ca lves fed HLA MR, as well as concentrations of cholesterol starting to differentiate after 16 d of age. Calves in our current study were fed MR at 0600 h and blood collection occurred within 1 to 2 h after this first feeding, hence concentrations reported h ere are postprandial in response to the type of MR fed. Fresh colostrum has been identified as a good source of endocrine factors and hormones such as insulin and IGF I ( Georgiev, 2008b, Blum and Baumrucker, 2008). Those compounds can exert effects on the gastrointestinal tract (GIT). The IGF I gets into the GIT in an active form and survives the digestion process, having an important role in the growth and development of the GIT and on the functional maturation of the calf and its adaptation to the new ex ternal environment after birth (Georgiev, 2008a; Flaga et al., 2011). Prepartum diets did not affect plasma concentrations of these two hormones after colostrum feeding (Chapter 3). However calves randomly assigned to LLA MR had lower concentrations of ins ulin than calves assigned to the HLA MR before colostrum feeding but, once colostrum was fed, concentrations of insulin did not differ between calves fed the two MR. However, calves fed HLA MR tended to have greater plasma concentrations of IGF I. Preweane d calves fed an increased amount of nutrients had an enhanced ADG and FE, with increased plasma concentrations of insulin, glucose, and IGF I as potent anabolic metabolites or hormones, with an additional reduction in PUN concentrations (Smith et al., 2002 ). This pattern of growth and metabolites are in complete agreement with our current findings. Similarly, Quigley et al. (2006) reported increased concentrations of glucose and IGF I when calves were fed increased amounts of MR which was reflected in a gre ater ADG and improved FE. However Quigley and

PAGE 193

193 coworkers (2006) observed an increase in PUN concentrations when calves had greater intake of MR which contrasts with Smith et al. (2002) and our current finding. Etherton and Bauman (1998) reviewed the main f unctions of growth hormone (GH), with the enhanced synthesis of IGF I in liver being one of the most important. Smith et al. (2002) administered external GH to calves and reported increased plasma concentrations of IGF I as a response. Authors concluded th at the GH:IGF I axis was functional in preweaned calves. Under this assumption, it can be hypothesized that other functions of GH might be happening also in calves with greater circulation concentrations of IGF I, one of which would be the reduction of ami no acid oxidation associated with greater efficiency of protein accretion. This should reduce the need for amino acid catabolism and, as a consequence, a reduction in PUN concentrations. Another role of GH is the reduction in clearance and oxidation of cir culating glucose, whereas in liver, GH should increase the output of glucose and might reduce the ability of insulin to inhibit gluconeogenesis. This should result in increased plasma concentrations of glucose as was reported by Smith et al. (2002) and in our current study. Recently, Piantoni et al. (2012) demonstrated the importance of the milk feeding period in mammary development (functionality and growth). Preweaned heifers that consumed more nutrients from birth to 65 d of age (a MR of 28% CP and 28% f at vs. a MR of 20% CP and 20% fat) had a dramatic change in expression of genes (genes associated with cell morphology, cell to cell signaling, and immune responses) in mammary parenchyma and fat pad tissues. In an earlier study (Daniels et al., 2008) usin g the same feeding treatments, authors reported that heifers fed increased amounts of nutrients (28% CP and 28% fat) had a numerically greater plasma concentration of

PAGE 194

194 IGF I. Piantoni et al., (2012) speculated that this increased concentration of IGF I migh t have been associated with some of the gene expression responses observed in their 2012 study. Cholesterol and BHBA are synthesized primarily in liver of preruminant calves, as products of lipid metabolism. Both metabolites were in greater concentrations in plasma of calves born from dams fed LLA MR; these greater plasmatic concentrations were accompanied by an increased accumulation of lipids in the liver of these calves (Chapter 5). Considering that the production of BHBA by the ruminal epithelium due t o minimal microbial activity is low, the increased plasma BHBA was likely due to incomplete oxidation of FA in the liver. Sato (1994) fed medium chain FA (C8 and C10) to neonatal calves causing a marked hyperketonemia within a few hours after feeding due t o preferential transport of these FA through the portal vein and greater availability for FA oxidation and synthesis of ketogenic products. This same biological process likely was happening in calves fed more medium chain FA coming from CCO in the current study. Incomplete oxidation of medium chain FA by the liver was likely responsible for elevated plasma concentrations of BHBA of calves fed more CCO. Polyunsaturated FA have been reported to reduce circulating concentrations of cholesterol whereas medium c hain FA such (C12:0, C14:0, and C16:0) have been identified as the most potent inducers of cholesterolemia (Fernandez and West, 2005). Cholesterolemic effect of CCO has been documented by Jenkins et al. (1985), Berr et al. (1993), and Chechi and Chema (200 6) in rats. Fernandez and West (2005) stated that upregulation of low density lipoprotein receptors and increased activity of

PAGE 195

195 cytochrome P450 7A are the most potential mechanisms by which n 6 FA reduces the concentrations of circulating cholesterol. Increa sed concentration of hematocrit commonly is caused by calf dehydration due to diarrhea whereas a reduced concentration is associated with anemic conditions (Moonsie Shageer and Mowat, 1993). Calves fed HLA MR tended to have a greater mean concentration of hematocrit as well as hemoglobin (data not shown). However it is unlikely that those calves were under hydrated since type of MR did not affect the incidence of diarrhea. Lower concentrations of hemoglobin in calves fed LLA MR might indicate an increased r isk of anemia. However hemoglobin concentrations were within the normal range for all preweaned calves (Brun Hansen et al., 2006). CD8+ T cells, B cells, and natural killer cell s; however their proportions change with calf newborn calves is between 20 to 25% and they decreased with age. This proportional decrease is not due to a change in the number of cells but due to an increase in absolute number of CD4+ and B cells, with the latter being remarkably lower in younger calves and reaching adult proportions at 11 to 12 wk of age. Calves fed the HLA MR had an increased concentration of circulating lymph ocytes. Therefore it could be assumed that these calves also had increased concentrations of CD4+, which are the precursors of T helper cells that have a regulatory function in the interaction of innate and adaptive immunity, as well as increased B cells, components of the humoral adaptive immunity responsible for antibody production. All of these cells are potential aiding factors for an improved immune response of calves fed HLA MR.

PAGE 196

196 Platelet concentrations in plasma of calves were within normal ranges rep orted for preweaned calves (Knowles et al., 2000). Platelet concentration increased by the second week in all calves whereas it was lower throughout the study in calves fed HLA MR. Platelets have been reported to enhance neutrophil migration by facilitatin g the endothelial membrane extravasation (Lam et al., 2011). However in vitro analysis of blood neutrophil expression of CD18 and CD62L was not affected by the type of MR fed indicating that platelet concentrations were not sufficiently depressed to affect the neutrophil adhesion molecule relationship in calves fed HLA MR. On the other hand, results might indicate an antinflammatory effect of LA considering studies that have related increased concentrations of platelets with development of various inflammat ory diseases (Smyth et al., 2009). switch from a preferential Th2 response to a Th1 response. The pattern of cytokine production is used to verify the predominant type of Th response. An increased concentratio n of IFN 4 is indicative of Th1 predominance (Chase et al., 2008). The in vitro stimulation of PBMC with concanavalin A produced greater concentrations of IFN HLA vs. LLA MR. Foote et al. (2007) reported that calves at a high growth rate (1.16 kg/d) due to greater DM intake compared with calves at a low growth rate (0.11 kg/d) had similar production of IFN the increased inta ke of LA rather than the improved growth was responsible for the better IFN human cells reported that LA inhibited production of IFN

PAGE 197

197 et al. (1997) reported n o change in IFN were cultured with lard, SO, or FO. However in vivo concentration of murine IFN during listeriosis infection was elevated when FO was fed. Acid soluble protein has dual inflammatory and immuno mod ulatory properties. One of the mechanisms by which ASP can exert its antinflammatory effect is by inhibiting platelet aggregation, hence platelet recruitment (Hochepied et al., 2003). In order to downreg ulate platelet aggregation and recruitment, a really high physiological concentration is needed (Costello et al., 1979). Based on this requirement, we hypothesize that the slight increased concentrations of ASP in calves fed LLA MR in comparison with calves fed HLA MR did not prevent platelet aggregation and migration that could lead to a potential negative effect of thrombosis and risk of exacerbated inflammatory responses of calves in this group. Moreover incidence of diseases was not different between groups of calves. In summary, feeding of MR enriched wi th LA changed the plasma FA profile of calves. Transfer of dietary FA to calf plasma was verified through increased proportions of LA and ALA in calves fed HLA MR while maintaining similar concentrations of DHA regardless of the MR fed. Calves fed HLA MR i mproved BW gain and feed efficiency. This enhanced performance was accompanied by increased concentrations of energy metabolites and anabolic hormones. Feeding HLA MR appeared to improve immune response by increasing the number of circulating lymphocytes a nd possibly by enhancing the switch from a Th2 to a Th1 response by the increased production of IFN

PAGE 198

198 Prepartum Supplementation of Fatty Acids Affects Calf Responses to a Linoleic Acid Enriched Milk Replacer No clear modification of the effect of MR on t he FA profile of plasma of calves at 30 to 60 d of age by prepartum diets was detected. In studies evaluating the synthesis of DHA from ALA deficient rats, the enzymes involved in this synthetic process were found to be up regulated in the liver but not in the brain, with enhanced activity under ALA deprivation (Rapoport et al., 2007; Igarashi et al., 2007). Therefore tissues can differ in their ability to synthesize longer chain FA from EFA. In the current study, the efficiency of conversion of LA to AA an d C20:3 and conversion of ALA to EPA and DHA were better when LA and ALA were in shorter supply. This is borne out by the fact that calves fed HLA had greater plasma concentrations of LA but lower concentrations of AA and C20:3 compared to calves fed LLA. Likewise calves fed HLA had greater plasma concentrations of ALA but lower concentrations of EPA and similar concentrations of DHA compared to calves fed LLA. Prepartum diets did not affect the growth and performance of calves fed a specific MR (no inter action of dam diet by MR). However, it has been stated that prepartum diets can induce a fetal programming event affecting the future performance of calves without affecting birth weight (Hess, 2003; Banta et al., 2066, 2011; Pettitt et al, 1987; Ferezou V iala et al., 2007 ). In addition, the preweaning period is another critical period where programming of future events might occur (Fowden et al., 2006). Recently Soberon et al. (2012) reported a potential epigenetic programming of lifetime productivity (mil k yield) due to an improved growth rate during the preweaning period. Severity of diarrhea was not affected by the single effect of prepartum diet or MR but it was affected by by their interaction. This interaction is actually expected if calves

PAGE 199

199 are fed co lostrum harvested from their respective dams and receive by passive transfer a pool of antibodies to be used to fight against potential pathogen invasion during the produce their own memory cells against invaders (Weaver et al., 2000; Heinrichs and Elizondo Salazar, 2009). In our current study, calves born from dams fed fat and supplemented with HLA instead of LLA MR had the fewer number of days of diarrhea. This improved re sponse of calves born from dams fed fat could be due to the fact that calves born from dams fed fat had a trend for greater serum total IgG after colostrum feeding (Chapter 3). In summary feeding fat prepartum may modify the ability of tissues to synthesiz e essential FA derivatives due to differential proportion of LA and ALA they had when they are born. No apparent effect of prepartum diets to modify performance of calves fed LA in MR was observed. Calves fed a MR enriched in LA and born from dams fed fat experienced fewer days of diarrhea and poor attitude. Summary Strategic feeding of EFA, both during the nonlactating pregnant period and in early life, can change the FA status of calves as evidenced by changes in plasma FA. These changes affected calf met abolism, health, and performance. Supplementing LA and ALA to dams increased plasma concentrations of LA but decreased those of EPA and DHA of neonatal calves. In addition, feeding a MR enriched in LA and ALA increased plasma concentrations of LA and ALA b ut decreased that of EPA but not DHA. Synthesis of DHA in the growing calf overcame the inhibiting effect of EFA in utero. Feeding fat prepartum did not have a negative influence on calf performance or health with the exception that plasma concentrations o f haptoglobin tended to be greater at 5

PAGE 200

200 and 9 d after birth suggesting that inflammation was increased but a tendency for lower expression of CD62L may suggest that the inflammatory process was not excessive in these calves. Increased intake of LA from app roximately 6.2 to 13.2 g/d on average over the 60 d period by partially replacing CCO with porcine lard in the MR increased BW gain by 3 kg over a 60 d period. Because feed intake was not changed, conversion of feed to gain was improved by 8%. This enhance d performance was accompanied by increased plasma concentrations of glucose and IGF I and lower plasma concentrations of urea N and cholesterol, which corroborate the enhanced anabolic process that these calves were undergoing during the preweaning period. Feeding more LA in the MR also influenced health and immunity as evidenced by greater hematocrit and blood lymphocyte concentrations, lower plasma concentrations of ASP, greater proportion of phagocytosis by blood neutrophils, and greater synthesis of IFN HLA MR may have improved the switch from a Th2 to a Th1 response based upon the increased in vitro production of IFN mediated immunity in these calves. Supplementing SFA prepartum resulted in calves consumi ng more DM (primarily grain) and gaining 2.6 kg more BW by 60 d of age compared to calves born from dams supplemented with EFA during the nonlactating period. These same calves also demonstrated improvements in immunity as evidenced by a greater concentrat ion of anti OVA IgG and greater synthesis of IFN calves were fed MR enriched in LA, they had lower fecal and better attitude scores at 2 wk of age.

PAGE 201

201 Table 4 1 Ingredient and chemical composition of milk replacers ( MR) and grain mix. Milk replacer 1 Grain mix LLA HLA Ingredients, % of DM Coconut base MR 2 100 ----Porcine lard base MR 3 100 --Barley, ground ----51.7 Peanut meal ----16.5 Beet pulp shreds ----24.5 Sugarcane molasses ---5.3 Mineral mix 4 ----2.0 Nutrient composition (DM basis) Lactose, % 34.1 34.2 --Protein, % 29.0 28.7 18.7 Fat, % 19.4 19.8 4.2 NDF, % 23.0 C % 0.8 0.8 0.5 P, % 0.8 0.8 0.5 Mg, % 0.1 0.1 0.4 K % 2.4 2.4 0.9 Na % 1.2 1.2 0 .2 S, % 0.4 0.4 0.2 Fe mg/kg 96.7 110.3 440.0 Zn mg/kg 40.3 41.3 55.5 Cu mg/kg 8.3 6.8 14.5 M n, mg/kg 49.0 47.8 46.5 Mo mg/kg 1.4 1.3 2.8 Co mg/kg 0.5 0.6 --1 Milk replacers were classified as low linoleic acid (LLA) or high linoleic acid (H LA). 2 homogenized coconut oil (30.5%), milk derivate products (68.6%), Neo Terramycin 100/50 (0.1%) and vitamin and mineral mixes (0.8%). Each kg contains 0.90% Ca, 0.87% P, 0.1 mg of Co, 10.1 mg of Cu, 1.0 mg of I 100 mg of Fe, 45,374 IU of vitamin A, 11,345 IU of vitamin D and 220 IU of vitamin E. 3 homogenized coconut oil (13.3%) and porcine lard (19.6%), milk derivate products (66.3%), Neo Terramycin 100/50 (0.1%) and vi tamin and mineral mixes (0.7%). Each kg contains 0.90% Ca, 0.87% P, 0.1 mg of Co, 10.1 mg of Cu, 1.0 mg of I, 100 mg of Fe, 45,374 IU of vitamin A, 11,345 IU of vitamin D and 220 IU of vitamin E. 4 Each kg contains 8.8% Ca, 4.2% P, 11.4% Mg, 12.4% Cl, 0.49 % K, 8.1% Na, 0.36% S, 58 mg of Co, 263 mg of Cu, 26 mg of I, 1933 mg of Fe, 923 mg of Mn, 8.46 mg of Se, 1109 mg of Zn, 259,000 IU of vitamin A, 70,000 IU of vitamin D, and 2,400 IU of vitamin E.

PAGE 202

2 02 Table 4 2 Fatty acid (FA) profile of milk replacers and grain mix. Milk replacer 1 Grain mix FA LLA HLA % of identified FA C8:0 8.5 6.1 ND 2 C10:0 6.1 4.5 0.0 C12:0 42.5 29.9 0.1 C14:0 15.9 11.9 0.2 C16:0 10.6 14.6 13.2 C16:1 0.3 0.7 0.1 C18:0 4.4 6.7 2.0 C18:1 8.9 15.7 47.1 C18:2 2.9 9.0 28.2 C ND 0.8 2.1 C20:1 ND ND 1.9 C22:0 ND ND 2.3 C24:0 ND ND 1.7 Others FA ND ND 1.1 1 Milk replacers are classified as low linoleic acid (LLA) or high linoleic acid (HLA). 2 ND = Not detected.

PAGE 203

203 Table 4 3 Mean concentration of total plasma fat ty acids (FA, mg/mL of plasma) and individual and group of FA expressed as % of total FA (g of FA/100 g of total FA) before colostrum feeding in calves born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential f atty acids (EFA) starting at 8 wk before expected calving date. Dam Diet 1 P values Control SFA EFA SEM FAT FA Parity FAT by P FA by P Parity (P) FA Null Parous Null Parous Null Parous FA mg/mL plasma 1.23 1.33 1.14 1.28 1.34 1.33 0 .07 0.91 0.09 0.21 0.81 0.33 C12:0 0.11 0.10 0.00 0.04 0.00 0.00 0.06 0.06 0.72 0.82 0.78 0.72 C14:0 1.83 2.12 1.52 1.58 1.99 1.58 0.23 0.15 0.30 0.92 0.27 0.31 C14:1 c9 0.56 0.44 0.50 0.40 0.50 0.32 0.07 0.26 0.54 0.02 0.93 0.54 C15:0 0.07 0.12 0.13 0.21 0.17 0.17 0.05 0.12 0.97 0.30 0.87 0.44 C16:0 29.6 30.4 30.0 29.3 30.5 29.9 0.54 0.80 0.30 0.66 0.12 0.95 C16:1 c9 5.26 4.82 5.46 5.21 4.89 5.21 0.42 0.69 0.50 0.72 0.53 0.50 C17:0 0.76 0.96 0.99 0.67 0.74 0.60 0.21 0.57 0.43 0.62 0.26 0.66 C17:1 c9 0.86 0.67 0.98 0.67 0.86 0.57 0.09 0.96 0.20 <0.01 0.47 0.86 C18:0 13.7 13.1 13.4 13.6 13.5 13.6 0.37 0.60 0.90 0.72 0.26 0.86 C18:1 c9 31.5 28.0 30.4 29.1 30.6 26.7 0.84 0.47 0.19 <0.01 0.56 0.12 C18:2 n 6 2.25 4.43 2.68 3.97 3.71 5.06 0.49 0.25 0.03 <0.01 0.34 0.95 C18:3 n 6 0.09 0.32 0.09 0.25 0.21 0.41 0.04 0.43 <0.01 <0.01 0.50 0.57 C18:3 n 3 0.00 0.11 0.02 0.03 0.11 0.04 0.04 0.85 0.16 0.53 0.03 0.26 C20:2 0.06 0.01 0.03 0.01 0.00 0.00 0.02 0.12 0.15 0.07 0.19 0.47 C20:3 n 6 1.31 2.03 1.8 0 2.08 1.63 2.91 0.20 0.01 0.09 <0.01 0.86 0.01 C20:4 n 6 4.13 5.23 4.27 5.32 3.67 5.77 0.29 0.78 0.80 <0.01 0.37 0.07 C20:5 n 3 0.37 0.07 0.44 0.14 0.34 0.03 0.05 0.73 0.03 <0.01 0.91 0.97 C22:4 n 6 0.02 0.12 0.00 0.15 0.00 0.25 0.03 0.34 0.16 <0.01 0. 12 0.16 C22:5 n 3 0.50 0.45 0.62 0.50 0.44 0.47 0.07 0.62 0.11 0.36 0.92 0.23 C22:6 n 3 0.88 0.51 0.99 0.61 0.75 0.44 0.07 1.00 <0.01 <0.01 0.77 0.55

PAGE 204

204 Table 4 3 Continued. Dam Diet 1 P values Control SFA EFA SEM FAT FA Parity FAT by P FA b y P Parity (P) Prim Mult Prim Mult Prim Mult Unknowns 6.18 5.97 5.66 6.21 5.37 6.02 0.41 0.48 0.55 0.33 0.27 0.90 46.1 46.8 46.1 45.4 47.0 45.8 0.72 0.56 0.37 0.53 0.19 0.76 38.1 33.9 37.3 35.4 36.8 32.8 1.00 0.61 0.13 <0.0 1 0.50 0.29 9.6 13.3 10.9 13.0 10.8 15.4 0.87 0.16 0.19 <0.01 0.83 0.16 6 7.9 12.1 8.9 11.8 9.22 14.4 0.78 0.13 0.06 <0.01 0.86 0.15 3 1.76 1.14 2.06 1.27 1.63 0.99 0.15 0.78 0.02 <0.01 0.72 0.61 1 Control = no fat supplement; SFA = Ener gy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 Null = nulliparous 3 P values for orthogonal contrasts and interactions. FAT = fat (SFA + EFA) vs. Control, FA = EFA vs. SFA.

PAGE 205

205 Table 4 4 Mean concent ration of total plasma fatty acids (FA, mg/mL of plasma) and individual and group of FA expressed as % of total FA (g of FA/100 g of total FA) of calves fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. All interactions with gender did not differ unless footnoted. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR A DD x A MR x A DD x MR x A Milk replacer 2 FA LLA HLA LLA HLA LLA HLA FA mg/mL plasma 2.04 1.95 2.12 1.95 2.12 1.93 0.07 0.62 0.88 0.02 0.48 0.90 0.91 0.36 0 .05 0.02 C10:0 0.03 0.00 0.06 0.01 0.02 0.03 0.02 0.36 0.32 0.06 0.61 0.06 0.34 0.61 0.96 0.41 C12:0 0.91 0.44 0.89 0.59 0.65 0.42 0.06 0.55 <0.01 < 0.01 0.07 0.63 0.89 0.41 0.53 0.78 C14:0 4.73 2.22 4.89 2.58 4.70 2.17 0.16 0.42 0.05 < 0.01 0.60 0.21 0 .04 0.63 0.46 0.73 C14:1 c9 0.18 0.24 0.25 0.22 0.24 0.20 0.02 0.43 0.62 0.81 0.04 0.90 <0.01 0.73 0.59 0.15 C15:0 0.41 0.48 0.45 0.43 0.46 0.45 0.05 0.96 0.75 0.71 0.35 0.90 0.03 0.96 0.77 0.97 C16:0 16.0 16.6 16.2 16.7 16.4 16.2 0.27 0.73 0.63 0.21 0 .41 0.17 <0.01 0.11 0.74 0.80 C16:1 c9 1.21 1.34 1.11 1.40 1.09 1.35 0.06 0.46 0.58 < 0.01 0.18 0.82 0.68 0.39 0.22 0.74 C17:0 0.37 0.36 0.34 0.37 0.34 0.37 0.03 0.68 0.92 0.47 0.34 0.97 <0.01 0.67 0.20 0.46 C17:1 c9 0.07 0.09 0.07 0.07 0.07 0.10 0.02 0.38 0.54 0.02 0.25 0.27 0.04 0.14 0.64 0.62 C18:0 13.8 13.3 13.9 13.9 13.7 13.2 0.25 0.47 0.11 0.10 0.71 0.38 <0.01 0.47 0.18 0.51 C18:1 c9 10.9 10.0 11.6 10.5 11.3 9.9 0.42 0.31 0.27 < 0.01 0.77 0.71 0.87 0.41 0.11 0.88 C18:2 n 6 41.6 46.8 39.9 45.1 41.1 47.0 0.89 0.23 0.09 < 0.01 0.79 0.75 0.00 0.42 0.08 0.52 C18:3 n 6 0.34 0.22 0.37 0.19 0.32 0.17 0.03 0.45 0.20 < 0.01 0.43 0.52 <0.01 0.90 0.30 0.24 C18:3 5 n 3 0.70 0.86 0.65 0.77 0.68 0.81 0.05 0.23 0.50 < 0.01 0.74 0.84 <0.01 0.53 0.55 0.78 C20 :2 0.19 0.25 0.21 0.25 0.21 0.29 0.03 0.41 0.28 0.01 0.97 0.46 <0.01 0.30 0.13 0.57 C20:3 6 n 6 1.34 0.90 1.38 0.98 1.32 0.98 0.06 0.39 0.59 < 0.01 0.48 0.68 <0.01 0.41 0.09 0.22 C20:4 n 6 3.21 2.82 3.14 3.03 3.17 3.17 0.11 0.23 0.45 0.05 0.07 0.58 <0.01 0.76 0.52 0.60 C20:5 n 3 0.12 0.08 0.13 0.07 0.12 0.07 0.02 0.88 0.74 < 0.01 0.74 0.83 0.11 0.49 0.55 0.96 C22:4 n 6 0.22 0.23 0.24 0.22 0.25 0.25 0.03 0.41 0.33 0.84 0.81 0.74 0.05 0.57 0.36 0.79 C22:5 n 3 0.29 0.33 0.30 0.30 0.28 0.36 0.02 0.78 0.24 < 0.01 0.76 0.01 <0.01 0.97 0.66 0.55 C22:6 n 3 0.26 0.20 0.25 0.23 0.19 0.22 0.02 0.85 0.10 0.43 0.10 0.20 <0.01 0.05 0.61 0.09

PAGE 206

206 Table 4 4 Continued. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR A DD x A MR x A DD x MR x A Milk replacer 2 LLA HLA LLA HLA LLA HLA Unknowns 3.54 3.03 3.53 2.91 3.43 2.97 0.27 0.75 0.94 0.02 0.95 0.77 <0.01 0.81 <0.01 0.17 36.3 33.4 36.8 34.6 36.4 32.9 0.44 0.42 0.01 < 0.01 0.94 0.14 0.19 0.70 0.30 0.71 11.9 10.8 13.1 11.3 12.5 10.8 0.41 0.11 0.20 < 0.01 0.30 0.95 0.89 0.28 0.17 0.91 48.3 52.7 46.5 51.2 47.7 53.3 0.88 0.28 0.07 < 0.01 0.60 0.57 <0.01 0.58 0.05 0.60 n 6 47.0 51.2 45.2 49.8 46.4 51.8 0.88 0.32 0.07 < 0.01 0.61 0.63 <0.01 0.49 0.0 5 0.57 n 3 1.37 1.47 1.33 1.37 1.26 1.47 0.06 0.26 0.80 0.02 0.86 0.17 0.04 0.25 0.77 0.65 1 Control = no fat supplement; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 LLA = 0.175 g of LA/B W 0.75 HLA = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 3 P values for orthogonal contrasts and interactions: FAT = fat (SFA + EFA) vs. Control, FA = EFA vs. SFA, MR = mil k replacer, A = age, DD = dam diet. Three way interactions were removed from the model if P > 0.25. 4 FA by gender, P = 0.02. 5 FAT by gender, P = 0.01; FAT by MR by gender, P = 0.05. 6 FA by gender P = 0.03.

PAGE 207

207 T able 4 5 Dry matter intake (DMI), body weight (BW) gain and feed efficiency ( FE) of Holstein calves fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR G DD x G MR X G Measure Milk replacer (MR) 2 LLA HLA LLA HLA LLA HLA Birth to 30d Birth weight, kg 38.7 41.6 40.6 42.4 41.7 40.3 1.31 0.32 0.71 0.30 0.23 0.23 <0.01 0.71 0.04 MR intake, kg of DM 14.7 15.6 15.3 16.0 15.3 15.2 0.42 0.43 0.35 0.15 0.41 0.35 <0.01 0.62 0.33 MR intake, % of BW 1.15 1.12 1.14 1.13 1.11 1.14 0.02 0.75 0.70 0.73 0.22 0.20 <0.01 0.86 0.34 BW gain, kg 7.59 9.42 8.16 10.2 8.07 8.39 0.72 0.75 0.19 0.02 0.61 0.24 <0.01 0.28 0.66 ADG, Kg/d 0.25 0.31 0.27 0.34 0.27 0.28 0.02 0.76 0.19 0.02 0.60 0.23 <0.01 0.28 0.68 FE, (kg BW g ain/kg MR intake) 0.51 0.60 0.53 0.63 0.52 0.56 0.05 0.99 0.34 0.05 0.87 0.54 0.19 0.42 0.81 31d to weaning MR intake, Kg of DM 18.8 20.1 19.5 20.6 19.4 19.6 0.47 0.50 0.28 0.03 0.42 0.30 <0.01 0.50 0.22 Grain mix intake, Kg of DM 10.4 11 .9 13.8 12.5 11.3 10.6 1.14 0.36 0.06 0.81 0.22 0.79 0.41 0.99 0.52 Total DMI, kg of DM 29.3 32.0 33.3 33.1 30.7 30.2 1.38 0.32 0.05 0.58 0.19 0.90 0.01 0.92 0.91 Total DMI, % of BW 1.75 1.74 1.87 1.75 1.75 1.70 0.05 0.61 0.10 0.19 0.37 0.50 0.07 0.76 0. 15 BW gain, Kg 19.0 20.3 20.2 21.4 17.8 20.5 1.07 0.71 0.13 0.05 0.76 0.50 <0.01 0.98 0.81 ADG, Kg/d 0.63 0.68 0.68 0.71 0.59 0.68 0.03 0.69 0.11 0.05 0.81 0.44 <0.01 0.97 0.81 FE, (kg BW gain/kg total DMI) 0.65 0.64 0.62 0.64 0.58 0.68 0.03 0.66 0.93 0 .09 0.13 0.19 0.40 0.81 0.96 Birth to weaning Final BW, Kg 65.3 71.7 69.0 74.1 67.6 69.3 1.97 0.36 0.12 0.01 0.40 0.39 <0.01 0.55 0.84 Total DMI, Kg 44.0 47.7 48.6 49.0 46.0 45.3 1.66 0.32 0.07 0.39 0.18 0.73 <0.01 0.85 0.90 Total DMI, % of BW 1.41 1.40 1.47 1.41 1.40 1.38 0.02 0.63 0.04 0.16 0.38 0.43 0.01 0.80 0.03 BW gain, Kg 26.6 29.6 28.4 31.6 25.9 28.9 1.27 0.55 0.04 <0.01 0.95 0.92 <0.01 0.78 0.91 ADG, Kg/d 0.44 0.49 0.47 0.53 0.43 0.48 0.02 0.49 0.04 <0.01 0.94 0.92 <0.01 0.77 0.87 FE, (kg BW gain/kg total DMI) 0.60 0.62 0.59 0.64 0.57 0.64 0.03 0.93 0.63 0.01 0.23 0.61 0.10 0.95 0.88 1 Control = no fat supplement; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ).

PAGE 208

208 2 LLA = 0.175 g of LA/BW 0.75 HLA = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 3 P values for orthogonal contrasts and interactions: FAT = fat (SFA + EFA) vs. Control, FA = EFA vs. SFA, DD = dam diet, MR = milk replacer, G = gender. Three way interactions were not significant.

PAGE 209

209 Table 4 6 Plasma concentrations of metabolites and hormones in Holstein calves fed milk replacer containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or esential fatty acids (EFA) starting at 8 wk before expected calving date. All interactions with gender did not di ffer unless footnoted. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR A DD x A MR x A DD x MR x A Measure Milk replacer 2 LLA HLA LLA HLA LLA HLA Glucose, mg/dL 91.4 92.8 88.8 93.2 89.5 92.0 1.52 0.35 0. 90 0.03 0.44 0.53 <0.01 0.07 0.99 0.35 PUN, mg/dL 7.59 7.63 8.95 7.88 8.52 7.74 0.38 0.05 0.48 0.06 0.16 0.74 <0.01 0.75 0.97 0.99 BHBA, mg/dL 1.08 0.80 1.52 0.88 1.49 0.94 0.17 0.06 0.93 <0.01 0.27 0.80 <0.01 0.40 0.11 0.98 180 170 171 165 169 166 7.4 0.25 0.98 0.28 0.67 0.82 <0.01 0.19 0.43 0.60 Total cholesterol, mg/dL 87.9 85.3 92.7 79.7 99.6 84.6 3.67 0.45 0.12 <0.01 0.08 0.83 <0.01 0.41 0.01 0.41 Insulin 5 ng/mL 1.21 1.46 1.30 1.45 1.33 1.41 0.13 0.69 0.98 0.14 0.52 0.78 <0.01 0.52 0. 42 0.10 IGF I, g/mL 57.0 63.7 50.7 62.1 52.0 53.2 4.33 0.12 0.40 0.08 0.99 0.25 <0.01 0.21 0.83 0.03 STP 6 g/dL 5.75 5.88 5.76 5.87 5.80 5.75 0.08 0.76 0.66 0.37 0.47 0.35 <0.01 0.73 0.13 0.35 1 Control = no fat supplement; SFA = Energy Booster 100 (Mil k Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 LLA = 0.175 g of LA/BW 0.75 HLA = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 3 P values for orthogonal contrasts and interactions: FAT = fat (SFA + EFA) vs. Control, FA = EFA vs. SFA, MR = milk replacer, A = age, DD = dam diet. Three way interactions were not significant. 4 Gender, P < 0.01, FAT by gender, P = 0.03. 5 MR by gender, P = 0.05. 6 Serum total protein. Gender, P = 0.02, FA by MR by gender, P = 0.01

PAGE 210

210 Table 4 7 Attitude and fecal scores and percentage of days with poor attitude and diarrhea in Holstein calves fed milk replacer containing low linoleic acid (LLA) or high linoleic aci d (HLA) from 1 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. All interactions with gender did not diff er unless footnoted. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR A DD x A MR x A DD x MR x A Measure Milk replacer 2 LLA HLA LLA HLA LLA HLA Health score 4 Attitude 1.03 1.06 1.03 1.02 1.05 1.03 0.01 0.29 0.32 0.89 0.06 0.85 <0.01 0.74 0.85 0.30 Fecal 1.12 1.22 1.24 1.14 1.19 1.20 0.04 0.48 0.96 0.85 0.03 0.14 <0.01 0.96 0.92 0.77 Percentage of days with 5 Poor attitude, 30 d 5.3 12.3 7.4 4.5 8.7 7.1 2 .0 0.28 0.33 0.63 0.01 0.74 Poor attitude, 60 d 3.3 6.4 4.2 2.0 5.0 3.7 1.1 0.24 0.27 0.89 0.02 0.67 Diarrhea 6 30 d 8.9 17.7 15.6 6.6 15.3 11.9 2.2 0.63 0.26 0.51 <0.01 0.21 Diarrhea, 60 d 4.5 8.9 8.3 3.5 7.5 6.2 1.1 0.76 0.37 0 .54 <0.01 0.11 1 Control = no fat supplement; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 LLA = 0.175 g of LA/BW 0.75 HLA = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusiv ely fed the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 3 P values for orthogonal contrasts and interactions. FAT = fat (SFA + EFA) vs. Control, FA = EFA vs. SFA, MR = milk replacer, A = age, DD = dam diet. Three way interactions were not sig nificant. 4 Scoring criteria for attitude was the following: 1 = responsive, 2 = non active, 3 = depressed, or 4 = moribund. Scoring criteria for feces was the following: 1 = feces of firm consistency, no diarrhea; 2 = feces of moderate consistency, soft no diarrhea; 3 = Runny feces, mild diarrhea; or 4 = watery feces, diarrhea. 5 Percentage of days with poor attitude (if score > 1) and diarrhea (if score > 2). 6 FA by gender, P = 0.04.

PAGE 211

211 Table 4 8 Mean concentration of blood cell number and white blo od cells percentages in Holstein calves fed milk replacer containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or esse ntial fatty acids (EFA) starting at 8 wk before expected calving date. All interactions with gender did not differ unless footnoted. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR A DD x A MR x A DD x MR x A Measure Milk repla cer 2 LLA HLA LLA HLA LLA HLA Blood cells number Total red 4 10 6 / 8.40 8.61 8.24 8.33 8.25 8.58 0.24 0.46 0.58 0.30 0.99 0.63 <0.01 0.24 0.75 0.35 Total white 5 10 3 8.46 8.75 8.60 9.35 8.17 8.58 0.49 0.88 0.23 0.23 0.73 0.77 <0.01 0.31 0.95 0.30 Neutrophils, 10 3 3.08 3.06 3.06 3.58 2.90 2.87 0.26 0.93 0.11 0.5 1 0.58 0.33 <0.01 0.17 0.91 0.31 Lymphocytes, 10 3 4.26 4.57 4.29 4.57 4.05 4.68 0.24 0.93 0.75 0.04 0.72 0.46 <0.01 0.76 0.96 0.81 Monocytes, 10 3 0.37 0.39 0.38 0.38 0.38 0.36 0.38 0.82 0.79 0.96 0.61 0.85 <0.01 0.45 0.42 0.41 Eosinophils 4 10 3 0.11 0.12 0.11 0.12 0.11 0.12 0.01 0.96 0.78 0.30 0.90 0.92 <0.01 0.60 0.07 0.01 Basophils, 10 3 0.11 0.11 0.11 0.12 0.10 0.11 0.01 0.95 0.44 0.37 0.54 0.62 <0.01 0.73 0.59 0.71 Platelets, 10 3 781 710 833 698 789 738 46.1 0.65 0.99 0.03 0.79 0.37 < 0.01 0.03 0.41 0.95 White Blood cells, % Neutrophils 39.0 36.8 38.5 40.5 38.5 35.5 1.57 0.78 0.11 0.39 0.54 0.12 <0.01 0.38 0.93 0.40 Lymphocytes 4 52.7 54.5 53.1 51.7 52.6 56.4 1.58 0.94 0.19 0.29 0.81 0.11 <0.01 0.45 0.91 0.34 Monocyte s 6 4.09 4.51 4.22 3.88 4.43 3.87 0.26 0.36 0.70 0.45 0.05 0.68 <0.01 0.62 0.65 0.61 Eosinophils 7 1.32 1.36 1.29 1.27 1.38 1.45 0.10 0.96 0.19 0.74 0.87 0.67 <0.01 0.14 0.21 <0.01 Basophils 1.30 1.26 1.28 1.25 1.23 1.33 0.06 0.89 0.77 0.88 0.48 0.27 <0.01 0.64 0.20 0.54 Hematocrit 8 % 34.9 35.8 33.8 35.5 34.6 36.3 0.97 0.72 0.39 0.08 0.63 0.97 <0.01 0.45 0.46 0.98 1 Control = no fat supplement; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 LL A = 0.175 g of LA/BW 0.75 HLA = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 3 P values for orthogonal contrasts and interactions: FAT = fat (SFA + EFA) vs. Control, FA = EF A vs. SFA, MR = milk replacer, A = age, DD = dam diet. Three and four way interactions with gender were removed from the model if P > 0.25. 4 Gender, P 5 FAT by gender, P = 0.05. 6 FAT by gender, P = 0.05. 7 Gender, P = 0.04, FA by MR by gender, P = 0.02. 8 Gender, P = 0.04.

PAGE 212

212 Table 4 9 Expression of adhesion molecules (CD18 and CD62L) on surface of blood neutrophils and phagocytic activity of blood neutrophils as in Holstein calves fed milk replacer containing low linoleic acid (LLA) or high lino leic acid (HLA) from 1 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. All interactions with gender did not differ unless footnoted. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR A DD x A MR x A DD x MR x A Measure Milk replacer 2 LLA HLA LLA HLA LLA HLA CD18 Expression CD18+, % 94.7 94.3 93.7 94.3 94.9 94.7 0.70 0.87 0.27 0.98 0.66 0.56 0.06 0.36 0.22 0.18 MFI 52.4 50.2 47.3 47.4 50.5 50.8 5.20 0.61 0.52 0.89 0.79 0.98 0.27 0.65 0.60 0.54 CD62L Expression CD62L+ 4 % 98.2 98.3 97.8 98.2 98.2 98.3 0.20 0. 49 0.23 0.23 0.50 0.50 0.23 0.12 0.36 0.22 MFI 376 389 329 301 357 364 31.2 0.10 0.13 0.85 0.65 0.56 <0.01 0.79 0.57 0.25 Phagocytic activity Phagocytosis, % 95.7 96.3 95.9 96.5 95.2 96.1 0.49 0.90 0.27 0.09 0.84 0.85 < 0.01 0.19 0.87 0.28 MFI 118 120 111 114 120 121 4.3 0.41 0.04 0.57 0.98 0.82 <0.01 0.65 0.46 0.95 Phagocytic neutrophils 5 10 3 3.19 3.26 3.24 3.88 3.06 2.99 0.29 0.84 0.07 0.41 0.71 0.27 <0.01 0.18 0.93 0.28 1 Control = no fat supplement; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 LLA = 0.175 g of LA/BW 0.75 HL A = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 3 P values for orthogonal contrasts and interactions: FAT = fat (SFA + EFA) vs. Control, FA = EFA vs. SFA, MR = milk replace r, A = age, DD = dam diet. Three and four way interactions were not significant. 4 Gender, P =0.04, FA by gender, P = 0.01, MR by gender, P = 0.01. 5 Gender, P =0.05.

PAGE 213

213 Table 4 1 0 Mean concentration of serum total protein, acute phase proteins, serum an ti OVA IgG and interferon gamma produced by peripheral blood mononuclear cells stimulated with concanavalin A in Holstein calves fed milk replacer containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Calves were born fr om cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. All interactions with gender did not differ unless footnoted. Dam Diet 1 P values 3 Measur e Control SFA EFA SEM FAT FA MR FAT x MR FA x MR A DD x A MR x A DD x MR x A Milk replacer 2 LLA HLA LLA HLA LLA HLA ASP 4 mg/L 94.1 72.3 90.0 75.7 90.0 88.3 4.01 0.34 0.11 <0.01 0.04 0.11 <0.01 0.90 0.09 0.59 Haptoglobin, OD x 10 0 0.94 0.96 1.04 1.02 1.02 1.05 0.03 0.06 0.89 0.88 0.78 0.65 <0.01 0.85 0.80 1.00 Anti OVA IgG, OD 0.87 0.86 0.87 0.94 0.82 0.84 0.04 0.99 0.07 0.51 0.39 0.55 <0.01 <0.01 0.38 0.99 IFN 15d, pg/mL 22.3 21.8 38.9 49.3 22.7 23.9 11.4 0.23 0.08 0.69 0.75 0.69 IFN 30d, pg/mL 19.9 48.7 35.5 61.5 21.5 34.2 13.67 0.74 0.14 0.05 0.69 0.63 1 Control = no fat supplement; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princeton, NJ). 2 LLA = 0.175 g of LA/BW 0.75 HLA = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30 d of life to provide 6.72 g of fat/kg of BW 0.75 3 P values for orthogonal contrasts and interactions: FAT = fat (SFA + EFA) vs. Control, FA = EFA vs. SFA, MR = milk replacer, A = age, D = dam diet. Three and four way interactions were not significant. 4 Acid soluble protein. FA by Gender, P = 0.04.

PAGE 214

214 A B Figure 4 1. Plasma fatt y acid c once ntrations in Holstein calves from 30 to 60 days of age. A) Concentrations of 14:0, C16:0, C18:0 and linoleic acid (LA) B) C oncentrations of linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (D HA ). C alves were fed milk replacers containing low or high linoleic acid and were born from dams fed diets su pplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. Grain was offered starting a t 31 d of life. Effect of age for all fatty acids (FA) was P < 0.01 except for C14:0( P = 0.04) and EPA ( P = 0.11). 0 10 20 30 40 50 60 C14:0 C16:0 C18:0 LA % of fatty acids 30 d 60 d 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ALA AA EPA DHA % of fatty acids 30 d 60 d

PAGE 215

215 Figure 4 2. Plasmatic concentrations of glucose in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 d ays of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Effect of dam diet b y age, P = 0.07 [slice effect, P = 0.08 (day 2), P = 0.05 (day 19) and P = 0.06 (day 26)]. 70 80 90 100 110 120 2 5 9 12 16 19 23 26 30 37 43 50 57 Glucose, mg/dL Day of age Control= 92.1 SFA= 91.0 EFA= 90.8

PAGE 216

216 Figure 4 3. Plasmatic concentrations of urea N in Holstein calves fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets su pplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. Grain was offer ed starting at 31 d of life. Effect of milk replacer, P = 0.03 and effect of age, P < 0.01 4 6 8 10 12 2 5 9 12 16 19 23 26 30 37 43 50 57 Plasma urea N, mg/dL Day of age LLA= 8.33 HLA= 7.75

PAGE 217

217 A B Figure 4 4. Plasmatic concentrations of hydroxybutyric acid (BHBA) and none sterified fatty acids (NEFA) in Holstein calves fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets su pplemented with no fat saturate d fatt y acids, or essential fatty acids starting at 8 wk before expected calving date. Grain was offered starting at 31 d of lif e. A) Effects of milk replacer, P < 0.01 and age, P < 0.01 B) Effect of age, P < 0.01. 0.0 0.4 0.8 1.2 1.6 2.0 2 9 16 23 30 37 43 50 57 hydorxybutyric acid, mg/dL Day of age LLA= 1.36 HLA= 0.87 50 100 150 200 250 300 350 2 9 16 23 30 37 43 50 57 NEFA, Eq/L Day of age LLA= 173 HLA= 167

PAGE 218

218 Figure 4 5. Plasmatic concentrations of to tal cholesterol in Holstein calves fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets su pplemented with no fat, saturated fatty acids or essential fat ty acids starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Effect of milk replacer by age, P = 0.01 [slice effect, P = 0.09 (day 16), P 0.01 (from 19 to 49 d), P = 0.06 (day 57)]. 20 40 60 80 100 120 140 160 0 2 5 9 12 16 19 23 26 30 37 43 50 57 Total cholesterol, mg/dL Day of age LLA= 93.3 HLA= 83.2

PAGE 219

219 A B Figure 4 6. Plasmatic concentrations of insulin in Holstein calves from 0 to 60 days of age. A) Calves were fed milk replacer containing low linoleic acid B) Calves were fed milk replacer containing low linoleic acid Calves were born from cows fed diets supplemented with no fat (Control), saturated fa tty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Interaction dam diet by milk replacer by age, P = 0.10 (slice effect at day 56, P = 0.07). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 14 28 42 56 Insulin, ng/mL Day of age Control = 1.21 SFA=1.30 EFA= 1.33 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 1 14 28 42 56 Insulin, ng/mL Day of age Control = 1.46 SFA= 1.45 EFA= 1.41

PAGE 220

220 A B Figure 4 7. Pl asmatic concentrations of insulin like growth factor I (IGF I) in Holstein calves from 0 to 60 days of age. A) Calves were fed milk replacer containing low linoleic acid B) Calves were fed milk replacer containing low linoleic acid Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Interaction dam diet by milk replacer by age, P = 0.03 (slice effect at day 56, P = 0.01). 0 20 40 60 80 100 120 0 1 14 28 42 56 IGF I, ng/mL Day of age Control = 57.0 SFA= 50.7 EFA= 52.0 0 20 40 60 80 100 120 0 1 14 28 42 56 IGF I, ng/mL Day of age Control = 63.7 SFA= 62.1 EFA= 53.2

PAGE 221

221 Figure 4 8. Serum to tal protein concentrations in Holstein calves fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets su pplemented with no fat, saturated fatty acids or essential fatty acids starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Effect of age, P < 0.01; effect of milk replacer by age, P = 0.13 (slice effect, P = 0.02 at day 2). 5.0 5.4 5.8 6.2 6.6 7.0 2 5 9 12 16 19 23 26 30 37 43 50 57 Serum Total protein, g/dL Day of age LLA= 5.77 HLA= 5.83

PAGE 222

222 A B Figure 4 9. Attitude score of Holstein calves from 0 to 60 days of age. A) Calves were fed milk replacer containing low linoleic acid B) Calves were fed milk replacer containing low linoleic acid Calves were born from cows fed diets supplemented wi th no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life Interaction of fat by milk replacer was P = 0.06 and of age was P < 0.01. 0.9 1.0 1.1 1.2 1.3 1 2 3 4 5 6 7 8 Attitude score Week of Age Control= 1.03 SFA= 1.03 EFA= 1.05 0.9 1.0 1.1 1.2 1.3 1 2 3 4 5 6 7 8 Attitude score Week of Age Control= 1.06 SFA= 1.02 EFA= 1.03

PAGE 223

223 A B Figure 4 10. Fecal score of Holstein calves from 0 to 60 days of age. A) Calves were fed milk replacer containing low linoleic acid B) Calves were fed milk replacer containing low linoleic acid Calves were born from cows fed diets supplemented with no f at (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life Interaction of fat by milk replacer, P = 0.01 and of age, P < 0.01. 0.0 0.5 1.0 1.5 2.0 2.5 1 2 3 4 5 6 7 8 Fecal score Week of Age Control= 1.12 SFA= 1.24 EFA= 1.19 0.0 0.5 1.0 1.5 2.0 2.5 1 2 3 4 5 6 7 8 Fecal score Week of Age Control= 1.22 SFA= 1.14 EFA= 1.20

PAGE 224

224 A B Figure 4 11. Blood concentrations of red and white blood cells in Holstein calves fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age Calves were born from cows fed diets su pplemented with no fat, saturated fatty acids, or esse ntial fatty acids starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Effect of age in red and white blood cells P < 0.01. 6.0 7.0 8.0 9.0 10.0 2 7 14 21 30 40 60 Red blood cells, 10 6 Day of age LLA= 8.30 HLA= 8.51 6.0 7.0 8.0 9.0 10.0 11.0 2 7 14 21 30 40 60 White blood cells, 10 3 Day of age LLA= 8.41 HLA= 8.89

PAGE 225

225 A B Figure 4 12. Blood c oncentrations of neutrophils and lymphocytes in Holstein calv es fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets supplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. Gr ain was offered starting at 31 d of life. A) Effect of age, P < 0.01 B) E ffect of milk replacer P = 0.04 and age P < 0.01. 0 1 2 3 4 5 6 2 7 14 21 30 40 60 Neutrophils, 10 3 Day of age LLA= 3.01 HLA= 3.17 0 1 2 3 4 5 6 2 7 14 21 30 40 60 Lymphocytes, 10 3 Day of age LLA= 4.20 HLA= 4.61

PAGE 226

226 A B Figure 4 13. Blood concentrations of monocytes and eosinophils in Holstein calves fed milk replacer containing low (LLA ) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets su pplemented with no fat saturated fat ty acids or essential fatty acids starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life A) Effect of age P < 0.01 B) E ffect of mil k replacer by age P = 0.01 (slice effect at day 7, P = 0.04 and at day 14, P = 0.06). 0.2 0.3 0.4 0.5 0.6 2 7 14 21 30 40 60 Monocytes, 10 3 Day of age LLA = 0.38 HLA= 0.38 0.00 0.05 0.10 0.15 0.20 0.25 2 7 14 21 30 40 60 Eosinophils, 10 3 Day of age LLA= 0.11 HLA= 0.12

PAGE 227

227 A B Figure 4 14 Blood concentrations of eosinophils in Holstein calves from 0 to 60 days of age A) Calves were fed milk replacer containing low linoleic acid. B ). Calves were fed milk replacer containing high linoleic acid. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain mix was offered starting at 31 d of life. Interaction of dam diet by milk replacer by age, P < 0.01 (slice effect at day 8, P = 0.05 ) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 2 7 14 21 30 40 60 Eosinophils, 10 3 Day of age Control= 0.11 SFA= 0.11 EFA= 0.11 0.00 0.05 0.10 0.15 0.20 0.25 0.30 2 7 14 21 30 40 60 Eosinophils, 10 3 Day of age Control= 0.12 SFA= 0.12 EFA= 0.12

PAGE 228

228 Figure 4 15. Blood concentrations of basophils in Holstein calves fed mi lk r eplacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diet s supplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. Grain was offer ed starting at 31 d of life. Effect of age, P < 0.01. 0.00 0.04 0.08 0.12 0.16 0.20 2 7 14 21 30 40 60 Basophils, 10 3 Day of age LLA= 0.11 HLA= 0.11

PAGE 229

229 A B Figure 4 16. Blood concentrations of platelets in Holstein calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age. Calves were born from cows fed diets suppl emented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. A) Effect of dam diet by age interaction, P = 0.03 (slice effect, P = 0.06 (day 7) and P = 0.05 (day 60). B) Effect of age, P < 0.01, effect of milk replacer, P = 0.03. 200 400 600 800 1,000 1,200 1,400 2 7 14 21 30 40 60 Platelets, 10 3 Day of age Control = 745 SFA = 764 EFA = 763 200 400 600 800 1,000 1,200 1,400 2 7 14 21 30 40 60 Platelets, 10 3 Day of age LLA= 801 HLA= 715

PAGE 230

230 Figure 4 17. Hematocrit concentrations in Holstein calves fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calve s were born from cows fed diets su pplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Effect of age, P < 0.01; effect of milk replacer on hematoc rit, P = 0.08. 30 32 34 36 38 40 2 5 9 12 16 19 23 26 30 37 43 50 57 Hematocrit, % Day of age LLA= 34.4 HLA= 35.9

PAGE 231

231 Figure 4 18. Mean fluorescence intensity (MFI) of n eutrophils positive to CD62L in blood of Holstein calves fed mil k replacer containing low or high linoleic acid from 0 to 60 days of age. Calves were born from cows fed diets supplement ed with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Interaction SFA vs. EFA, P = 0.10. 200 250 300 350 400 450 500 550 600 1 8 15 22 40 60 CD62L MFI Day of age Control= 382 SFA= 315 EFA= 361

PAGE 232

232 A B Figure 4 19. Mean Fluorescence in tensity (MFI) and concentration of phagocytic blood neutrophils (B) in Holstein calves fed mil k replacer containing low or high linoleic acid from 0 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. A) Interaction SFA vs. EFA, P = 0.04. B) Interaction SFA vs. EFA, P = 0.08. 70 90 110 130 150 1 8 15 22 40 60 Phagocytic neutrophil, MFI Day of age Control= 119 SFA= 113 EFA= 121 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 1 8 15 22 40 60 Phagocytic neutrophils, 10 3 /uL Day of age Control= 3.10 SFA= 3.40 EFA= 2.89

PAGE 233

233 Figure 4 20. Percentage of blood neutr ophils undergoing phagocytosis in Holstein calves fed milk replacer containing low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets su pplemented with no fat, saturated fatty acids, or essential fatty acids st arting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Effect of milk replacer, P = 0.09; effect of age, P < 0.01. 92 93 94 95 96 97 98 2 7 14 21 40 60 Phagocytosis, % Day of age LLA= 95.6 HLA= 96.3

PAGE 234

234 Figure 4 21. Plasmatic concentration of acid soluble protein in Holstein calves fed milk replacer con taining low (LLA) or high linoleic acid (HLA) from 0 to 60 days of age. Calves were born from cows fed diets su pplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life. Effect of milk replacer and age, P < 0.01. Effect of milk replacer by age interaction, P = 0.09 (slice effect starting at 16 d of age 0.10). 0 30 60 90 120 150 180 210 240 270 2 5 9 12 16 19 23 26 30 37 43 50 57 Acid Soluble protein, mg/L Day of age LLA= 91.4 HLA= 78.8

PAGE 235

235 A B Figure 4 22. Plasmatic concentration of haptoglobin and serum anti OVA IgG in Holste in calves fed milk replacer containing low or high linoleic acid from 0 to 60 days of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Grain was offered starting at 31 d of life A) Effect of fat P = 0.06) and of age P < 0.01. B) Effect of dam diet by age interaction, P < 0.01 (slice effect at days 2, 10, and 20, P < 0.04). 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2 5 9 12 16 19 23 26 30 37 43 50 57 Haptoglobin, OD x 100 Day of age Control= 0.95 SFA= 1.03 EFA= 1.04 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 2 10 20 30 40 50 60 Serum anti OVA IgG, OD Day of age Control= 0.87 SFA= 0.91 EFA= 0.83 Ovalbumin immunization

PAGE 236

236 CHAPTER 5 EFFECT OF SUPPLEMENT AL ESSENTIAL FAT TY ACIDS TO PREGNANT HOLSTEIN COWS AND TH EIR PREWEANED CALVES ON CALF HEPATIC FATT Y ACID PROFILE AND GEN E EXPRESSION Background Raising good quality replacement heifers, able to calf between 22 to 24 months and reaching at least 80% of adult weight is crit ical to gradually improve overall herd performance. Raising heifers is the most challenge area of management on dairy farms. After birth, dairy calves are removed immediately from their dams and transferred to a different unit to initiate the preweaning pe riod which can take a few weeks to approximately 8 wk. The preweaning period which starts with an adequate passive transfer of immunity is considered one of the most critical periods affecting future performance (Beam et al., 2009; Furman Fratczac, 2011). In addition, optimized feeding management of heifers during the preweaning period has a positive impact on future milk production (Soberon et al., 2012). A relative new concept adopted from human lking performance of calves could be affected by the type of diet fed to their dams during late gestation (Fowden et al., 2006; Gicquel et al, 2008). During the preweaning period, newborn calves have to cope with different environmental challenges such as adaptation to an external uterine life, pathogens, and different nutritional value of feeds. An appropriate management of all these factors should result with in calves able to overcome health problems, increase feed intake, and maintain a rapid growth rat e. Feeding high energy diets for rapid growth during the pre weaning period has reduced both the age to reach the target breeding weight and costs

PAGE 237

237 associated with raising of replacement heifers (Radcliff et al., 2000; Raeth Knight et al., 2009). The rapid growth rate of calves during the preweaning period implies a need for their bodies to have an efficient utilization of nutrients. The liver plays a key role in nutrient utilization due to its strategic position in the circulatory system; hence a profound u nderstanding of its mechanism of nutrient utilization is needed. Pioneer studies (Jenkins et al., 1985; Jenkins et al., 1986; Jenkins and Kramer, 1986) supplemented the MR of newborn calves with different sources of fat and reported that concentration of e ssential EFA in liver and plasma reflected the composition of FA in the MR. Hence selective supplementation of FA would be expected to modify FA profile of different tissues and by that means its functionality. Early studies (Mashek et al. 2002; Mashek and Grummer, 2003, 2004) cultured preruminant calf hepatocytes with different FA to evaluate oxidative and gluconeogenic activity. Authors reported that different SFA, MUFA or PUFA did not affect gluconeogenesis as they did in liver of ruminant calves wherea s LA, CLA c 9 t 11 and CLA t 10 c 12 did not affect propionic acid metabolism to produce glucose. However, regardless the type of FA, the formation of both glucose and glycogen were decreased when FA concentrations increased from 0.1 to 1.0 mM. Limited informa tion has been generated regarding the role dietary EFA might have in modifying the expression of genes in liver of preweaned calves. However, no study had evaluated the effect of supplementing EFA prepartum and continued supplementation of EFA during early life of the calf on liver metabolism through global gene expression analysis. The hypothesis was that feeding increased amounts of LA during late gestation and the preweaning period would modify the FA

PAGE 238

238 expression of hepatic genes. An additional hypothesis was that early strategic feeding would have a long term effect on to evaluate the supplementation of EFA to prepartum cows during the la st two months of pregnancy and during the preweaning period on hepatic FA profile and global gene expression in liver of 30 d of age with MR as only feed. An additional objective was to evaluate productive and reproductive responses of heifers at their fir st lactation. Materials and Methods Prepartum Management FL) from October 2008 to June 2009. All procedures for animal handling and care were approved by the University of Flori nulliparous (n = 35) and previously parous (n = 61) Holstein cattle were sorted according to calving date, parity, body weight (BW), and body condition score (BCS) and assigned to one of the three treatments at 8 wk before their expected calving date. Prepartum treatments: supplementation (Control), 1.7% of dietary dry matter (DM) of 2.0% of dietary DM as Ca salts of FA enriched w Church and Dwight, Princeton, NJ) as well as cattle general management were the same as those indicated in chapter 3. Calves Dietary Treatments, Feeding Management and Analyses All procedures regarding calving managemen t at birth and colostrum feeding were done according details presented in Chapter 3. Calves were blocked by gender (n = 56 females and 40 males) and dam diet and randomly assigned to receive a MR containing

PAGE 239

239 low (LLA, 0.56% LA, DM basis) or high concentrati ons of LA (HLA, 1.78% LA, DM basis) for 60 d starting at birth. Milk replacers and grain mix fed in this study were similar to that used in study reported in chapter 4. Similarly, all feeding management of calves was performed as indicated for calves in ch apter 4. Procedures for housing, weighting and immunization of newborn calves were performed according to that indicated in chapter 4. Liver Biopsy Liver biopsies were performed at 30 2 d using a percutaneous liver biopsy needle (Aries Surgical, Davis, C A). Briefly, an ultrasound imaging on the right flank was used to determine the optimal intercostal liver biopsy location. The area that was previously shaved and disinfected was anesthetized with 10 mL of 2% lidocaine HCl (Pfizer Inc., New York, NY). A 1 cm stab incision was made through the skin, after a thorough re sterilization of the target zone. The biopsy instrument was inserted through the incision crossing the muscle layer reaching the liver and a liver sample (approximately 500 mg) was obtained. The open skin was closed with a surgical disposable sterile skin stapler (Oasis Inc., IL). Biopsied calves were subcutaneously injected with 1 mL of antibiotic at the base of the ear (Excede, Pfizer Inc., New York, NY), and their post surgical behavior wa s monitored for the following 12 h. The liver sample was rinsed immediately with sterile saline, sample was split into 2 vials and snap frozen in liquid N, and stored at 80 o C until analyzed for liver FA profile and mRNA abundance. Calves Liver Fatty Aci d Profile Liver samples (~250 mg) were freeze dried for 48 h (Labconco Kansas City, MO) and delivered to Michigan State University for analysis of FA profile. Briefly, total FA

PAGE 240

240 from freeze dried liver samples were extracted using the standard procedure of Bligh and Dyer (1959) and then extracted FA were methylated by the 2 step procedure of Nuerberg et al. (2007) with some modifications. The FA methyl esters were quantified using a GC 2110 Plus gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a spli t injector (1:100 split ratio) and a flame ionization det ector using a CP Sil 88 WCOT fused silica column (100 m 0.25 mm i.d. 0.2 Forest, CA). Total RNA isolation Total cellular RNA was isolated from liver tissue (n = 18) using Qiazol reagent (Qiagen, Valencia, CA) and p urified (RNA MIDI isolation kit, Qiagen, Valencia, CA) were immersed in 3 mL of Qiazol (quiagen, Valencia, CA) just prior to their homogenization in a conventional Rotor Stator homogenizer. Homogenated solution in each tube was left for 5 min at room temperature and then 0.6 mL of chloroform was added to each tube and maintained at room temperature for 3 min. Tubes were centrifuged at 5000 g at 4C for 10 min. After the upper phase containing RNA (1.5 mL) was transferred from each tube to a tube containing 1.5 mL of ethanol (70%) and mixed immediately to suspend the precipitates, the mixed solution was added to the RNeasy midi spin column. Column tubes with the RNA suspe nsion were centrifuged at 5000 g at 23C for 5 min. The flow through was discarded, and 2 mL of RW1 buffer was added to the column and centrifuged at 5000 g at 23C for 5 min. After the flow through was discarded, 160 uL of DNase working solution (12.5 % of DNase stock solution in RDD buffer) was added carefully on the membrane of the column to ensure complete DNA digestion. A series of three additional washing steps with corresponding

PAGE 241

241 buffers followed by centrifugation were performed, before a final 1.5 mL of RNase free water was added to collect the RNA after centrifugation. Integrity and concentration of the RNA was then analyzed using a micro volume spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA ). Purified RNA was aliquoted an d then stored at 80C. Affymetrix Array Hybridization, washing, staining and scanning Isolated RNA samples were delivered to the Interdisciplinary Center for Biotechnology Research (ICBR) of the University of Florida. Briefly, amplification and biotin lab eling were performed with an initial 200 ng of RNA by using MessageAmp III Samples were then tested in the Bioanalyzer for quality determination (all samples had an RNA integrity number > 7.5) and subsequently submitted for fragmentation and Array, Affymetrix Inc., Santa Clara, CA). Arrays were washed on a fluidics station 450 (Affymetrix, I nc., Santa Clara, CA) with the hybridization wash and stain kit from Affymetrix. Fluorescent signals were measured with the Affymetrix GeneChip scanner 3000 7G. Affymetrix Data Analysis The Affymetrix GeneChip Bovine Genome array contains 24,027 probe sets corresponding to approximately 23,000 transcripts including assemblies from ~19,000 UniGene clusters. The Affymetrix CEL files, obtained after the fluorescence signal measure of each Affymetrix chip, were loaded into an AffyBatch object using R/Bioconduct or environment (Gentleman et al., 2004).

PAGE 242

242 Data normalization and background correction were performed using guanine cytosine Robust Multichip Average (gcRMA) function as described by Wu et al. (2004). All Affymetrix control probes (AFFX prefix) were exclud ed as n on informative probes using the informative/ non informative (I/NI) calls from the enhanced FARMS algorithm (Talloen et al., 2007). Differential gene expression was analyzed using linear models for microarray (LIMMA) as described by Smyth (2005). Tr eatments were arranged in a 3 x 2 factorial design that included the evaluation of 5 contrasts, as detailed in the Statistical design section. Enrichment analysis of DEG was evaluated using the functional annotation clustering method within the Data Base for Annotation, Visualization and Integrated Discovery (DAVID, Huang et al., 2009) bioinformatics resource. The enriched DEG were grouped according to their biological process (BP) and molecular function (MF) terms based on the gene ontology (GO, Ashburner et al., 2000) and Kiotto Encyclopedia of Genes and Genomens (KEGG, Kanehisa and Goto, 2000) pathways. Statistical Analysis Dam diets (n = 3) and MR diets (n = 2) were arranged in a 3 x 2 factorial randomized block design. On a weekly basis, a cohort of H olstein cows at 8 wk before the expected calving date was blocked by parity (nulliparous and parous) and BCS. Within each block, cattle were assigned randomly to one of the three dietary treatments. Calves after birth were blocked by dam diet and gender an d randomly assigned to one of the two MR. A total of 40 male and 56 female calves were enrolled. Liver FA profile and all productive and reproductive measures were analyzed using the MIXED procedure of SAS (Release 9.2) according to the following model: Y ijk j ij ijK Where Y ijk i is the fixed

PAGE 243

243 j is the fixed effect of MR (LLA and HLA), ij al error. The following orthogonal contrasts were performed for all variables [1) FAT: dam diet of fat (SFA + EFA) vs. no fat (control), 2) FA: dam diet EFA vs. SFA, 3) MR: milk replacer HLA vs. LLA, 4) FAT by MR: interaction of contrasts 1 and 3, 5) FA by MR: interaction of contrasts 2 and 3]. For FA profile and all productive and reproductive measures, a P Analysis with the LIMMA package (Smyth, 2005) was used for Identification of DEG after using the method of Benjamini and Hoechberg (BH) to adjust for multiple tests and control the false discovery rate (FDR) up to 5%, a cut off for adjusted P value of comparing DEG in pre determined contrasts of experimental groups, the appropriate reference group was defined for each comparison per contrast as follows: Arrangement of treatments Treatment Dam Diet Milk replacer Number of samples 1 Control LLA 3 2 Control HLA 3 3 SFA LLA 3 4 SFA HLA 3 5 EFA LLA 3 6 EFA HLA 3 1. Contrast FAT: Dam diet (SFA + EFA) /2 Control (reference). 2. Contrast FA: Dam diet EFA SFA (reference). 3. Contrast MR: Milk replacer HLA LLA (reference)

PAGE 244

244 4. Interaction FAT by MR: [(SFA HLA + EFA HLA) /2 : Control HLA (reference)] [(SFA LLA + EFA LLA) /2 : Control LLA (reference)] 5. Interaction FA by MR: [EFA HLA : SFA HLA (reference)] [EFA LLA : SFA LLA (reference)] Binary data were all analyzed by logistic regression using the LOGISTIC pr ocedure of SAS (SAS Inst. Inc., Cary, NC). The models included the effects of dam diet and milk replacer. Adjusted odds ratio and the 95% confidence interval (CI) were calculated. Differences discussed in the text were significant at P The modified terms and KEGG pathways within the DAVID annotation tool. Results Liver Fatty Acid Content and Profile Mean FA concentration on liver of male calves was not affected by dam diet. but by the type of MR fed. Calves fed the HLA MR had a lower mean concentration of total FA in liver (7.56 vs. 8.47% of total DM, Tabl e 5 1). Mean proportions of SFA, MUFA, and PUFA across treatments were 42.6, 15.4, and 39.3% respectively. These groups of FA were affected only by the type of MR fed to calves. Calves fed HLA MR had a lower proportion (of total FA) of SFA (40.0 vs. 45.1%) and MUFA (14.3 vs.16.4%) but greater proportions of PUFA (43.6 vs. 35.56%, Table 5 1). As expected, most of the individual FA in liver of calves also were affected by the MR fed. Calves fed LLA MR, whose only source of fat was CCO, had increased proporti ons ( P < 0.01, Figure 5 1) of C12:0, C14:0, and C16:0, with the greatest proportional difference detected for C14:0 (5,22 vs. 1.30%). Regarding MUFA, OA

PAGE 245

245 represented 75% of total MUFA in liver of calves fed LLA MR, followed by C16:1 which occurred in minor proportions of total MUFA but also occurred in greater proportions in liver of males fed LLA vs. HLA MR (0.48 vs. 39%, P = 0.02). Of the six identified n 6 FA, four were increased or tended to be increased in liver of calves fed HLA MR, namely LA (22.1 vs. 15.9% of total FA, P < 0.01), C20:2 (1.01 vs. 0.54% of total FA, P < 0.01), AA (10.78 vs. 10.17% of total FA, P = 0.09), and C22:4 (1.27 vs. 1.13% of total FA, P = 0.03, P = 0.03) whereas proportions of GLA (0.03 vs. 0.07% of total FA, P < 0.01) and C20:3 (2.70 vs. 3.36% of total FA, P = 0.01) were decreased in liver of calves fed HLA MR. Proportions of LA and AA accounted for ~76% of total n 6 FA in calves fed HLA MR, hence proportions of total n 6 FA were greater in calves fed HLA as compared to those f ed LLA MR (37.6 vs. 31.1% of total FA, P < 0.01, Table 5 1, Figure 5 2 A). Four n 3 FA were identified in the liver of calves. Of these ALA (0.99 vs. 0.70% of total FA, P < 0.01) and DPA (2.06 vs. 1.57% of total FA, P < 0.01) were greater in liver of calve s fed HLA MR whereas EPA was greater in liver of calves fed LLA MR (0.24 vs. 0.19% of total FA, P < 0.01) and proportions of DHA did not differ due to the type of MR fed. Because ALA and DPA accounted for 60% of total n 3 FA in liver of calves fed HLA MR, the total proportion of n 3 FA was greater in this group of calves compared to calves fed LLA MR (5.08 vs. 4.17% of total FA, P < 0.01, Table 5 1, Figure 5 2 B). However the effect of MR on the proportions of n 3 FA of liver was influenced by the type of f at fed to their dams. If the dam was fed SFA, the effect of MR on the shorter chain n 3 FA (ALA and EPA) was magnified; that is, the increase in ALA proportions due to the feeding of HLA MR was greater if SFA (1.03 vs. 0.65%) rather than EFA (0.91 vs. 0.7 1%) was fed prepartum (FA by MR interaction, P = 0.04, Table 5 1). Similarly EPA proportions were

PAGE 246

246 increased in liver fat of calves by HLA MR only if SFA (0.30 vs. 0.18%) and not EFA (0.22 vs. 0.22%) was fed to their dams prepartum (FA by MR interaction, P = 0.04, Table 5 1). Lastly, only HLA and not LLA MR increased DHA proportions in liver fat of calves if their dams were fed EFA (1.90 vs. 1.34%) rather than SFA (1.84 vs. 2.05%) prepartum (FA by MR interaction, P = 0.03, Table 5 1). Feeding fat to prepartu m cows produced some minor effects on liver FA profiles of their calves such as greater proportions of AA (10.73 vs. 9.97% of total FA, P = 0.05) and DPA (1.87 vs. 1.70% of total FA, P = 0.02) but lower proportions of ALA (0.82 vs. 0.89% of total FA, P = 0 .05) compared to calves from dams not supplemented with fat. Differential Expression of Genes in Liver A total of 58 transcripts were up regulated according to the criteria of false discovery rate (Figure 5 3) in liver of calv es born from dams fat (EFA + SFA) compared to that of calves born form dams not fed fat, but only 41 transcripts were either annotated or identified with the bovine DAVID annotation tool. Feeding a specific type of fat during the prepartum period resulted in the up regulation of 75 transcripts (Figure 5 3) in liver of calves born from dams fed EFA compared to those fed SFA. From these 75 transcripts, only 63 were recognized by bovine David. Those 2 contrasts of dam diet effects shared a total of 7 transcript s (Figure 5 3) that were differentially expressed in the same manner and 2 of them were not annotated. Regarding the effect of feeding a HLA MR, 53 transcripts were up regulated when HLA rather than LLA MR was fed (Figure 5 3). From those transcripts only 4 2 were recognized by bovine DAVID. A t otal of 208 transcripts were up regulated differentially in liver from calves fed HLA instead of LLA MR in a manner that differed between dams fed or not fed fat

PAGE 247

247 prepartum (interaction of FAT by MR, Figure 5 3). Of the 208 transcripts, 167 were read by bovine DAVID. A specific comparison between type of fats fed prepartum indicated that a t otal of 132 transcripts were up regulated in liver of calves born from dams fed EFA instead of SFA in a manner that differed due to t he type of MR fed (interaction of FA by MR, Figure 5 3). Of these 132 differentially expressed transcripts, 107 were read by bovine DAVID. Among both interactive contrasts, 13 of the differentially express ed transcripts were commonly up regulated. It is str ikingly clear that distinct differences in gene expression are detected, and the differences are much more pronounced when looking at the interactive effects between prepartum supplementation and type of MR fed preweaning. In contrast, feeding fat prepartu m downreg ulated 51 transcripts in liver of calves, with 39 of these transcripts being read by DAVID (contrast FAT, Figure 5 4). Liver of calves born from EFA fed dams had 56 downreg ulated transcripts compared to calves born from SFA fed dams (contrast FA, Figure 5 4). From these 56 transcripts, 51 were read by DAVID. These two dam diet contrasts had 5 common downreg ulated genes with 4 of them read by DAVID. If calves were fed HLA instead of LLA MR, 31 transcripts were downreg ulated with 19 being read by DAV ID. A total of 187 genes were differentially downreg ulated in liver of calves if they were fed HLA instead of LLA while born from fat fed dams (interaction FAT by MR, Figure 5 4). From these 197 transcripts, 132 were read by DAVID. When comparing the effec t of feeding a specific profile of FA prepartum, liver of calves born from dams fed EFA instead of SFA had a differential downreg ulated response when fed HLA instead of LLA MR. These calves had 182

PAGE 248

248 downreg ulated transcripts with 134 being read by DAVID. A mong both interactive contrasts, 17 of the differentially expressed transcripts were commonly downreg ulated. Enriched Gene Ontology Terms The groups of DEG within GO terms were identified using the DAVID analysis of functional annotation clusters with med ium stringency. The enrichment score (ES) of each cluster represents the log 10 value of the geometric mean of all adjusted Fisher P values for each GO within a cluster. Hence the greater the ES the smaller is the P value. Authors of DAVID annotation too l recommend giving more attention to clusters with ES 1.3 and adjusted Fisher P values for GO terms 0.10. However they also recommend evaluating ES with lower values in terms of the expected biological meaning according to the experimental condition (Huang et al., 2009). Therefore, after analyzing all clu sters and GO (only BP and MF) terms within each cluster for all five contrasts evaluated in the current study, it was decided to present only clusters with ES 1.00 ( P 0.10) and within cluster, only GO terms with an adjusted fisher P value of 0.10. A single exception was done for a cluster with an ES = 0.97 (contrast FA by MR) due to its significant biological meaning. F rom the 41 up regulated and recognized genes for the effect of feeding fat prepartum, the analysis with bovine DAVID resulted in 3 enri ched clusters. Yet not one of these clusters or GO terms fit within the selected enrichment criteria (ES adjusted Fisher P On the other hand the enrichment analysis of up regulated genes within the contrast of FA resulted in a total of 9 enriched clusters but only 1 cluster including 2 BP met the criteria of selection (Table 5 2). The enri ched biological processes were 1) negative regulation of metabolic and transcription processes which included 4 genes and 2) negative regulation of transcription, which

PAGE 249

249 resulted in the enrichment of 3 genes. Feeding HLA MR instead of LLA MR resulted in the enrichment of 4 clusters but only 3 of them met the enrichment criteria (Table 5 2). The first enriched cluster included the MF calcium ion binding with 7 enriched genes; the second included 2 MF, namely actin binding and motor activity and the BP striate d muscle tissue development; the last cluster included 2 MF, namely cation binding and calcium ion binding, and included 1 BP, namely, proteolysis involved in cellular protein catabolic process. The interaction contrasts of dam diet and MR resulted in a gr eater number of DEG and hence in a greater number of enriched clusters. For the interaction FAT by MR, only 7 clusters were selected from a total of 27 clusters enriched wi th up regulated genes (Table 5 3). The top enriched cluster included the highest numb er of DEG involved in electron carrier activity, oxidation reduction, and iron ion binding. The other clusters had at most 2 GO terms involved in processes such as binding, transport, and metabolic processes. The other combined effect of feeding a specific type of fat and HLA MR (interaction of FA by MR) resulted in the enrichment of 22 clusters with only 3 meeting the criteria assumed in this current study (Table 5 4). The enriched clusters included different BP terms involved in catabolic processes to gen erate energy intermediates, phospholipid biosynthetic process, organophosphate metabolic processes, and protein complex assembly. The analysis of downreg ulated DEG resulted in a few enriched clusters affected by dam diets or MR but a greater number of enr iched clusters affected by the interaction of dam diet and MR. This pattern was similar to that observed with the up regulated DEG. The two contrasts involving dam diets (FAT and FA) resulted in a total of 6 enriched

PAGE 250

250 clusters within each contrast, but usi ng the enrichment criteria set for this study, only 2 clusters were selected for the contrast of FAT which included actin binding, striated muscle tissue development, and motor activity (Table 5 5). Only 1 cluster was selected for the contrast of FA which included the genes involved in catabolic processes. Calves fed HLA instead of LLA MR had 3 enriched clusters with their downreg ulated DEG but only 1 cluster met the criteria of enrichment. The GO included iron ion binding and oxidation reduction. Similar to the upregulated DEG enrichment analysis, prepartum diets influenced the effect of HLA feeding on the enrichment of clusters for downreg ulated DEG in the liver of calves. Feeding HLA MR to calves born from dams fed fat instead of control diets resulted i n the enrichment of 19 clusters but only 4 clusters met the criteria of enrichment (Table 5 6). The main enriched GO terms in this interaction group were different binding activities, striated muscle development, and heart morphogenesis. Calves fed HLA MR and born from dams fed EFA instead of SFA resulted in the enrichment of 25 clusters with downreg ulated DEG but only 3 clusters met the criteria used in the current study (Table 5 7). The top enriched cluster included GO terms involved in proteolysis, pepti dase activity, and thiolesterase mediated by ubiquitin. Other clusters included genes involved in different signaling pathways and different immune activities. Enriched KEGG Pathways Enriched pathways within each contrast of evaluation were identified wit h DAVID using cut off criteria to contain at least 3 genes in a given pathway and have an adjusted Fisher P value these cut off settings, the up regulated DEG in dam diet contrasts (FAT and FA) did not enriche any KEGG pathway. However, feedin g

PAGE 251

251 MR enriched 4 pathways (Table 5 8). Two up regulated pathways shared the same genes that encode for sarcomeric proteins (Tajsharghi, 2008) and were re lated to cardiomyopathy. The up regulation of t he PPAR pathway included the up regulation of the gene codi ng for PPAR receptor and 2 of its target genes (OLR1 and ANGPTL4; Table 5 8). T he up regulation of this PPAR pathway is an indicator of enhanced lipid transport (OLR1) and adipocyte differentiation (ANGPTL4, also labeled as PGAR) in calves fed HLA MR (Figure 5 5 so d esignated by diamond symbol ). The last up regulated KEGG pathway in liver of calves fed HLA MR was the tight junction pathway which included 3 genes (MYL2, MYH7, and ACTN2) which encode for two sarcomeric proteins, actin and myosin, that might be related to handling of cardiomyopathy disorders (Tajsharghi, 2008). The enriched KEGG pathways of liver of calves fed MR was influenced greatly by the prepartum diet fed to their dams. Calves fed HLA instead of LLA MR and born from dams fed fat instead of the contr ol diet (interaction of FAT by MR) experienced an upregulation of 8 KEGG pathways (Table 5 8). One of the upregulated pathways was the PPAR signaling pathway. The gene coding for PPAR upregulated but 6 PPAR target genes were up re gulated (Table 5 8 and Figure 5 5, so designated by star symbol). In addition to the PPAR pathway, well known for its regulatory process in lipid oxidation, other catabolic KEGG pathways, involved in metabolism of lipids, carbohydrates, and drugs also we re up regulated (Table 5 8). In contrast, when calves were fed HLA MR instead of LLA MR and were born from dams fed EFA instead of SFA (interaction of FA by MR), 4 KEGG pathways were enriched (Table 5 8). The enriched pathways are involved in catabolic proce sses and generation

PAGE 252

252 of intermediate products to ge nerate energy, with a marked up regulation of the oxidative phosphorylation pathway. Interestingly, adipocytokine pathway was up regulated that included 3 genes involved in regulation of insulin sensitivity ( ADIPOR2, STAT3, and ACSL5 Iabeled as FACS so designated by star symbol, Figure 5 6). Neither the type of fat fed prepartum nor the type of MR fed preweaning downreg ulated any KEGG pathways. Four KEGG pathways were downregulated due to feeding of FAT prepar tum (Table 5 9). Of these 4 pathways, 3 pathways were downreg ulated mainly due to HLA rather than to LLA MR (FAT by MR interaction). For 2 of the pathways, the 3 genes affected were identical, namely MYL2, TNNC1, and TPM2 for hydrotrophic cardiomyopathy an d dilated cardiomyopathy. For the tight junction pathway involved in maintaining the impermeable integrity of all cell membranes, the main effect of FAT feeding influenced 3 genes (MYL2, MYH7, and ACTN2) whereas the interaction of FAT by MR influenced thes e same 3 genes plus MYH1 and CASK (Table 5 9). Genes MYL2, MYH1, and MYH7 help code for the myosin protein and ACTN2 codes for actinin protein as illustrated in Figure 5 7 (so designated with arrow symbol). The CASK (calcium/calmodulin dependent serine p rotein kinase) protein functions as a scaffolding protein. In addition, calves born from dams fed fat instead of control diet had 3 downreg ulated genes (ICAM1, MYL2, and ACTN2) within the leukocyte transendothelial migration KEGG pathway (Table 5 9 and Fi gure 5 8 so designated with star symbol, MYL2 is shown as MLC). Lastly liver from calves fed HLA MR and born from dams fed fat had 5 genes (SOCS1, UBA7, PML, HERC4, and BIRC3) downreg ulated from the ubitquitin mediated proteolysis KEGG pathway (Table 5 9). This same KEGG pathway (ubitquitin mediated proteolysis) was influenced in liver

PAGE 253

253 of calves fed HLA and born from dams fed EFA but 3 different (CUL3, KLHL9, and ITCH) and 1 common (BIRC3) genes were downreg ulated (Table 5 9). The other KEGG pathway affec ted in the liver of calves fed HLA and born from dams fed EFA was the pyrimidine metabolism pathway involving 4 genes (UPP2, ENTPD4, EPYD, and NME7). Heifers Productive and Reproductive Performance Performance of experimental heifers was evaluated until the end of their first lactation. A total of 56 heifers participated in the experiment, however only 33 heifers were included in the data set because 23 heifers were culled before finishing at least 150 d in their first lactation. The effect of MR and its interaction with dam diet had minimal impact on all productive and reproductive variables measured (Table 5 10). In contrast, prepartum feeding of fat had major influences on future outcomes. Age at first insemination did not differ due to dam diet (mean o f 13.1 mo). However heifers born from dams fed fat during the last 8 wk prior to calving had a greater number of inseminations at first conception (2.6 vs. 1.7, P = 0.04, Table 5 10). In agreement with a greater number of inseminations it was an older age at first calving (24.2 vs. 22.9 mo, P = 0.02, Table 5 10) in heifers born from dams fed fat. Because heifers born from fat fed dams were older at first calving, they also were heavier (548 vs. 512 kg, P = 0.04, Table 5 10) and had greater BCS (3.3 vs. 3.1 P = 0.04, Table 5 10) than heifers born from control fed dams. The length of lactation did not differ with diets (296 vs. 302 days, P = 0.56). Heifers from fat fed dams tended to have a greater BCS (3.53 vs. 3.43, P = 0.08, Table 5 10) at the end of lact ation. Days in milk at peak of lactation tended to be earlier for heifers born from fat fed dams (80.5 vs. 96.3 P = 0.08, Table 5 10). Heifers born from dams fed fat prepartum

PAGE 254

254 produced more mature equivalent milk during their first lactation (12,004 vs. 1 0,605 kg, P = 0.02, Table 5 10). Concentrations of fat and protein in milk did not differ due to prepartum diets but lactose concentration tended to be greater for heifers born from dams fed fat (4.81 vs. 4.78, P = 0.08, Table 5 10). The type of FA fed pre partum did not affect any of the variables except BCS at dry off. Heifers fed the LLA MR and born from dams fed EFA prepartum tended to have more body condition than those fed the HLA MR (3.8 vs. 3.5) whereas the type of MR fed did not affect BCS at dry o ff if dams were fed SFA prepartum (3.4 vs. 3.4, ), P = 0.07, Table 5 10). Culling incidence was evaluated as total incidence and additional, the most frequent reasons for culling. No effect of any diet was observed on the incidence of culling (Table 5 11) Mean culling rate was 27.8% (5/18), 50% (11/22), and 43.8% (7/16) for heifers born from dams fed control, SFA, or EFA diets, respectively. Regarding MR diets, heifers fed the LLA MR had a culling rate of 46.4% (13/28) whereas that of heifers fed HLA MR w as 35.7% (10/28) but the difference was not significant. The most common reasons for culling were reproductive problems (n = 5), poor growth (n = 8), and mastitis and low production (n = 5). Neither prepartum dam diet nor preweaning calf diet affected the incidence of a particular reason of culling. Discussion Regulation of Hepatic Total and Individual Fatty Acid Concentration Fatty liver is a critical condition that can lead to impairment of liver function. The negative effects and etiology of this conditi on have been well documented in humans ( Reddy and Rao, 2006; Cave et al., 2007; Semple et al., 2009; Thomson and Knolle, 2010) and in dairy cows (Bobe et al., 2004). Fat concentration of liver in preweaned dairy calves was increased by feeding CCO in the M R by Jenkins and Kramer (1986).

PAGE 255

255 Upon in vitro incubation of liver tissue from calves fed CCO or tallow in MR, Gruffat Mouty et al. (2001) reported that the liver from CCO fed calves had reduced concentration of Apo protein B and reduced in vitro secretion of VLDL. In the current study, calves fed LLA MR had 12% greater proportions of FA in liver compared to calves fed HLA MR but it is unlikely that excessive steatosis occurred. Jenkins and Kramer (1986) documented an increase of 48% in proportion of FA in f resh liver when feeding a CCO based MR compared to a MR containing 95% CCO and 5% corn oil (% of fat), however, calves fed CCO MR had a better performance, which might indicate that the liver was not affected by this increase in fat. In vitro studies using liver of calves fed CCO reported a reduction in FA oxidation which suggests an increase in esterification in liver (Graulet et al., 2000). Coconut oil is composed by MCFA which leave the enterocyte and directly arrives to the liver by portal vein, greater and faster availability of these MCFA are partially oxidized and elongated to synthesize longer chain FA and TG that due cannot leave the liver at same rate as they are synthesized ending up accumulating (Sato, 1994). Hence calves fed LLA MR rich in CCO, might follow same mechanism. Studies performed by Jenkins and Kramer (1986, 1990) and Leplaix Chalat et al. (1996) suggested that the amount of fat provided to calves and more important the type of dietary FA can impact the accumulation of fat in liver. T his agrees with the results of the current study. As stated in previous studies, CCO, a fat rich in medium chain FA, has been associated with a steatotic condition, whereas LA, and other PUFA, are potent inducers of lipid oxidation in liver (Clarke et al., 1977; Sampath and Ntambi, 2005). The primary mechanism by which PUFA enhance fat oxidation is by the activation of PPAR

PAGE 256

256 regulator of key genes involved in lipid oxidation (Forman et al., 1997; Hostetler et al., 2005). In the current study LA and A A (as well as all PUFA) were present in greater proportions in liver fat of calves fed HLA MR. This increased proportion of natural ligands of PPAR might account for the up regulation of PPARA gene when calves were fed HLA MR. Up to the time of liver biop sy (30 1 d of age), calves were fed only MR. As a result, microbial activity in the rumen was not fully active which likely limited hydrolysis and biohydrogenation of dietary FA. Consequently, the FA profile of the liver reflected the FA profile of the t ype of MR fed. Concentrations of C12:0 and C14:0 were greater in liver of calves fed LLA MR. Even though C12:0 was dramatically greater in CCO compared to porcine lard (42.5 vs. 29.9%), the differences in liver proportions of C12:0 were small but signific ant (1.23 vs. 0.29%). This because much of the C12:0 would have been readily oxidized for energy by the liver, leaving little to accumulate in hepatic tissue. Concentration of C16:0 was greater in liver of calves fed LLA vs. HLA (16.5 vs. 13.9%) despite b eing in greater concentration in the HLA MR (14.6 vs. 10.6%). Palmitic acid is the longest chain FA in CCO and would be the predominant FA absorbed in the lymphatic system as part of the chylomicron matrix from the LLA MR. As the liver takes up these C16: 0 dominated lipoproteins, they would be synthesized into triglycerides and stored by hepatic tissues and likely be found in greater concentrations compared to calves storing the longer chain C18 FA from the HLA MR. As expected concentration of LA was incr eased in liver of calves fed more LA. However the other 18 carbon FA in liver tissue did not follow exactly the MR pattern. Concentrations of C18:0 in liver matched those in MR but that of C18:1 did not. The HLA MR contained 76% more

PAGE 257

257 C18:1 than the LLA MR but concentrations of C18:1 in liver were lower in calves fed HLA MR (10.1 vs. 11.3%). This may have occurred if biohydrogenation of some C18:1 by ruminal microorganism took place, although it is more likely that ruminal activity was minimal due to feedi ng of MR alone. However, some of the ingested MR would have ended up in the rumen rather than the abomasum, providing a substrate for the population of anaerobic microbes established there. The conversion of some C18:1 to C18:0 in calves fed HLA MR which have more C18:1 may have caused the decreased concentration of C18:1 in hepatic tissues. Regarding the n 6 FA derivatives, the desaturases/elongase enzymes were operational, since the proportion of AA, C20:2, and 22:4 increased in liver of calves fed HLA M R. Only 1 n 3 derivative (DPA) was increased in liver of calves fed HLA despite the fact that the parent FA (ALA) was greater from HLA feeding. This indicates that these same desaturase/elongase enzymes were less active in PUFA metabolism. Interestingly so me studies have documented a preferential use of the desaturase / elongase enzymes by a parent FA when it is in greater proportion and conversely limiting the synthesis of longer chain FA from the parent FA found in lower concentrations (Chan et al., 1993; Goyens et al., 2006; Liou et al., 2007). Feeding of High Linoleic Aci d in Milk Replacer Up regulated PPAR and its Target Genes Based upon DEG analysis, The HLA MR fed to calves greatly influenced the PPAR signaling pathway thus potentially impacting FA oxidation at the tissue level and delivering net energy for cell functions. Up regulation of PPARA should be expected to enhance some hepatic catabolic processes such as lipid oxidation and gluconeogenesis (Rakhshandehroo et al., 2010). However response was not clear cut. Upregulation of

PAGE 258

258 OLR1 and ANGPTL4 genes accompanying the up regulation of the PPAR A in calv es fed HLA MR have an opposite effect to reduce the clearance of lipid from liver tissue. Expression of OLR1, the receptor responsible for binding to oxidized low density lipoprotein cholesterol (ox LDL) in order to prevent its elimination from the liver, is constitutively in low concentrations, but its activation can be induced under pathological conditions such as diabetes mellitus, hypertension, myocardial ischemia, and atherosclerosis (Mehta et al., 2006). In addition, OLR1 also can be induced by elevat ed amounts of ox LDL and reactive oxygen species (Khaidakov et al., 2011). Based on the metabolic profile of calves fed HLA, there was no evidence that calves were undergoing any of the above pathological conditions. However the gene expression of the anti oxidant enzyme, SOD2, was downreg ulated in calves fed HLA MR (fold change of 1.40, P < 0.01, Appendix 4). Because SOD is a member of the reactive oxygen species family, reactive oxygen species were not likely responsible for OLR1 inducement. Moreover, pat hways related to catabolic processes that generate reactive oxygen species (i.e., mitochondrial respiration, peroxisomal FA oxidation, microsomal cytochrome P450 metabolism) were not enriched by any KEGG pathway or GO term in liver of calves fed HLA MR. It is well documented that activation of PPAR will en hance oxidative processes by up regulating the expression of several t arget genes, among them the CYP4 family (Rakhshandehroo et al., 2009). In addition, enhanced oxidative processes have been associated with increased production of reactive oxygen species. These oxygen species are known to increase tissue damage (West, 200 0; Sun et al., 2002). However, the oxidative process was apparently reduced in calves fed HLA MR

PAGE 259

259 since 3 genes in addition to SOD2 (CYP4A22, CYP2C19, and HAAO) involved in oxidative / reduction processes were downreg ulated (Appendix 4). The other gene upre gulated in the PPAR signaling pathway was ANGPTL4. It is directly up regulated by PPAR ANGPTL4 gene (Zhu et al., 2012). A role of PPAR is to help clear TG from plasma by up regulating the activity of dif ferent lipoprotein lipases. The gene, ANGPTL4, has an inhibitory effect on lipoprotein lipase (Duval et al., 2007). As expression of ANGPTL4 was upregulated by feeding of more LA, plasma concentrations of TG should have increased. However, total FA in plas ma of calves fed HLA MR was lower than that of calves fed the LLA MR (Chapter 4). These 3 identified upregulated genes in the PPAR signaling pathway seem to be exerting pro and anti lipolytic effects. The option to exert a pro or an anti lipolytic effect may allow the calf to better adapt to the immediate energy circumstances. The activation of PPAR is required for normal adaptive responses to starvation (Inagaki et al., 2007). However, calves fed HLA MR were under normal feeding conditions and undergoin g increased anabolic processes, verified by the greater BW gain and plasmatic IGF I concentrations (Chapter 4). Therefore, although increased availability of PUFA in calves fed HLA MR might increased the activity of PPARA gene, but the not urged need to sy nthesis energy intermediate products as well as glucose might prevented further activations of other catabolic enzymes by PPARA Feeding Fat Prepartum and High Linoleic Aci d in Milk Replacer Up regulated PPAR Target Genes In the previous section, the effect of HLA on expression of genes of PPAR signaling pathway has both anti and pro lipolytic effects. In this section, all upregulated genes associated with PPAR had a clear pro lipolytic function. Expression of CYP4A

PAGE 260

260 genes is sensitive to PPAR dams fed any source of fat and supplemented with HLA MR had 6 up regulated genes (CYP4A11, CYP4A22, CYP27A1, APOA5, ACADL, and ACAA1) within the PPARA signaling pathway. However expression of the specific PPARA gene did not change. All of the 6 upregulated genes have well defined functions regarding lipid transport, cholesterol synthesis, and lipid oxidation. The CYP4A11 and CYP4A22 genes act through enhancing mic rosomal oxidation (Savas et al., 2003). Synthesis of bile acids from cholesterol is a catabolic process to eliminate excess cholesterol and CYP27A1 has a clear role in this process (Chen and Chiang, 2003). Clearance of TG from circu lation is aided by the activity of APOA5 which has high affinity for lipids. Metabolic studies using mice documented that APOA5 can lower plasma TG by reducing the hepatic VLDL TG production rate and by enhancing the lipolytic conversion of TG rich lipopro teins (Pennacchio and Rubin, 2003). Finally oxidation oxidation, respectively (Rakhshandehroo et al., 2010). A significant number of additional pathways were up regulat ed in liver of calves fed HLA instead of LLA and born from dams fed fat instead of control diets (FAT by MR). The pathways included FA metabolism, glycerolipid metabolism, arachidonic acid metabolism, and drug metabolism pathways (Table 5 12). The up regula tion of these pathways are indicative that these groups of calves were certainly undergoing a hydroxylation and mitochondrial and oxidation and, by these means, might be generating energy intermediate produ cts such as NADPH. Electron carrier activity, oxidation/reduction, transmembrane

PAGE 261

261 transport, NAD/NADPH binding, and coenzyme metabolic processes include genes that are related closely with the processing and further metabolism of lipid cataboli c products or iginated by the up regulated activity of the aforementioned genes. Genes associated with the PPAR signaling pathway were upregulated due to supplementing of fat during prepartum. Furthermore, the stimulatory effect of HLA MR occurred in calves born from da ms fed either SFA or EFA prepartum (i.e., no FA by MR interaction was detected, Appendix 1). Certain nutritional conditions occurring during the fetal period or early life have a more marked effect on fetal programming occurrence (Fowden et al., 2006; Gic quel et al, 2008). Any of these prepartum and preweaning the later life of offspring. The potential effects of dam diet on fetal programming could be modified by the p reweaning diet offered. Feeding of Essential Fatty Acids Prepartum and High Linoleic Acid in Milk Replacer Enhanced Catabolic Processes and ATP Generation Neither feeding EFA nor HLA MR alone as main effects influenced catabolic processes and ATP genera tion. Although the interaction of FA and MR was essentially not significant for FA profile of liver (Table 5 5), these diets influenced gene expression in the liver. Feeding HLA MR to calves modified the effect of EFA fed prepartum (interaction FA by MR). It is possible that provision of greater amounts of LA and ALA deal with continued feeding of greater amounts of LA once they are born. The upregulated pathways in the contrast of FA by MR support this hypothesis. Although the PPARA pathway, which has a big role in FA oxidation, was not up regulated, other pathways that are indicative of oxidation of nutrients were up regulated such as

PAGE 262

262 glycolysis and most importantly, oxi dative phosphorylation, the end point pathway to generate high energy compounds. Glycolysis and alcohol catabolic processes were two up regulated BP within the top enriched cluster in the interaction response of FA by MR (Table 5 8). Feeding HLA MR to calve s born from dams fed EFA promote the upregulation of genes within a cluster as compared to calves fed LLA MR and born from dams fed SFA. These 2 BP shared the same set of genes, namely ALDOA, TPI1, ENO1, OGDH, and MDH2. The first three genes code for enzym es within the glycolytic pathway whereas OGDH synthesizes succinyl CoA from ketoglutarate within the Krebs cycle and MDH2 exports oxaloacetate from mitochondria through conversion to malate in a reversible reaction (Hartsock and Nelson, 2008). Another e nriched BP within the top enriched cluster was before plus UQCR1, COX10, ATP6V1E1, ATP5B, and NDUFS2. These genes likewise are listed for the up regulated oxidative phos phorylation pathway. All of these latter 5 genes are enzymes involved in four of the five complexes of the oxidative respiratory chain responsible for the intermediate products of oxidation (NADPH, FADPH) to be converted to ATP (Osellame et al., 2012). Th e enhanced catabolic processes in this group of calves (interaction of FA by MR) are indicative of greater glucose availability to be used as a source of energy. Indeed calves fed HLA MR had greater plasma concentrations of glucose and IGF I (Chapter 4). T his greater availability of glucose in liver is derived from the diet, specifically lactose, which was the only source of glucose to these calves at the time the liver biopsy was performed. In fact, the glycolysis pathway in these calves was

PAGE 263

263 up regulated wi th the greater expression of GALM in calves fed HLA MR and born from dams fed EFA (FA by MR interaction, Table 5 12), the gene that encodes for the first enzyme of four needed to get glucose 1 P from D galactose. Specifically, the GALM gene mutarotates the configuration to D galactose, which follows a series of conversion steps to UDP glucose (Thoden et al., 2004) In addition, because several key genes of the oxid ative respiratory chain were up regulated, several intermediate energy produ cts would have been diverted to mitochondria for ATP synthesis. Oxidation of lipids through oxidation is another important contributor to intermediate energy products. It is speculative that this mechanism was also active in this group of calves. Althoug h no individual gene involved in the oxidation process was found up regulated, there was a linear fold change increase of 1.94 ( false discovery rate = 0.14) in expression of PPAR relative to calves born from cows fed EFA but fed LLA MR. Synthesis of phos were up regulated via 2 BP enriched in liver of calves fed HLA instead of LLA MR and born from dams fed EFA instead of SFA diets (interaction of FA by MR). Both of these BP had the common enriched genes CDIPT, LPCAT3, and ALG12 and the organophosphate metabolic process also had TPI1 enriched. Phospholipids are not just structural components of the cell membrane but are critical to such functions as second messenger molecules, membrane receptors for the recruitment of specific proteins, chaperones to aid in protein folding, and modulators of protein function (McMaster and Jackson, 2004). Thus an up regulation of phospholipid synthesis is an indirect indicator of modified functionality of liver cells.

PAGE 264

264 Regulation of Carbohydrate Metabolism One of the main roles of activated PPAR is to upregulate genes that increase synthesis of glucose during fasting conditions (Rakhshandehroo et al., 2009). Based on the DEG upregulated genes detected in the current study, neither prepartum diets nor MR nor the interaction between them up regulat ed expression of gluconeogenic genes (Appendices 1, 2, 3, 4, and 5). Increased need for gluconeogenesis due to fasting conditions in calves of the current study was not expected to occur under the feeding regimen used for calves in this study. When mice we re fasted for 12, 24, 48, or 72 h, genes that code for enzymes aiding in the production of energy in the early fasting period were upregulated, but gluconeogenesis per se was not initiated until after prolonged fasting (Sokolovic et al., 2008). Calves in t he current study were experiencing constant growth and appropriate feeding conditions thus there would have been little to no utilization of aminoacids for potential synthesis of glucose via gluconeogenesis. However, since the main source of carbohydrate i disaccharide composed by 1 mole of glucose and 1 mole of galactose, an enhancement in the mechanism of galactose isomerization was logical and perhaps needed. Liver genes of calves within the interaction groups of FAT by MR an d FA by MR experienced an up regulatory effect on expression of several genes involved in galactose metabolism to conversion into glucose (Tables 5 4 and 5 8). It is possible that calves fed HLA and born from dams fed SFA or EFA were having a more efficient conversion of galactose into glucose. However, the overall better response of calves fed HLA MR in regard to ADG and feed efficiency was not affected by the supplemental fat when compared to control diets (no FAT by MR interaction; Chapter 4).

PAGE 265

265 Regulation of Protein Turnover Degradation of proteins occurs through the ubiquitin proteasome pathway and involves the following two successive steps:1) tagging of the substrate by covalent attachment of multiple ubiquitin molecules and 2) degradation of the tagged protein by the 26S proteasome complex with release of free and reusable ubiquitin (Glickman and Ciechanover, 2002). The roles of protein ubiquitination include intra cellular controls over a wide range of biological processes including: protein degradation DNA repair, endocytosis, autophagy, transcription, immunity, and inflammation (Husnjak and Dikic, 2012). Thus, a tight regulation of ubiquitinization processes will ensure appropriate balance between degradation and maintenance of activity of many active proteins within cells. Regulation of ubiquitin mediated protein degradation can happen at any point of the three enzymatic reactions occurring in the cascade via up down regulation of any of the several enzymes of the cascade (Gao and Karin, 2005). Calves fed HLA instead of LLA MR and born either from dams fed fat (interaction FAT by MR, genes: SOCS1, UBA7, PML, HERC4, and BIRC3) or from dams fed EFA (interaction FA by MR, genes: CUL3, KLHL9, ITCH, and BIRC3) had a different set of downreg ulated genes codi ng for enzymes involved in the activation of ubiquitin mediated proteolysis in one of the three enzymatic reactions (Glickman and Ciechanover, 2002). Massive degradation of sk eletal muscle proteins could up regulate the activity of the ubiquitin proteosome pathway. Calves in this study did not undergo prolonged fasting periods (2 feedings per day). In addition, instead of degradation of muscle they were under muscle accretion conditions (Chapter 4). Calves in the current study had greater BW gain and plasmat ic IGF I concentrations at least in calves fed HLA MR ( P < 0.08, Table 4 6) regardless of

PAGE 266

266 the diet fed prepartum. Hence it should be expected that ubiq uitinization of proteins, by up regulation of its coding genes, should not be of high activity. An interes ting gene, USP 2, was found to be up and down regulated by the interactions FAT by MR and FA by MR, respectively (Appendices 5 5 and 5 6). The differential regulation of this gene is primarily due to the greater up regulation (greater mean expression value) in liver of calves fed HLA and born from dams fed SFA diets (Appendix 1). The USP2, is another proteolytic enzyme that has been found over expressed in human prostate cancer and has been associated to increase the half life of FASN, an enzyme associated w ith the malignancy of aggressive prostate cancer (Renatus et al., 2006). Metzig et al. (2011) documented that downreg ulation of USP2 inhibited TNF / NF k B signaling, hence reducing the risk of inflammation. The current finding that calves fed HLA MR and b orn from dams fed EFA instead of SFA were able to downreg ulate the expression of USP2 might mean that these calves had an improved ability to cope with inflammatory processes. Generally LA is considered a proinflammatory FA compared with ALA or other n 3 F A (Calder, 2006; Whelan, 2008; Weaver et al., 2009) However the current finding indicates that when compared to SFA supplementation, supplementation of LA during the prepartum and preweaning periods could prevent excessive inflammatory processes. Regulati on of Inflammation and other immune processes Ubiquitinization of proteins can modify the activity of immune cells or immune metabolites as it clearly alters gene expression of USP2, potentially leading to the downreg ulation of the TNF ich is a critical pathway enhancing inflammatory conditions (Harhaj and Dixit, 2012). Although n 6 FA are mostly considered proinflammatory FA, some studies have reported that n 6 FA also can have

PAGE 267

267 antinflammatory activities (Fritsche, 2008; Bjermo et al., 2012). The principal mechanism by which the inflammatory response is implemented is through activation of NFkB transcription factor, the key mediator of the inflammatory response (Weaver et al., 2009) Other mechanisms may include cessation of neutrophil re cruitment by reduction of migration and increased apoptosis of neutrophils and other leukocytes (Lawrence et al., 2002). from calves born from cows fed dams fed fat (contras t FAT), regardless of the subsequent MR fed, were downreg ulated (Table 5 9). One gene was ICAM1, a gene that directly regulates leukocyte migration, as it is an intracellular adhesion molecule critical to moving leukocytes from the circulation and allowing transmigration into the infected tissue for subsequent phagocytic activity (Lawson and Wolf, 2009). On the other hand, MYL2 and ACTN2 are involved in structural support of the leukocyte, by formation of cytoskeleton, regulation of leukocyte movement allow ing the leukocyte to move forward and finally enhancing migration (Sanchez Madrid and Del Pozo, 1999). Leukocyte migration to infected or damaged tissues is a necessary process to aid in the healing of cellular damage from pathogens. Liver has a high deman d for leukocytes to migrate into hepatocytes and help fight potential microbial infections and tissue trauma. However an excessive migration of leukocytes to hepatocytes could be detrimental and increase hepatocyte damage leading to chronic liver injury (J aeschke, 2006). Although liver was examined at only one point in time (30 d of age), it cannot be ruled out that a ability of leukocytes to migrate into the hepatocytes was reduced over a long period of time and could negatively impact the stability of the hepatic tissue.

PAGE 268

268 The apparent mechanism of the downregulated response of leukocyte migration in calves born from dams fed fat (contrast FAT) is not clear. It would be expected that only calves born from dams fed EFA, regardless of the MR fed (contrast FA) would have a better and more effective resolution of inflammation, leading to a downregulation of inflammatory mechanisms, than calves born from dams fed SFA. This assumption is based on the mechanism that PUFA (preferentially n 3 followed by n 6 FA) can induce the inactivation of the TNF apparently downreg ulated in calves fed HLA but only when born from EFA fed and not SFA fed dams (interaction FA by MR) as presented in a previous section. However, a re cent study (Bjermo et al, 2012) fed obese subjects with supplements rich in SFA or PUFA reported no differential expression of inflammatory and oxidative stress genes, which might indicate that both sources of FA had similar regulatory effect on expression of genes within the inflammatory process. Based on the down regulation of UPS2 as evidenced in the FA by MR interaction, increased activation of the TNF controlled inflammatory processes. Another mechanism could be the down regulation of leukocyte transendothelial migration; however this mechanism was not directly influenced by the interaction of FA by MR but only affected in calves born from dams fed fat regardless of the MR fed (contrast FAT). However, in addition to UPS2 which was exclusively downregulated by the interaction FA by MR, 3 additional DEG were downregulated (Appendix 6) but not enriched i n any GO term or KEGG pathway. This might indicate that calves of this interaction were able to better resolve inflammation. The genes were BCL10, CASP3, and ITCH. The BCL10 gene encodes the B cell

PAGE 269

269 lymphoma 10. An over expressed BCL10 induces a constitutiv e activation of the NFkB / JNK resulting in the over activation, differentiation, and proliferation of specific T and B cells (Thome, 2004). The CASP3 gene when up regulated is a potent inducer of apoptosis of immune cells such as lymphocytes. An upregulat ed CASP3 could be detrimental to lymphocyte function during sepsis conditions and result in death (Hotchkiss et al., 2000). An over expressed ITCH gene was reported to inhibit TNF mediated NFkB mice cells (Shembade et al., 2008). The listed functions of these 3 genes appear to be antagonistic; the BCL10 (+) and ITCH ( ) both have roles in activation of NFkB but in different directions; whereas the CASP3 by being downregulated prevented the excessive apoptosis of leukocytes that could prevent them from per forming under inflammatory processes. Under the circumstances of the current study and considering that LA is well known to have proinflammatory effects, these genes acting in different ways to resolve inflammatory processes confirm our aforementioned hypo thesis of a potential greater ability of calves fed HLA and born from dams fed EFA (interaction FA by MR) to resolve inflammation. Feeding of Essential Fatty Acids Prepartum and High Linoleic Acid in Milk Replacer Improved Insulin Sensitivity Adipocytokine s are soluble factors namely cytokines which are produced by the adipose tissue. The most common adipocytokines are adiponectin, leptin, resistin, and visfatin, all of which have important roles in regulating insulin resistance (Tilg and Moschen, 2006). Ad iponectin prevents insulin resistance, acting intracellularly, by binding to its receptor, ADIPOR2, which is the most abundant receptor of adiponectin in liver tissue (Kadowaki and Yamauchi, 2005). Although adiponectin per se was not up regulated, the expre ssion of its rec eptor, ADIPOR2 was certainly up regulated in

PAGE 270

270 calves fed HLA MR and born from dams fed EFA (interaction FA by MR, Table 5 8). Consequently, it can be postulated that calves of this interaction were less likely to develop insulin resistance. T he mechanisms by which adiponectin performs an insulin sensitizing action have been discovered recently. One mechanism is by activating AMPK, thus downreg ulating the expression of gluconeogenic genes (Kadowaki et al., 2006). In fact, calves in this group h ave the KEGG glycolysis pathway upregulated with five genes (ALDOA, TPI1, GALM, PGM1, and ENO1, Table 5 8) as well as five genes in the BP of glycolysis (3 shared with the KEGG pathway ALDOA, TPI1, and ENO1 and 2 different genes OGDH and MDH2, Table 5 4). Unfortunately some key genes regulating glycolysis (phosphofructokinase and piruvate kinase) or gluconeogenesis ( phosphoenolpyruvate carboxykinase and glucose 6 phosphatase ) were not up or downregulated respectively (Appendix 6), which could have pro vided a clearer picture about the prevalence of glycolysis or gluconeogenesis. Another postulated mechanism of adiponectin sensitizing insulin is via increased oxidation and energy consumption, in part via PPAR (Kadowaki et al., 2006). However, regarding fat content in liver, similar concentrations of total FA were found in calves fed MR if they were born from dams fed SFA or EFA (interaction FA by MR), which might indicate that the most probably mechanism of .insulin sensitization was though reduction of gluconeogenesis rather than change in the proportion of FA in liver by enhancing their oxida tion. Another up regulated gene in this adipocytokine signaling pathway was STAT3 (Table 5 8). This gene can inhibit SREBP 1c promoter activity. By inhibiting the expression of SREBP I, the synthesis of FA may be reduced thus preventing steatosis

PAGE 271

271 and dysl ipidemia, hence reducing the risk of insulin resistance (Ueki et al ., 2004). In addition to the up regulation of STAT3, SOCS6 was downreg ulated by the interaction of FA by MR (Appendix 5 6), although it was not enriched in any GO term or KEGG pathway. The S OCS6 gene has been reported to reduce the active form of STAT3 protein (Hwang et al., 2007). Therefore the down regulation of SOCS6 gene could be associated with the increased expression of STAT 3 which would support the reduced risk of insulin resistance in calves born from cows fed EFA and supplemented with HLA MR. Fat and Fatty Acid Supplementation and its Risk and Prevention of Cardiomyopathic Diseases The sarcomere is the fundamental unit of cardiac and skeletal muscle contraction. Recent studies have identified mutations in genes coding for these proteins as the main drivers of different cardiomyopathic disorders (Tajsharghi 2008). The four identified up regulated genes in calves fed HLA MR coding for sarcomeric proteins related to hypertonic cardiomy opathy and dilated cardiomyopathy (Table 5 8) indicate a potential accumulation of those proteins which have been indicated as one of the reasons for incidence of myopathy (Fielitz et al., 2007). Mutation of sarcomeric genes are one of the most common etio logies for cardiomyopathic diseases (Probst et al., 2011), and this mutation is commonly accompanied by an over expression of the up regulated genes found in liver of calves fed HLA MR. However, the microarray analysis does not indicate whether a gene has m utated. Additional work would be required to verify gene mutation. Bovine dilated cardiomyopathy is a terminal myocardial disease with common age at onset between 2 4 years (Owczarek Lipska et al., 2011). Heifers in this study (n = 56) were followed throu ghout their first 45 mo of life. Only 1 death was reported due to

PAGE 272

272 endocarditis and that was fo r a calf not in the group of up regulated genes for cardiomyopathies. Feeding HLA instead of LA MR (contrast MR) also up regulated genes from the tight junction pa thway (Table 5 8) as well as genes from BP and MF related to actin and calcium binding, as well as striated muscle tissue development (Table 5 2). Cardiac and skeletal muscle contractions are regulated by calcium dependent interactions with the thick and t hin filaments of tropomyosin and troponin of sarcomeric proteins. Thus, when intracellular calcium concentrations increase, it binds to troponin C resulting in regulation of muscle con traction (Lee et al., 2010). Up regulation of calcium binding might be a result of a change in its sensitivity to troponin C. Karibe et al. (2001) reported that a mutation of tropomyosin modified the affinity to calcium. The tight junction pathway is responsible for regulating the paracellular movement of Ca, ions and solutes b etween cells (Hartsock and Nelson, 2008). Some genes of the tight junction pathway coding for sarcomeric proteins also were up regulated The up regulation of this pathway could potentially increase the risk of heart disease, but as stated early, heart probl ems were not reported in heifers fed HLA MR throughout their first 45 months of age. Perhaps the increased gene expression in the liver of tight junction responses associated with feeding of HLA MR contributed to a greater cardiac function if also expresse d in the heart (not determined in the present study). This may be associated with increased milk production in the first lactation due to increased cardiac output and blood flow to mammary gland In the previous section, the up regulation of sarcomeric genes due to feeding HLA MR was discussed. Interestingly, these same genes were downreg ulated in livers of

PAGE 273

273 calves born from dams fed fat but only if the calves were fed HLA. as well as have down regulated pathways related to this condition such as tight junctio n, including BP and MF such as actin and calcium binding. The reason for this interaction is unclear. It can be hypothesized that a potential fetal programming may have occurred in this group of calves born from cows fed fat prepartum, which may pre condit ion the calves in this group to respond differentially to either high or low levels of LA in the preweaned diet. Feeding increased amounts of fat, primarily saturated fat, has been reported to induce cardiomyopathies in obese mice (Fang et al., 2008). Howe ver, feeding PUFA, primarily n 3 FA, reduced the risk of cardiomyopathies in mice (Takahashi et al., 2005). A recent study reported that feeding FO to sheep induced cardiac dysfunction after infusion of doxorubicin, as displayed by a greater level of ventr icular dilatation compared with placebo sheep (Carbone et al., 2012). The aforementioned studies have led to different conclusions regarding the influence of fat on risk of cardiac problems. However the most common postulation is that PUFA have a protectiv e effect, hence it is not clear why calves fed HLA instead of LLA MR (contrast MR) had a potential increased risk of cardiac problems thorough up regulation of some genes involved in these pathogenesis. Current results warrant further investigation of under standing potential interactions of prenatal dam diets with neonatal diets of the newborn on subsequent development and metabolic/endocrine regulation of productivity and health traits. Prepartum Fat Feeding Influenced Future Adult Performance Late gestat ion and preweaning periods have been identified as two of the most critical periods during which nutritional management could have long term effects in future offspring performance (Fowden et al., 2006). Studies conducted using humans have documented a det rimental effect on birth weight and health of offspring born from

PAGE 274

274 undernourished women (Barker, 1997; Pettitt et al., 1987). In the current study calves fed the HLA MR during the preweaning period had a marked improved performance with a greater body weigh t gain and feed efficiency (Chapter 4). However, increased intake of LA via MR had no effect on all post pubertal measures of production and reproduction, with the exception of a trend for greater concentration of milk lactose when calves were fed HLA MR. There was a numerical difference of 552 kg of mature equivalent milk for heifers fed HLA MR. Soberon et al. (2012) recently reported that every 1 kg increase in ADG by heifers during the preweaning period resulted in an additional 850 kg of mature equivale nt milk during the first lactation. In the current study, heifers born from dams fed SFA had better ADG during the preweaning period than heifers born from dams fed EFA (Chapter 4). This advantage in BW gain due to prepartum fat type did not translate into better lactation performance. In fact, heifers from dams fed EFA had numerically greater (517 kg) mature equivalent milk. The most significant change in milk yield was observed as result of supplementing fat prepartum, regardless of the type of FA provide d (even though supplementing fat prepartum did not influence the performance of heifers during the preweaning period, Chapter 4). Heifers fed fat prepartum resulted in the most dramatic increase in milk yield at first lactation, producing ~13% more milk th an heifers born from dams not supplemented with fat. Because these same heifers conceived later, they were ~45 d older and 36 kg heavier at calving. Since heavier heifers can consume more feed DM, milk yield may have been increased partly due to greater f eed intake based on body size but it is unlikely that an additional 1400 kg of milk (4.6 kg/d in a 305 d lactation period) would be

PAGE 275

275 produced by heifers that have a 36 kg BW advantage. Moreover, the mature equivalent milk is corrected by age and BW at calvi ng. Most of the first studies documented that additional intake of nutrients during the prepubertal period had a negative impact on future milk production (Foldager and Sejrsen, 1987). More recent studies have documented increased intake of nutrients durin g the preweaning period improved future milk production [Shamay et al., 2005 (n = 40, 5.1% increase in milk); Moallem et al., 2010 (n = 46, 10.3% increase in milk); Soberon et al., 2012 (1244 kg);]. However, in the current study, heifers did not have an in creased intake of nutrients as heifers from all other studies, but ADG was improved during the preweaning period die to feeding of HLA MR. Feeding of fat to dams during prepartum, increased milk production of their calves during their subsequent lactation as first calf heifers. Although not significant, heifers fed HLA MR during the first 60 d of life had a non significant 4.9% greater milk production as first calf heifers. The increased number of inseminations to initiate a first pregnancy and the greater milk production during first lactation due to fat feeding of the dam in late gestation on subsequent heifer performance suggests some alteration in neonatal programming that influences subsequent heifer performance. Certainly future studies should focus on the potential long lasting effect of prepartum dam diets and postnatal calf diets on programming subsequent heifer performance reproductive and lactational performances. Summary Supplementing greater amounts of LA and ALA during the prepartum and prewea ning periods modified the response of liver to different metabolic processes. This differential profile of liver FA might have modified the activity of the liver regarding

PAGE 276

276 expression of hepatic genes. Similarity of liver dietary FA profile, depended more o n the MR. Greater effect of MR were verified by the increased proportions of C12:0 and C14:0 in calves fed a MR formulated with CCO, whereas calves supplemented with porcine had liver with greater proportions of LA and three of its derivative FA. The analy sis of representative genes within a cluster or metabolic pathway resulted d in fewer enriched genes due to prepartum diets and MR as compared to the amount of enriched genes obtained by the interaction of prepartum diets and MR. The DEG identified in all preplanned interaction were related to processes such as lipid and carbohydrate metabolism, protein metabolism, and inflammatory processes. Polyunsaturated FA, such as LA, are potent ligands of PPAR mechanism FA can exert their function in different metabolic processes. C alves fed MR containing porcine lard, regardless of prepartum diets, enhanced the expression of PPARA gene and two PPAR lipolytic effects. Howeve r greater lipolytic effect of prepartum diets was observed in liver of calves fed MR containing porcine lard and born from dams fed fat instead control diet. Calves of this group had 6 upregulated genes, targeted for activation by the PPAR ame calves had upregulated other group of genes involved in FA metabolism, glycerolipid metabolism and AA metabolism. The up regulation of genes in all aforementioned pathways might indicate that these calves were undergoing a preferential degradation of li pids. Interestingly, the key enzymes in the gluconeogenic pathway were neither upregulated by prepartum diets nor by MR or their combined effect. The different profile of FA provided prepartum also affected the expression of genes in liver of calves fed a particular MR. Calves fed porcine lard and born from dams

PAGE 277

277 fed EFA instead of SFA had up regulated genes involved in glycolysis and oxidative phosphorylation. Although increased oxidative phosphorylation could negatively impact the liver by excessive generat ion of free radicals. Calves in this group had more down regulated genes involved in inflammatory response as compared to the other four preplanned contrasts. This effect could have a positive impact limiting exaggerated inflammatory response that could ne gatively impact liver function. However, a potential attenuated inflammatory response, which could negatively impact calf survival, could not be ruled out. A long term effect of preweaning diets on performance of heifers at first lactation, regardless its considerable impact in liver gene expression at 30 d of calf age was not apparent. However the effect of prepartum diets appeared to impact more dramatically the future performance of heifers. Heifers born from dams supplemented with fat had ~13% greater m ilk production at first lactation compared to those born from dams not supplemented with fat. Other studies have reported positive impact of improved ADG during the preweaning on future milk production. In the current study a numerical increase of 5.3% in milk production was observed for calves fed MR containing porcine lard instead of CCO. Findings in this study reveal a strong effect of prepartum diet during the fetal period to modify the response of calves to strategic supplementation of FA during the p reweaning period. However, the greater long term effect of prepartum diets versus preweaning diet, might indicate that the most critical period of programming effect of diets occurs during the late gestation rather than the preweaning period. Future resear ch should focus on detailing the mechanisms by which strategic lipid

PAGE 278

278 supplementation actually modifies the production and activity of proteins encoded for the DEG. Moreover, more efforts should be attained to evaluate different nutritional strategies duri ng the late gestation period that would positively impact the future performance of dairy cattle.

PAGE 279

279 Table 5 1 Mean concentrati on of liver fatty acid (FA, g of FA/100g of total FA) of Holstein male calves fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Calves were born from dams fed diets supplemented with no fat (Control), saturated fatty acids (SFA), and essential fatty acid (EFA) Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR Fatty acid Milk replacer (MR) 2 LLA HLA LLA HLA LLA HLA Total FA, % DM 8.49 6.76 8.6 7.85 8.32 8.06 0.44 0.13 0.94 0.02 0.12 0.59 C12:0 1.25 0.36 1.25 0.25 1.21 0.27 0.20 0.74 0.94 <0.01 0.81 0.88 C14:0 5.29 1.52 5.08 1.1 5 5.28 1.24 0.71 0.72 0.84 <0.01 0.86 0.94 C16:0 16.76 14.05 16.36 14.09 16.31 13.56 0.57 0.51 0.63 <0.01 0.8 0.68 C16:1 c 9 0.50 0.43 0.48 0.39 0.45 0.36 0.04 0.26 0.53 0.02 0.84 0.98 C17:0 0.38 0.39 0.40 0.43 0.46 0.41 0.53 0.62 0.40 0.05 0.76 0.23 C1 8:0 20.48 22.69 21.84 23.82 21.68 24.10 0.94 0.13 0.95 0.01 1.00 0.82 C18:1 t 6 8 0.02 0.01 0.05 0.02 0.02 0.02 0.01 0.39 0.26 0.24 0.74 0.26 C18:1 t 9 0.07 0.07 0.10 0.07 0.06 0.06 0.01 0.96 0.10 0.47 0.60 0.21 C18:1 t 10 0.13 0.08 0.15 0.11 0.14 0.18 0.0 6 0.42 0.68 0.73 0.67 0.55 C18:1 t 11 0.19 0.16 0.21 0.20 0.22 0.24 0.04 0.19 0.55 0.85 0.69 0.68 C18:1 t 12 0.09 0.09 0.10 0.10 0.10 0.13 0.01 0.08 0.25 0.31 0.30 0.08 C18:1 c 9 12.37 10.94 12.33 10.00 11.47 9.98 0.60 0.17 0.48 <0.01 0.64 0.50 C18:1 c 11 2.62 2.67 2.57 2.61 2.38 2.57 0.09 0.17 0.21 0.21 0.67 0.40 C18:2 n 6 15.87 23.00 15.20 22.04 16.57 21.27 0.74 0.30 0.70 <0.01 0.30 0.17 C18:3 n 6 0.07 0.03 0.08 0.01 0.05 0.04 0.05 0.62 0.97 <0.01 0.77 0.05 C18:3 n 3 0.74 1.04 0.65 1.03 0.71 0.91 0.04 0.05 0.41 <0.01 0.87 0.04 CLA 9c, t 11 0.04 0.02 0.02 0.02 0.04 0.03 0.01 0.45 0.13 0.15 0.71 0.74 C20:2 n 6 0.56 1.05 0.50 1.00 0.56 0.99 0.04 0.23 0.58 <0.01 0.76 0.50 C20:3 n 6 3.13 2.53 3.78 2.48 3.17 3.09 0.28 0.22 1.00 0.01 0.84 0.04 C20:4 n 6 9.94 10.02 10.09 10.84 10.48 11.49 0.43 0.05 0.24 0.09 0.28 0.76 C20:5 n 3 0.21 0.16 0.30 0.18 0.22 0.22 0.02 <0.01 0.28 <0.01 0.65 <0.01 C22:4 n 6 1.20 1.24 0.96 1.28 1.23 1.30 0.08 0.68 0.07 0.03 0.27 0.12 C22:5 n 3 1.47 1.92 1.59 2.09 1.64 2.16 0.08 0.02 0.52 <0.01 0.64 0.90 C22:6 n 3 1.51 1.61 2.05 1.84 1.34 1.90 0.16 0.13 0.06 0.27 0.77 0.03 3.93 2.73 2.52 3.90 3.70 2.32 0.29 0.38 0.50 <0.01 0.72 0.99

PAGE 280

280 Table 5 1 Continued Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR Fatty acid Milk replacer (MR) 2 LLA HLA LLA HLA LLA HLA 2.82 1.94 2.13 1.66 2.24 1.84 0.46 0.30 0.75 0.47 0.25 0.36 44.64 39.68 45.38 40.32 45.37 40.00 0.66 0.29 0.80 <0.01 0.82 0.82 cis 16.96 15.12 16.66 14.00 15.57 13.88 0.79 0.14 0.46 <0.01 0.81 0.55 cis 34.94 42.75 35.53 42.92 36.13 43.50 1.11 0.48 0.61 <0.01 0.82 1.00 4 0.04 0.02 0.02 0.02 0.04 0.03 0.01 0.45 0.13 0.15 0.71 0.74 trans 0.09 0.07 0.09 0.11 0.10 0.07 0.01 0.28 0.37 0.12 0.98 0.83 trans 0.52 0.43 0.65 0.53 0.57 0.68 0.13 0.24 0.80 0.77 0.72 0.41 n 3 3.97 4.8 4.61 5.21 3.93 5.24 0.23 0.07 0.17 <0.01 0.75 0.14 6 30.76 37.88 30.62 37.64 32.05 38.19 1.03 0.73 0.36 <0.01 0.76 0.68 1 Control = no fat supplemented; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & D wight, Princeton, NJ). 2 LLA= 0.175 g LA/BW 0.75 HLA=.562 g LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30d of life to provide 6.72 g fat / kg BW 0.75 3 P values for orthogonal contrasts and interactions; FAT: contrast of dam diet (SF A+EFA) vs. control,; FA: contrast of dam EFA vs. SFA; MR= milk replacer 4 Concentration of CLA t 10, c 12 were 0 for all treatments.

PAGE 281

281 Table 5 2 Functional annotation clusters for main effects of up regulated enriched GO terms in liver of Hols tein male calves fed milk replacer containing low or high linoleic acid from 1 to 30 days of age. Calves were born from dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Cluster 2 # GO 3 Term Fold E 4 Count 5 P value 6 Contrast FA 7 1 (ES = 1.44) BP_GO:0045934 negative regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 6.8 4 0.019 BP_GO:0016481 negative regulation of transcription 5.5 3 0.098 Contrast MR 8 1 ( ES = 2.81) MF _GO:0005509 calcium ion binding 4.5 7 0.003 2 (ES = 2.39) MF_GO:0003779 actin binding 10.0 4 0.006 BP_GO:0014706 striated muscle tissue development 21.3 3 0.008 MF_GO:0003774 motor activity 9.4 3 0.037 3 (ES = 1.23) MF_GO:0043169 cation binding 2.1 1 5 0.002 MF_GO:0005509 calcium ion binding 4.5 7 0.003 BP_GO:0051603 proteolysis involved in cellular protein catabolic process 5.6 3 0.090 1 Calves were fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. 2 Clustering of GO terms using the DAVID functional annotation cl ustering option. Table presents the most relevant GO terms within each cluster. Each cluster shows the group enrichment score (ES= log 10 scale) that represents the geometric mean of all P values of each annotation term in the group. 3 Gene ontology codes prefix BP = biological process and MF= molecular function. The GO terms cellular component and the detail of each GO terms is present in supplemental material. 4 Fold enrichment of each GO term within a cluster. 5 The gene members which belong to an annot ation term. Some genes can be repeated in different GO terms within each cluster. 6 Fisher exact P value. 7 Main effect of FA: Effect of feeding EFA prepartum with SFA diet as reference (Contrast FA). 8 Main effect of MR: Effect of feeding HLA milk replace r to newborn calves with LLA milk replacer as reference (Contrast MR).

PAGE 282

282 Table 5 3 Functional annotation clusters for the interac tion fat by milk replacer of up regulated enriched GO terms in liver of Holstein male calves fed milk replacer containing low or high linoleic acid from 1 to 30 days of age. Calves were born from dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Cluster 2 # GO 3 Term Fold E 4 Count 5 P value 6 Interaction FAT by MR 7 1 (ES = 2.88) MF_GO: 0009055 electron carrier activity 8.6 13 0.000 BP_GO:0055114 oxidation reduction 3.7 22 0.000 MF_GO:0005506 iron ion binding 4.5 11 0.000 2 (ES = 2.78) BP_GO:0055085 transmembrane transport 2.0 9 0.078 3 (ES = 2.54) MF_GO:0051287 NAD or NADH binding 11.8 4 0.004 4 (ES 1.84) BP_GO:0006732 coenzyme metabolic process 5.5 6 0.004 BP_GO:0019362 pyridine nucleotide metabolic process 13.6 3 0.020 5 (ES = 1.78) BP_GO:0042364 water soluble vitamin biosynthetic process 15.2 3 0.016 6 (ES = 1.68) BP_GO:0006 869 lipid transport 5.6 5 0.012 7 (ES = 1.49) BP_GO:0005996 monosaccharide metabolic process 4.2 6 0.014 1 Calves were fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calv es were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk be fore expected calving date. 2 Clustering of GO terms using the DAVID functional annotation clustering option. Table presents the m ost relevant GO terms within each cluster. Each cluster shows the group enrichment score (ES= log 10 scale) that represents the geometric mean of all P values of each annotation term in the group. 3 Gene ontology codes prefix BP = biological process and M F= molecular function. The GO terms cellular component and the detail of each GO terms is present in supplemental material. 4 Fold enrichment of each GO term within a cluster. 5 The gene members which belong to an annotation term. Some genes can be repeate d in different GO terms within each cluster. 6 Fisher exact P value. 7 Interaction fat by milk replacer: ( [(SFA HLA + EFA HLA) /2 : Control HLA (reference)] [(SFA LLA + EFA LLA) /2 : Control LLA (reference)]

PAGE 283

283 Table 5 4 Functional annotation clust ers for the interaction fa tty acid by milk replacer of up regulated enriched GO terms in liver of Holstein male calves fed milk replacer containing low or high linoleic acid from 1 to 30 days of age. Calves were born from dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Cluster 2 # GO 3 Term Fold E 4 Count 5 P value 6 Interaction FA by MR 7 1 (ES = 2.87) BP_GO:0006091 generation of precursor metabolites and energy 6.1 10 0.000 BP_GO:0006096 glycolysis 17.0 5 0.000 BP_GO:0046164 alcohol catabolic process 11.1 5 0.001 2 (ES = 1.22) BP_GO:0008654 phospholipid biosynthetic process 9.0 3 0.042 BP_GO:0019637 organophosphate metabolic process 4.9 4 0.047 3 (ES = 1.21) BP_GO:0006461 protein complex assembly 4.3 6 0.0 12 1 Calves were fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calv es were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (E FA) starting at 8 wk before expected calving date. 2 Clustering of GO terms using the DAVID functional annotation clustering option. Table presents the most relevant GO terms wit hin each cluster. Each cluster shows the group enrichment score (ES= log 10 s cale) that represents the geometric mean of all P values of each annotation term in the group. 3 Gene ontology codes prefix BP = biological process and MF= molecular function. The GO terms cellular component and the detail of each GO terms is present in su pplemental material. 4 Fold enrichment of each GO term within a cluster. 5 The gene members which belong to an annotation term. Some genes can be repeated in different GO terms within each cluster. 6 Fisher exact P value. 7 Interaction fatty acid by milk r eplacer: [EFA HLA : SFA HLA (reference)] [EFA LLA : SFA LLA (reference)]

PAGE 284

284 Table 5 5 Functional annotation cl usters for main effects of down regulated enriched GO terms in liver of Holstein male calves fed milk replacer containing low or high linol eic acid from 1 to 30 days of age. Calves were born from dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Cluster 2 # GO 3 Term Fold E 4 Count 5 P value 6 Contrast FAT 7 1 (ES = 2.44) MF_GO:0003779 actin binding 14.2 4 0.002 BP_GO:0014706 striated muscle tissue development 20.3 3 0.008 MF_GO:0003774 motor activity 13.4 3 0.019 2 (ES = 2.29) MF_GO:0005509 calcium ion binding 4.6 5 0.017 Contrast FA 8 1 (ES = 1.19) BP_GO:0051603 proteolysis involved in ce llular protein catabolic process 5.9 3 0.081 BP_GO:0044257 cellular protein catabolic process 5.9 3 0.082 Contrast MR 9 1 (ES = 1.08) MF_GO:0005506 iron ion binding 11.9 3 0.020 BP_GO:0055114 oxidation reduction 5.3 4 0.029 1 Calves were fed mil k replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk befo re expected calving date. 2 Clustering of GO terms using the DAVID functional annotation clustering option. Table presents the most relevant GO terms wit hin each cluster. Each cluster shows the group enrichment score (ES= log 10 scale) that represents th e geometric mean of all P values of each annotation term in the group. 3 Gene ontology codes prefix BP = biological process and MF= molecular function. The GO terms cellular component and the detail of each GO terms is present in supplemental material. 4 F old enrichment of each GO term within a cluster. 5 The gene members which belong to an annotation term. Some genes can be repeated in different GO terms within each cluster. 6 Fisher exact P value. 7 Main effect of FAT: Effect of feeding fat prepartum (SFA + EFA) /2 with control diet as reference (contrast FAT). 8 Main effect of FA: Effect of feeding EFA prepartum with SFA diet as reference (contrast FA). 9 Main effect of MR: Effect of feeding HLA milk replacer to newborn calves with LLA milk replacer as ref erence (contrast MR).

PAGE 285

285 Table 5 6 Functional annotation clusters for the interaction fat by milk replacer of down regulated enriched GO terms in liver of Holstein male calves fed milk replacer containing low or high linoleic acid from 1 to 30 days of age. Calves were born from dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Cluster 2 # GO 3 Term Fold E 4 Count 5 P value 6 Interaction FAT by MR 7 1 (ES = 2.62) MF_GO:0003779 actin binding 5.4 5 0.013 BP_GO:001470 6 striated muscle tissue development 8.4 3 0.047 2 (ES = 1.75) MF_GO:0030554 adenyl nucleotide binding 1.9 14 0.022 MF_GO:0005524 ATP binding 1.9 13 0.032 3 (ES = 1.66) BP_GO:0003007 heart morphogenesis 18.9 3 0.010 4 (ES = 1.41) MF_GO:0005509 calcium ion binding 1.4 5 0.463 1 Calves were fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calv es were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or es sential fatty acids (EFA) starting at 8 wk before expected calving date. 2 Clustering of GO terms using the DAVID functional annotation clustering option. Table presents the most relevant GO terms wit hin each cluster. Each cluster shows the group enrichmen t score (ES= log 10 scale) that represents the geometric mean of all P values of each annotation term in the group. 3 Gene ontology codes prefix BP = biological process and MF= molecular function. The GO terms cellular component and the detail of each GO terms is present in supplemental material. 4 Fold enrichment of each GO term within a cluster. 5 The gene members which belong to an annotation term. Some genes can be repeated in different GO terms within each cluster. 6 Fisher exact P value. 7 Interactio n fat by milk replacer: ( [(SFA HLA + EFA HLA) /2 : Control HLA (reference)] [(SFA LLA + EFA LLA) /2 : Control LLA (reference)]

PAGE 286

286 Table 5 7 Functional annotation clusters for the interaction fatty a cid by milk replacer of down regulated enriched GO ter ms in liver of Holstein male calves fed milk replacer containing low or high linoleic acid from 1 to 30 days of age. Calves were born from dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Cluster 2 # GO 3 Term Fold E 4 Count 5 P value 6 Interaction FA by MR 7 1 (ES = 1.97) BP_GO:0051603 proteolysis involved in cellular protein catabolic process 4.4 8 0.002 BP_GO:0006511 ubiquitin dependent protein catabolic process 7.1 5 0.005 MF_GO:0070011 peptidase activity, acting on L amino acid peptides 3.1 9 0.008 MF_GO:0004221 ubiquitin thiolesterase activity 10.0 3 0.035 2 (ES = 1.01) BP_GO:0007179 transforming growth factor beta receptor signaling pathway 19.6 3 0.010 BP_GO:0007178 transmembrane receptor protein se rine/threonine kinase signaling pathway 9.8 3 0.036 3 (ES = 0.97) BP_GO:0050863 regulation of T cell activation 6.5 3 0.075 BP_GO:0051249 regulation of lymphocyte activation 5.4 3 0.103 1 Calves were fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. 2 Clustering of GO terms using the DAVID functional annotation clustering option. Table presents the most relevant GO terms within each cluster. Each cluster shows the group enrichment score (ES= log 10 scale) that represents the geometric mean of all P values of each annot ation term in the group. 3 Gene ontology codes prefix BP = biological process and MF= molecular function. The GO terms cellular component and the detail of each GO terms is present in supplemental material. 4 Fold enrichment of each GO term within a cluste r. 5 The gene members which belong to an annotation term. Some genes can be repeated in different GO terms within each cluster. 6 Fisher exact P value. 7 Interaction fat ty acid by milk replacer: [EFA HLA : SFA HLA (reference)] [EFA LLA : SFA LLA (refere nce)]

PAGE 287

28 7 Table 5 8 Functional annotati on chart for enriched up regulated KEGG pathways for main factors and interactions in liver of Holstein male calves fed milk replacer containing low or high linoleic acid from 1 to 30 days of age. Calves were born f rom dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Entry ID 2 Pathway Fold E 3 Count 4 P Value 5 Genes 6 Contrast MR 7 bta05410 Hypertrophic cardiomyopathy 15.2 4 0.002 DES, MYL2, TNNC1, TPM2 bta05414 Dilated cardiomyopathy 14.3 4 0.002 DES, MYL2, TNNC1, TPM2 bta03320 PPAR signaling pathway 12.3 3 0.022 PPARA, OLR1, ANGPTL4 bta04530 Tight junction 6.7 3 0.065 MYL2, MYH7, ACTN2 Interaction FAT by MR 8 bta00071 Fatty acid metabolism 9.4 5 0.002 CYP4A11, C YP4A22, ACADL, DCI, ACAA1 bta03320 PPAR signaling pathway 6.4 6 0.002 CYP4A11, CYP4A22, CYP27A1, APOA5, ACADL, ACAA1 bta00561 Glycerolipid metabolism 8.7 5 0.002 GLYCTK, AKR1A1, PPAP2A, LIPC, AGPAT2 bta03010 Ribosome 5.1 6 0.006 RPL13, RPL34, RPL8, RPS9 RPS4Y1, RPS8 bta00520 Amino sugar and nucleotide sugar metabolism 6.8 4 0.020 GALK1, PGM1, HEXB, GALT bta00590 Arachidonic acid metabolism 5.2 4 0.039 CYP4A11, CYP4A22, LTA4H, CYP2E1 bta00052 Galactose metabolism 8.9 3 0.042 GALK1, PGM1, GALT bta0098 3 Drug metabolism 6.5 3 0.075 CES2, DPYD, GMPS Interaction FA by MR 9 bta00010 Glycolysis / Gluconeogenesis 10.7 5 0.001 ALDOA, TPI1, GALM, PGM1, ENO1 bta00190 Oxidative phosphorylation 4.5 5 0.022 UQCRC1, COX10, ATP6V1E1, ATP5B, NDUFS2 3bta00260 Gl ycine, serine and threonine metabolism 10.7 3 0.030 GCAT, PSAT1, GLDC bta04920 Adipocytokine signaling pathway 5.6 3 0.096 ADIPOR2, STAT3, ACSL5 1 Calves were fed milk replacer (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. 2 Entry ID for the corresponding Kyoto encyclopedia for gene and genomes (KEGG) pathway. 3 Fold enrichment for each corresponding pathway. 4 The number of gene members for each corresponding pathway. 5 Fisher exact P value. 6 List of genes in each corresponding KEGG pathway. 7 Main effect of MR, comparing the effec t of feeding HLA milk replacer to newborn calves with LLA milk replacer as reference. 8 Interaction fat by milk replacer: ( [(SFA HLA + EFA HLA) /2 : Control HLA (reference)] [(SFA LLA + EFA LLA) /2 : Control LLA (reference)] 9 Interaction fat ty acid by mi lk replacer: [EFA HLA : SFA HLA (reference)] [EFA LLA : SFA LLA (reference)]

PAGE 288

288 Table 5 9 Functional ann otation chart for enriched down regulated KEGG pathways for main factors and interactions in liver of Holstein male calves fed milk replacer containin g low or high linoleic acid from 1 to 30 days of age. Calves were born from dams fed diets supplemented with no fat, saturated fatty acids, and essential fatty acids 1 Entry ID 2 Pathway Fold E 3 Count 4 P Value 5 Genes 6 Contrast FAT 7 bta05410 Hypertrop hic cardiomyopathy 12.9 3 0.019 MYL2, TNNC1, TPM2 bta05414 Dilated cardiomyopathy 12.0 3 0.022 MYL2, TNNC1, TPM2 bta04670 Leukocyte transendothelial migration 8.3 3 0.044 ICAM1, MYL2, ACTN2 bta04530 Tight junction 7.6 3 0.052 MYL2, MYH7, ACTN2 Interac tion FAT by MR 8 bta04530 Tight junction 5.8 5 0.009 MYH1, MYL2, CASK, MYH7, ACTN2 bta04120 Ubiquitin mediated proteolysis 5.3 5 0.013 SOCS1, UBA7, PML, HERC4, BIRC3 bta05410 Hypertrophic cardiomyopathy (HCM) 5.9 3 0.087 MYL2, TNNC1, TPM2 bta05414 D ilated cardiomyopathy 5.5 3 0.098 MYL2, TNNC1, TPM2 Interaction FA by MR 9 bta00240 Pyrimidine metabolism 5.7 4 0.030 UPP2, ENTPD4, DPYD, NME7 bta04120 Ubiquitin mediated proteolysis 3.9 4 0.078 CUL3, KLHL9, ITCH, BIRC3 1 Calves were fed milk replac er (MR) containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 30 d of age. Calves were born from cows fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expec ted calving date. 2 Entry ID for the corresponding Kyoto encyclopedia for gene and genomes (KEGG) pathway. 3 Fold enrichment for each corresponding pathway. 4 The number of gene members for each corresponding pathway. 5 Fisher exact P value. 6 List of ge nes in each corresponding KEGG pathway. 7 Main effect of MR, comparing the effect of feeding HLA milk replacer to newborn calves with LLA milk replacer as reference. 8 Interaction fat by milk replacer: ( [(SFA HLA + EFA HLA) /2 : Control HLA (reference)] [ (SFA LLA + EFA LLA) /2 : Control LLA (reference)] 9 Interaction fat ty acid by milk replacer: [EFA HLA : SFA HLA (reference)] [EFA LLA : SFA LLA (reference)]

PAGE 289

289 Table 5 10 Productive and reproductive parameter of Holstein heifer s fed milk replacer conta ining low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Heifers were born from dams fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calvi ng date. Dam Diet 1 P values 3 Control SFA EFA SEM FAT FA MR FAT x MR FA x MR Milk replacer (MR) 2 LLA HLA LLA HLA LLA HLA Age at 1st Insemination, months 13.2 13.1 13.2 12.8 13.2 13.1 0.2 0.69 0.35 0.20 0.66 0.35 N of Insemination s 1.6 1.9 2.4 2.3 2.8 2.8 0.5 0.04 0.39 0.92 0.66 0.99 Age 1st calving, years 1.9 1.9 2.1 2.0 2.0 2.0 0.1 0.02 0.76 0.44 0.43 0.45 BW at calving, kg 515 508 54 5 545 565 538 19.5 0.04 0.75 0.49 0.85 0.51 BCS at calving 3.1 3.0 3.3 3.3 3.4 3.2 0.1 0.04 0. 89 0.64 0.86 0.35 BW at drying, kg 606 635 637 645 715 650 32.4 0.14 0.23 0.72 0.29 0.29 BCS at drying 3.4 3.5 3.4 3.4 3.8 3.5 0.1 0.08 0.02 0.55 0.03 0.07 Length of lactation d 301 302 302 30 1 276 304 12.4 0.56 0.38 0.37 0.54 0.25 DIM at peak of act ation, d 107.4 85.3 76.4 89.5 78.0 78.0 10.2 0.08 0.64 0.72 0.11 0.54 Mature equivalent Milk, kg 10 107 11 103 11 542 11 948 12 136 12 389 694 0.02 0.48 0.34 0.57 0.92 Fat, % 3.65 3.64 3.67 3.63 3.63 3.53 0.10 0.71 0.53 0.56 0.75 0.80 Protein, % 3.09 3.05 3.08 3.07 3.05 3.03 0.04 0.69 0.31 0.48 0.68 0.93 Lactose, % 4.78 4.78 4.77 4.85 4.80 4.83 0.02 0.08 0.71 0.07 0.16 0.30 1 Control = no fat supplement; SFA = Energy Booster 100 (Milk Specialties, Dundee, IL); EFA = Megalac R (Church & Dwight, Princ eton, NJ). 2 LLA = 0.175 g of LA/BW 0.75 HLA = 0.562 g of LA/BW 0.75 Milk replacer (20% fat) was exclusively fed the first 30d of life to provide 6.72 g fat/kg BW 0.75 3 P values for orthogonal contrasts and interactions. FAT = fat (SFA + EFA) vs. Control FA = EFA vs. SFA, DD= dam diet, MR = milk replacer.

PAGE 290

290 Table 5 11 Incidenc e and main causes of culling of Holstein heifers fed milk replacer containing low linoleic acid (LLA) or high linoleic acid (HLA) from 1 to 60 days of age. Heifers were born from dams fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Item Factor % (n/n) AOR 2 95% CI P Total culling Dam Diet Control 27.8 (5/18) Ref. SF A 50.0 (11 /22) 2.6 0.69 10.0 0.28 EFA 43.8 (7/16) 2.0 0.49 8.6 0.71 Milk Replacer LLA 46.4 (13/28) Ref. HLA 35.7 (10/28) 0.63 0.21 1.9 0.41 Reproductive Dam Diet Problems Control (0/18) Ref. SFA 13.6 (3/22) EFA 12.5 (2 /16) Milk Replacer LLA 10.7 (3/28) Ref. HLA 7.1 (2/28) 0.63 0.09 4.25 0.63 Poor growth Dam Diet Control 11.1 (2/18) Ref. SFA 13.6 (3/22) 1.27 0.18 8.91 0.53 EFA 18.8 (3/16) 1.90 0.26 13.7 0.14 Milk Replacer LLA 21.4 (6/28) Ref. HLA 7.1 (2/28) 0.28 0.05 1.54 0.14 Mastitis and Dam Diet Low production Control 5.6 (1/18) Ref. SFA 9.1 (2/22) 1.72 0.14 21.5 0.92 EFA 12.5 (2/16) 2.51 0.20 31.9 0.52 Milk Replacer LLA 3.6 (1/28) Ref. HLA 14.3 (4/28) 4.56 0.47 44.0 0.19 Ot hers 2 Dam Diet Control 11.1 (2/18) Ref. SFA 13.6 (3/22) 1.26 0.19 8.52 0.53 EFA 3.3 (1/16) 0.53 0.04 6.51 0.51 Milk Replacer LLA 10.7 (3/28) Ref. HLA 10.7 (3/28) 1.0 0.18 5.48 1.0 1 Adjusted o dds ratio, Control was reference (Ref.) for treatment dam diets and LLA was reference for milk replacer. 2 Includes: dead (2), accidentally ill (2), pneumonia (1), and foreign body (1).

PAGE 291

291 Figure 5 1. Concentrations of C12:0, C14:0 and C16:0 in liver of Holstein calves fed milk replacer containing low (LLA) or high LA (HLA) from 1 to 30 days of age. Calves were born from cows fed diets su pplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. For all fatty acids, e ffect of m ilk replacer P < 0.01. 0 2 4 6 8 10 12 14 16 18 C12:0 C14:0 C16:0 % of total fatty acid LLA HLA

PAGE 292

292 A B Figure 5 2. Concentrations of omega 3 and 6 fatty acids in liver of Holstei n calves fed milk replacer containing low (LLA) or high LA (HLA) from 1 to 30 days of age. Calves were born fro m cows fed diets supplemented with no fat, saturated fatty acids, or essential fatty acids starting at 8 wk before expected calving date. A) Concentrations of linoleic acid (LA), arachidonic acid (AA) and total n 6 FA; e ffect of milk replacer on LA and tot al n 6, P < 0.01, on AA, P = 0.09. B) Concentrations of linolenic acid (ALA), docosapentaenoic acid (DPA) and total n 3 FA ; effect of milk replacer, for all fatty acids, P < 0.01. 0 5 10 15 20 25 30 35 40 45 LA AA n 6 % of total fatty acid LLA HLA 0 1 2 3 4 5 6 ALA DPA n 3 % of total fatty acid LLA HLA

PAGE 293

293 Figure 5 3. Venn diagram of the up regulated differential expressed genes in liver of male calves fed milk replacer (MR) contai ning low (LLA) or high (HLA) from 1 to 30 d ays of age. Calves were born from cows fed either control diets (no fat), saturated fatty acids (SFA) or essential fatt y acids (EFA) starting at 8 wk of expected calving date. 1) Contrast of FAT: [(SFA + EFA) /2 vs control (reference)]. 2) Contrast of FA: [EFA vs. SFA (reference)]. 3) Contrast of MR: [HLA vs. LLA (reference)]. 4) Interaction FAT by MR : ([(SFA HLA + EFA HLA) /2 : Control HLA (reference)] [(SFA LLA + EFA LLA) /2 : Control LLA (reference)]. 5) Intera ction FA by MR: [EFA HLA : SFA HLA (reference)] [EFA LLA : SFA LLA (reference)].

PAGE 294

294 Figure 5 4. Venn d iagram of the downreg ulated differential expressed genes in liver of male calves fed milk replacer (MR) containing low (LLA) or high (HLA) from 1 to 30 d ays of age. Calves were born from cows fed either control diets (no fat), saturated fatty acids (SFA) or essential fatty acids (EFA) starting at 8 wk e expected calving date. 1) Contrast of FAT: [(SFA + EFA) /2 vs. control (reference)]. 2) Contrast of FA: [EFA vs. SFA (reference)]. 3) Contrast of MR: [HLA vs. LLA (reference)]. 4) Interaction FAT by MR : ([(SFA HLA + EFA HLA) /2 : Control HLA (reference)] [(SFA LLA + EFA LLA) /2 : Control LLA (reference)]. 5) Interaction FA by MR: [EFA HLA : SFA HLA (refe rence)] [EFA LLA : SFA LLA (reference)].

PAGE 295

295 Figure 5 5. Upregulated genes of the PPARA KEGG pathway in Diamond symbol corresponds to upregulated genes by the contrast high linoleic acid milk replacer vs. low linoleic acid milk replacer ( reference) G enes are: peroxisome proliferator rec epto r (PPARA), oxidized lipoprotein receptor 1 (OLR1) and angiopoietinin like 4 (ANGPTL4 = PGAR). Star symbol corresponds to genes up regulated by the contrast FAT by milk replacer. Genes are: cytochrome P450 subfamily 27A1 (CYP27A1), cytochrome P450 subfamily 4A11 (CYP4A11), cytochrome P450 subfamily 4A22 (CYP4A22), acyl CoA dehydrogenase, long chain (LCAD), apolipoprotein A5 (APO A5) and thiolase B.

PAGE 296

296 Figure 5 6. Upregulated genes of the adipocytoki ne KEGG pathway in Star symbol corresponds to up regulated genes by the contrast FA by MR. Genes are: fatty acyl CoA synthetase (FACS), signal transducer and activator of transcription 3 (STAT3), and adiponectin receptor 2 (ADIPOR).

PAGE 297

297 Figure 5 7. Differentially expressed genes within the tight junction KEGG pathway in Star symbol corresponds to downreg ulated by the contrast of FAT vs. control (reference) G enes are: two myosin subfamilies, myosin regulatory light chain 2 (MYL2) and myosin heavy chain 7 (MYH7), and actinin (ACTN2). Arrow symbol corresponds to genes downreg ulated by the interaction FAT by milk replacer. Genes are: three myosin subfamilies, heavy chain 1 (MYH1), MYL2, and MYH7; calcium/calmodulin dependent serine protein kinase (CASK), and actinin (ACTN2).

PAGE 298

298 Figure 5 8. Downregulation of genes in the l eukocyte transendothelial migration KEGG pathway in Star symbol corresponds to downreg ulated genes by the interaction FA by milk replacer. Down regulated genes are marked with start a nd are: intracellular adhesion molecule 1 (ICAM1), myosin heavy light chain 2 (MLC = MYL2), and actinin (ACTN2).

PAGE 299

299 CHAPTER 6 EFFECT OF FEEDING MI LK REPLACER ENRICHED WITH INCREASING LINOLEIC ACID ON HOLSTEIN CAL F PERFORMANCE, IMMUNE RESPONSE AND HEALTH Background Essentiality of certain long chain fatty acids (FA) was discovered by Burr and Burr (1929, 1930, 1932) in pioneer studies performed with rats fed fat free diets and supplemented with purified FA or mixtures of them. These authors identified the symptoms of linoleic aci d (LA) deficiency, namely poor growth, dermatitis, poor reproduction, and death. A LA requirement was documented later using swine, poultry, and guinea pigs (Hill et al., 1961; Bieri and Prival, 1966; Reid et al., 1964). Authors also concluded that linolenic acid (ALA) was able to prevent these signs of deficiency. determined by identifying the role of their derivatives, eicosapentaenoic acid (EPA) and docosahex aenoic acid (DHA), in brain development, as reviewed by Innis (1991). Although the dietary essentiality of LA and ALA has been clearly demonstrated, specific requirements of LA have only been established for mice and rats. The National Research Council (19 95) recommends a daily intake of 1.3 and .0.55% of LA from the total daily intake of metabolizable energy for laboratory rats. An adjustment for metabolic body weight (BW = 100 g) results in a daily intake of 0.551 and 0.212 g of LA/BW 0.75 Some recommenda tions have been released for humans, but most of these recommendations have focused on groups (e.g. n 3 or n 6) or ratios (e.g. LA:ALA and n 6:n 3) of FA instead of single FA. In a review paper, Palmquist (2009) pointed out the inaccuracy of recommending i ntake of LA and ALA in terms of their ratio, particularly because this practice leads to reduction of the absolute intake of ALA. Czernichow et al.

PAGE 300

300 (2010) reviewed studies on intake of n 6 FA and risk of cardiac diseases and recommended an intake of n 6 FA above 10% of the total energy intake in order to reduce the risk of cardiac diseases. Ramsden et al. (2010) used the same studies reviewed by Czernichow et al. (2010) in another analytical approach. They concluded that if dietary intake of n 6 are increas ed without a parallel increase of n 3 intake, a greater risk of cardiac diseases would result. Calder and Deckelbaum (2011) agreed with the analysis of Ramsden et al. (2010) but pointed out that both studies grouped FA without considering their individual effects which could potentially lead to confounding effects. A limited number of studies have evaluated the supplementation of fat sources Canada was among the first to evaluate the replacement of milk fat with sources of less expensive fat such as vegetable oils. Their studies (Jenkins et al., 1985, 1986; Jenkins and Kramer, 1986) are the foundation to evaluate the effects of total or partial replacement of milk fat with vegetable oil in order to enrich the milk replacer (MR) with essential FA (EFA). They evaluated calf responses in terms of growth, diarrhea incidence, and FA profile of most of the important tissues and organs involved in lipid metabolism. Authors conclud ed that commonly supplemented milk contains enough LA to avoid signs of deficiency, but that the requirement of EFA might be greater under conditions of high stress on preweaned or newly weaned calves. To the best of our knowledge no studies have been alth ough The most recent studies, in preweaned calves, supplementing EFA to evaluate the growth response and the activity of different markers of immune responses have been

PAGE 301

301 conducted supplementing n 3 FA. The hypothesis was that immune responses would be impr oved by increasing intakes of LA. This improved immune response could positively affect calf productive performance. Therefore the objective was to evaluate the effect of supplementing increasing amounts of LA in a MR to newborn calves during the first 60 d of life on calf growth, health and different markers of immune responses. Materials and Methods Enrollment and Management of Pregnant Cows FL) from October 2010 to June 2011. All procedures for animal handling and care were weekly cohort of pregnant nulliparous (n = 39) and previously parous (n = 64) Holstein cattle were enrolled in the stud y starting at 8 wk before expected calving day. Experimental cattle were fed once daily (0800 h) with a single diet prepared as a totally mixed ration formulated to have low concentrations of total and essential FA (Table 6 1). Offered feed was adjusted d aily to achieve 5 to 10% orts. Orts were collected and weighed daily. A bermudagrass silage sample was collected once a week and dried for 1 h using a Koster (Koster Crop Tester, Inc., Strongsville, OH) for determination of dry matter (DM). Proportions o f forages and concentrates in the diet were adjusted weekly based on the weekly DM values in order to maintain the formulated forage to concentrate ratio (55.3:44.7). Weekly samples of silage, hay, and concentrate were ground to pass through a 1 mm screen using a Wiley Mill (Arthur H. Thomas, Company, Philadelphia, PA). Samples were composited monthly, pooled in a single sample and analyzed (Dairyland Laboratories, Inc., Arcadia, WI) for crude protein

PAGE 302

302 (CP), ash free acid detergent fiber, ether extract, ash, and individual minerals (Ca, P, Mg, K, S, Na, Cl, Mn, Zn, Cu, Fe, and Mo). Calving Management at Birth and Colostrum Feeding Calves were born from January 4 th 2011 through April 5 th 2011. Pregnant cows gave birth to calves in a sod based pen. All cows were monitored for signs of parturition initiation every 30 min between 0530 to 1530 h and then every 2 hours between 1530 and 0530 hours. Ease of calving was scored according to Sewallem et al. (2008) as unassisted (1), easy pull (2), hard pull (3), and s urgery (4). Within 2 h of birth calves were weighed, ear tagged, and the umbilical cord was disinfected with 10% Betadine solution (Purdue Frederick Co., Norwalk, CT). Parturient cows were milked within 6 h of calving and colostrum was harvested. Concentr ation of total immunoglobulin G (IgG) in colostrum was measured using a colostrometer. Colostrum of good quality (> 50g/L of IgG) was frozen ( 20C) in 4 L amounts. Immediately after weighing, calves were given 4 L of thawed and warmed colostrum having a m inimum IgG concentration of 55 g/L using an esophageal feeder. Calves were housed temporarily in individual hutches (1 x 1 m) equipped with a heat lamp and moved to individual wire hutches (1 x 1.5 m) on sand bedding when they were between 2 to 16 h of age Appropriate Passive Immune Transfer Identification Blood samples were collected via jugular venipuncture before colostrum feeding, and again within 24 to 30 h after feeding colostrum. Calf blood samples were collected in a clot activated tube (Vacutainer Becton Dickinson, Franklin Lakes, NJ) and serum was separated at room temperature. Tubes were centrifuged for 15 min at 2095 x g (Allegra X 15R centrifuge, Beckman Coulter, Inc). Serum total protein (STP)

PAGE 303

303 concentrations were determined using an automatic temperature compensated hand refractometer (Reichert Jung; Cambridge Instruments Inc. Buffalo, NY). Sera and colostral total IgG concentrations were measured using a single radial immunodifusion method (Triple J Farms, Bellingham, WA) following the manuf protocol with some modifications. Briefly, sera and colostral samples were diluted with sterile saline (0.9% NaCl) at a ratio of 7:10 and 1:15 respectively. Diluted samples (5 ose gel with anti bovine IgG. Plates were left undisturbed for 27 h at room temperature and resulting ring diameters were measured with a monocular comparator (VMRD, Inc., Pullman WA). A standard curve was plotted with reference sera supplied by the manufa cturer (1.96, 14.02, and 27.48 g/L of IgG). Concentrations of IgG in diluted samples were read from the standard curve and the corresponding correction factor, due to dilution, was applied afterwards. Samples were run in singlet, but a control sample, incl uded in each plate, was run in duplicate resulting in a 3.6% intra assay variation. Calves were considered as having an appropriate passive transfer (APT) if they W eaver et al., 2000). Alternatively, STP was another measure to evaluate APT by colostrum feeding (Donovan et al., 1998, Calloway et al., 2002). The apparent efficiency of IgG absorption (AEA, %) was calculated according to (Quigley et al., 1998) assuming that serum was 9.9% of calf body weight (BW) using the following equation: (IgG concentration in serum at 24 to 30 h of colostrum feeding (g/L) [0.099 BW (kg) at birth) I gG intake (g) 100%.

PAGE 304

304 Dietary Treatments, Feeding Management and Analyses Calves were blocked by parity of the dam and gender (females = 60, males = 43) and assigned randomly to receive one of four MR from 0 to 60 d of life. Treatments of differing LA conc entrations were prepared by mixing preplanned ratios of hydrogenated coconut oil (CCO; (Welch, Holme & Clark Co., Inc, Newark, NJ)) and soybean oil (SO; Winn Dixie Co.). The treatment (T) ratios of CCO and SO were the following: T1 = 100:0, T2 = 96.0:4.0 T3 = 87.9:12.1, and T4 = 71.8:28.2 and the FA profile is described in Table 6 2. Reconstitution of MR was done consistently throughout the experiment. Briefly, amounts of each fat source and emulsifier (3% of the oils, GRINDSTED MONO DI HV 52 K A Dan isco, USA Inc.) needed to feed the number of calves assigned to each treatment were calculated. Fats were kept in a walk in cooler (4 o C). Every day at 0530 h, the needed amounts of each fat source and emulsifier per treatment were weighed (Ohaus TAJ4001 series, 0.1 g resolution). The required amount of CCO was melted to just reach the liquid form using a conventional microwave oven followed by the addition of the required amounts of SO. Oils were warmed to 70 to 80C which is the required temperature for proper dissolution of the emulsifier. Immediately, the blend of fats and emulsifier were transferred into insulated containers and transferred to the calf area. Animal Milk Product s Co., Shoreview, MN) and warm water (40 to 43 o C) were weighed and (11% DM solution) mixed for 5 to10 min using an electric drill with a wire wisk attachment (12.5 cm diameter). Then the blend of oils and emulsifier were added and mixed again with the elec tric drill. Surface oil droplets were not observed. Immediately

PAGE 305

305 upon mixing, individual calves were offered amounts (L) of MR to achieve LA intakes of 0.144 (T1), 0.206 (T2), 0.333 (T3), or 0.586 (T4) g of LA per kg of BW 0.75 Targeted intakes of LA formul ated in the current study were selected with reference to the recommended intake of LA in rats (NRC, 1995) and from results of our previous study (Chapter 4) in which 2 rates of LA (0.487 vs. 0.149 g/kg WB 0.75 ) were provided with the MR. Laboratory rats ha ve a LA requirement of 0.5 and 1.3% of the metabolizable energy for females and males, respectively (NRC for Laboratory Animals (1995). The LA requirement of rats expressed in relation to BW 0.75 was calculated for a 100 g BW growing rat consuming 16.4 g/d (Kennedy and Mitra, 1963) of a 4 kcal of ME/g of DM diet. Gross energy value of LA and its digestibility was considered to be 9 kcal/g and 96.7% (NRC, 1995). Using the previous specifications the LA requirement of male and female rats were 0.551 and 0.212 g/kg of BW 0.75 respectively. The LA intake rates formulated for the current study diets were below and above those calculated for rats on a metabolic BW basis and within the range of LA rates used in Chapter 4. An attempt was made to feed the minimum rate feasible using feedstuffs commonly available to the dairy industry. The temperature of the liquid MR placed in front of calves was always between 35 to 38 C. At each feeding, each calf was monitored to ensure that the MR was consumed within 10 min of off er. Those calves not willing to drink quickly were fed using a nipple bottle preferentially or an esophageal feeder alternatively. Temperature of MR was verified and warmed in a hot water bath if needed for these calves. Calves were fed MR exclusively dur ing the first 30 d of life and supplemented with a single grain mix of low concentration of LA starting at 31 d of age (Table 6 3). Amounts

PAGE 306

306 of MR offered were increased weekly according to BW measured weekly throughout the 60 d of the experimental period w hereas grain mix was offered in ad libitum amounts. Clean water was available in ad libitum amounts at all times. Powdered MR and grain mix were sampled weekly. Weekly samples were composited monthly and then composited in a single sample. Samples were ana lyzed (Dairyland Laboratories, INC., Arcadia, WI) for CP, ash free acid detergent fiber (only for grain mix), ether extract (grain mix), mojonier fat (MR), ash, and individual minerals (Ca, P, Na, Cl, Mg, K, S, Mn, Zn, Cu, Fe, and Mo). Milk replacer was fe d at a constant rate per kg of BW 0.75, and adjusted weekly based on a new BW; however, calves that lost BW in a 7 d period were offered the same amount of MR as that offered the previous week. If calves did not consume all of their morning MR within a few minutes of offer, the remaining MR was given using an esophageal feeder whereas the afternoon feeding was replaced with electrolytes if calves were not willing to drink voluntarily. Body Weight and Immunizations Calf BW was measured at birth before colost rum intake. This measure was used to assign the amount of MR each calf was offered until the next weekly BW measure. Weekly BW measures were done every Monday at 1700 h (about 4 h after the second MR feeding) and the new intakes were adjusted starting on e very Wednesday of the same week. Body weight and whither and hip heights (as measures of growth) also were recorded at 0, 30 and 60 d of age. The 30 and 60 d BW were measured before the morning milk feeding (0530 h). All immunization protocols were done ac cording details in chapter 4.

PAGE 307

307 Calf Scoring for Health Assessment and Incidence of Health Disorders Calves were scored daily using the calf health scoring system from the University of Wisconsin (http://www.vetmed.wisc.edu/dms/fapm/fapmtools/calves.htm). At titude, fecal consistency, nasal discharge, ocular discharge, and cough were scored daily after the first feeding of MR (0830 to 0930 h) using a 0 to 3 scale. For attitude, calves were categorized as 0 when alert and responsive, 1 when non active, 2 when d epressed, and 3 when moribund. Fecal consistency was scored as 0 when firm, 1 when soft or of moderate consistency, 2 when runny or mild diarrhea, and 3 when watery and profuse diarrhea. For nasal score, 0 was normal serous discharge, 1 was when a small am ount of unilateral cloudy discharge was present, 2 was when bilateral cloudy or excessive mucus discharge was present, and 3 was when copious bilateral mucopurulent discharge was present. Ocular discharge was scored as 0 when normal, 1 when a small amount of ocular discharge was present, 2 when moderate amount of bilateral discharge was present, and 3 when heavy ocular discharge was present. Cough was scored after pressing the trachea as 0 when absent, 1 when a single cough was induced, 2 when repeated coug h or ocacional spontaneous cough was induced, and 3 when repeated spontaneous cough was detected. Weekly averages of all scores were generated per calf for statistical analysis. Calves with fecal score > 1 were considered to have diarrhea and severe diarrh ea when score = 3, whereas calves with score > 0 for other occurrences were considered as being abnormal for that measure. Incidence of health disorders were recorded daily for individual calves. Rectal temperature was measured daily during the first 14 d of age, and on days when the calf displayed clinical signs of disease such as diarrhea, bloat, coughing, increased respiratory frequency, depr

PAGE 308

308 39.5C were categorized as febrile. Day when disease was first diagnosed was recorded and duration of each illness event was determined. Number of episodes of fever, diarrhea, and pneumonia were determined. To distinguish between different episodes, an interval of 4, 4, and 10 d between diagnoses of fever, diarrhea and pneumonia, respectively, had to elapse to characterize a new event. Calves with digestive and respiratory problems were treated b y farm personnel according to protocols established by the herd veterinarian. Hormone and Productive Metabolite Analyses Before colostrum was fed, a jugular blood sample was collected from each calf and again after 24 to 30 h of feeding colostrum into clot activated tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ). Serum was separated at room temperature and tubes were centrifuged for 15 min at 2095 x g (Allegra X 15R centrifuge, Beckman Coulter, Inc). Weekly samplings of blood into clot activated and K 2 EDTA tubes were centrifuged for 15 min at 2095 x g for harvesting of serum and plasma, respectively. Before storing of serum, STP was measured using an automatic temperature compensated hand refractometer (Reichert Jung; Cambridge Instruments Inc. B uffalo, NY). Plasma samples for all productive metabolites were analyzed once a week at approximate ages of 1, 8, 15, 22, 29, 36, 43, 50, and 57 1 d whereas analyses of hormones were done on sera sample from d 0 and in plasma samples at 1, 15, 29, 43, an d 57 1 d of age. A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to measure plasma glucose (Bran and Luebbe Industrial Method 339 19; Gochman and Schmitz, 1972) and PUN (Bran and Luebbe Industrial Method 339 01; Marsh et al ., 1965). A total of twelve runs (each balanced for treatment and gender)

PAGE 309

309 were performed. Each run included a common control sample which was run in duplicate with a final intra and interassay variations of 1.0 and 1.3% and 3.0 and 4.2% for glucose and PU N, respectively. hydroxybutyric acid (BHBA) were determined using a commercial kit (Wako Autokit 3 HB; Wako Diagnostics, Inc., Richmond, VA). Unknown samples were run in singlet including a control sample which was run in duplicate. A total of tw elve plates, balanced for same number of calves per treatment, were run. Intra and inter plate variations were 5.6 and 7.8%, respectively. Total cholesterol concentrations were determined using a commercial kit (Cholesterol E kit, Wako Diagnostics Inc., R ichmond, VA). Each sample was analyzed in duplicate, including a common control sample in each of the 24 plates. Intra and inter assay variations were 3.2 and 6.8%, respectively. Plasma concentrations of insulin like growth factor I (IGF I) were analyzed I Immunoassay, R&D Systems Inc.) with some modifications in sample preparation. Briefly, serum and plasma samples were run in singlet. The pre treatment of samples, to release the IGF I from their binding proteins, was done with half of the indicated volumes for sample pre treatment reagents to maintain the final suggested dilution of samples (1:100); control sample was included in duplicated wells per plate. The intra plate variation for con trol sample was 3.6%, whereas the inter plate variation was 8.1%. Insulin concentrations were analyzed by a double antibody radioimmunoassay (Badinga et al., 1991). Intra and interassay variations were 7.3 and 14.6%, respectively.

PAGE 310

310 Markers of Immunity Ana lyses Blood was collected from puncture of the jugular vein into heparinized vacutainer tubes at 7, 14, 28, and 42 2 d of age. Samples were transported at ambient temperature with constant gentle inversion. Quantification of individual cells and cell pop ulations were performed using a ProCyte Dx hematology analyzer (IDEXX Laboratories, Inc., Westbrook, ME). Tubes were kept at room temperature with gentle inversion and analyzed within 2 h of collection. Neutrophil phagocytosis and oxidative burst were meas ured on blood of calves at 7, 14, 28, and 42 d of age using a dual color flow cytometry assay using methodology modified from Smits et al. (1997). Whole blood samples were collected in replicate for this analysis and for quantification of cell populations. Tubes were kept at room temperature with gentle inversion and assayed immediately after the hematologic results were done. Briefly, whole blood (100 L) was transferred into each of 3 polystyrene round bottom tubes (12 x 75 mm) and 10 L of 50 M dihydror hodamine 123 (DHR, Sigma Aldrich, Saint Louis, MO) was added to all tubes. Tubes were vortexed slowly and incubated at 37C for 10 min with constant rotation using a nutator (BD, San Jose, CA). A 10 L solution of 20 g/L of phorbol myristate acetate (PMA, Sigma Aldrich) was added into tube number 2 (positive control for oxidative burst). A pathogenic E. coli bacterial suspension (10 6 CFU/mL) isolated from a case of bovine mastitis and labeled with propidium iodide (Sigma Aldrich) was added to tube number 3 to establish a 40:1 ratio of bacteria to neutrophil, using the concentration of neutrophils in blood provided by the hematologic results. Tubes were slowly vortexed and incubated at 37C for 30 min with constant rotation. After incubation, tubes were plac ed immediately on crushed ice to stop neutrophil activity. Tubes were processed into a Q

PAGE 311

311 Prep Epics immunology workstation (Coulter Corp., Miami, FL) on a 35 sec cycle using three lysing reagents, followed by the addition of 500 uL of cold distilled water to complete the hemolysis and 10 L of 0.4% tryphan blue to quench extracellular oxidized DHR. Tubes were vortexed slowly and kept on crushed ice until flow cytometry analysis within 2 h of fixation at the University of Florida Flow Cytometry Core Lab. For each sample the optical features of 10,000 neutrophils were acquired using a Facsort flow cytometer equipped with a 488 nm argon ion laser for excitation at 15 mW (BD Biosciences, San Jose, CA) and CellQuest software (Becton Dickinson, San Jose, CA). Forw ard (roughly proportional to the diameter of the cell) and side (proportional to membrane irregularity) scatters were used for preliminary identification of neutrophil cells on dot plots (Jain et al., 1991). Density cytograms were generated by linear ampli fication of the signals in the forward and side scatters. Parameters analyzed included the percentage of neutrophils that phagocytized bacteria and the percentage of neutrophils with a phagocytosis induced oxidative burst. Also, mean fluorescence intensity (MFI) of green (DHR oxidation) and red (PI labeled bacteria) wave lengths were used as an estimation of the total gated neutrophil mean oxidative burst intensity (interpreted as the mean number of reactive oxygen species produced per neutrophil) and mean phagocytic activity (indicator of mean number of bacteria engulfed per neutrophil), respectively. Before harvesting of plasma from the blood collected in K 2 EDTA tubes, concentrations of hematocrit were determined by centrifuging (Microspin 24 tube micro h ematocrit centriguge, Vulcon Technologies, Grandview, Mo) heparinized micro hematocrit capillary tubes (Fisherbrand, Thermo Fisher Scientific Inc.) for 3 min and

PAGE 312

312 read in a micro hematocrit tube reader (Model CR, Damon/IEC, Needham Heights, MA). Concentrati ons of STP and acute phase proteins were determined on weekly plasma samples at approximate ages of 1, 8, 15, 22, 29, 36, 43, 50, and 57 1 d whereas determination of IgG against ovalbumin (OVA) were done in sera sample at 1, 22, 43, and 57 1 d of age Blood was collected into heparinized tubes at 7, 14, 28, and 42 2 d of age for in vitro analysis of neutrophil activity whereas proliferation of lymphocytes and production of cytokines were done at 14, 28, and 42 2 d of age. These analyses were perfo rmed within 2 h of blood harvest. Calves were injected s.c. with 0.5 mg of OVA (Sigma Aldrich, Saint Louis, MO) diluted in Quil A adjuvant solution (0.5 mg of Quil A in 1 mL of phosphate buffered saline (PBS), Accurate Chemical & Scientific Corp., Westbury NY) using sterile procedures at 1, 22, and 43 d of age. Serum concentrations of bovine anti OVA IgG were measured on the same days of injection and at 57 d of age by the method described by Mallard et al. (1997) and detailed in chapter 3. Intra and inte rassay coefficients of variation based on the positive control were 3.6 and 3.8%, respectively. Concentrations of plasma haptoglobin (Hp) were determined by measuring the differences of H 2 O 2 activity with haptoglobin hemoglobin (Hb) as described previously (Mikamura and Suzuki, 1982). Concentration of Hp is reported as arbitrary units (optical density x 100). Intra and interassay coefficients of variation were 5.6 and 6.8%, respectively. Plasma concentrations of ASP were determined according to Nakajima e t al. (1982) with some modifications. Plasma samples (50 L) were incubated with PCA solution (1 mL, 6 M perchloric acid, Fisher Scientific, Hampton, NH, USA). (The intra and interassay coefficients of variations were 4.6 and 8.6%, respectively.

PAGE 313

313 In orde r to determine whether blood lymphocytes from experimental calves contained detectable amounts of cytokines in a preliminary study, whole blood was stimulated for cell proliferation with phytohaemagglutinin (PHA, L1668; Sigma Aldrich) + lipopolysaccharide (LPS, E. coli 0111:B4; Sigma Aldrich) at a dose of 0.2 + 1 g/mL. This dose was selected from three doses tested (PHA + LPS: 0.2 + 1 g/L; 1 + 5 g/mL; 5 + 25 mg/mL) based upon earlier work using human whole blood cell proliferation (De Groote et al., 199 2). Stimulated and non stimu lated blood samples from four pre weaned calves at the University of Florida dairy herd were analyzed (Aushon Biosystems, Billerica, MA ) for tumo (IFN 2 (IL 2), and IL 4. The non stimulated samples had very low concentrations of all cytokines (TNF 2 at < 12, and IL 4 at < 40 pg/mL), whereas stimulated samples had g reatly increased concentrations of all cytokines with the lowest stimulation dose of mitogens selected for use with all samples collected for the experimental calves in the current study. The analysis of whole blood lymphocyte proliferation was performed following the protocol of Hulbert et al. (2011) with some modifications. Briefly, whole blood was diluted at 1:5 with RPMI 1640 (Invitrogen) containing 1% antibiotics (Gibco Antibiotic Antimycotic, Invitrogen). Whole blood was stimulated with a combination of 0.2 g/mL of PHA + 1 g/mL of LPS. Stimulated and non stimulated samples were incubated in sterile 24 well cell culture plates (2mL wells) for 48 h in a humidified 5% CO 2 chamber. The cell culture plates were centrifuged for 12 min at 1455 x g (Allegra X 15R Centrifuge, Beckman Coulter, Inc). The supernatant fraction from 3 wells was pooled

PAGE 314

314 and aliquoted into 200 L microtubes and stored at 80 o C until analyzed for bovine TNF Quantification of TNF supernatant samples, based on the preliminary results from the validation test in which the concentrations of cytokines of non sti mulated cells were very low. Bovine TNF and IFN including a pool of stimulated samples as a contr ol. Standards were diluted in RPMI with 4% BSA and 1% antibiotics; stimulated samples were not diluted. The intra and interassay coefficients of variation were 2.0 and 11.4% and 8.4 and 13.2% for TNF and IFN was 78 and 125 pg/mL for TNF and IFN Cell mediated hypersensitivity to epidermal injection of PHA (L1668; Sigma Aldrich) was done in calves at 29 and 59 2 d of age. The treated shoulder was shaved and sterilized with 78% alcohol. The injected area was identified by circling it with a isotonic saline solution) was made in the middle of the created circle using insulinic syringes. The skin fold thickness was measured before injection at 6, 24, and 48 h after injection using a digital caliper (Mitutoyo, Kawasaki, Kanagawa, Japan). Delayed type Hypersensitivity (DTH) response to PHA injection was determined by the increase in the diameter of the skin fold t hickness related to the diameter before injection as a proportion (%) of increase with respect to the baseline (diameter before injection).

PAGE 315

315 Statistical Analysis The experiment was of a completely randomized design. Calves were stratified by gender and ran domly assigned to one of the four MR on the day of birth. Nearly all dependent variables were measured repeatedly and analyzed using the PROC GLIMMIX procedure of SAS (Release 9.2) using the following model: Y ijkl i j ij + Cl( ij ) + W l ) il jl ijl jkl Where Y ijkl i is the fixed effect of MR (T1, j ij is the interaction of MR and gender; Cl( ij ) is the random eff ect of calf nested within MR and l il is the jl ijm is the ijkl is the residual error. Repeated measures data were tested to determine the structure of best fit, namely compound symmetry, compound symmetry heterogeneous, autoregressive 1, and autoregressive 1 heterogeneous as indicated by a Schwartz Bayesian informati on criteria value closest to zero (Littell et al., 1996). If repeated measures were taken on unequally spaced intervals, the sp(pow) covariance structure was used. For non repeated measures, the same model was used after removing the age effect and their i nteractions. All variables were tested for normality of residuals using the Shapiro Wilk test of SAS version 9.2 (SAS Inst. Inc., Cary, NC). Non normally distributed data were transformed as suggested using the guided data analysis of SAS and back transfor med using the link and ilink function of PROC GLIMMIX procedure. Different temporal responses to treatments were further examined using the SLICE option of the GLIMMIX procedure.

PAGE 316

316 Coefficients for testing of orthogonal contrasts when using unequally spaced quantitative treatments were generated using PROC IML of SAS. Orthogonal contrasts performed were the following: 1) linear effect of treatment, 2) quadratic effect of treatment, 3) cubic effect of treatment, 4) gender effect, 5) interaction of contrasts 1 and 4, 6) interaction of contrasts 2 and 4, and 7) interaction of contrasts 3 and 4. If a 3 way interaction of time with the main effects of treatment and gender or interaction of age with gender had a P > 0.25 (Bancroft, 1968), the interactions were drop ped from the model and the model was rerun. Binary data were analyzed by logistic regression using the LOGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). The models included the effects of treatment and gender of calf. Adjusted odds ratio and the 95% c onfidence interval (CI) were calculated. Birth weight and height deviations within each gender were covariates for analysis of BW gain and growth, respectively. First day measure of plasma metabolites was used as covariate for the same metabolites. Finally serum total IgG concentration at 1 d of life was used as a covariate for health measures. Differences discussed in the text were significant at P P another probability is indicated. Results A t otal of 103 calves were enrolled in the study (n = 43 males and 60 females), born from nulliparous (n = 39) and parous (n = 64) Holstein animals fed a low fat and low EFA diet during the last 8 wk of expected calving date. Six male and 2 female calves were removed from the study at an average of 13 d of age due to death from causes other than the treatments or unwillingness to drink the MR. A total of 95 calves completed the study, however 7 calves were removed (T1: 1, T2: 2, T3: 3, and T4: 1)

PAGE 317

317 from the data set because they lost BW during the first 30 d of life regardless of severity of disease. Treatment effects on calf performance were not affected by including or excluding these 7 calves from the data set. Distribution of genders to treatments was as foll ows: T1: 7 males and 14 females, T2: 9 males and 13 females, T3: 9 males and13 females, and T4: 9 males and 14 females. Birth weight of calves assigned to treatments did not differ and averaged 38.9, 41.1, 39.3, and 40.2 kg for calves assigned to T1, T2, T 3, and T4 respectively (Table 6 4), however males were heavier than females (42.0 vs. 37.7 kg, P < 0.01). The IgG concentration of colostrum fed to male calves decreased linearly as LA intake increased (95, 97, 77, and 79 g/L for T1, T2, T3, and T4, respec tively) whereas IgG concentration of colostrum fed to female calves was unchanged across LA treatments (82, 72, 76, and 87 g/L for T1, T2, T3, and T4, respectively, gender by linear LA interaction, P = 0.05). Because target intake of colostrum was 4 L of c olostrum, intake of IgG from colostrum followed the same pattern, namely (379, 382, 308, and 317 g for males and 330, 286, 304, and 346 g for females for T 1, 2, 3, and 4, respectively, gender by linear LA interaction, P = 0.07). Serum concentration of tot al IgG after consumption of colostrum did not differ among treatments or genders (mean of 2.14 g/dL). The AEA of colostral IgG was unchanged by LA intake in female calves (26.1, 26.3, 22.5, and 24.4% for T1, T2, T3, and T4, respectively), whereas males as signed to T1 had the lowest AEA (21.8, 26.3, 28.3, and 27.4% for T1, T2, T3, and T4, respectively, gender by quadratic LA interaction, P = 0.04). Two calves assigned to T2 and one calf assigned to T3 failed to attain APT after colostrum feeding (serum tota l IgG < 1 g/dL) which was corroborated by

PAGE 318

318 their low (< 5 g/dL) STP concentration. Concentrations of serum total protein after colostrum consumption did not differ among treatments or genders (mean of 5.8 g/dL). Measures of Growth and Feed Efficiency Male calves assigned to T2 tended to consume more MR DM than males on other treatments due to less MR refusal whereas female calves consumed similar amounts of MR (gender by LA cubic interaction, P = 0.09, Table 6 5). As a result of greater intake of MR by male calves fed T2, BW gain ( P = 0.02, Figure 6 1A), ADG ( P = 0.02), and FE ( P = 0.04) had quadratic patterns whereas female calves tended to linearly increase in, BW gain ( P = 0.07), ADG ( P = 0.07), and FE ( P = 0.10) with increasing intake of LA (gender by li near LA interaction, Figures 6 1A and B). During the period when MR and grain were offered together (31 to 60 d of age), no effect of LA intake on BW gain or FE was detected. This lack of LA treatment effect held true for the total 60 d period. During the first 30 d of life, wither height (cm) and wither growth rate (cm/d) of females tended to increase linearly with increasing intake of LA whereas wither height and growth rate of males did not differ among LA treatments (gender by linear LA interaction, P = 0.09, Table 6 6). During these same 30 d, hip height (cm) and hip growth rate of both genders tended to increase as intake of LA increased from 0.144 to 0.333 g/kg of BW 0.75 before decreasing for calves fed T4 (quadratic effect, P = 0.09). For the follow ing 30 d of life, this same quadratic pattern was detected for height of both withers (P = 0.05) and hips (P = 0.04) as well as growth rate of both withers (P = 0.04) and hips (P = 0.04, Table 6 6, Figure 6 2). Metabolic and Hormonal Profile in Plasma Co ncentrations of plasma glucose were greatest at the first d of age, exceeding 100 mg/dL, and decreased to between 80 and 90 mg/dL for 5 wk, then rose during the

PAGE 319

319 last 3 wk (effect of age, P < 0.01, Figure 6 3A). Increased feeding of LA from T1 to T3 to fema le calves resulted in decreasing plasma concentrations of glucose before rebounding in female calves fed T4 (89.2, 87.6, 84.9, and 90.3 mg/dL) whereas that of male calves did not differ according to LA treatment (90.1, 87.8, 90.2, and 86.6 ng/mL, gender by quadratic LA interaction, P = 0.02, Table 6 7). Concentrations of PUN gradually increased during the first 30 d of age peaking between 10 and 11 mg/dL and then gradually decreased once grain intake commenced (effect of age, P < 0.01, Figure 6 3B). Mean co ncentrations of PUN of female calves tended to follow a quadratic response to LA feeding being lowest when fed T2 and greatest when fed T3 (8.0, 7.6, 8.5, and 7.9 mg/dL) whereas PUN concentrations of male calves were steady for T1, T2, and T3 until increas ing whenT4 was consumed (7.7, 7.7, 7.3, and 8.5 mg/dL, gender by quadratic LA interaction, P = 0.07, Table 6 7). Plasma concentrations of BHBA were low the first 30 d of life (below 0.7 mg/dL) before gradually increasing when grain mix intake commenced (e ffect of age, P < 0.01, Figure 6 4A). Mean concentrations of plasma BHBA tended to decrease as intake of LA increased (T1 = 0.88, T2 = 0.80, T3 = 0.76, T4 = 0.76 mg/dL, linear effect of LA treatment, P = 0.06, Table 6 7) for both genders. Plasma concentrat ions of cholesterol increased with age starting with values around 40 mg/dL during the first 8 d of age and increasing gradually until grain was offered after which concentrations held steady (90 to 110 mg/dL, effect of age, P < 0.01, Figure 6 4B) till the study ended. Mean concentrations of plasma cholesterol increased quadratically as intake of LA increased (T1 = 77.3, T2 = 82.5, T3 = 89.2, T4 = 86.9 mg/dL, quadratic effect of LA treatment, P = 0.04, Table 6 7). Male calves, regardless of treatment, had g reater mean concentrations

PAGE 320

320 of BHBA (0.84 vs. 0.75 mg/dL, P = 0.04) and total cholesterol (87.3 vs. 80.7 mg/dL, P = 0.03, Table 6 7). Mean concentrations of the anabolic hormones insulin and IGF I did not differ due to LA treatment. Concentrations of plasma insulin were low at birth, increasing one d after feeding of colostrum, decreasing at 2 wk of age, and then increasing steadily thereafter (effect of age, P < 0.01, Figure 6 5A). On the other hand, IGF I had its greatest concentration at birth, decreasing dramatically until d 15 of age when, similar to insulin, concentrations steadily increased thereafter (effect of age, P < 0.01, Figure 6 5B). Compared to female calves, male calves had greater mean concentrations of insulin (2.7 vs. 2.0 ng/mL, P < 0.01) a nd IGF I (42.0 vs. 39.0 ng/mL, P = 0.06). Mean of STP concentrations were about 5.8 g/dL the first wk of life after colostrum feeding but decreased at ~15 d to 5.5 to 5.6 g/dL throughout the remainder of the study (age effect, P < 0.01, Figure 6 6). Treatm ent with LA did not affect STP concentration. Incidence of Diarrhea and Other Diseases Calves were generally responsive and without signs of diseases except for diarrhea. Mean scores for attitude and ocular discharge were 0.15 and 0.01 and were not affecte d by LA treatments (Table 6 8). Severity (greater mean attitude score) of poor attitude and diarrhea increased at the second wk of age (effect of age, P < 0.01, Figures 7A, B). Severity of diarrhea (lower mean fecal score) tended to decrease as intake of L A increased (0.70, 0.66, 0.66, and 0.60, linear effect of LA treatment, P = 0.07, Table 6 8). increase linearly as the intake of LA increased (7.0, 7.3, 7.5, and 7.6 d, linear effect of LA treatment, P = 0.10, Table 6 8). Mean score of nasal discharge tended to be greater in calves fed T3 and lowest when calves were fed T1 or T2 (quadratic effect of LA

PAGE 321

321 treatment, P = 0.10). Rectal temperature was lowest at the first d ay of age (mean of 38.3C) and gradually increased until peaking around d 8 (39.0C, effect of age, P = 0.01, Figure 6 8). Males had lower or tended to have lower mean scores for attitude (0.13 vs. 0.18, P = 0.02), fecal consistency (0.62 vs. 0.69, P = 0.0 8), and nasal discharge (0.03 vs. 0.06 P = 0.09), as well as lower mean rectal temperature during the first 14 d of age (38.9 vs. 38.8C, P = 0.02). When abnormal scores or days with fever were calculated as percentage of days of life (Table 6 8), no effe ct of treatment was detected except for percentage of days with nasal discharge (Table 6 8) which peaked for calves on T3 ( P = 0.04). Treatment did not affect the risk of pneumonia (18.1% incidence), navel infection (4.5% incidence), bloody diarrhea (43.1% incidence), or fever (62.1% incidence).Gender also was not a risk factor for disease with the exception of fever. Female calves had a 2.9 fold increase ( P = 0.03) in risk of developing fever compared to male calves apart from dietary treatment (Table 6 9) Blood Cell Populations Concentrations of red blood cells increased with age the first 30 d of life and then decreased at 42 d (effect of age, P < 0.01, Figure 6 9A). Mean concentration of red blood cells tended to decrease as intake of LA increased start ing at T2 (T1 = 8.16, T2 = 8.71, T3 = 8.32, and T4 = 7.90 10 3 P = 0.10, Figure 6 9B). However hematocrit measures were not affected by LA treatments but by age in a similar pattern as to red blood cell concentrations (e ffect of age, P < 0.01, Figure 6 9B). Concentrations of total white blood cells were greatest at 7 d (mean of 11.5 10 3 3

PAGE 322

322 (effect of age, P < 0.01, Figure 6 10). Mean concentra tion of total white blood cells increased in males consuming increasing amounts of LA between T1 and T3 before decreasing in males fed T4 (8.7, 9.2, 10.0, and 8.9 10 3 demonstrated the opposite effect (10.4, 10.1, 9.4, and 11.0 10 3 quadratic LA interaction, P = 0.04, Table 6 10). These changes in white blood cells were primarily due to changes in neutrophils as treatment effects on neutrophils mimicked that effect on white blood cells (gender by quadratic LA intera ction, P = 0.04, Table, 6 10). Concentrations of neutrophils accounted for about 42% of the total population of white blood cells (Table 6 10). Therefore as expected the pattern due to age mimicked that pattern for total white blood cell concentrations. Ne utrophil concentrations were greatest at 7 d of age (effect of age, P < 0.01, Figure 6 11A). Lymphocytes were ~50% of total white blood cells (Table 6 10) and their mean concentrations were not affected by treatment or gender. Calves at 7 d of age had low er concentrations of lymphocytes and concentrations increased gradually with age of the calf (effect of age, P < 0.01, Figure 6 11B). Similarly mean concentrations of blood monocytes (mean of 483/L), eosinophils (mean of 57/L) and platelets (mean of 497 x10 3 /L) were not affected by LA treatment but by age (effect of age, P < 0.01, Table 6 10, Figures 6 12A, 6 12 B and 6 13B, respectively). Mean concentration of blood basophils decreased by about 50% with increasing age (effect of age, P < 0.01, Table 6 10, Figure 6 13A). Males fed T3 or T4 had greater mean concentrations of basophils than males fed T2 (32, 26, 54, and 43/ L) whereas LA treatment did not have an effect on basophils of female calves (34, 44, 32, and

PAGE 323

323 43/L, gender by cubic LA interaction, P = 0.03 Table 6 10). A similar response was detected for the proportion of basophils in total white blood cells ( P = 0.02, Table 6 10). Neutrophil Phagocytosis and Oxidative Burst Proportion of blood neutrophils undergoing phagocytosis did not change wit h age (effect of age, P = 0.12, Figure 6 14A) but throughout the study, mean proportion of phagocytic neutrophils tended to be greater in calves fed T2 or T3, with proportions in calves fed T1 or T4 not differing from each other (T1 = 62.1, T2 = 66.6, T3 = 64.2, and T4 = 62.8 %, cubic effect of treatment, P = 0.09, Table 6 11). Proportion of neutrophils producing oxidative radicals did not differ due to treatment or age. Mean fluorescence intensity for phagocytic activity and production of oxidative radical s was not affect by treatment but by age, with greater proportions at 7 d of age (effect of age, P < 0.01, Figure 6 14B). Concentration of Acute Phase Proteins Age had a big impact ( P < 0.01) on concentrations of both acute phase proteins evaluated. Plasma concentrations of ASP were greater the first wk of age, decreasing gradually to a nadir from 29 d of age (effect of age, P < 0.01, Figure 6 15A). Changes in plasma concentrations due to LA treatments were detected at different ages (age by treatment inter action, P < 0.01; d 9, 16, and 23, P P Figure 6 15A), the differences among treatments were minimal. Plasma concentrations of Hp followed the same pattern as that for fecal and attitude scores. Haptoglobin concentrations reached the highest values at 8 d of life, with calves fed T1 having the greatest concentration, and falling to nadir values from 15 d till the experiment ended (treatment by age interaction, P = 0.02, Figure 6 15B). Females had greater mean concentrations of plasma ASP throughout the study (91.3

PAGE 324

324 vs. 83.8 mg/L, P = 0.02, Table 6 12) and similarly, mean Hp concentrations tended to be greater in females compared to males (0.87 vs. 0.78 OD x 100, effect of gender, P = 0.06, Table 6 12). Humoral and Cell Media ted Immune Responses Plasma concentrations of anti OVA IgG at 1 d of age were high which was unexpected considering that dams of the current study were not injected with OVA; however, they might have retained some circulating antibodies from injection of O VA in values prior to OVA injection, concentration of anti OVA IgG at day 1 were used as a covariate for each calf. Calves, regardless of LA treatment, were not respo nsive to the first and second OVA injection but were responsive to the third injection (d 1 = 0.16, d 22 = 0.14, d 43 = 0.13, and d 57 = 0.27, effect of age, P = 0.01, Figure 6 16A, B). Males fed T2 and T3 were responsive to the second and third injections of OVA, hence had the greater mean anti OVA IgG concentration throughout the study whereas females had similar responses throughout the study regardless of LA treatment (gender by quadratic LA interaction, P = 0.04). Lymphocyte proliferation in whole bloo d after 48 h of stimulation with PHA and LPS differed due to calf age. Proliferation of stimulated lymphocytes characterized as an increase above proliferation of nonstimulated cells (stimulation index) was similar at 14 and 28 d of age but was greater at 42 d of age (effect of age, P < 0.01, Figure 6 17A). Stimulated blood lymphocytes proliferated 23 to 36 times greater than that of nonstimulated blood lymphocytes collected from calves at 14 and 28 d of age. Proliferation was greater at 42 d of age, rangin g between a 28 and 48 fold increase. Proliferation of stimulated lymphocytes from calves fed T1 or T4 did not change much

PAGE 325

325 from 14 to 28 to 42 d but proliferation was dramatically changed at 42 d compared to earlier time points when blood lymphocytes were s timulated from calves fed T2 or T3 (Figure 6 17A). Lymphocytes from calves fed T2 demonstrated greater proliferation at all 3 measuring days of age as reflected in stimulation index means of 26.3, 39.5, 28.9, and 28.2 for T1, T2, T3, and T4, respectively (cubic effect of LA, P = 0.01, Table 6 12). If response is measured as number of lymphocytes proliferated per number of greatest increase occurring between 28 and 42 d ( effect of age, P < 0.01, Figure 6 17B). Again, when averaged across days, lymphocytes from calves fed T2 proliferated to a greater degree than calves fed other LA treatments (3.0, 4.7, 3.5, and 3.8, cubic effect of LA, P = 0.01, Table 6 12) and this was mo st apparent at d 42 (Figure 6 17B). Mean lymphocyte proliferation was lower when T1 was compared to the other treatments as a group (3.31 vs. 4.33 counts per minute, P = 0.04). Concentration of TNF PHA decreased at 28 d of age (14 d = 416, 28 d = 294, and 42 d = 415 pg/mL, effect of age, P < 0.01, Figure 6 18A). Although LA treatments did not have an effect on mean concentra tions of TNF age of calves fed T3. Mean concentrations of IFN increased at 42 d of age (14 d = 227, 28 d = 268, and 42 d = 373 pg/mL, effect of age, P < 0.01, Figure 6 18B). Mean concentrations of IFN blood cells tended to be greater in male calves fed T3 (T1 = 260, T2 = 357, T3 = 411, and T4 = 209 pg/mL, Figure 6 19A) whereas females fed T2 had the greater IFN

PAGE 326

326 production compar ed to females fed the other LA diets (T1 = 256, T2 = 327, T3 = 227, and T4 = 265 pg/mL, gender by quadratic effect of LA interaction, P = 0.09, Figure 6 19B, Table 6 12). Not all calves were responsive to an intradermal injection of PHA, hence delayed ty pe hypersensitivity (DTH) to PHA injection was evaluated using only the responsive calves (calves having an increase in skin fold thickness after PHA injection on any of the 3 measuring times post injection). The adjusted risk ratio analysis at 30 and 60 d of age indicated that neither of treatments 2, 3, or 4 differed from T1 (reference, P > 0.40) and averaged 91% (73/80) and 78% (68/78) at 30 and 60 d of age, respectively. At 30 d of age, response at each hour of measurement decreased with h post injecti on ( P < 0.01) with means of 15.2, 11.7, and 9.5% for 6, 24, and 48 h, respectively (Figure 6 20A). Mean skin fold thickness increased linearly with increasing intake of LA (7.7, 11.0, 14.4, and 15.6% for T1, T2, T3, and T4, respectively, linear effect of t reatment, P = 0.03, Table 6 13). However this pattern differed when gender was considered. Extent of response of female calves peaked when fed T3 and T4 whereas that of male calves peaked when fed T2, T3, and T4 (gender by LA diet interaction, P < 0.01, T able 6 13). When PHA was injected at 60 d of age, skin fold change likewise decreased ( P < 0.01) with hours after injection (10.8, 5.2, and 5.5%, for 6, 24, and 48 h, respectively, Figure 6 20B). However at 60 d of age, calves fed T3 tended to have the sma llest mean skin fold change (8.2, 9.0, 5.8, and 10.0% for T1, T2, T3, and T4, respectively, quadratic effect of LA treatment, P = 0.09, Table 6 13). Discussion Serum total IgG or STP concentrations are used as estimators of APT. The use of STP concentratio n after colostrum feeding is preferred by commercial farms because it

PAGE 327

327 is a cheaper and faster tool to estimate APT than serum IgG. In the current study those calves identified as failing to achieve APT using the minimum serum concentrations of IgG also wer e so identified by failing to achieve the minimum STP concentration. A positive correlation of serum IgG and STP at 24 h of colostrum was detected in this study (r = 0.74, P < 0.01, data not shown), which agrees with results of others (Colloway et al., 200 2; Campbell et al., 2007). Dairy calves reared by commercial farms usually are fed milk or MR at fixed amounts per calf. Some farms use a step down method which consist in gradually reducing the liquid feed offered in order to encourage intake of grain mix generally after the first 4 wk of age, with grain mix offered free choice starting the first day of life. In the current study the MR (29.7% CP, 18.7% fat) was fed in increasing amounts weekly as a 0.75 during the whole prewean ing period and intake of grain mix was delayed until 31 d of age. Accordingly, it was expectable that calves would not perform similarly to commercial calves. Consequently, calf performance in the first 30 d of life was poor with ADG averaging 111 g/d and FE at 175 g of gain/kg of DMI. This first 30 d period was the only period in which LA intake affected gain, namely, males fed T2 having a better ADG (176 vs. 93 g/d) and FE (268 vs. 146 g of gain/kg of DMI) than males fed the other LA diets (gender by cub ic LA diet interaction). This positive response of male calves fed T2 was not replicated, even numerically, in the second 30 d of life. Greenberg et al. (1950) and Pudelkewicz et al. (1968) concluded that male rats have a greater requirement for LA than fe male rats when using BW gain, skin lesions, and accumulation of tetraene FA as measures of response to LA supplementation. The National Research Council (1995)

PAGE 328

328 recommends a minimum intake of LA in rat diets (0.5% of ME as energy from LA for females and 1.3 % of ME as energy from LA for males) based on results from previous studies ( Greenberg et al., 1950; Pudelkewicz et al., 1968). In the current study it is difficult to explain why T2 stimulated BW gai n and feeding whereasT3 and T4 had similar gains to calv es fed T1. Slightly more MR was consumed by male calves fed T2 but the 17 to 23 g/d increase in MR intake would not account for nearly doubling the BW gain for this treatment group. Body weight gain and FE in the first 30 d by female calves tended to incr ease linearly with increasing LA intake (2.6, 3.1, 3.3, and 3.4 kg for T1, T2, T3, and T4, respectively) as did FE (0.15, 0.17, 0.18, and 0.19 g of gain/kg of DMI). In a previous study (Chapter 4) in which 2 intakes of LA (0.149 or 0.487 g/kg of BW 0.75 ) in MR were tested, calves fed the greater amount of LA, regardless of gender, had better ADG and FE. Hence, a LA feeding rate of 0.149 g/kg of MBW was deficient. The current lower feeding rate of 0.144 g of LA per kg of BW 0.75 is below that recommended for g rowing female rats of (0.212 g of LA per kg of BW 0.75) by 33%. It may be that the LA requirement for female Holstein calves is at least 0.206 g of LA per kg of BW 0.75 (T2) which equals 3.0 g of LA/d for a 35 kg calf. If a 20% fat MR is fed at 454 g of DM daily, LA concentration is 0.66% of DM or 3.3% of fat. A 100% tallow based MR containing 3.8% LA would supply 3.5 g of LA per day and meet the proposed LA requirement. However if the LA requirement is closer to that supplied by T3, (0.333 g of LA per kg of MBW), a 20% fat MR fed at 454 g of DM/d to a 35 kg calf would need to supply 4.8 g of LA/d. This would require the MR to contain 1% LA (DM basis) or 5.3% LA (fat basis)

PAGE 329

329 and the fat source would need to be a mix of approximately 85% tallow and 15% porci ne lard (13.9% LA). All calves in the current study suffered from diarrhea starting at a mean of 7 d of age in calves fed T1, with the onset tending to be linearly delayed slightly with increasing intake of LA. Episodes of disease in preweaned calves are the main drivers of reduced performance. Morrison et al. (2009) fed 900 g/d of MR DM (27% CP, 17% fat) to Holstein female calves and reported an ADG of 320 g during the first 28 d of life but calves also were fed free choice a commercial grain mix. Authors did not report incidence of diseases in these calves. On the other hand, Jenkins et al. (1985) fed a 24% CP, 20% fat MR (DM basis) as the only feed fed the first 4 wk of age to male calves using tallow, CCO, or corn oil (CO) as sources of MR fat. Intake o f DM from MR averaged 800 g/d. Calves fed CO had the poorest ADG (392 g/d) which was associated respectively. In a later study in which only MR was fed from 3 to 31 d of life, Jenkins et al. (1986) fed male calves a MR (24% CP, 20% fat, DM basis) with tallow, canola oil, or reclaimed restaurant cooking fat as fat sources. Mean DMI of MR was 823 g/d and mean ADG was 570 g and diarrhea was not detected, when half the tallow was r eplaced with CO, severe scours was observed and ADG decreased to 310 g. Jenkins and Kramer (1986) fed MR containing one of 4 sources of fat, namely 100% CCO, 95% CCO + 5% CO, 92.5% CCO + 7.5% canola oil, and 100% tallow to male calves for 42 d without grai n mix. Mean DMI was 979 g/d and ADG of 660 g. Mean intake of LA was 1,

PAGE 330

330 Certainly performance of calves in the current study was inferior to that of calves in aforementioned studies but major differences exist between these studies. Calves in the 4 aforementioned studies consumed much more DM (875 vs. 618 g) and were housed in a warm, insulated build ing vs. outside during the winter season. Differences in diarrhea severity may have occurred in the previous studies but this was not tested. Mean fecal scores of calves in the current study peaked at 2 (mild diarrhea) during wk 2 of life whereas previous authors often indicated that diarrhea was not a problem in their studies. Nevertheless, ADG of calves in the current study in the first 30 d was 90 g apart from male calves fed T2 whereas ADG of all calves in chapter 4 managed in a similar fashion was 288 g. Fat density of MR used in chapter 4 was a bit greater (19.6 vs. 18.7%) but intake of DM from MR was actually greater in the current study (618 vs. 512 g/d). It may have been that the fat in the MR in the current study was not emulsified properly leadi ng to reduced digestibility of MR fat even though a proven emulsifier was used at the correct amount and mixing was extensive. If fat digestibility was reduced in the current study, it was not reflected by greater incidence of diarrhea. Incidence and seve rity of diarrhea were similar between the two studies. Jenkins (1988) repeated a previous study from 1985 using CCO or CO as sources of fat. Even though the exact same diets were fed, calves fed CO had appreciably less diarrhea than in their previous stu dy. The only difference between those studies was the fat dispersion method, low pressure dispersion in first study and homogenization in the second study, with the latter producing smaller sized fat globules (< 1 m vs. 10 to 20 m). In the current study a commercial emulsifier was used and the solution was vigorously stirred using an electric drill. The size of fat globules was not measured.

PAGE 331

331 However globule size in this study might be lower than 10 m based on the findings of Jenkins et al. (1985) and Jenkins (1988) who reported that fat globules greater than 10 m resulted in increased incidence of diarrhea when feeding CO. In contrast in the current study inclusion of SO decreased the severity of diarrhea. Theref ore, the size of fat globule in the MR of the current study should not be a big risk factor for poor fat digestibility considering that the normal size of fat globules in raw milk ranges from 0.15 to 15 m (Michalski et al., 2006). In the current study, SO replaced up to 24% of CCO, however, mean fecal score was actually reduced linearly as intake of LA increased. The main cause of diarrhea in calves of the current study was likely of environmental (infection) rather than nutritional (size of fat globule) o rigin, because another study conducted at the same location (Perdomo, 2011) also reported a 100% incidence of diarrhea by experimental calves fed pasteurized milk. In that study, ADG for the first 28 d of age was 350 g but these calves were fed 1 kg of mil k DM of high nutrient density (28.5% CP, 26.8% fat, DM basis) and were offered a commercial grain mix in ad libitum amounts. Calves of the current study were fed 618 g of MR DM (29.7% CP, 18.7% fat, DM basis) as the only feed for the first 30 d. Body weigh t gain of female calves between 31 and 60 d of age (630 g/d) was somewhat typical of that of commercial dairy farms. Soberon et al. (2012), aiming to evaluate the effect of ADG during the preweaned period on future milk production, evaluated heifer growth on 2 farms. The Cornell University farm with a population of 1244 heifers reported an ADG of 820 g/d (range of 100 to 1580 g/d), whereas that from a commercial farm was 660 g/d (range of 320 to 1270 g/d) for 623 heifers. Heifers at

PAGE 332

332 both farms were fed comm ercial MR (28% CP and 15 to 20% fat) at a rate of ~900 g/d (DM basis) and were offered a commercial grain mix in ad libitum amounts. No effect of treatment was detected for ADG or FE for the second 30 d of life and the whole 60 d period. In contrast, hip a nd wither growth for the overall preweaning period was better for calves fed T2 and T3. Deficiency of LA led to impaired growth of rats as reported by (Burr and Burr, 1929, 1930). Plasma concentrations of metabolites and hormones in the current study were estimated in the postprandial period because calves always were bled within 1 to 2 h after their morning feeding. Also important to remember is that the gross nutritional composition of the MR in terms of concentrations of protein, fat, lactose, minerals, and vitamins was the same for all LA treatments. Differences in plasma concentrations of anabolic metabolites and hormones normally are seen when groups of calves experience different growth rates. Smith et al. (2002) fed preweaned calves with increased a mount of nutrients resulting in enhanced ADG and FE with parallel increased concentrations of insulin, glucose, and IGF I but a reduction in PUN concentrations. Similarly, Quigley et al. (2006) reported increased concentrations of glucose and IGF I when ca lves were fed increased amounts of MR which was reflected in a greater BW gain and FE. Better ADG and FE of calves fed greater intake of LA in chapter 4, also resulted in increased plasma concentrations of glucose and IGF I but reduced PUN, and even though calves fed low or high amounts of LA were fed diets of similar nutrient density. In the current study a lack of effect of LA treatment in ADG and FE was accompanied by a lack of difference in all aforementioned metabolites and hormones.

PAGE 333

333 Linoleic acid is k nown to have a potent effect activating the peroxisome proliferator activator receptor oxidation and regulating cholesterol synthesis in many species (Forman et al., 1997; Li and Chiang, 2009). Lower plasma concent rations of oxidation, with increasing intake of LA might indicate that the amount of LA evaluated in the present oxidation, leading to a complete oxidation of FA. More likely, calves with lowe r intakes of LA (thus greater intake of CCO) had greater intakes of medium chain FA (C10 and C12) which resulted in calves with greater concentrations of BHBA. Results from the current study are in agreement with the findings from Sato (1994) who fed mediu m chain FA (C8 and C10) to neonatal calves and caused a marked hyperketonemia a few hours after feeding. It was thought that this was due to preferential transport of these FA through the portal vein and greater availability for oxidation and synthesis of ketogenic products. Likewise, in the previous study (Chapter 4), calves fed greater amounts of CCO and lower amounts of porcine lard had increased plasma concentrations of BHBA. Medium chain SFA such as C12:0, C14:0, and C16:0 have been identified as the most potent inducers of cholesterolemia in laboratory animals (Fernandez and West, 2005). In a previous study (Chapter 4), calves fed a MR with a greater proportion of CCO had greater plasma concentrations of cholesterol. In contrast, feeding polyunsaturat ed FA (PUFA) to rats resulted in reduced concentrations of circulating plasmatic cholesterol compared to rats fed CCO ( Berr et al., 1993; Chechi and Chema, 2006 ). Authors agreed that increased concentrations of cholesterol in plasma were related to greater concentrations of LDL cholesterol and vise versa. A review article by

PAGE 334

334 Fernandez and West (2005) proposed different mechanisms by which n 6 FA might lower plasma cholesterol. First n 6 FA may upregulate LDL receptors and secondly, they may increase the ac tivity of cytochrome P450 7A, hence increasing the synthesis of bile acid as a means to remove cholesterol from circulation. Strong evidence exists for diets rich in saturated FA to induce an increase in plasmatic cholesterol, with diets rich in n 6 FA doi ng the opposite. Findings in this current study contradict the general acceptance of n 6 FA as reducers of cholesterol in plasma. At this point, we cannot offer a potential reason why may this have occurred. Plasma concentration of red blood cells was mea sured only at 4 d of age during the experimental period, and calves fed T2 had the greatest mean concentration at the time. However, hematocrit concentration was measured once a week and the values did not differ due to LA treatment. Increased concentratio n of red blood cells usually is related to calf dehydration, often caused by increased incidence or severity of diarrhea whereas a reduced concentration of red blood cells is associated with anemic conditions (Moonsie Shageer and Mowat, 1993). Severity of diarrhea (using mean fecal scores) decreased linearly with intake of LA, hence greater red blood cells in calves in T2 could not be due to dehydration, otherwise calves in T1 should have had greater concentrations of red blood cells or hematocrit. Mean va lues of hematocrit were within normal ranges for preweaned calves (Brun Hansen et al., 2006). If these calves were experiencing nutritional stress based upon low BW gain the first 30 d of life, increased feeding of LA may not have been able to optimize gai n but may have been able to influence immune responses. Concentrations of white blood cells were greater at 7 d of age falling thereafter and a similar pattern was observed for

PAGE 335

335 blood neutrophil concentrations. In current study we did not analyze the expres sion of receptors on neutrophil surfaces. It is known that although neutrophil receptors (CD18, CD62L) are expressed constitutively, their expression could be downregulated by immunosuppresion. Therefore, the number of receptors expressed per neutrophil co uld be reduced, this was found in cows after parturition and in calves abruptly weaned (Weber et al. 2001; Lynch et al., 2010). Fewer receptors expressed per neutrophil was associated with neutrophilia, possibly indicating the inability of neutrophils to m igrate to the infection zone, hence increasing the risk of infections (Weber et al. 2001). If it is assumed that the lower mean concentration of neutrophils detected in female calves fed T2 or T3 was due to increased migration from the blood to sites of in flammation, it would indicate that these calves were better able to mount an attack against infection; however a decreased production of neutrophils in bone marrow could not be ruled out. Regardless of gender, a greater proportion of blood neutrophils from calves fed T2 or T3 performed phagocytosis and produced oxidative radicals, which indicates a more efficient activity of neutrophils in these calves. If these neutrophils were in lower concentrations due to increased migration to the sites of inflammation and had improved immune activity, calves would be more efficient to respond to inflammatory processes to resist pathogen invasions. Studies evaluating the effect of different stressors on neutrophil phagocytic activity of calves have reported variable res ults (Pang et al., 2009; Hulbert et al., 2011). Concentrations of Hp are absent in healthy calves but elevated under subclinical inflammatory disorders (Ganheim et al., 2007; Cray et al. 2009). Experimental models of respiratory and digestive tract infecti on in calves reported increased plasma

PAGE 336

336 concentrations of Hp in sick calves as compared to healthy ones (Deignan et al. 2000; Heegaard et al., 2000; da Silva et al. 2011). Concentrations of Hp peaked at 8 d of age when episodes of diarrhea were greatest. Ca lves fed T1 had greater plasma concentration of Hp at this point time. However fecal score at this time did not differ among LA treatments so calves fed T1 had a greater immune reaction to inflammation of the small intestine suggesting the feeding more LA reduced the inflammatory response as compared to the feeding of saturated FA. Acid soluble protein is identified as having dual inflammatory and immunomodulatory properties. One of the mechanisms by which ASP can exert its antinflammatory effect is by inhi biting the proliferation of blood lymphocytes after mitogen stimulation (Hochepied et al., 2003). At d 15, calves fed T3 had lower concentrations of ASP which matched with the lower in vitro proliferation of lymphocytes for T3 calves collected at d 14. Se lective proliferation of T cells after 48 h in vitro stimulation with LPS + PHA was greater in calves fed T2 and this held true at every time of measure whereas calves fed T3 responded well only at 42 d of age. Some human studies however failed to detect a n effect of LA on cell proliferation but this was due likely to the short duration of the studies and or to minimal or no change in the profile of FA in blood cells which may have prevented LA from having an opportunity to exert an effect on cell prolifera tion ( Kelley et al., 1989, 1992; Yaqoob et al., 2000). In contrast, Thanasak et al. (2005) cultured bovine PBMC with 2 doses (125 or 250 uM) of LA or ALA and reported that the higher concentration of LA inhibited proliferative response of PBMC to mitogens. Later Gorjao et al. (2007) evaluated the proliferative response of human lymphocytes to IL 2 stimulation and reported that lower concentrations of LA stimulated proliferation of

PAGE 337

337 lymphocytes preventing apoptosis and necrosis (< 75 uM) but that greater conc entrations of LA reduced the proliferation of lymphocytes with respect to the control media. Based upon the results of Gorjao et al. (2007), it can be hypothesized that none of the current treatments had toxic effects so as to induce apoptosis and necrosi s of lymphocytes which would have prevented their proliferation because all LA treatments stimulated lymphocytes equal to or than that of T1. Another reason why LA intakes greater than that of T2 would not have toxic effects on immune cells that could pre vent their proliferation was that the production of IFN was increased by stimulated cells especially from males fed T2 and T3 and from females fed T2, whereas T4 and T3 and T4 from and males and females, respectively did not differ from that of calves fe d T1. These results contrast with those of Wallace et al. (2001) who fed mice diets, of low fat or high fat supplemented with CCO (2.3% of LA), safflower oil (SAO, 61% of LA) or FO (9% of LA). The FA profile of the phospholipids in spleen lymphocytes refl ected the dietary FA but IFN production was decreased when mice were fed SAO or FO. The current study, however agrees with the previous study (Chapter 4) where stimulated PBMC of calves fed greater amounts of LA (between the amount offered with T3 and T4) produced more IFN One of the goals T helper 2 (Th2) response towards a Th1 response. The pattern of cytokine production is used to verify the predominant type of Th response. An increased concentration of IFN 4 is indicative of Th1 predominance (Chase et al., 2008). Greater mea n production of IFN fed T2 and T3 or

PAGE 338

338 females fed T2 might indicate an improved ability of these calves to swi tch to the Th1 response. Interferon presentation, cell cycle growth and apoptosis, leukocyte trafficking, and B cell depletion (Arens et al., 2001; Chen and Liu, 2009). Hence, it should be expected that as long as IFN production of anti OVA IgG was greater by male calves when fed T2 or T3, which also matched with an increased production of IFN latter just numerically). Foote et al. (2007) fed increased amounts of nutrients to preweaned calves and reported better growth but production of TNF of anti OVA IgG were not affected by treatment. The finding of Foote et al. (2007) could be interpreted as that the activation or inactivation of a cell type response (T cells producing IFN IgG). Hence, it can be concluded that male calves fed T2 and T3 had an overall better function of T and B cells, whereas female calves only had improved T cell function. Delayed type hypersensitivity tests the ability of mononuclear immune cells to infiltrate and/or accumulate into regions of antigen deposition. It is strictly a cell mediated response and not an antibody mediated response (Berhagen et al. 1996). The DTH skin test produces a characteristic response which includes induration, swelling, and monocytic infiltration into the site of the lesion within 24 t o 72 h (Black, 1999). Use of antigen rather than mitogens is the best approach to evaluate DTH responses; however, the use of antigens has been reported to cross react with Mycobacterium tuberculosis leading to false positives (Hernandez et al., 2005). On the other hand,

PAGE 339

339 mitogens such as PHA, without being a strong inducer of DTH, has been demonstrated to induce a moderate response after an intradermal injection in calves (Stanton et al., 2000; Ballou and DePeters, 2008) and in cows (Hernandez et al., 2005; Caroprese et al., 2009). Previous research suggests that increasing LA intake would increase the proliferation of lymphocytes up to a point beyond which further increases in LA would suppress lymphocyte proliferation. However this was not documented in t he current study. Ballou and DePeters (2008) hypothesized that a positive correlation exists between in vitro lymphocyte proliferation and a DTH response of cells to explain their results. However Hernandez et al. (2005) reported that the main cells infilt rating into the skin of co ws challenged with PHA were eosinophils, macrophages, and neutrophils but not lymphocytes, whereas in the skin of sheep intradermally challenged with avidin, the cells, neutrophils, macrophages, and CD45R + B c ells (Lofthouse et al., 1995). The current findings at 30 d of age indicate that skin thickness responded linearly to PHA injection with increasing intake of LA (Table 6 13) but without a concomitant increase in lymphocyte proliferation (Figure 6 17). The refore current results may support the work of Hernandez et al. (2005). Measures of DTH at both 30 and 60 d measures were affected by time, with the greater response at 6h after PHA injection at both measurement times. This differential response due to t ime after injection is in agreement to Staton et al. (2000) and Hernandez et al. (2005) who reported that the largest responses to a PHA challenge were seen at 8 and 6 h post injection respectively. Hernandez et al. (2005) concluded that PHA is not a viabl e alternative to determine true DTH. Responses to PHA injection

PAGE 340

340 in current study were variable; this might be due to other factors that could influence the response of calves, with the exception of gender which did not affect the results in this study. Mor eover the lack of ability of PHA to maintain a true DTH (greater skin fold changes at 24 or 48 h) in the current study might indicate that other alternative mitogens or true antigens should be used to test DTH response in dairy calves. Summary Intake of LA was progressively adjusted by partially replacing hydrogenated CCO with SO in MR. Male calves fed LA at 0.206 g/kg of BW 0.75 had better ADG and FE during the first 30 d of age and this was accompanied by a tendency for greater intake of MR. However ADG re turned to baseline when male calves were fed LA at the greater rates of 0.333 and 0.586 g/kg of BW 0.75 Female calves tended to improve ADG and FE with increasing intake of LA in the first 30 d of life. However these responses to increasing LA intake after initiation of grain feeding at 31 d of life. On the other hand, wither and hip growth was greater by calves consuming LA at or exceeding 0.206 g/kg of BW 0.75 during the 60 d study. These changes in gain and growth were not accompanied by increases in circ ulation concentrations of glucose, insulin or IGF 1. Circulating concentrations of white blood cells, neutrophils, and monocytes were generally greater in female compared to male calves but gender effects on white blood cells and neutrophils were modified by LA intake. Similarly, some of the measured immune markers differed with gender and intake of LA. Males fed LA at 0.206 and/or 0.333 g/kg of BW 0.75 had increased production of anti OVA IgG, production of IFN stimulated blood cells, and DTH in response to an intradermal injection of PHA at 30 d. Female calves fed LA at 0.206 g/kg of BW 0.75 had increased production of IFN stimulated blood cell s and DTH in response to an intradermal injection of PHA at 30 d

PAGE 341

341 were increased in females fed 0.333 or 0.586g/kg of BW 0.75 Regardless the gender calves fed 0.206 and/or 0.333 g/kg of BW 0.75 had greater phagocytosis activity by blood neutrophils, prolifer ation of stimulated whole blood cells, and DTH in response to an intradermal injection of PHA at 60 d. Diarrhea affected all calves. Mean score of feces and age at first outbreak of diarrhea decreased and increased linearly respectively with increasing int ake of LA. Plasma concentrations of haptoglobin were lower in calves fed LA at or > 0.206 g/kg of BW 0.75 at 8 d of age when diarrhea was most evident. Risk of diseases (pneumonia, naval infection, bloody diarrhea, or fever) was not reduced by increased fe eding of LA. Feeding T2 or T3 diets to preweaned Holstein calves increased responses for most of the markers of immunity evaluated in this study and improved wither and hip growth and feces and attitude scores. Hence under the conditions of the present st udy, intakes of LA of between 0.206 and 0.333 g g/kg of BW 0.75 promoted better productive performance possibly by improving the immune status of calves. Future research should seek to clarify the mechanisms by which increased intake of LA might differentia lly modify the response of healthy and unhealthy female and male calves.

PAGE 342

342 Table 6 1. Ingredient and chemical composition of diet fed to nonlactating, pregnant Holstein animals. Prepartum diet Ingredient, % of DM Bermudagrass silage 46.50 Corn si lage 8.80 Citrus pulp 31.70 Soybean meal 9.20 Mineral mix 1 3.80 Nutrients, DM basis Crude protein, % 13.80 NE L 2 Mcal/kg 1.46 NDF, % 39.85 Ether extract, % 3.20 Ash, % 8.12 Ca, % 1.26 P, % 0.34 Mg, % 0.40 K, % 1.52 S, % 0.34 Na, % 0.18 Cl, % 0.87 Mn, mg/kg 64.00 Zn, mg/kg 59.00 Cu, mg/kg 20.00 Fe, mg/kg 212.00 Mo, mg/kg 0.62 DCAD, mg/100 g 20.07 1 Contains (DM basis) 34.5% corn meal, 5.0% dicalcium phosphate, 16.0% calcium carbonate, 10% cal cium sulfate, 5% magnesium oxide, 10% magnesium sulfate, 4% sodium chloride, 1.7% Zinpro 4 plex (Zinpro, Minneapolis, MN), 0.4% Rumensin 80 (Elanco Animal Health, IN), 0.35% Sel Plex 2000 (Alltech Biotechnology, Nicholasville, KY), 0.002% Ca iodate, and a vitamin premix. Each kg contains 24.5% CP, 9.8% Ca, 1.5% P, 4.2% Mg, 3.2% S, 1.7% Na, 10.7% Cl, 475 mg of Zn, 160 mg of Cu, 456 mg of Mn, 7.4 mg of Se, 37.4 mg of Co, 13.2 mg of I, 118,000 IU of vitamin A, 27,500 IU of vitamin D, 2,600 IU of vitamin E, and 770 mg of monensin. 2 Calculated from the estimation of energetic values of individual ingredients using the NRC software (2001) and considering intake at 3X of maintenance.

PAGE 343

343 Table 6 2. Fatty acid (FA) profile of sources if fat, emulsifier and basal milk replacer FA Coconut oil 1 Soybean oil 2 Emulsifier 3 Milk replacer 4 C6:0 0.6 ND 5 ND 0.3 C8:0 7.8 ND ND 2.0 C10:0 6.2 ND ND 4.2 C12:0 50.0 ND ND 30.6 C14:0 18.1 0.1 0.1 14.9 C16:0 8.4 11.4 11.1 17.6 C16:1 ND 0.1 ND 0.7 C18:0 8.7 4.0 87.9 7 .6 C18:1 0.1 20.5 0.1 15.2 C18:2 0.0 55.3 0.1 5.9 0.0 8.1 0.0 0.1 C20:0 0.1 0.3 0.5 ND Other FA 0.0 0.2 0.2 0.9 1 Welch, Holme & Clark Co., Inc, Newark, NJ 2 Winn Dixie Co.. 3 Grindsted mono di HV 52, Gillco Ingredients, San Marcos, CA. 4 oil, vitamins, and minerals. Each kg contains 1.03% Ca, 0.80% P, 0.11 mg of Co, 10.9 mg of Cu, 1.1 mg of I, 110 mg of Fe, 49,560 IU of vitamin A, 12,400 IU o f vitamin D, and 241 IU of vitamin E. 5 Non detected

PAGE 344

344 Table 6 3. Ingredient and chemical composition of milk replacers and grain mix fed to preweaned Holstein calves. Milk replacer Grain mix Ingredients, % of DM 31:7 milk replacer 1 89.80 Oil combination 2 9.68 Emulsifier 3 0.48 Steam rolled barley 51.7 Soybean meal 16.5 Beet pulp shreds 24.5 Sugarcane molasses 5.3 Mineral mix 4 2.0 Nutrients, DM basis Lactose, % 39.70 Crude protein, % 29.70 18.30 Ether extract, % 18.70 2.10 Ash, % 6.08 5.42 Ca, % 0.77 0.57 P, % 0.72 0.45 Mg, % 0.13 0.35 K, % 2.12 0.92 S, % 0.39 0.26 Na, % 0.76 0.16 Cl, % 1.27 0.32 Mn, mg/kg 49.50 55.00 Zn, mg/kg 53.00 57.00 Cu, mg/kg 11.70 16.00 Fe, mg/kg 132.00 362.00 Mo, mg/kg 0.60 1.40 1 oil, vitamins, and minerals. Each kg contains 1.03% Ca, 0.80% P, 0.11 mg of Co, 10.9 mg of Cu, 1.1 mg of I, 110 mg of Fe, 49,560 IU of vitamin A, 12,400 IU of vitamin D, and 241 IU of vitamin E. 2 Contains proportions of coconut oil:soybean oil according to treatments (T), T1 = 100:0, T2 = 95.99:4.01, T3 = 87.93:12.07, and T4 = 71.77:28.23. 3 Grindsted mono d i HV 52, Gillco Ingredients, San Marcos, CA. 4 Each kg of DM contains 8.8% Ca, 4.2% P, 11.4% Mg, 12.4% Cl, 0.49% K, 8.1% Na, 0.36% S, 58 mg of Co, 263 mg of Cu, 26 mg of I, 1933 mg of Fe, 923 mg of Mn, 8.46 mg of Se, 1109 mg of Zn, 259,000 IU of vitamin A, 70,000 IU of vitamin D, and 2,400 IU of vitamin E.

PAGE 345

345 Table 6 4. Passive immunity related measures of newborn male (M) and female (F) Holstein calves assigned to treatments with increasing amounts of linoleic acid (LA) Treatments 1 Contrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G M F M F M F M F Total calves 7 14 9 13 9 13 9 14 Colostrum fed Quantity, L 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 0.02 0.73 0.88 0.63 0.61 0.29 0.71 0.24 Total IgG, g/L 94.6 82. 4 96.5 71.5 77.0 76.1 79.1 87.4 7.49 0.55 0.14 0.89 0.17 0.05 0.97 0.22 Total IgG intake, g 379 330 382 286 308 304 317 346 30.3 0.52 0.15 0.94 0.17 0.07 0.94 0.25 Birth Body weight, kg 41.1 36.8 43.4 38.8 40.3 38.2 43. 2 37.2 1.57 0.76 1.00 0.13 <0.01 0.61 0.32 0.57 STP, g/dL 4.87 4.55 4.86 4.58 4.83 4.59 4.64 4.83 0.11 0.86 0.90 0.88 0.04 0.01 0.48 0.87 24 h after birth Serum total IgG, g/dL 3 1.99 2.30 2.26 2.01 2.06 1.91 2.24 2.34 0 .21 0.46 0.24 0.67 1.00 0.97 0.26 0.36 AEA 4 % 21.8 26.1 27.1 26.3 28.3 22.5 27.4 24.4 2.00 0.61 0.60 0.24 0.36 0.12 0.04 0.87 STP, g/dL 5.90 5.75 6.10 5.72 5.91 5.59 5.72 5.96 0.19 0.87 0.68 0.48 0.26 0.16 0.28 0.73 STP increase, g/dL 1.03 1.22 1.24 1. 14 1.08 1.00 1.08 1.13 0.18 0.76 0.69 0.53 0.91 0.94 0.47 0.62 1 Targeted intakes of linoleic acid from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/k g of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and treatment by gender interactions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender. 3 Four out of 88 calves had an IgG con centration < 10 g/dL, two fed treatment 2 and one fed treatment 3. 4 % AEA = [IgG concentration in serum at 24 h of life (0.099 x body weight at birth)] / IgG intake 100%.

PAGE 346

346 Table 6 5. Dry matter intake (DMI), body weight (BW) gain, and feed effic iency (FE, kg of BW gain/kg of DMI) of preweaned male (M) and female (F) Holstein calves fed increasing amounts of linoleic acid (LA) Treatments 1 Contrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G M F M F M F M F Birth to 30 d Birth weight 3 kg 41.1 36.8 43.4 38.8 40.3 38.2 43.2 37.2 1.57 0.76 1.00 0.13 <0.01 0.61 0.32 0.57 BW gain, kg 2.75 2.62 5.26 3.09 2.80 3.30 2.70 3.44 0.54 0.66 0.44 0.01 0.49 0.07 0.98 0.02 ADG, kg/d 0.09 0.09 0.18 0.10 0.09 0.11 0.09 0.11 0.02 0.65 0.47 0.01 0.41 0.07 1.00 0.02 MR intake, kg DM 19.2 17.6 19.7 17.8 19.0 18.0 19.2 17.9 0.17 0.96 0.54 0.03 <0.01 0.17 0.27 0.09 LA intake, g 95 87 143 129 212 208 393 358 5.85 <0.01 0.76 0.21 <0.01 0.02 0.16 0.35 FE 0.14 0.15 0.27 0.17 0 .15 0.18 0.15 0.19 0.03 0.84 0.44 0.01 0.93 0.10 0.93 0.04 31 to 60 d Weight at 30 d, kg 44.9 40.3 47.4 40.8 44.9 41.0 44.8 41.1 0.54 0.66 0.44 0.01 <0.01 0.07 0.98 0.02 BW gain, kg 23.5 19.2 21.3 18.9 23.7 19.1 24.1 19.3 1.39 0.51 0.84 0.34 <0.01 0.58 0.95 0.44 ADG, kg/d 0.78 0.64 0.71 0.63 0.79 0.64 0.80 0.64 0.05 0.51 0.83 0.34 <0.01 0.59 0.95 0.44 MR intake, kg DM 22.9 20.9 23.6 21.0 23.1 21.1 22.9 21.1 0.22 0.88 0.24 0.17 <0.01 0.17 0.67 0.13 Grain intake, kg DM 16.3 13.7 13.4 13. 3 16.1 13.5 15.6 13.3 1.66 0.92 0.88 0.30 0.11 0.82 0.95 0.40 FE 0.58 0.56 0.57 0.55 0.60 0.55 0.63 0.56 0.02 0.14 0.86 0.54 <0.01 0.18 0.96 0.70 Birth to weaning BW at 60 d, kg 68.3 59.5 68.7 59.7 68.6 60.1 68.9 60.4 1.35 0.61 0.91 0.9 2 <0.01 0.88 0.95 0.90 Total BW gain, kg 26.2 21.9 26.6 22.0 26.5 22.4 26.8 22.7 1.35 0.61 0.92 0.92 <0.01 0.88 0.95 0.90 ADG, kg/d 0.44 0.36 0.44 0.37 0.44 0.37 0.45 0.38 0.02 0.64 0.94 0.88 <0.01 0.89 0.95 0.90 MR intake, kg DM 42.1 38.5 43.3 38.8 42 .1 39.1 42.0 39.0 0.34 0.89 0.28 0.05 <0.01 0.12 0.77 0.07 Total DMI, kg 58.4 52.2 56.7 52.1 58.2 52.6 57.7 52.4 1.76 0.94 0.95 0.55 <0.01 0.93 0.90 0.66 FE 0.44 0.42 0.47 0.42 0.45 0.43 0.47 0.43 0.01 0.23 0.86 0.30 <0.01 0.93 0.99 0.45 1 Targeted inta kes of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and treatment by gen der interactions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender. 3 Birth weight deviations from the mean birth weight within each gender were covariates for analysis of BW gains. Hence birth w e ight added to any later variable of gain will not give the expected body weight.

PAGE 347

347 Table 6 6. Wither and hip height and growth of preweaned male (M) and female (F) Holstein calves fed increasing amounts of linoleic acid (LA) Treatments 1 Contrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G M F M F M F M F Height, cm Day 0 withers 76.9 74.2 76.7 74.6 75.9 73.3 76.9 73.7 0.98 0.67 0.35 0.54 <0.01 0.66 0.83 0.74 Day 0 hip 81.1 78.5 81.2 79.5 80.3 78.1 81.5 78.2 1.03 0.83 0.56 0.37 <0.01 0.56 0.67 0.73 Day 30 withers 79.1 76.5 79.3 76.3 79.6 77.9 78.7 77.6 0.54 0.41 0.12 0.35 <0.01 0.09 0.83 0.45 Day 30 hip 83.2 81.6 84.3 80.9 84.3 82.5 83.4 82.2 0.53 0.44 0.09 0.58 <0.01 0.22 0.55 0.08 Day 60 withers 84.5 81.2 84.9 82.7 85.1 83.4 84.7 82.4 0.66 0.54 0.05 0.58 <0.01 0.56 0.27 0.69 Day 60 hip 89.8 85.9 90.2 87.7 90.3 88.1 89.3 87.1 0.69 0.97 0.04 0.48 <0.01 0.32 0.38 0.55 Growth, cm/d Wither, 1 st 30 d 0.08 0.09 0.09 0.08 0.10 0.13 0.07 0.12 0.02 0.38 0.13 0.39 0.13 0.09 0.85 0.41 Hip, 1 st 30 d 0.07 0.10 0.11 0.08 0.11 0.13 0.08 0.12 0.02 0.48 0.09 0.62 0.18 0.21 0.52 0.07 Wither, 2 nd 30 d 0.18 0.15 0.19 0.21 0.18 0.18 0.20 0.16 0.02 0.96 0.43 0.11 0.45 0.32 0.29 0.25 Hip, 2 n d 30 d 0.22 0.14 0.20 0.23 0.20 0.19 0.20 0.16 0.02 0.46 0.35 0.18 0.08 0.92 0.10 0.02 Wither, all period 0.13 0.12 0.14 0.15 0.14 0.16 0.13 0.14 0.01 0.55 0.04 0.63 0.71 0.48 0.29 0.61 Hip, all period 0.15 0.12 0.15 0.15 0.15 0.16 0.14 0.14 0.01 0.90 0. 04 0.44 0.57 0.34 0.38 0.56 1 Targeted intakes of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and treatment by gender interactions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender.

PAGE 348

348 Table 6 7. Plasma concentrations of glucose, plasma urea nitrogen (PUN), B hydro xybutyrate (BHBA), total cholesterol, insulin, and insulin like growth factor I (IGF I) of preweaned male (M) and female (F) Holstein calves fed increasing amounts of linoleic acid (LA). All interactions with age did not differ unless footnoted Treatments 1 Contrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G Age M F M F M F M F Glucose, mg/dL 90.1 89.2 87.8 87.6 90.2 84.9 86.6 90.3 1.46 0.70 0.18 0.46 0.53 0.12 0.02 0.23 <0.01 PUN, mg/dL 7.69 8.02 7.74 7.64 7.32 8.49 8.47 7.86 0.35 0.24 0.70 0.62 0.41 0.27 0.07 0.15 <0.01 BHBA, mg/dL 0.92 0.84 0.81 0.78 0.81 0.71 0.82 0.69 0.06 0.06 0.17 0.58 0.04 0.35 0.96 0.64 <0.01 Total cholesterol, mg/dL 78.6 76.0 83.8 81.2 92.3 86.0 94.4 79.4 4.08 0.03 0.04 0.98 0.03 0.09 0.81 0.90 <0.0 1 Insulin, ng/mL 2.66 2.20 2.66 1.72 2.48 2.03 2.92 2.04 0.29 0.63 0.40 0.44 <0.01 0.75 0.89 0.26 <0.01 IGF I 3 g/mL 39.5 38.4 45.1 38.6 41.1 39.5 42.3 39.4 2.22 0.68 0.66 0.25 0.06 0.93 0.94 0.21 <0.01 STP 4 g/dL 5.59 5.60 5.52 5.74 5.57 5.67 5.56 5.66 0.07 0.93 0.75 0.67 0.03 0.90 0.54 0.15 <0.01 1 Targeted intakes of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for o rthogonal contrasts of treatments, gender, and treatment by gender interactions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender. 3 Gender by age P = 0.10. 4 Serum total protein, gender by age, P < 0.01.

PAGE 349

349 Table 6 8. Health scores and percentage of days with poor attitude, fever, diarrhea and nasal discharge of preweaned male (M) and female (F) Holstein calves fed increasing amounts of linoleic acid (LA). All interactions with age did not diffe r unless footnoted Treatments 1 Contrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G Age M F M F M F M F Scores 3 Feces 0.66 0.73 0.64 0.68 0.59 0.73 0.60 0.60 0.05 0.07 0.92 0.60 0.08 0.53 0.32 0.41 <0.01 At titude 0.14 0.19 0.11 0.18 0.12 0.20 0.13 0.15 0.03 0.53 0.78 0.53 0.02 0.51 0.57 0.92 <0.01 Nasal discharge 4 0.02 0.03 0.02 0.06 0.04 0.09 0.04 0.05 0.02 0.44 0.10 0.83 0.09 0.77 0.30 0.64 0.01 Ocular discharge 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0. 01 0.86 0.81 0.89 0.49 0.58 0.82 0.46 0.06 Rectal temp, C 38.8 38.9 38.8 38.9 38.8 38.9 38.8 38.9 0.05 0.36 0.93 0.22 0.02 0.99 0.56 0.68 <0.01 Days to diarrhea 7.21 6.82 7.18 7.49 7.68 7.38 7.75 7.52 0.35 0.10 0.39 0.72 0.54 0.85 0.86 0.30 Days with 5 % Poor attitude 12.9 16.0 9.6 15.1 12.0 16.4 10.9 13.3 2.54 0.53 0.81 0.36 0.04 0.72 0.69 0.71 Nasal discharge 2.05 3.02 1.54 5.37 4.99 8.25 3.19 4.34 1.85 0.51 0.04 0.54 0.08 0.79 0.46 0.60 Ocular discharge 1. 44 1.29 0.55 2.19 1.30 2.07 1.48 1.41 0.90 0.91 0.74 0.86 0.39 0.71 0.51 0.41 Cough 0.01 0.35 0.18 0.27 0.00 0.91 0.37 0.46 0.30 0.38 0.52 0.78 0.10 0.84 0.25 0.30 Fever, first 14 d 4.86 8.54 5.69 5.12 3.93 6.61 4.06 7.05 2.30 0.74 0.61 0.75 0.18 0.8 1 0.78 0.36 Diarrhea 14.7 18.6 15.7 15.8 14.2 18.3 15.1 13.7 2.00 0.28 0.81 0.61 0.25 0.30 0.59 0.24 Severe diarrhea 3.92 5.10 5.64 4.54 3.69 5.49 4.31 3.33 0.97 0.29 0.61 0.57 0.75 0.49 0.48 0.12 1 Targeted intakes of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and treatment by gender interactions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender. 3 Nasal score scale: 0 = normal serous discharge, 1 = small amount of unilateral cloudy discharge, 2 = bilateral cloudy or ex cessive mucus discharge, and 3 = cop ious bilateral mucopurulent discharge. Attitude score scale: 0 = responsive, 1 = non active, 2 = depressed, and 3 = moribund. Feces score scale: 0 = firm feces, no diarrhea; 1 = soft feces, no diarrhea, 2 = mild diarrhea and 3 = watery diarrhea, sever e diarrhea. Ocular score scale: 0 = normal, 1 = small amount of ocular discharge, 2 = moderate amount of bilateral discharge, 3 = heavy o cular discharge. Cough score scale: 0 = none, 1= induced single cough, 2 =: induced repeated cough or occasional spo ntaneous cough, 3 = repeated spontaneous cough. 4 Treatment by age, P = 0.08

PAGE 350

350 5 Percentage of days with health issue over a total of a 60 d period unless another time period is indicated. Poor attitude, nasal, and ocular discharges if score scale > 0. Fever if temp C (103 F). Diarrhea if score scale > 1 and severe diarrhea if score scale = 3.

PAGE 351

351 Table 6 9. Incidence of diseases in preweaned Holstein calves fed increasing amounts of linoleic acid (LA) Item Treatment 1 %(n/n) AOR 2 95% CI P Pneumonia T1 14.3 (3/21) Ref. T2 18.2 (4/22) 1.42 0.20 7.36 0.95 T3 18.2 (4/22) 1.42 0.27 7.36 0.95 T4 21.7 (5/23) 1.75 0.36 8.59 0.59 Male 11.8 (4/34) Ref. Female 22.2 12/54) 2.19 0.64 7.49 0.21 Navel infection T1 4.8 (1/21) Ref. T2 4.5 (1/22) 0.83 0.05 14.90 0.96 T3 0.0 (0/22) 0.96 T4 8.7 (2/23) 1.77 0.14 22.10 0.95 Male 8.8 (3/34) Ref. Female 1.9 (1/54) 0.18 0.02 1.88 0.15 Bloody diarrhea 3 T1 47.6 (10/21) Ref. T2 40.9 (9/22) 0.69 0.21 2.24 0.80 T3 31.8 (7 /22) 0.46 0.14 1.51 0.21 T4 52.2 (12/23) 1.18 0.37 3.79 0.31 Male 50.0 (17/34) Ref. Female 38.9 (21/54) 0.60 0.26 1.40 0.29 Fever T1 68.2 (14/21) Ref. T2 58.3 (14/22) 0.94 0.26 3.42 0.99 T3 72.0 (16/22) 1.47 0.38 5.52 0.29 T4 50.0 (12/23) 0.56 0.16 1.97 0.18 Male 51.4 (17/34) Ref. Female 69.0 (39/54) 2.86 1.07 6.70 0.03 1 Targeted intakes of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the onl y feed during the first 30 d of life. 2 Adjusted odds ratio, T1 was reference (Ref.) for treatment diets and male was reference for gender. 3 Diarrhea occurred in all calves.

PAGE 352

352 Table 6 10. Mean concentration of blood cell number and white blood cells perc entages in preweaned male (M) and female (F) Holstein calves fed increasing amounts of linoleic acid (LA). All interactions with age did not differ unless footnoted. Treatments 1 Contrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G Age M F M F M F M F Blood cells Total Red, 10 6 /uL 8.28 8.03 8.85 8.56 8.41 8.22 8.22 7.57 0.32 0.10 0.24 0.11 0.13 0.50 0.69 0.85 <0.01 Total white, 10 3 /uL 8.7 10.4 9.2 10.1 10.0 9.4 8.9 11.0 0.62 0.64 0.91 0.59 <0.01 0.99 0.04 0.6 7 <0.01 Neutrophils, 10 3 /uL 3.26 4.14 3.35 3.89 3.61 3.43 3.27 4.45 0.31 0.57 0.43 0.94 0.01 0.55 0.04 0.71 <0.01 Lymphocytes, 10 3 /uL 4.11 4.74 4.67 4.71 4.63 4.73 4.60 4.47 0.28 0.95 0.36 0.55 0.41 0.28 0.60 0.45 <0.01 Monocytes, 10 3 /uL 0.42 0.61 0.44 0.49 0.41 0.57 0.33 0.65 0.09 0.84 0.83 0.51 <0.01 0.25 0.60 0.56 <0.01 Eosinophils 3 10 3 /uL 48.9 75.2 56.7 50.6 59.2 64.6 43.4 54.0 8.21 0.18 0.54 0.21 0.13 0.81 0.24 0.12 <0.01 Basophils, #/uL 31.8 34.5 26.1 43.9 54.5 32.5 43.1 43.3 7.04 0.12 0.36 0.68 0.95 0.42 0.10 0.03 <0.01 Platelets, 10 3 /uL 488 521 527 515 561 415 518 447 49.8 0.48 0.84 0.56 0.15 0.27 0.13 0.66 <0.01 White cells, % Neutrophils 41.6 43.3 39.7 42.7 41.4 39.4 40.2 45.7 2.05 0.64 0.22 0.90 0.17 0.41 0.17 0.36 <0.01 Lymphocytes 51.2 48.1 53.1 49.6 51.6 52.0 53.6 45.3 1.93 0.70 0.18 0.71 0.01 0.18 0.12 0.46 <0.01 Monocytes 6.21 7.14 5.43 6.24 5.22 7.18 4.98 7.19 0.99 0.73 0.72 0.49 0.04 0.42 0.80 0.79 <0.01 Eosinophils 4 0.59 0.69 0.61 0.51 0.65 0.69 0.50 0.50 0.09 0 .19 0.31 0.18 0.86 0.86 0.79 0.25 <0.01 Basophils 0.35 0.32 0.26 0.46 0.54 0.35 0.51 0.37 0.07 0.11 0.35 0.78 0.44 0.09 0.51 0.02 <0.01 Hematocrit, % 33.1 32.3 32.8 32.9 32.3 32.2 32.1 32.1 0.65 0.23 0.80 0.62 0.62 0.74 0.69 0.65 <0.01 1 Targeted intake s of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and treatment by gende r interactions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender. 3 Gender by age, P = 0.05. 4 Gender by age, P <0.01

PAGE 353

353 Table 6 11. Phagocytosis, oxidative burst, and mean fluorescence intensity (MFI) of neutrophils in peripheral blood of preweaned male (M) and female (F) Holstein calves fed increasing amounts of linoleic acid (LA) Treatments 1 Contrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G Age M F M F M F M F Ph agocytosis, % of neutrophils 60.2 64.0 67.4 65.8 64.3 64.1 62.9 62.7 2.28 0.58 0.27 0.09 0.81 0.64 0.50 0.38 0.14 Phagocytosis, MFI 21.2 22.8 23.2 24.6 21.2 24.5 23.0 21.4 2.10 0.78 0.62 0.42 0.45 0.41 0.45 0.71 <0.01 Oxidative burst %, of neutrophils 48.9 53.9 55.8 55.9 53.4 53.8 50.5 50.4 2.52 0.24 0.18 0.15 0.46 0.46 0.55 0.53 0.95 Oxidative burst, MFI 32.2 33.1 35.5 35.5 31.5 36.8 36.9 34.6 2.90 0.45 0.88 0.36 0.63 0.62 0.29 0.52 <0.01 1 Targeted intakes of LA f rom milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and treatment by gender interac tions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender.

PAGE 354

354 Table 6 12. Mean concentration of plasma acute phase proteins, serum anti OVA IgG, cytokines produced by whole blood cells stimulated with LPS + PHA, and proliferation of whole blood cells by thymidine incorporation in preweaned male (M) and female (F) Holstein calves fed increasing amounts of linoleic acid (LA). All interactions with age did not differ unless footnoted Treatments 1 C ontrasts 2 P values Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q*G Cb*G Age M F M F M F M F ASP 3 mg/L 88.7 96.1 81.8 98.1 85.0 85.0 93.4 94.8 4.70 0.07 0.83 0.58 0.05 0.25 0.70 0.15 <0.01 Haptoglobin 4 OD x 100 0.82 0.86 0.75 0.84 0.77 0.90 0.7 7 0.88 0.10 0.06 0.49 0.94 0.91 0.64 0.62 0.84 <0.01 Anti OVA IgG, OD 0.10 0.17 0.22 0.18 0.24 0.18 0.13 0.22 0.04 0.71 0.33 0.58 0.08 0.47 0.04 0.47 <0.01 TNF 311 362 424 321 457 390 345 389 64.2 0.68 0.94 0.72 0.20 0.66 0.26 0.40 <0.01 IFN 260 256 357 327 411 227 209 265 66.4 0.39 0.38 0.38 0.19 0.62 0.09 0.57 <0.01 Whole blood cell proliferation Cont rol 5 0.50 0.52 0.56 0.56 0.55 0.58 0.59 0.65 0.08 0.71 0.65 0.23 0.81 0.75 0.93 0.87 <0.01 Stimulated 5 12.8 14.7 23.5 20.4 14.0 18.8 16.2 19.0 2.83 0.33 0.01 0.67 0.44 0.63 0.71 0.22 <0.01 Difference 5 12.1 14.1 22.8 19.8 13.3 17.9 15.5 18.0 2.78 0.33 0. 01 0.71 0.44 0.65 0.72 0.22 <0.01 Stimulation index 6 25.5 28.4 41.7 36.5 25.3 32.7 27.6 29.4 4.47 0.47 0.01 0.47 0.51 0.80 0.61 0.22 <0.01 Stimulated per Lymphocyte 7 2.87 3.13 4.98 4.36 3.00 4.04 3.56 4.11 0.59 0.37 0.01 0.61 0.50 0.57 0.58 0.26 <0.01 1 Targeted intakes of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.333, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and t reatment by gender interactions. L= linear effect of treatment, Q = quadratic effect of treatment, Cb = cubic effect of treatment, G = gender. 3 Treatment by age, P < 0.01 4 Treatment by age, P = 0.02. 5 Proliferation is expressed as KCPM (1000 counts per minute of thymidine incorporation). 6 CPM of stimulated cells divided by CPM of nonstimulated cells. 7 CPM of stimulated cells divided by the number of lymphocytes in whole blood.

PAGE 355

355 Table 6 13. Skin fold change measured after 6, 24, and 48 h of intrade rmal injection of 150 ug of phytohaemagglutinin in preweaned male (M) and female (F) Holstein calves. All interactions with age did not differ unless footnoted Treatments 1 Contrasts 2 P values Hour after injec tion Measure T1 T2 T3 T4 SEM L Q Cb G L*G Q *G Cb*G M F M F M F M F Total calves 7 14 9 13 9 13 9 14 Responsive calves At 30 d, n = 7 13 7 9 8 12 5 11 At 30 d 6.5 8.8 16.3 5.7 11.8 17.0 15.6 15.6 3.22 0.03 0.21 0.85 0.73 0.60 0.68 0.01 <0.01 At 60 d, n = 5 11 6 10 7 11 8 11 At 60 d 6.6 7.0 8.5 7.4 3.5 5.8 11.7 6.6 1.92 0.33 0.09 0.18 0.54 0.17 0.25 0.42 <0.01 1 Targeted intakes of LA from milk replacer was as follows: T1 = 0.144, T2 = 0.206, T3 = 0.33 3, T4 = 0.586 g of LA/kg of BW 0.75 Milk replacer was the only feed during the first 30 d of life. 2 P values for orthogonal contrasts of treatments, gender, and treatment by gender interactions. L= linear effect of treatment, Q = quadratic effect of treat ment, Cb = cubic effect of treatment, G = gender.

PAGE 356

356 A B Figure 6 1. Body weight gain and feed efficiency (gain : intake) during the first 30 d of life of preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to o ne of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Cubic effect of treatment by gender, P = 0.02, linear effect of treatment by gender, P = 0.07. B) Cubic effect of treatment by gender, P = 0.04, linear effect of treatment by gender, P = 0.10. 2.0 3.0 4.0 5.0 6.0 T1 T2 T3 T4 Body weight gain, kg Treatment Male Female 0.1 0.2 0.2 0.3 0.3 0.4 T1 T2 T3 T4 Feed efficiency Treatment Male Female

PAGE 357

357 Figure 6 2. Averages daily wither and hip growth during first 60 d of life of preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). Quadratic effect of treatment in wither growth, P = 0.04. Quadratic effect of treatment in hip growth, P = 0.04. 0.10 0.12 0.14 0.16 0.18 T1 T2 T3 T4 Growth, cm/d Treatment Wither Hip

PAGE 358

358 A B Figure 6 3. Plas ma concentrations of glucose and urea N (PUN ) of preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Quadratic effect of treatment by gender, P = 0.02 and e ffect of age, P < 0.01. B) Quadratic effect of treatment by gender, P = 0.07 andf effect of age, P < 0.01 75 80 85 90 95 100 105 110 115 1 8 15 22 29 36 43 50 57 Glucose, mg/dL Day of Age T1 = 89.7 T2 = 87.7 T3 = 87.5 T4 = 88.5 4 6 8 10 12 14 1 8 15 22 29 36 43 50 57 PUN, mg/dL Day of Age T1 = 7.85 T2 = 7.65 T3 = 7.90 T4 = 8.20

PAGE 359

359 A B Figure 6 4. P lasma concentrations of BHBA and total cholesterol in preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T 2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Linear effect of treatment, P = 0.06 and effect of age, P < 0.01. B) Linear effect of treatment, P = 0.03 and effect of age, P < 0.01. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1 8 15 22 29 36 43 50 57 BHBA, mg/dL Day of Age T1 = 0.88 T2 = 0.80 T3 = 0.76 T4 = 0.76 0 20 40 60 80 100 120 1 8 15 22 29 36 43 50 57 Total cholesterol, mg/dL Day of Age T1 = 77.3 T2 = 82.5 T3 = 89.2 T4 = 86.9

PAGE 360

360 A B Figure 6 5. Plas ma concentrations of in sulin an d IGF I in preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/W B 0.75 ). A) Effect of age, P < 0.01. B) Effect of age, P < 0.01. 0 1 2 3 4 5 6 0 1 15 29 43 57 Insulin, ng/mL Day of Age T1 = 2.45 T2 = 2.20 T3 = 2.25 T4 = 2.45 0 10 20 30 40 50 60 70 80 90 0 1 15 29 43 57 IGF, ng/mL Day of Age T1 = 39.0 T2 = 41.8 T3 = 40.3 T4 = 40.9

PAGE 361

361 Figure 6 6. Total serum protein (STP) in preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). Effect of age, P < 0.01. 5.2 5.4 5.6 5.8 6.0 6.2 1 8 15 22 29 36 43 50 57 STP, mg/dL Day of Age T1 = 5.60 T2 = 5.63 T3 = 5.62 T4 = 5.61

PAGE 362

362 A B Figure 6 7. Attitude and fecal average weekly scores of preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Effect of age, P < 0.01. B) Linear effect of treatment, P = 0.05, and eff ect of age, P < 0.01. 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1 2 3 4 5 6 7 8 Attitude score Week of Age T1 = 0.17 T2 = 0.15 T3 = 0.17 T4 = 0.14 0.0 0.5 1.0 1.5 2.0 2.5 1 2 3 4 5 6 7 8 Fecal score Week of Age T1 = 0.88 T2 = 0.80 T3 = 0.76 T4 = 0.76

PAGE 363

363 Figure 6 8. Rectal temperature first 14 days of life of preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). Effect of age, P < 0.01. 38.0 38.2 38.4 38.6 38.8 39.0 39.2 39.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Rectal temperature, C Day of Age T1 = 38.9 T2 = 38.9 T3 = 38.8 T4 = 38.8

PAGE 364

364 A B Figure 6 9. Red blood cells and hematocrit concentration in Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of fou r treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Linear effect of treatment, P = 0.10 and effect of age, P < 0.01. B) Effect of age, P < 0.01. 6 7 8 9 10 7 14 28 42 Red blood cells, 10 6 /uL Day of Age T1 = 8.16 T2 = 8.71 T3 = 8.32 T4 = 8.00 26 28 30 32 34 36 38 40 1 8 15 22 29 36 43 50 57 Hematocrit, % Day of Age T1 = 32.6 T2 = 32.7 T3 = 32.1 T4 = 32.0

PAGE 365

365 Figure 6 10. Concentrations of w hite blood cells of Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T 4= 0.586 g LA/WB 0.75 ). Effect of age, P < 0.01. 7 8 9 10 11 12 13 14 7 14 28 42 White blood cells, 10 3 /uL Day of Age T1 = 9.53 T2 = 9.67 T3 = 9.68 T4 = 9.93

PAGE 366

366 A B Figure 6 11. Concentrations of neutrophil and lymphocyte in blood of Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Effect of age, P < 0.01. B) Effect of age, P < 0.01. 2 3 4 5 6 7 8 7 14 28 42 Neutrophils, 10 3 /uL Day of Age T1 = 3.70 T2 = 3.62 T3 = 3.75 T4 = 3.86 3.0 3.5 4.0 4.5 5.0 5.5 6.0 7 14 28 42 Lymphocytes, 10 3 /uL Day of Age T1 = 4.39 T2 = 4.65 T3 = 4.64 T4 = 4.44

PAGE 367

367 A B Figure 6 12. Concentrations of monocytes and eosinophils in blood of Holstein ca lves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Effect of age, P < 0.01. B) E ffect of age, P < 0.01. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 7 14 28 42 Monocytes, 10 3 /uL Day of Age T1 = 0.52 T2 = 0.47 T3 = 0.49 T4 = 0.52 0.00 0.03 0.06 0.09 0.12 0.15 7 14 28 42 Eosynophils, 10 3 /uL Day of Age T1 = 0.07 T2 = 0.06 T3 = 0.06 T4 = 0.05

PAGE 368

368 A B Figure 6 13. Concentrations of basophils and platelets in blood of Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Effect of age, P < 0.01. B) Effect of a ge, P < 0.01. 0.00 0.02 0.04 0.06 0.08 0.10 7 14 28 42 Basophils, 10 3 /uL Day of Age T1 = 0.04 T2 = 0.04 T3 = 0.05 T4 = 0.04 300 400 500 600 700 7 14 28 42 Platelets, 10 3 /uL Day of Age T1 = 505 T2 = 521 T3 = 488 T4 = 483

PAGE 369

369 A B Figure 6 14. Neutrophil phagocytosis and mean fluorescence intensity (MFI) of neutrophils in blood of Holste in calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Cubic effect of treatmen t, P = 0.09. B) Effect of age, P < 0.01. 55 60 65 70 75 7 14 28 42 Neutrophil phagocytosis, % Day of Age T1 = 62.2 T2 = 66.6 T3 = 64.2 T4 = 62.8 15 20 25 30 35 7 14 28 42 Neutrophil phagocytosis MFI Day of Age T1 = 22.1 T2 = 24.1 T3 = 23.0 T4 = 22.2

PAGE 370

370 A B Figure 6 15. Concentrations of a cid s oluble protein (A SP) and Haptoglobin in plasma of preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Effect of treatment by age, P < 0.01 [slice effect at 8, 15, and 22 days ( P 0.01) at 29, 36, and 57 days ( P 0.08)] B) Ef fect of treatment by age, P = 0.02 [slice effect at days 8 and 43 ( P < 0.05)]. 40 70 100 130 160 190 220 250 280 1 8 15 22 29 36 43 50 57 ASP, mg/L Day of Age T1 = 89.6 T2 = 88.6 T3 = 82.1 T4 = 89.4 0 1 2 3 4 5 6 1 8 15 22 29 36 43 50 57 Haptoglobin, OD x 100 Day of Age T1 = 0.88 T2 = 0.80 T3 = 0.76 T4 = 0.76

PAGE 371

371 A B Figure 6 16. Serum Anti OVA IgG concentrations in preweaned Holstein calves fed increased intake of linoleic acid. A) Males. B) Females. Day 1 was used as covariate. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). Quadratic effect of treatment by gender, P = 0.04, effect of age, P < 0.01. 0.05 0.05 0.15 0.25 0.35 0.45 1 22 43 57 Anti OVA IgG, OD Day of Age T1 = 0.10 T2 = 0.22 T3 = 0.24 T4 = 0.13 Ovalbumin immunization COV 0.05 0.05 0.15 0.25 0.35 0.45 1 22 43 57 Anti OVA IgG, OD Day of Age T1 = 0.17 T2 = 0.18 T3 = 0.18 T4 = 0.22 Ovalbumin immunization COV

PAGE 372

372 A B Figure 6 17. Lymphocyte proliferation in Holstein calves fed increased intake of linoleic acid. A) Lymphocyte proliferation measured as counts per minute (CPM) of thymidine incorpor ation respect to non stimulated cells B) Lymphocyte proliferati on measured as respect to number of blood lymphocytes. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). For both variables, c ubic effect of treatment, P = 0.01 and effect of age, P < 0.01. 20 25 30 35 40 45 50 14 28 42 Stimulation index (Stimulated/non stimulated), CPM Day of Age T1 = 26.32 T2 = 39.51 T3 = 28.93 T4 = 28.21 0 2 4 6 8 10 14 28 42 CPM per lymphocyte (CPM per ul of blood lymphocyte Day of Age T1 = 3.00 T2 = 4.66 T3 = 3.48 T4 = 3.83

PAGE 373

373 Figure 6 18. Tumor necrosis factor produced by stimulated whole blood cells of preweaned Holstein calves fed increased intake of linoleic acid. Calves were assigned to one of four tr eatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). Effect of age, P< 0.01 150 250 350 450 550 14 28 42 Tumor necrosis factor pg/mL Day of Age T1 = 337 T2 = 373 T3 = 424 T4 = 367

PAGE 374

374 A B Fi gure 6 19. Interferon produced by stimulated whole blood cells of prewean ed Holstein calves fed increased intake of linoleic acid. A) Males. B) Females. Calves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.7 5 ). Quadratic effect of treatment by gender, P = 0.09, effect of age, P = 0.01. 50 100 250 400 550 700 850 14 28 42 IFN pg/mL Day of Age T1 = 260 T2 = 357 T3 = 411 T4 = 209 50 100 250 400 550 700 850 14 28 42 Interferon pg/mL Day of Age T1 = 256 T2 = 327 T3 = 227 T4 = 265

PAGE 375

375 A B Figure 6 20. Skin fold change after phytohaemagglutinin injection as percentage of the baseline measure in responsive Holstein calves fed increased intake of linolei c acid. A) Measured at 30 days of age. B) Measured at 60 days of age. C alves were assigned to one of four treatments with increased intake of linoleic acid ( T1= 0.144 g LA/WB 0.75 T2= 0.206 g LA/WB 0.75 T3= 0.333 g LA/WB 0.75 T4= 0.586 g LA/WB 0.75 ). A) Lin ear effect of treatment, P = 0.03, effect of h post challenge, P < 0.01. B) Quadratic effect of treatment, P = 0.09, effect of h post challenge, P < 0.01. 0 5 10 15 20 25 6 24 48 Skin fold change, % increase respect to baseline Hours after challenge T1 = 7.7 T2 = 11.0 T3 = 14.4 T4 = 15.6 0 3 6 9 12 15 18 6 24 48 Skin fold change, % increase respect to baseline Hours after challenge T1 = 6.8 T2 = 8.0 T3 = 4.7 T4 = 9.2

PAGE 376

376 CHAPTER 7 GENERAL DISCUSION AND CONCLUSIONS The experiments presented here were conducted with two o bjectives. The first general objective was to evaluate the effect of supplementing essential fatty acids (FA) to prepartum Holstein cattle and to their newborn calves during the first 60 days of life on calf growth and development. The transfer of FA fed p repartum to colostrum was influenced by the dietary FA profile and by its metabolism in the rumen of the pregnant cattle Colostrum of nulliparous heifers had greater proportions of ALA, AA, EPA, DPA, and DHA whereas LA was greater in colostrum of fat from parous cows The major individual CLA detected in the current study was CLA c 9, t 11, whereas CLA t10, c12 was detected only in cows fed EFA but in limited concentrations. Increased proportions 6 derivatives indicate that elongase / desatu rase activities in the mammary gland were taking place. However, increased proportions of total and individual CLA, as well as total C18:1 trans FA in colostrum of dams fed EFA, indicate that the Ca salt of EFA were not completely effective in preventing t he processes of biohydrogenation by ruminal microbes. Intake of IgG did not differ due to dietary treatments but serum concentrations of total IgG (2.83 vs. 2.44 g/dL) and anti OVA IgG (1.13 vs. 0.90 OD) after colostrum feeding were greater in calves born from cattle supplemented with SFA vs. EFA. Feeding of fat prepartum improved AEA across parities from 23.3 to 27.9% regardless of type of fat supplemented. It is possible that cattle fed fat gave birth to calves that had a more efficient mechanism to tran sfer IgG into circulation, possibly by modifying the activity of FcRn receptors in the intestinal tract due to the likely differential composition of FA in the cell membrane.

PAGE 377

377 The second study involved the strategic feeding of EFA, both during the nonlacta ting pregnant period and in early life. The FA status of newborn calves was affected by the type of fat supplemented prepartum. C alves born from cows fed EFA had increased concentrations of LA in plasma but AA concentration was unaffected by type of diet; however GLA and C20:3 n 6, which are precursors of AA in the elongation desaturation steps, were greater in plasma of calves born from dams fed EFA. The increased proportions of these intermediate FA might indicate that the enzymatic activity of FA desatur ases and elongases that are shared by both n 6 and n 3 groups of FA was preferentially metabolizing LA over ALA in dams supplemented with fat enriched in LA, although final end products of AA and C22:4 were not increased significantly. Interestingly, suppl ementing SFA prepartum increased the proportions of EPA and DHA in plasma of newborn calves. Another important finding is the parity effect on proportion of EFA and their derivatives Calves born from nulliparous heifers had increased plasma concentration s of n 3 FA such as EPA, DPA, and DHA but decreased LA and AA. Although the plasma of dams was not analyzed for FA, the FA profile of colostrum was analyzed. This result is in agreement with the FA profile reported for colostrum of nulliparous heifers that had concentrations of ALA, AA, EPA, DPA, and DHA whereas LA was greater in colostrum of parous cows (Chapter 3). Calves born from dams fed SFA, although not statistically different, were 0.5 kg heavier than calves born from dams fed EFA. In addition dams fed SFA ate more DM than dams fed EFA (Greco et al., 2010). Calves of SFA fed dams had greater grain mix intake during 31 to 60 d of age. This greater intake resulted in a better ADG. Whether a direct relation between dam and calf performance exists is not clear. The increased

PAGE 378

378 intake of grain did not change the plasma concentrations of energy and protein metabolites as reported by (Laarman et al., 2012) because calves born from SFA or EFA fed dams did not differ in plasma concentrations of glucose or PUN. Moreover, calves born from dams fed SFA demonstrated i mprovements in immunity as evidenced by a greater concentration of anti OVA IgG and greater synthesis of IFN 15 d of life. When these calves were fed MR enriched in LA, they had lower fecal and better attitude scores at 2 wk of age. Plasma con centrations of LA in newborn calves increased markedly at 30 and 60 d of life from that at birth (~11.5 fold increase). The concentration of fat in plasma was less in calves fed the HLA MR which may result from a greater digestibility of the FA in porcine lard compared to CCO (Murley et al., 1949). Feeding a MR containing a highly saturated FA fat source (CCO) resulted in elevated plasma concentrations of C10:0, C12:0, and C14:0, as reported by others (Jenkins and Kramer, 1986). Likewise, calves fed a MR c ontaining a combination of CCO and a highly unsaturated FA fat source (porcine lard) had increased plasma concentrations of LA and ALA, similar to the findings of Wrenn et al. (1973) and Jenkins and Kramer (1986, 1990). Calves fed HLA MR (0.487 g of LA/kg of BW 0.75 ) had an improved ADG and FE during throughout the 60 d preweaning period. Increased ADG was not accompanied by greater DMI. However, this better growth was accompanied by increased concentrations of anabolic metabolites and hormones, which agree s with studies reporting increased concentrations of plasma anabolic metabolites and hor mones in faster growing calves (Smith et al., 2002; Quigley et al., 2006). On the other hand, plasma concentrations of cholesterol and BHBA were in lower concentrations in plasma of calves fed HLA MR.

PAGE 379

379 concentrations of total lipids and cholesterol by regulating their metabolism at the liver by enhancing lipid oxidation and reducing lipid acc umulation and export. Thus reduced circulating concentrations of cholesterol, BHBA and total lipid in plasma might indicate a better efficiency of nutrient utilization. Moreover, feeding HLA MR appeared to improve immune responses by increasing the number of circulating lymphocytes and possibly by enhancing the switch from a Th2 to a Th1 response by the increased production of IFN The combined effect of feeding fat prepartum and a LA enriched MR during the prew eaning period appeared to modify the ability of tissues to synthesize essential FA derivatives due to differential proportion of LA and ALA calves had when they were born. No apparent effect of prepartum diets to modify performance of calves fed LA in MR w as observed. However calves fed a MR enriched in LA and born from dams fed fat experienced fewer days of diarrhea and poor attitude. This interaction effect might be mediated by the passive transfer of IgG which tended to be in greater concentrations in ca lves born from dams fed fat as compared to those born from control dams. In Chapter 5, strategic feeding of FA during the prepartum and preweaning periods modified the response of liver to different metabolic processes. This differential profile of liver F A might have modified the activity of liver regarding expression of hepatic genes. Ability of liver to mimic dietary FA profile, was markedly affected by the MR fed rather than by prepartum diets or its combined effect with MR. Greater effect of MR were ve rified by the increased proportions of C12:0 and C14:0 in calves fed a MR formulated with CCO, whereas when CCO was partially replaced by porcine lard, the liver

PAGE 380

380 contained greater proportion of LA and three of its derivative FA. Concentrations of total FA was greater in calves fed LLA MR, which was expected based on the results in Chapter(4 where calves fed LLA MR had increased concentrations of circulating BHBA, cholesterol and total FA. First calves fed LLA MR had also greater proportions of total FA in l iver as compared to calves fed HLA MR. Liver of calves fed HLA MR had upregulated the expression of PPARA gene, which is a potent inducer of lipid oxidation and utilization in liver. However an interesting interaction was observed in calves fed HLA MR when they were born from dams fed FAT instead of control diet. A greater number of genes (n = 6) coding for enzymes involved in lipid utilization might indicate that the prepartum feeding of fat increased the effect of MR per se. In addition calves in this int eraction also had up regulated another group of genes involved in FA metabolism, glycerolipid metabolism and AA metabolism. The up regulation of genes in all aforementioned pathways might indicate that these calves were certainly undergoing a preferential de gradation of lipids. Feeding a specific profile of FA in the late gestation period also modified the response of calves fed a MR enriched in LA. Liver of calves fed porcine lard and born from dam s fed EFA instead of SFA had up regulated genes involved in gl ycolysis and oxidative phosphorylation. The increased oxidative phosphorylation could have a negative impact on tissue stability if excessive amount of free radicals are produced. On the other hand, calves in this group d more downreg ulated genes involved in regulation of inflammatory responses. This effect could have a positive impact limiting exaggerated inflammatory responses that could negatively impact liver function. However, a potential

PAGE 381

381 attenuated inflammatory response that could negatively impact ca lf survival could not be ruled out. First lactation milk yield by heifers born and used in this study was not influenced by the MR fed. However feeding fat during late gestation instead of a control no fat diet resulted in13% greater milk production at fir st lactation (12,004 vs. 10,605 kg). Other studies have reported positive impacts of improved ADG during preweaning on future milk production but in the current study only a numerical increase of 5.3% in milk yield was observed for calves having a faster g rowth rate due to consumption ofa MR containing porcine lard instead of CCO. Findings in this study reveal a strong effect of prepartum diets during the fetal period to modify the response of calves to strategic supplementation of FA during the preweaning period. However, long term effects of prepartum diets, regardless of the preweaning diet, suggests that the more critical period of programming through nutrition occurred during late gestation. Future research should focus on detailing the mechanisms by wh ich designated expressed genes (DEG) due to strategic lipid supplementation modify the production and activity of the proteins encoded by the DEG. Moreover, more efforts should be made to evaluate nutritionally strategies that would positively impact fetus and newborn calves so as to improve their future performance. The last study aimed to determine the requirement of LA in preweaned Holstein calves. Four dietary intakes of LA were formulated (0.144, 0.206, 0.333 and 0.586 g of LA/kg of BW 0.75) based on th e recommendation of LA for laboratory rats. Calves in this study were exclusively fed MR during the first 30 d of life. However, body weight gain in this study was poor, ADG averaged 111 g/d and FE at 180 g of gain/g of DMI. The first

PAGE 382

382 30 d of life was the only period in which LA intake affected BW gain. Male calves fed 0.206 g of LA/kg of BW 0.75 had the greater ADG whereas females linearly increased the ADG as intake of LA increased Studies performed in rats reported that female rats have about one third o f the male requirement for LA (0.5% vs. 1.3% of ME;(Greenberg et al., 1950; Pudelkewicz et al., 1968) The current results, based on the performance obtained in the first 30 d, seem to oppose of the findings observed in rats. All calves in this study suffe red from diarrhea starting at a mean of 7 d of age in calves fed T1, with the onset tending to be linearly delayed slightly with increasing intake of LA. Episodes of disease in preweaned calves are the main drivers of reduced performance. Early studies rep laced milk fat with vegetable oils such as coconut oil, corn oil, and tallow. Feeding CO resulted in calves with greater episodes of diarrhea and hence resulted in more attenuated BW gain ( Jenkins et al., 1985). However in the current study, increasing int ake of LA linearly reduced the severity of diarrhea. Body weight of female calves between 31 to 60 d of age, was similar to that obtained by commercial farms but did not differ with increased intake of LA. As expected due to poor BW the first 30 d of life and recovered BW the second 30 d of life, but without effect of treatments, plasma concentrations of metabolites, glucose, PUN, and hormones, insulin, and IGF I did not differ along the 60 d period. However, concentrations of BHBA increased linearly with intake of LA whereas concentrations of total plasma cholesterol surprisingly increased linearly with intake of LA. In a previous study, presented in chapter 4, calves fed increased intake of LA had reduced concentrations of total cholesterol in plasma. Fee ding PUFA have been well documented to reduce circulating levels of cholesterol

PAGE 383

383 and triglycerides, hence it is not clear why in the current study this mechanism did not work. If these calves were experiencing nutritional stress based upon low BW gain the first 30 d of life, increased feeding of LA may not have been able to optimize gain but may have been able to influence immune responses. Population of blood cells were not influenced by increased intake of LA but it changed along calf age, in a pattern ex pected for their age. However, regardless of the treatment, concentrations of neutrophils and haptoglobin peaked around d 7 to 8 which was the period in which episodes of diarrhea began. Proliferation of T cells after 48 h of in vitro stimulation with LPS + PHA was greater in calves fed 0.206 g of LA/kg of BW 0.75) and this held true at 14, 28 and 42 d of age whereas calves fed 0.333 g of LA/kg of BW 0.75 responded well only at 42 d of age. Linoleic acid is co mmonly identified as having pro inflammat ory acti vities. However an ant inflammatory activity, by reducing the proliferation of lymphocytes has been reported when increased concentrations of LA were added to media containing PBMC ( Thanasak et al., 2005; Gorjao et al., 2007). Based on the proliferative res ponses of calves fed 0.333 or 0.586 g of LA/kg of BW 0.75 as compared to 0.144 g of LA/kg of BW 0.75 although the rate of proliferation was minimal, it can be inferred that the different intakes of LA provided in the current study would not have toxic effect s on lymphocytes that could prevent its proliferation. The production of IFN by stimulated lymphocytes in whole blood was increased in calves fed 0.333 and 0.586 g of LA/kg of BW 0.75 This result also corroborates the postulation that the intakes of LA w ere not preventing lymphocytes to proliferate. Moreover, the delayed type hypersensitivity analysis indicated that 60 d old calves fed 0.586 g of LA/kg of BW 0.75

PAGE 384

384 had the greatest skin response to an intradermal injection of PHA. Overall, f eeding diets of 0 .333 or 0.586 g of LA/kg of BW 0.75 to preweaned Holstein calves increased responses for most of the markers of immunity evaluated in this study and improved wither and hip growth and severity of feces and attitude scores, hence a minimum intake of LA by da iry calves should be at least 0.206 g/kg of BW 0.75 Strategic feeding of LA during the first 60 d (0.487 g/kg of BW 0.75 ) of life improved overall performance in a first study; however intakes at and above 0.206 g/kg of BW 0.75 improved the response of cal ves in a second study. Future research should clarify the mechanisms by which targeted intake of LA might differentially modify the response of healthy and unhealthy calves.

PAGE 385

385 APPENDIX A LIST OF DIFFERENTIAL Y EXPRESSED GENES List of differential express ed genes in liver of Holstein male calves fed milk replacer containing low or high linoleic acid from 1 to 30 days of age. Males were born from dams fed diets supplemented with no fat (CTL), saturated fatty acids (SFA), or essential fatty acids (EFA) start ing at 8 wk before expected calving date. Genes are ranked for alphabetical order according gene symbol Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt .6156.1.S1_at 3290025600 apoptosis related protein 3 1187 642 958 974 899 1074 Bt.9298.1.S1_at AARSD1 alanyl tRNA synthetase domain containing 1 71.51 58.26 96.24 63.98 51.55 87.66 Bt.20249.1.S1_a_at ABCD3 ATP binding cassette, sub family D (ALD), member 3 1480 1044 1380 1383 1445 1445 Bt.10387.1.S1_at ABCF1 ATP binding cassette, sub family F (GCN20), member 1 181 262 234 165 114 233 Bt.20453.1.S1_at ABHD14A abhydrolase domain containing 14A 116 66.12 91.61 100 71.98 95.25 Bt.2858.1.S1_at ABHD6 abhydro lase domain containing 6 44.06 46.67 69.33 38.77 29.14 44.11 Bt.5188.1.S1_at ABTB1 ankyrin repeat and BTB (POZ) domain containing 1 133 147 129 155 169 76.64 Bt.2050.1.A1_at ACAA1 acetyl CoA acyltransferase 1 7042 4021 6667 7640 5981 7710 Bt.27073.1.S1_ at ACADL acyl CoA dehydrogenase, long chain 802 493 731 980 985 1050 Bt.28278.1.S1_at ACE2 angiotensin I converting enzyme (peptidyl dipeptidase A) 2 867 247 1177 1326 859 1016 Bt.21101.1.A1_at ACMSD aminocarboxymuconate semialdehyde decarboxylase 355 15 3 426 128 454 293 Bt.6177.1.S1_at ACOT8 acyl CoA thioesterase 8 146 40.99 103 83.20 74.73 141 Bt.5193.1.S1_at ACP5 acid phosphatase 5, tartrate resistant 354 192 225 267 210 316 Bt.5193.2.S1_a_at ACP5 acid phosphatase 5, tartrate resistant 2197 1213 142 6 1685 1385 1999 Bt.15886.1.S1_at ACSL5 acyl CoA synthetase long chain family member 5 7508 8409 9820 8272 4538 9079 Bt.4604.1.S1_a_at ACSM1 acyl CoA synthetase medium chain family member 1 6824 7425 7465 6766 4089 7292 Bt.19544.1.A1_at ACSM2A acyl CoA synthetase medium chain family member 2A 4049 3041 5609 4253 5104 4881 Bt.8435.1.S1_at ACTA1 actin, alpha 1, skeletal muscle 4.76 402 4.94 4.75 4.93 4.94 Bt.20557.1.S1_at ACTN2 actinin, alpha 2 5.09 41.28 4.76 4.89 5.17 5.01 Bt.12030.2.S1_at ACTN4 actin in, alpha 4 79.59 83.85 98.96 79.15 58.56 97.26 Bt.19723.1.A1_at ACTR10 actin related protein 10 homolog (S. cerevisiae) 2390 2135 1489 2163 2827 2019 Bt.26992.1.A1_at ADAM10 ADAM metallopeptidase domain 10 938 1017 641 917 1055 798 Bt.805.1.S1_at ADIPO R2 adiponectin receptor 2 410 223 372 357 202 520 Bt.22590.1.S1_at AGPAT2 1 acylglycerol 3 phosphate O acyltransferase 2 (lysophosphatidic acid acyltransferase, beta) 108 47.38 71.25 88.94 35.16 103 Bt.22170.1.S1_a_at AGPAT5 1 acylglycerol 3 phosphate O acyltransferase 5 (lysophosphatidic acid acyltransferase, epsilon) 394 303 350 404 428 479

PAGE 386

386 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EF A HLA Bt.2048.1.S1_at AGPS Alkylglycerone phosphate synthase 195 190 142 204 216 196 Bt.6813.1.A1_at AKAP5 A kinase (PRKA) anchor protein 5 61.18 54.75 111 74.24 124 83.74 Bt.4449.1.S1_at AKR1A1 aldo keto reductase family 1, member A1 (aldehyde reducta se) 1176 490 1050 913 834 1062 Bt.11078.2.S1_at AKR7A2 aldo keto reductase family 7, member A2 (aflatoxin aldehyde reductase) 69.41 45.06 68.41 61.05 33.40 75.85 Bt.24662.1.S1_at AKT1S1 AKT1 substrate 1 (proline rich) 70.89 64.02 64.05 53.06 37.41 67.76 Bt.3248.1.S1_at ALDH4A1 aldehyde dehydrogenase 4 family, member A1 284 217 350 300 164 321 Bt.16137.1.S1_at ALDH9A1 aldehyde dehydrogenase 9 family, member A1 387 288 700 508 346 688 Bt.22533.1.S1_at ALDOA aldolase A, fructose bisphosphate 751 928 804 6 41 501 1120 Bt.20207.1.A1_at ALG12 asparagine linked glycosylation 12, alpha 1,6 mannosyltransferase homolog (S. cerevisiae) 40.21 37.37 35.21 28.79 23.52 32.68 Bt.18435.3.A1_at ANGEL1 angel homolog 1 (Drosophila) 156 103 81.28 153 125 127 Bt.24203.1.S1 _at ANGPTL3 angiopoietin like 3 3778 3232 3045 3139 4713 2926 Bt.4816.1.S1_at ANGPTL4 angiopoietin like 4 58.16 83.01 62.49 70.46 60.56 135 Bt.9069.1.S1_at ANKRD10 ankyrin repeat domain 10 414 499 456 575 493 412 Bt.22626.1.A1_at ANKRD12 ankyrin repeat domain 12 132 232 160 166 198 159 Bt.28798.1.A1_at ANKRD22 Ankyrin repeat domain 22 6.61 10.77 5.99 5.63 7.92 5.74 Bt.21981.3.S1_at ANTXR1 anthrax toxin receptor 1 109 199 164 134 195 183 Bt.12745.1.A1_at ANTXR2 anthrax toxin receptor 2 66.95 72.80 79.8 3 89.73 97.62 149 Bt.27322.1.S1_at AP1AR adaptor related protein complex 1 associated regulatory protein 160 150 138 188 302 157 Bt.8775.1.S1_at AP1B1 adaptor related protein complex 1, beta 1 subunit 426 452 520 430 338 452 Bt.2056.1.S1_at APEH N acyla minoacyl peptide hydrolase 474 318 501 519 412 484 Bt.26604.1.S1_at APLNR apelin receptor 335 170 214 325 159 245 Bt.22694.1.A1_at APOA5 apolipoprotein A V 3686 1726 3750 3676 2751 3798 Bt.17961.1.S1_at APOC4 apolipoprotein C IV 6157 4066 5987 5874 4642 6016 Bt.9735.1.S1_at APOM apolipoprotein M 1541 586 1348 884 1157 1132 Bt.9735.2.A1_at APOM apolipoprotein M 2499 1100 2211 1653 1850 2086 Bt.19980.2.S1_at ApoN ovarian and testicular apolipoprotein N 1313 764 1257 1305 978 1320 Bt.28934.1.S1_at AREG amphiregulin 6.04 7.72 7.62 5.70 7.77 61.27 Bt.14075.1.S1_at ARHGAP5 Rho GTPase activating protein 5 223 176 209 242 381 222 Bt.20329.2.S1_at ARL4D ADP ribosylation factor like 4D 221 149 221 247 132 290 Bt.17432.1.S1_at ARL5B ADP ribosylation factor li ke 5B 277 348 281 312 394 277 Bt.8078.1.S1_at ARPC4 actin related protein 2/3 complex, subunit 4, 20kDa 61.30 60.51 78.09 53.70 33.96 54.87 Bt.16276.1.A1_at ARSK arylsulfatase family, member K 402 234 300 478 688 447 Bt.18330.2.S1_at ASGR2 asialoglycopr otein receptor 2 580 322 582 543 479 685 Bt.18037.2.A1_at ASPDH aspartate dehydrogenase domain containing 125 45.29 96.25 65.81 72.86 78.18 Bt.24211.1.A1_at ASPN asporin 1700 2582 1779 1689 2802 1850 Bt.8053.1.S1_at ATAD1 ATPase family, AAA domain conta ining 1 1136 1913 1202 1480 1236 1154 Bt.20514.1.S1_at ATG2B similar to ATG2 autophagy related 2 homolog B 226 258 361 344 333 351

PAGE 387

387 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title C TL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.20206.1.A1_at ATP11B ATPase, class VI, type 11B 502 394 377 626 629 501 Bt.1059.3.S1_a_at ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 429 543 628 455 267 553 Bt.4431.1.S1_a_at ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide 4577 4319 4634 3881 3743 4903 Bt.1753.1.S1_at ATP6V1E1 ATPase, H+ transporting, lysosomal 31kDa, V1 subunit E1 828 875 855 683 601 850 Bt.25471.1.S1_at ATXN3 ataxin 3 12.07 21.97 1 3.02 18.84 33.42 14.13 Bt.25471.2.A1_at ATXN3 ataxin 3 56.98 70.51 55.05 75.07 117 64.05 Bt.14059.1.A1_at AUH AU RNA binding protein/enoyl CoA hydratase 2639 2349 2087 2764 3512 2901 Bt.4898.1.S1_at BASP1 brain abundant, membrane attached signal protein 1 689 932 787 806 928 699 Bt.22524.2.A1_at BBS5 Bardet Biedl syndrome 5 190 186 117 203 198 181 Bt.5412.1.S1_at BCKDHB branched chain keto acid dehydrogenase E1, beta polypeptide 2811 1938 2420 2569 2871 2533 Bt.11445.1.A1_at BCL10 B cell CLL/lymphoma 10 518 430 290 516 659 437 Bt.11043.1.S1_a_at BCL2L12 BCL2 like 12 (proline rich) 4.51 6.45 4.63 4.63 4.63 4.51 Bt.9391.2.S1_at BIRC3 baculoviral IAP repeat containing 3 170 241 191 242 282 181 Bt.2824.1.S1_at BLOC1S1 biogenesis of lysosomal organelles complex 1, subunit 1 1098 661 853 977 829 931 Bt.29823.1.S1_x_at BOLA MHC class I heavy chain 15.02 148 15.35 16.98 23.70 27.24 Bt.29823.1.S1_at BOLA MHC class I heavy chain 14.45 111 20.85 15.79 26.79 20.57 Bt.8121.1.S1_x_at BOLA MHC class I heavy chai n 4138 2983 3645 2467 1999 3497 Bt.4762.1.S1_at BOLA NC1 non classical MHC class I antigen 49.15 60.96 46.95 29.48 29.65 29.37 Bt.1048.1.S1_at BORA aurora borealis 48.39 72.93 52.36 47.06 71.65 74.67 Bt.21099.1.A1_at BRMS1L breast cancer metastasis supp ressor 1 like 101 112 101 114 180 116 Bt.26364.1.A1_at BTBD8 BTB (POZ) domain containing 8 16.46 19.91 11.39 16.05 46.71 15.96 Bt.19064.1.A1_at BTD biotinidase 354 195 256 265 205 283 Bt.22510.1.S1_at C11H2ORF7 chromosome 2 open reading frame 7 ortholog 477 290 317 365 236 465 Bt.8903.1.S1_at C14H8ORF70 chromosome 8 open reading frame 70 ortholog 280 201 175 181 301 221 Bt.9310.1.S1_at C16orf5 chromosome 16 open reading frame 5 54.91 38.46 38.30 49.40 37.32 45.14 Bt.26522.2.S1_at C1H3ORF34 chromosome 3 open reading frame 34 ortholog 47.12 37.52 46.77 34.56 62.48 53.69 Bt.19274.1.A1_at C1QTNF7 C1q and tumor necrosis factor related protein 7 4.65 4.60 4.65 4.65 11.76 4.68 Bt.2481.2.S1_at C23H6ORF105 Chromosome 6 open reading frame 105 ortholog 1172 596 769 1187 888 1072 Bt.3865.3.S1_a_at C25H16orf14 chromosome 16 open reading frame 14 ortholog 396 207 326 403 188 354 Bt.20997.1.S1_at C2H1orf144 chromosome 1 open reading frame 144 ortholog 57.10 44.93 83.49 50.31 27.07 59.60 Bt.19664.1.A1_at C3H1ORF21 0 chromosome 1 open reading frame 210 ortholog 168 79.30 77.95 121 73.45 139 Bt.4507.1.S1_at C4A complement component 4A 8023 15537 10673 8089 6089 8548 Bt.16789.1.A1_at C5H12orf11 chromosome 12 open reading frame 11 ortholog 76.27 75.02 79.71 85.26 141 76.27 Bt.25752.1.A1_at C7H5orf24 chromosome 5 open reading frame 24 ortholog 53.96 36.64 72.85 69.09 66.32 54.91

PAGE 388

388 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.5164.1.S1_at CA14 carbonic anhydrase XIV 82.25 24.90 41.91 89.65 22.29 46.59 Bt.23960.1.S1_at CA5B carbonic anhydrase VB, mitochondrial 44.81 40.52 34.29 57.14 66.45 43.96 Bt.16382.1.A1_at CALCRL calcitonin receptor like 235 215 169 219 396 289 Bt.26832.1.S1_at CANT1 calcium activated nucleotidase 1 72.90 38.14 32.06 82.11 22.15 61.95 Bt.6686.1.S1_at CASK calcium/calmodulin dependent serine protein kinase (MAGUK family) 123 160 134 109 198 132 Bt.10084.1.S1_at CASP3 ca spase 3, apoptosis related cysteine peptidase 233 205 180 210 287 174 Bt.13989.1.A1_at CAV2 caveolin 2 161 150 117 156 232 155 Bt.15971.1.S1_at CCAR1 cell division cycle and apoptosis regulator 1 402 571 451 459 658 413 Bt.4405.1.S1_s_at CCDC104 coiled coil domain containing 104 241 246 230 241 339 234 Bt.18220.1.A1_at CCDC112 coiled coil domain containing 112 26.11 29.07 23.00 35.79 63.76 26.54 Bt.29506.1.S1_at CCDC82 coiled coil domain containing 82 65.79 66.53 54.94 79.09 117 52.48 Bt.26562.2.S1_at CCDC86 coiled coil domain containing 86 4.75 7.60 4.89 4.84 4.72 4.79 Bt.9974.1.S1_at CCL3 chemokine (C C motif) ligand 3 83.61 127 80.02 216 169 82.50 Bt.9974.1.S1_a_at CCL3 chemokine (C C motif) ligand 3 20.20 22.73 17.36 25.69 24.05 12.33 Bt.154.1.S 1_at CCL8 chemokine (C C motif) ligand 8 9.56 35.58 11.23 20.96 9.82 11.16 Bt.23572.1.S1_at CCNDBP1 cyclin D type binding protein 1 908 569 528 713 471 829 Bt.20977.3.S1_at CCPG1 cell cycle progression 1 97.36 95.49 87.88 95.34 180 92.65 Bt.22069.1.A1_a t CCPG1 Cell cycle progression 1 278 266 221 248 370 220 Bt.5415.1.S1_at CCS copper chaperone for superoxide dismutase 522 299 441 445 138 161 Bt.5096.1.S1_at CCT3 chaperonin containing TCP1, subunit 3 (gamma) 485 454 691 381 313 555 Bt.16580.1.S1_at CD 2AP CD2 associated protein 16.88 30.42 23.17 48.32 48.85 28.77 Bt.13864.1.A1_at CDC26 cell division cycle 26 homolog (S. cerevisiae) 689 445 587 655 553 565 Bt.1667.1.S1_at CDC34 cell division cycle 34 homolog (S. cerevisiae) 846 498 644 771 434 709 Bt. 20490.1.S1_at CDC42EP4 CDC42 effector protein (Rho GTPase binding) 4 1397 1417 1406 2103 828 1261 Bt.23366.1.S1_at CDIPT CDP diacylglycerol -inositol 3 phosphatidyltransferase 254 222 299 201 159 286 Bt.2.1.S1_at CDK1 cyclin dependent kinase 1 19.63 20.5 4 20.86 17.60 47.67 22.80 Bt.27042.1.S1_at CENPC1 centromere protein C 1 54.53 79.14 53.38 65.42 109 53.18 Bt.14213.1.A1_at CES2 carboxylesterase 2 (intestine, liver) 2528 1830 2596 2389 2099 3155 Bt.4336.1.S1_at CFD complement factor D (adipsin) 1905 7 92 1613 2077 1539 2023 Bt.13556.1.S1_at CFH complement factor H 1346 1065 1317 1358 2035 704 Bt.17612.2.S1_at CFHR4 complement factor H related 4 4578 6584 4003 2920 4835 4204 Bt.24506.2.A1_at CHIC2 cysteine rich hydrophobic domain 2 25.22 24.93 18.72 2 4.76 34.92 25.85 Bt.11411.1.S1_at CIAPIN1 cytokine induced apoptosis inhibitor 1 228 250 254 151 119 199 Bt.13381.1.S1_at CIDEC cell death inducing DFFA like effector c 5.03 4.65 4.85 4.73 4.88 9.09 Bt.10007.1.A1_at CKAP2 cytoskeleton associated protein 2 73.40 75.56 54.64 56.17 144 78.75 Bt.12980.3.S1_a_at CL43 collectin 43 10800 9177 13456 9711 4595 9921 Bt.11279.1.A1_at CLCN4 chloride channel 4 94.58 57.27 101 83.43 60.42 129 Bt.27474.1.S1_at CLEC4F C type lectin domain family 4, member F 23.63 130 32.44 147 17.24 60.25

PAGE 389

389 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.2113.1.S1_at CNDP2 CNDP dipeptidase 2 (metallopeptidase M 20 family) 1399 1447 1738 1273 955 1606 Bt.11256.1.S1_at CNOT1 CCR4 NOT transcription complex, subunit 1 1032 1321 1503 1095 682 1081 Bt.19218.2.S1_at CNOT6 CCR4 NOT transcription complex, subunit 6 368 341 326 404 509 365 Bt.8617.1.S1_at CNRIP1 cannabi noid receptor interacting protein 1 178 110 118 132 115 151 Bt.26828.1.S1_at CNTLN centlein, centrosomal protein 84.17 94.10 56.66 108 182 66.68 Bt.21467.1.S1_at COG4 component of oligomeric golgi complex 4 141 171 197 114 125 163 Bt.4141.1.S1_at COPE c oatomer protein complex, subunit epsilon 406 283 386 331 247 414 Bt.1332.1.S1_a_at COX10 COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase (yeast) 90.90 93.65 131 87.06 65.56 85.72 Bt.395.1.S1_at COX8B cytochrome c oxidase subunit VIII H (heart/muscle) 4.55 8.58 4.54 4.55 4.54 4.55 Bt.22479.1.S1_at CPEB4 cytoplasmic polyadenylation element binding protein 4 13.95 14.04 14.73 15.06 28.78 16.89 Bt.25663.1.A1_at CPNE8 copine VIII 107 109 138 176 196 199 Bt.24779.2.S1_at CRE M cAMP responsive element modulator 5.34 5.08 10.63 5.17 12.38 6.84 Bt.1927.1.S1_at CRISPLD2 /// TIMM13 cysteine rich secretory protein LCCL domain containing 2 /// translocase of inner mitochondrial membrane 13 homolog (yeast) 69.73 156 109 95.87 96.37 9 9.39 Bt.23143.2.S1_at CSDE1 cold shock domain containing E1, RNA binding 2118 1503 1814 2085 2360 2163 Bt.22563.1.A1_s_at CSDE1 cold shock domain containing E1, RNA binding 1228 889 1062 1257 1456 1173 Bt.6646.1.S1_at CTDSP1 CTD (carboxy terminal domain RNA polymerase II, polypeptide A) small phosphatase 1 60.17 45.44 48.46 61.50 23.83 52.55 Bt.5240.1.S1_at CTGF connective tissue growth factor 57.00 136 113 95.23 78.91 82.21 Bt.4150.1.S1_at CTNNBL1 catenin, beta like 1 618 390 572 647 431 619 Bt.4902 .1.S1_at CTSZ cathepsin Z 3142 2751 3730 2462 2973 3733 Bt.18003.1.S1_at CUL3 cullin 3 9.34 9.10 9.64 15.51 23.48 11.33 Bt.23998.1.A1_a_at CUX2 cut like homeobox 2 100 176 69.49 160 173 94.93 Bt.21216.1.S1_at CXorf56 chromosome X open reading frame 56 o rtholog 336 350 416 313 245 396 Bt.10609.2.A1_at CYP20A1 cytochrome P450, family 20, subfamily A, polypeptide 1 623 392 360 604 552 605 Bt.9699.1.S1_at CYP26A1 cytochrome P450, family 26, subfamily A, polypeptide 1 3801 2270 1496 4297 754 3765 Bt.16001. 1.S1_at CYP27A1 cytochrome P450, family 27, subfamily A, polypeptide 1 3443 1869 2926 2793 2367 2772 Bt.12255.1.A1_at CYP2C19 cytochrome P450, family 2, subfamily C, polypeptide 19 24.47 19.90 34.35 24.68 38.41 20.55 Bt.23912.1.A1_a_at CYP2E1 cytochrome P450, family 2, subfamily E, polypeptide 1 1650 1346 884 2057 753 1838 Bt.14369.1.A1_at CYP39A1 cytochrome P450, family 39, subfamily A, polypeptide 1 110 131 126 87.39 185 168 Bt.4126.1.A1_at CYP4A11 cytochrome P450, family 4, subfamily A, polypeptide 1 1 6763 5881 6267 7116 5348 7182

PAGE 390

390 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.27036.1.S1_at CYP4F2 cytochrome P450, family 4, subfamily F, polypeptide 2 3251 1464 1955 3247 1338 2597 Bt.13530.1.S1_at DCI dodecenoyl CoA isomerase 3449 2063 3493 3439 2770 3376 Bt.23178.1.S2_at DCN decorin 4023 4244 3077 4081 5309 4139 Bt.18792.1.S1_at DCTN6 Dynactin 6 27.36 28.49 25.51 27.88 104 29.94 Bt.12508.1.S1_at DCTPP1 dCTP pyrophosphatase 1 70.20 57.73 78.96 46.72 42.61 79.22 Bt.22199.1.S1_at DDIT4L DNA damage inducible transcript 4 like 5.68 14.18 5.90 5.41 5.84 5.93 Bt.9047.1.S1_at DDT D dopachrome tautomerase 5288 3157 4816 4503 4453 4461 Bt.8323.1.S1_at DDX21 DEAD (Asp Glu Ala Asp) box polypeptide 21 668 921 668 657 682 664 Bt.6334.1.A1_at DEGS1 degenerative spermatocyte homolog 1, lipid desaturase (Drosophila) 1237 1083 950 1269 921 1180 Bt.6141.1.S1_at DES desmin 14.09 20.47 14. 61 16.01 10.63 19.86 Bt.16832.1.A1_at DHDPSL dihydrodipicolinate synthase like, mitochondrial 438 214 482 498 362 395 Bt.13376.1.S1_at DHRS1 dehydrogenase/reductase (SDR family) member 1 488 264 710 348 351 542 Bt.8915.1.A1_at DHTKD1 dehydrogenase E1 an d transketolase domain containing 1 67.28 125 80.98 112 115 170 Bt.2506.1.S1_at DKK3 dickkopf homolog 3 (Xenopus laevis) 48.86 63.94 43.82 39.24 64.12 92.21 Bt.27889.1.S1_at DLD Dihydrolipoamide dehydrogenase 49.94 52.11 50.58 49.55 125 53.07 Bt.9632.2. S1_at DMBT1 deleted in malignant brain tumors 1 3594 6452 5969 3592 3012 4917 Bt.27589.1.A1_at DNAH12L /// LOC781795 dynein, axonemal, heavy chain 12 like /// similar to ciliary dynein heavy chain 7 23.39 34.57 27.74 23.09 29.16 26.19 Bt.6341.1.S1_at DNA JC1 DnaJ (Hsp40) homolog, subfamily C, member 1 82.57 79.23 70.50 83.54 80.89 44.67 Bt.6020.1.S1_at DNAJC11 DnaJ (Hsp40) homolog, subfamily C, member 11 149 140 201 129 90.56 125 Bt.211.1.S1_at DNAJC3 DnaJ (Hsp40) homolog, subfamily C, member 3 1323 897 1347 965 1925 1134 Bt.869.1.S1_at DPM1 dolichyl phosphate mannosyltransferase polypeptide 1, catalytic subunit 1234 1688 1423 1277 1621 1166 Bt.2110.1.S1_at DPP3 dipeptidyl peptidase 3 601 649 853 516 368 665 Bt.2424.1.S1_at DPYD dihydropyrimidine dehyd rogenase 5585 3430 4807 5765 7575 5005 Bt.15705.1.S2_at DSTN destrin (actin depolymerizing factor) 406 336 334 462 385 449 Bt.15705.1.S1_at DSTN destrin (actin depolymerizing factor) 1945 1417 1067 1517 1707 1774 Bt.28523.1.S1_at DTX3L deltex 3 like (Dr osophila) 1322 5711 1991 2159 1390 1156 Bt.13768.1.S1_at DYNLT3 dynein, light chain, Tctex type 3 642 739 662 773 1117 693 Bt.27286.2.S1_at ECD ecdysoneless homolog (Drosophila) 50.97 50.46 65.06 40.71 50.16 80.81 Bt.20265.1.A1_at ECD ecdysoneless homol og (Drosophila) 692 653 688 519 571 797 Bt.7963.1.S1_at EHD1 EH domain containing 1 264 207 236 229 112 181 Bt.11769.2.S1_at EID3 EP300 interacting inhibitor of differentiation 3 13.70 12.08 16.66 12.16 22.94 11.22 Bt.18928.1.A1_at EIF4E3 eukaryotic tra nslation initiation factor 4E family member 3 216 228 119 206 315 224

PAGE 391

391 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.19745.1.S1 _at ELL2 elongation factor, RNA polymerase II, 2 414 282 309 346 633 440 Bt.1983.1.S1_at EMR1 egf like module containing, mucin like, hormone receptor like 1 302 316 364 247 164 394 Bt.3857.1.S1_at ENDOG endonuclease G 456 245 334 390 259 422 Bt.22783.1 .S1_at ENO1 enolase 1, (alpha) 2064 1860 2691 1498 1069 2132 Bt.22169.1.S1_at ENO3 enolase 3 (beta, muscle) 11.31 40.81 10.10 9.72 9.36 10.10 Bt.16000.1.S1_at ENTPD4 ectonucleoside triphosphate diphosphohydrolase 4 343 314 297 542 336 304 Bt.22737.1.S1_ at ERBB2IP erbb2 interacting protein 1658 2095 1899 1736 2630 1800 Bt.18026.1.A1_at ERBB2IP erbb2 interacting protein 20.04 20.82 20.05 27.46 30.22 21.38 Bt.28586.1.S1_at ERMP1 endoplasmic reticulum metallopeptidase 1 169 144 202 143 165 183 Bt.17415.3. A1_at ERRFI1 ERBB receptor feedback inhibitor 1 6.11 5.90 21.72 5.90 6.16 6.10 Bt.23905.1.A1_at ERRFI1 ERBB receptor feedback inhibitor 1 3683 2726 6077 3458 4158 2775 Bt.24361.1.S1_at ESF1 ESF1, nucleolar pre rRNA processing protein, homolog (S. cerevis iae) 35.95 55.32 39.31 38.44 66.56 33.94 Bt.5350.1.S1_at ETFA electron transfer flavoprotein, alpha polypeptide 2268 1831 1770 2511 2021 2394 Bt.4555.1.S1_at ETFB electron transfer flavoprotein, beta polypeptide 455 302 447 421 308 513 Bt.1817.1.S1_at E TV1 ets variant 1 21.96 24.37 24.70 15.85 55.16 20.89 Bt.4758.1.S1_at FABP3 fatty acid binding protein 3, muscle and heart (mammary derived growth inhibitor) 7.46 12.17 8.33 6.44 8.13 8.13 Bt.22869.1.S2_at FABP5 fatty acid binding protein 5 (psoriasis as sociated) 10.26 22.25 11.81 10.39 32.80 19.19 Bt.7023.1.S1_at FAHD2A fumarylacetoacetate hydrolase domain containing 2A 545 383 583 752 564 751 Bt.26318.1.S1_a_at FAIM Fas apoptotic inhibitory molecule 16.67 19.90 18.29 26.78 55.85 16.67 Bt.28623.1.S1_a t FAT1 FAT tumor suppressor homolog 1 (Drosophila) 364 751 503 523 302 556 Bt.6449.1.S1_at FBLN5 fibulin 5 90.70 155 85.46 109 74.70 115 Bt.20361.2.A1_at FBXL20 F box and leucine rich repeat protein 20 130 85.78 138 72.46 109 203 Bt.24950.1.S1_at FBXL5 F box and leucine rich repeat protein 5 1619 1148 1329 1391 1161 1521 Bt.24205.1.A1_at FGB fibrinogen beta chain 3765 2393 1656 2819 2867 2386 Bt.22730.1.S1_at FGFR1OP2 FGFR1 oncogene partner 2 41.21 54.92 46.90 65.21 63.91 51.63 Bt.2587.2.S1_a_at FH fu marate hydratase 293 235 364 366 277 545 Bt.19999.1.A1_at FICD FIC domain containing 159 35.33 60.98 91.15 114 160 Bt.2899.1.S2_at FOS FBJ murine osteosarcoma viral oncogene homolog 75.66 111 75.64 244 119 82.66 Bt.21181.1.S1_at FOXK2 forkhead box K2 81 .32 116 89.57 89.57 60.02 62.61 Bt.10777.1.S1_at FOXP1 forkhead box P1 45.11 47.25 52.62 65.01 89.05 88.03 Bt.6180.1.S1_at FRG1 FSHD region gene 1 333 354 300 352 617 336 Bt.121.1.S1_at FRZB frizzled related protein 24.42 29.78 38.54 29.23 30.14 75.30 Bt.18415.1.A1_at FTSJD1 FtsJ methyltransferase domain containing 1 409 928 186 455 403 342 Bt.15854.1.A1_at FUBP1 far upstream element (FUSE) binding protein 1 626 892 754 585 826 560 Bt.2190.1.S1_at FUBP3 far upstream element (FUSE) binding protein 3 62 8 669 453 680 784 571

PAGE 392

392 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.2169.1.S1_at FUCA1 fucosidase, alpha L 1, tissue 1146 825 883 916 885 1034 Bt.26635.2.S1_at FZD1 frizzled homolog 1 (Drosophila) 102 103 74.98 67.34 144 125 Bt.5197.1.S1_at G3BP1 GTPase activating protein (SH3 domain) binding protein 1 873 1267 925 790 1200 918 Bt.20252.2.S1_a_at GALK1 galactokinase 1 163 83. 08 128 168 87.18 150 Bt.2580.1.S1_at GALM galactose mutarotase (aldose 1 epimerase) 832 454 921 571 711 902 Bt.21464.2.S1_a_at GALT galactose 1 phosphate uridylyltransferase 264 123 188 275 154 276 Bt.21464.3.S1_a_at GALT galactose 1 phosphate uridylylt ransferase 227 107 160 210 116 202 Bt.21464.1.S1_at GALT galactose 1 phosphate uridylyltransferase 859 562 625 957 480 821 Bt.28744.1.S1_at GBP4 guanylate binding protein 4 123 737 363 210 215 127 Bt.16350.2.A1_s_at GBP5 guanylate binding protein 5 5.23 7.62 4.94 5.37 5.57 5.11 Bt.14207.1.S1_at GCAT glycine C acetyltransferase 340 229 633 327 285 332 Bt.20267.1.S1_at GCLM glutamate cysteine ligase, modifier subunit 184 165 157 129 180 244 Bt.25088.1.A1_at GCSH Glycine cleavage system protein H (aminom ethyl carrier) 29.44 18.55 27.03 28.52 34.62 27.87 Bt.21798.1.S1_at GIMAP6 GTPase, IMAP family member 6 31.19 235 34.98 93.29 35.67 150 Bt.13777.2.S1_at GIMAP7 GTPase, IMAP family member 7 14.19 34.78 54.54 30.10 72.62 32.93 Bt.13777.1.S1_at GIMAP7 GTPa se, IMAP family member 7 221 322 357 251 406 243 Bt.26769.1.S1_at GIMAP8 GTPase, IMAP family member 8 4.59 4.86 4.86 4.86 4.58 94.75 Bt.12579.1.A1_at GK5 glycerol kinase 5 1700 914 2422 2730 2291 2281 Bt.13486.1.A1_at GLDC glycine dehydrogenase (decarbo xylating) 2512 2731 3104 1967 1424 2129 Bt.24597.1.S1_at GLG1 golgi apparatus protein 1 31.31 53.49 42.15 33.24 13.26 45.51 Bt.11167.1.S1_at GLRX5 glutaredoxin 5 978 485 581 369 365 588 Bt.12240.1.A1_at GLYATL3 glycine N acyltransferase like 3 1853 1021 1242 1171 1199 1328 Bt.13942.1.S1_at GLYCTK glycerate kinase 439 203 351 454 260 477 Bt.22350.1.A1_at GMCL1 germ cell less homolog 1 (Drosophila) 507 482 416 490 867 483 Bt.9140.1.S1_at GMNN geminin, DNA replication inhibitor 206 182 132 180 305 211 B t.25097.1.S1_at GMPS guanine monphosphate synthetase 359 244 319 321 346 406 Bt.18321.1.A1_at GNB4 guanine nucleotide binding protein (G protein), beta polypeptide 4 302 283 139 163 181 155 Bt.20919.2.A1_at GNMT glycine N methyltransferase 99.05 25.32 77 .59 64.64 45.20 71.71 Bt.29268.1.S1_at GOLT1A golgi transport 1 homolog A (S. cerevisiae) 526 343 396 502 333 499 Bt.11178.1.S1_at GPC3 glypican 3 19510 11064 19030 14169 15175 16879 Bt.14464.1.A1_at GPHN gephyrin 176 261 239 198 280 184 Bt.22676.1.A1_ at GPN3 GPN loop GTPase 3 436 498 357 444 619 414 Bt.7575.1.A1_at GPT2 glutamic pyruvate transaminase (alanine aminotransferase) 2 212 134 312 237 440 218 Bt.5170.1.S1_at GRHPR glyoxylate reductase/hydroxypyruvate reductase 1365 686 1267 1416 904 1465 B t.7413.1.S1_at GRN granulin 266 295 300 227 188 271 Bt.27623.2.S1_a_at GRTP1 growth hormone regulated TBC protein 1 324 164 226 331 187 283

PAGE 393

393 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gen e Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.3201.1.S1_at GRWD1 glutamate rich WD repeat containing 1 72.42 79.82 74.21 73.93 35.30 54.35 Bt.227.3.A1_x_at GSTA1 glutathione S transferase A1 10042 10167 11208 7377 7898 10169 Bt.227.2.A1_at GSTA1 glutathione S transferase A1 15853 15223 16920 13053 8989 13963 Bt.28076.1.A1_at GSTO1 glutathione S transferase omega 1 1683 1051 1551 1472 1321 1797 Bt.13641.1.S1_at GSTZ1 glutathione transferase zeta 1 6300 5074 7227 5706 4463 6512 Bt.20241.1. S1_at HAAO /// LOC786774 3 hydroxyanthranilate 3,4 dioxygenase 785 331 708 439 494 514 Bt.7237.2.S1_a_at HADHA hydroxyacyl CoA dehydrogenase 420 326 608 327 254 568 Bt.15687.1.S1_at HERC4 hect domain and RLD 4 1158 1491 1615 1184 1778 1193 Bt.27463.1.A 1_at HERC6 hect domain and RLD 6 4.57 14.16 4.60 4.62 5.47 4.55 Bt.22498.2.S1_at HES4 Hairy and enhancer of split 4 (Drosophila) 10.53 27.49 9.87 37.05 11.62 11.31 Bt.2183.1.A1_at HEXB hexosaminidase B (beta polypeptide) 1182 754 817 1200 852 1101 Bt.19 899.1.A1_at HGD homogentisate 1,2 dioxygenase 15581 12993 18375 12777 12975 15842 Bt.6171.1.A1_at HIBADH 3 hydroxyisobutyrate dehydrogenase 3357 2026 3695 2779 3696 3699 Bt.1738.1.S1_at HIBCH 3 hydroxyisobutyryl CoA hydrolase 521 483 469 496 734 472 Bt. 19519.1.S1_at HLTF Helicase like transcription factor 1229 1110 1067 1238 2034 1272 Bt.6397.2.S1_at HMGB2 high mobility group box 2 1392 1372 769 1016 1949 1159 Bt.3928.1.S1_at HNRNPAB heterogeneous nuclear ribonucleoprotein A/B 1627 1945 1668 1838 1033 1452 Bt.21801.2.S1_at HNRNPL heterogeneous nuclear ribonucleoprotein L 200 277 268 221 209 217 Bt.19922.1.S1_at HPD 4 hydroxyphenylpyruvate dioxygenase 2248 1521 2019 1082 1161 2144 Bt.22672.1.A1_at HPGD hydroxyprostaglandin dehydrogenase 15 (NAD) 785 3 84 434 924 1351 863 Bt.20399.1.S1_at HSD17B13 hydroxysteroid (17 beta) dehydrogenase 13 788 646 531 452 790 1340 Bt.23179.1.S1_at HSP90AA1 heat shock 90kD protein 1, alpha 1688 1448 3975 1519 1973 2218 Bt.19575.1.S1_at HSPA14 heat shock 70kDa protein 14 445 454 450 580 517 385 Bt.19575.2.S1_at HSPA14 heat shock 70kDa protein 14 46.40 39.89 40.53 65.45 65.99 44.45 Bt.5372.1.S1_at ICAM1 intercellular adhesion molecule 1 202 253 153 165 125 179 Bt.1730.1.A1_at ID1 inhibitor of DNA binding 1, dominant neg ative helix loop helix protein 1379 1839 777 2417 546 635 Bt.2415.1.S1_at ID2 inhibitor of DNA binding 2, dominant negative helix loop helix protein 2031 1638 1323 1585 1270 1439 Bt.13324.4.S1_at IDH1 isocitrate dehydrogenase 1 (NADP+), soluble 6527 3234 6119 5994 5185 7139 Bt.13324.1.S1_a_at IDH1 isocitrate dehydrogenase 1 (NADP+), soluble 734 346 652 625 614 915 Bt.27759.2.S1_at IDO1 indoleamine 2,3 dioxygenase 1 5.28 12.35 6.39 6.18 7.37 6.02 Bt.22021.1.S1_at IFI16 interferon, gamma inducible protei n 16 257 905 426 458 581 240 Bt.17223.1.S1_at IFI35 interferon induced protein 35 169 266 201 247 92.98 158 Bt.20785.2.S1_at IFI44 interferon induced protein 44 301 1913 536 1363 377 207 Bt.20785.1.A1_at IFI44 interferon induced protein 44 474 2854 815 1825 569 305 Bt.19620.1.A1_at IFI44 interferon induced protein 44 459 2885 709 1263 529 315

PAGE 394

394 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.8436.1.S1_at IFI6 interferon, alpha inducible protein 6 524 5793 4070 1704 771 589 Bt.24098.1.A1_at IFIH1 interferon induced with helicase C domain 1 96.55 505 98.70 128 163 114 Bt.14054.1.A1_at IFRD1 interferon related developmental regulat or 1 354 587 393 374 444 352 Bt.14054.2.S1_at IFRD1 interferon related developmental regulator 1 24.46 60.30 45.61 38.18 44.91 37.13 Bt.8829.1.S1_a_at IFT122 Intraflagellar transport 122 homolog (Chlamydomonas) 149 120 113 146 165 229 Bt.11379.1.S1_at I FT52 intraflagellar transport 52 homolog (Chlamydomonas) 35.01 75.79 50.26 53.39 45.49 47.17 Bt.190.1.A1_at IGFBP1 insulin like growth factor binding protein 1 43.74 18.58 26.62 20.71 116 105 Bt.3843.1.S1_at IGJ immunoglobulin J chain 767 540 584 735 109 2 896 Bt.22116.1.A1_at IL18BP interleukin 18 binding protein 10.64 33.21 14.48 10.46 10.06 8.56 Bt.12760.1.S1_at INHBA inhibin, beta A 41.73 371 252 146 400 189 Bt.24767.1.S1_at INTS3 integrator complex subunit 3 176 225 209 198 314 193 Bt.5768.1.S1_at IRF7 interferon regulatory factor 7 91.67 330 94.29 190 59.20 71.28 Bt.11259.1.S1_at ISG12(A) putative ISG12(a) protein 1811 13433 8824 6225 1113 1745 Bt.9779.1.S1_at ISG12(B) similar to TLH29 protein precursor 6.28 59.92 7.26 7.25 7.00 5.92 Bt.12304.1 .S1_at ISG15 ISG15 ubiquitin like modifier 1388 14191 2938 8522 785 485 Bt.3212.1.S1_at ISOC2 isochorismatase domain containing 2 1397 873 1275 1408 1021 1530 Bt.8905.1.S1_at ITCH itchy E3 ubiquitin protein ligase homolog (mouse) 120 149 123 141 174 98.7 9 Bt.5536.1.S1_at ITGB5 integrin, beta 5 785 623 692 923 559 846 Bt.21565.1.S1_at IWS1 IWS1 homolog (S. cerevisiae) 305 433 340 287 378 315 Bt.29879.1.S1_at KAT2B K(lysine) acetyltransferase 2B 69.81 48.30 55.08 94.07 133 73.94 Bt.6972.1.S1_at KBTBD10 kelch repeat and BTB (POZ) domain containing 10 4.89 11.65 4.74 4.74 5.80 4.89 Bt.16187.1.A1_at KBTBD6 kelch repeat and BTB (POZ) domain containing 6 169 174 137 136 389 279 Bt.15691.1.S1_at KCNK5 potassium channel, subfamily K, member 5 67.82 112 182 14 1 92.77 152 Bt.9170.1.A1_at KIAA1147 KIAA1147 456 228 289 454 281 378 Bt.9527.2.S1_at KLF10 Kruppel like factor 10 11.69 11.70 11.21 16.94 28.77 14.76 Bt.11751.1.A1_at KLHL23 kelch like 23 73.58 61.33 42.76 67.03 112 62.99 Bt.3191.1.A1_at KLHL24 kelch like 24 (Drosophila) 602 386 541 354 808 1091 Bt.19212.1.S1_at KLHL9 kelch like 9 (Drosophila) 942 863 723 905 1058 864 Bt.16496.1.A1_at KNTC1 kinetochore associated 1 199 167 230 324 191 256 Bt.12663.1.S1_at KRT19 keratin 19 5.19 9.77 8.67 5.00 5.22 6 .01 Bt.26150.1.A1_at L2HGDH L 2 hydroxyglutarate dehydrogenase 195 175 145 153 313 206 Bt.14129.1.S1_at LACTB2 lactamase, beta 2 1245 1108 954 1141 1548 1128 Bt.27891.1.S1_at LARS2 leucyl tRNA synthetase 2, mitochondrial 59.33 103 124 66.65 95.88 84.29 Bt.19614.1.A1_at LIPC lipase, hepatic 3438 2059 3810 3490 3957 3843 Bt.4643.1.S1_at LMAN2 lectin, mannose binding 2 2328 1912 2616 2040 1793 2463 Bt.20934.1.S1_at LOC100137763 hypothetical protein LOC100137763 145 121 46.45 95.35 177 89.72 Bt.8724.1.S1 _at LOC100299281 --788 402 751 902 515 995 Bt.5692.1.S1_at LOC100425208 --141 124 132 218 215 137 Bt.24749.1.S1_at LOC100430496 --522 393 370 727 753 461 Bt.24001.1.A1_at LOC100433242 --3253 1408 1757 2740 1309 2494 Bt.28945.1.A1_at LOC10044046 1 --186 174 152 210 242 169 Bt.29398.1.S1_at LOC100582155 --722 384 576 613 824 656

PAGE 395

395 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.16058.2.S1_at LOC100583040 --30.49 16.08 42.24 29.81 91.77 46.50 Bt.8421.2.S1_at LOC100623159 --2695 1769 2344 3395 2863 3117 Bt.17814.1.A1_at LOC100736585 --799 730 922 1081 518 912 Bt.26804.1.S1_at LOC100847122 --190 278 274 164 299 2 06 Bt.18114.1.A1_at LOC100851000 --96.56 43.59 40.79 36.06 55.50 41.03 Bt.18577.2.A1_at LOC472962 --346 400 272 386 583 327 Bt.6556.1.S1_at LOC504773 regakine 1 2726 1984 1969 1527 850 2097 Bt.4937.1.S1_at LOC505941 similar to KIAA1398 protein 2285 5253 3167 2494 1521 2436 Bt.15796.1.S1_at LOC508226 similar to CDC42 binding protein kinase beta 53.46 71.60 62.98 51.13 29.51 44.06 Bt.25111.1.A1_at LOC508347 Similar to interferon induced protein 44 like 299 2041 410 755 314 253 Bt.12586.1.A1_at LOC5 08439 similar to CG2943 CG2943 PA 371 417 480 312 341 379 Bt.643.1.S1_at LOC508666 Similar to MPIF 1 3295 2008 2772 3001 3207 6186 Bt.21461.1.S1_at LOC509034 similar to Feline leukemia virus subgroup C receptor related protein 2 (Calcium chelate transpor ter) (CCT) 20.22 11.18 5.79 6.58 5.79 5.79 Bt.26538.1.S1_at LOC509420 similar to chromosome 9 open reading frame 61 19.93 17.08 60.64 18.18 32.62 27.17 Bt.23696.1.A1_at LOC509457 WD repeat domain 73 like 4.56 370 4.56 4.56 4.56 369 Bt.18323.1.A1_at LOC5 09506 similar to Cytochrome P450, family 4, subfamily F, polypeptide 2 203 89.68 152 178 122 164 Bt.18440.2.S1_at LOC510382 similar to guanylate binding protein 4 5.19 6.92 6.25 6.93 25.65 5.48 Bt.18440.3.A1_at LOC510382 similar to guanylate binding prot ein 4 18.94 15.41 10.99 28.88 113 17.86 Bt.2049.1.S1_at LOC510634 hypothetical LOC510634 1101 448 717 665 492 839 Bt.27118.1.A1_at LOC510651 hypothetical LOC510651 799 1515 852 803 1263 839 Bt.3300.1.S1_at LOC511523 similar to SLC2A4 regulator 447 249 3 69 487 289 371 Bt.18316.1.A1_at LOC513587 Similar to UPF0474 protein C5orf41 137 89.08 83.41 91.93 231 118 Bt.12704.1.S1_at LOC514801 similar to retina copper containing monoamine oxidase 11.96 37.45 20.07 17.94 16.23 12.61 Bt.10371.1.S1_at LOC516241 si milar to cysteine sulfinate decarboxylase 107 68.04 49.00 84.64 76.19 79.90 Bt.8736.1.S1_at LOC520588 similar to chromosome 1 open reading frame 9 763 898 886 865 1234 891 Bt.28626.2.S1_at LOC521363 similar to GC rich sequence DNA binding factor (GCF) (T ranscription factor 9) (TCF 9) 8.44 10.65 11.44 9.93 16.15 9.95 Bt.13184.1.S1_at LOC523126 similar to ATP binding cassette, sub family C, member 4 12.71 111 55.67 152 9.61 22.34 Bt.22421.1.A1_at LOC530325 similar to signal peptide peptidase like 2A 1717 1873 1028 1825 2482 1465 Bt.26568.2.S1_a_at LOC531049 similar to Putative eukaryotic translation initiation factor 3 subunit (eIF 3) 131 150 215 116 127 154 Bt.12665.1.A1_at LOC531600 similar to AAT1 alpha 49.38 69.10 60.44 57.80 69.34 52.35 Bt.1785.1.A 1_at LOC532189 similar to carboxypeptidase D 245 172 244 309 347 305 Bt.19937.1.S1_at LOC532189 similar to carboxypeptidase D 1221 938 1102 1274 1189 1349 Bt.27966.1.S1_at LOC532789 similar to PAWR 57.99 36.56 38.17 47.53 69.63 57.01 Bt.21869.1.S1_at LO C537017 similar to CMP N acetylneuraminic acid hydroxylase 334 431 301 534 580 446

PAGE 396

396 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.24881.1.S1_at LOC539690 similar to Complement component C1q receptor precursor (Complement component 1 q subcomponent receptor 1) (C1qR) (C1qRp) (C1qR(p)) (C1q/MBL/SPA receptor) (Matrix remodeling associated protein 4) (CD93 antigen) (CDw93) 329 168 23 2 297 204 308 Bt.2859.1.A1_at LOC540253 hypothetical LOC540253 210 159 141 221 393 185 Bt.27403.1.S1_at LOC540987 similar to Uncharacterized protein C5orf5 (GAP like protein N61) 251 344 297 371 424 288 Bt.20758.1.S1_at LOC541014 hypothetical protein LO C541014 233 211 172 249 257 189 Bt.6162.1.S1_at LOC613560 similar to putative c Myc responsive 69.40 32.13 60.19 69.92 51.05 72.51 Bt.28139.1.S1_at LOC614107 similar to Hexokinase 2 (Hexokinase type II) (HK II) 12.77 36.13 13.06 11.70 15.62 11.11 Bt.117 72.2.S1_at LOC614339 hypothetical protein LOC614339 73.28 43.28 59.86 64.83 151 88.54 Bt.10797.2.S1_a_at LOC615093 hypothetical protein LOC615093 1229 746 965 1116 1205 1203 Bt.2965.1.A1_at LOC618434 hypothetical LOC618434 1631 891 1280 1652 1016 1406 B t.16672.1.A1_at LOC698727 --22.50 17.91 11.17 25.88 51.93 13.31 Bt.1978.3.S1_at LOC780933 cationic trypsin 80.98 15.67 41.51 43.49 38.59 64.96 Bt.5466.2.S1_a_at LOC783142 ribosomal protein S4, Y linked 1 /// ribosomal protein S4, Y linked 2 /// simila r to ribosomal protein S4 /// hypothetical protein LOC783463 9895 7390 8909 9955 8466 9879 Bt.2999.1.A1_at LOC783843 similar to seven transmembrane helix receptor 135 148 90.95 116 180 123 Bt.22065.1.S1_at LOC783920 similar to mCG1046517 5.10 11.69 5.03 4.59 4.85 5.03 Bt.15530.1.S1_at LOC784762 similar to 60S ribosomal protein L12 /// ribosomal protein L12 3946 3157 3318 3740 2995 4049 Bt.6899.1.S1_at LOC784769 similar to MGC127725 protein 529 511 448 624 632 498 Bt.17352.1.A1_at LOC785119 similar to programmed cell death 10 179 189 128 227 193 181 Bt.23566.2.S1_at LOC785936 Hypothetical protein LOC785936 18.98 23.60 17.74 14.68 63.34 30.42 Bt.28764.1.A1_at LOC787057 similar to zinc finger protein 415 39.28 57.52 46.44 36.75 88.81 56.30 Bt.18080.2.S 1_at LOC787094 similar to tescalcin 5.47 8.94 13.00 5.95 6.13 5.99 Bt.11233.1.S1_at LOC787143 /// TOP2B similar to DNA topoisomerase II, beta isozyme /// topoisomerase (DNA) II beta 180kDa 1453 1312 1494 1748 1903 1516 Bt.19994.1.S1_at LOC789597 similar to PDZ domain containing guanine nucleotide exchange factor PDZ GEF2 351 385 368 450 621 392 Bt.9655.2.S1_at LOC790332 similar to enterocytin 117 73.85 21.73 17.89 20.37 110 Bt.27204.1.S1_at LPCAT3 lysophosphatidylcholine acyltransferase 3 150 89.18 168 67.33 38.15 95.31 Bt.8135.1.S1_at LRAT lecithin retinol acyltransferase (phosphatidylcholine -retinol O acyltransferase) 103 90.47 62.69 85.81 192 104

PAGE 397

397 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.6143.1.S1_at LTA4H leukotriene A4 hydrolase 661 417 491 579 429 584 Bt.13257.2.A1_at LTV1 LTV1 homolog (S. cerevisiae) 146 249 216 174 208 128 Bt.22150.1.A1_at LZTFL1 leucine zipper transcription factor like 1 277 316 262 285 449 276 Bt.21336.1.S1_a_at MAD2L2 MAD2 mitotic arrest deficient like 2 (yeast) 128 131 118 184 76.89 102 Bt.24258.2.S1_at MAN1A1 mannosidase, alpha, class 1A, member 1 566 506 435 659 869 815 Bt.6774.2.S1_at MAP1LC3B microtubule associated protein 1 light chain 3 beta 564 360 448 583 438 579 Bt.25957.1.S1_at MAVS mitochondrial antiviral signaling protein 71.93 83.44 61.87 45.41 31.74 61.43 Bt.20529.1.A1_at MBLAC1 metallo beta lactamase domain containing 1 31 6 194 291 281 261 310 Bt.21433.1.S1_at MCM6 minichromosome maintenance complex component 6 252 260 190 230 379 292 Bt.7915.1.S1_at MDH2 malate dehydrogenase 2, NAD (mitochondrial) 4161 3775 5167 3727 3564 4191 Bt.13251.1.S1_at MFNG MFNG O fucosylpeptide 3 beta N acetylglucosaminyltransferase 156 111 94.87 120 117 125 Bt.7327.2.S1_a_at MGC133692 hypothetical LOC506714 6266 6082 4934 6281 8276 5715 Bt.17517.1.S1_at MGC134574 hypothetical LOC505226 527 469 321 457 622 468 Bt.18540.1.A1_at MGC165715 Hypot hetical LOC530484 427 302 403 362 710 533 Bt.9774.1.S1_a_at MGC165862 hypothetical LOC614805 265 222 146 314 455 315 Bt.3678.1.S1_at MKI67IP MKI67 (FHA domain) interacting nucleolar phosphoprotein 417 492 341 509 426 385 Bt.12370.1.S1_at MLF2 myeloid le ukemia factor 2 395 405 500 265 185 375 Bt.24793.1.S1_at MN1 meningioma (disrupted in balanced translocation) 1 5.27 9.94 5.01 5.27 5.03 10.45 Bt.15685.1.A1_at MOSC2 MOCO sulphurase C terminal domain containing 2 11838 10364 9867 11363 13442 9858 Bt.172 19.1.A1_at MPDU1 mannose P dolichol utilization defect 1 402 457 467 436 282 469 Bt.27187.1.S1_at MPHOSPH10 M phase phosphoprotein 10 (U3 small nucleolar ribonucleoprotein) 197 286 219 261 395 158 Bt.11135.1.S1_at MPV17 MpV17 mitochondrial inner membrane protein 726 511 643 602 549 726 Bt.4985.1.S1_at MRPL23 mitochondrial ribosomal protein L23 7129 4393 6496 6411 4345 5934 Bt.4985.1.S1_a_at MRPL23 mitochondrial ribosomal protein L23 4430 2490 4062 3992 2836 3656 Bt.26953.1.A1_at MRPL36 mitochondrial ri bosomal protein L36 138 92.73 119 124 99.84 121 Bt.3811.1.S1_at MRPS18B mitochondrial ribosomal protein S18B 263 226 343 221 228 299 Bt.20270.1.S1_at MSL1 male specific lethal 1 homolog (Drosophila) 285 486 356 333 322 350 Bt.4503.1.S2_at MTCH2 mitochon drial carrier homolog 2 (C. elegans) 2956 2025 2709 2545 2336 2883 Bt.26410.1.A1_at MTERF mitochondrial transcription termination factor 159 184 120 211 223 174 Bt.18045.1.S1_at MTPAP mitochondrial poly(A) polymerase 130 183 195 149 172 143 Bt.8143.1.S1 _at MX2 myxovirus (influenza virus) resistance 2 (mouse) 5.36 32.14 5.83 6.29 5.29 4.96 Bt.8090.2.S1_at MYBBP1A MYB binding protein (P160) 1a 65.02 101 88.07 68.53 32.47 54.73

PAGE 398

398 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.10310.1.S1_at MYBPC1 myosin binding protein C, slow type 4.76 10.74 4.71 4.71 4.66 4.76 Bt.12300.1.S1_at MYH1 myosin, heavy chain 1, skeletal muscle, adult 4. 51 7.02 4.51 4.67 4.67 4.67 Bt.12300.2.S1_at MYH2 myosin, heavy chain 2, skeletal muscle, adult 4.52 579 4.52 4.52 4.52 4.55 Bt.6620.1.S1_at MYH7 myosin, heavy chain 7, cardiac muscle, beta 4.56 30.59 4.60 4.63 4.56 4.63 Bt.4922.1.S1_at MYL1 myosin, lig ht chain 1, alkali; skeletal, fast 4.52 377 4.52 4.53 4.53 4.53 Bt.1905.1.S1_at MYL2 myosin, light chain 2, regulatory, cardiac, slow 4.53 109 4.52 4.52 4.53 4.72 Bt.11199.1.S1_at MYOZ1 myozenin 1 5.38 10.03 5.30 5.30 5.37 5.38 Bt.5399.1.S2_at NADK NAD kinase 1501 1374 2014 1591 979 1448 Bt.5399.1.S1_at NADK NAD kinase 86.34 86.01 103 85.83 54.21 95.69 Bt.3999.1.S1_at NAGA N acetylgalactosaminidase, alpha 281 198 220 217 210 245 Bt.5542.2.S1_at NAP1L1 nucleosome assembly protein 1 like 1 2074 1580 14 53 2265 2607 2009 Bt.26892.1.S1_at NBN nibrin 963 1330 1005 1035 1325 818 Bt.2905.1.S1_at NDRG2 NDRG family member 2 1507 1397 2268 2027 1756 2422 Bt.4475.1.S1_at NDUFS2 NADH dehydrogenase (ubiquinone) Fe S protein 2, 49kDa (NADH coenzyme Q reductase) 2 585 1794 2887 2602 1933 2691 Bt.653.1.S1_at NEK6 NIMA (never in mitosis gene a) related kinase 6 3586 4248 4811 3629 2713 4099 Bt.17428.1.A1_at NHLRC3 NHL repeat containing 3 390 230 304 311 308 513 Bt.3023.1.S1_at NIT1 nitrilase 1 318 218 365 229 219 3 43 Bt.9705.1.S1_at NKTR natural killer tumor recognition sequence 355 481 465 504 467 342 Bt.6993.2.A1_a_at NME7 non metastatic cells 7, protein expressed in (nucleoside diphosphate kinase) 360 311 214 328 406 324 Bt.12285.3.S1_a_at NMI N myc (and STAT) interactor 792 1711 767 864 868 786 Bt.5129.1.S1_a_at NNAT neuronatin 80.39 19.67 7.77 34.99 32.49 23.36 Bt.5129.2.A1_at NNAT neuronatin 259 65.21 27.99 123 105 75.81 Bt.7381.1.S1_at NPLOC4 nuclear protein localization 4 homolog (S. cerevisiae) 134 133 148 142 97.72 109 Bt.3599.1.S1_at NPM1 nucleophosmin (nucleolar phosphoprotein B23, numatrin) 5466 5845 4659 5714 6540 5290 Bt.6316.1.S1_at NR2F6 nuclear receptor subfamily 2, group F, member 6 1185 1073 1104 1418 683 963 Bt.20373.1.S1_at NRP1 neuropil in 1 941 918 590 830 1096 948 Bt.20932.1.S1_at NSA2 NSA2 ribosome biogenesis homolog (S. cerevisiae) 1236 1250 806 1387 1323 1031 Bt.1946.1.S1_at NSFL1C NSFL1 (p97) cofactor (p47) 168 162 190 144 111 160 Bt.20677.1.S1_at NSL1 NSL1, MIND kinetochore comp lex component, homolog (S. cerevisiae) 64.52 53.98 36.70 51.89 92.81 53.78 Bt.17805.2.A1_at NUDT12 nudix (nucleoside diphosphate linked moiety X) type motif 12 90.41 83.95 82.08 79.85 174 124 Bt.26961.1.S1_at NUDT14 nudix (nucleoside diphosphate linked m oiety X) type motif 14 83.89 43.42 71.23 82.65 63.31 109 Bt.17124.1.A1_s_at NUDT14 nudix (nucleoside diphosphate linked moiety X) type motif 14 255 157 265 324 219 366 Bt.20891.1.S1_at OAS2 2' 5' oligoadenylate synthetase 2, 69/71kDa 1105 4606 2338 2372 579 653

PAGE 399

399 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.27143.1.A1_at ODF2L Outer dense fiber of sperm tails 2 like 133 164 141 131 232 131 Bt.12910.1.S1_at OGDH oxoglutarate (alpha ketoglutarate) dehydrogenase (lipoamide) 74.36 72.23 107 63.77 46.93 79.52 Bt.367.1.S1_at OLR1 oxidized low density lipoprotein (lectin like) receptor 1 13.35 34.27 12.06 52.91 16.24 11.74 Bt.17777.1 .S1_at OPTN optineurin 663 1436 900 726 881 707 Bt.17777.3.S1_at OPTN optineurin 97.72 217 156 121 167 119 Bt.17777.2.S1_at OPTN optineurin 331 691 531 461 586 400 Bt.13189.1.A1_at ORC4L Origin recognition complex, subunit 4 like (yeast) 153 202 165 170 209 165 Bt.28245.1.S1_at OSTBETA organic solute transporter beta 1072 1112 983 1581 481 894 Bt.15997.1.S1_at P2RX4 purinergic receptor P2X, ligand gated ion channel, 4 311 169 258 324 192 383 Bt.5360.1.S1_a_at PAPOLA poly(A) polymerase alpha 315 411 47 8 331 478 331 Bt.6521.1.A1_at PARD6B par 6 partitioning defective 6 homolog beta (C. elegans) 57.79 39.38 65.58 78.98 67.40 57.62 Bt.18116.1.S1_at PARP12 poly (ADP ribose) polymerase family, member 12 6.73 13.78 11.87 9.25 7.93 8.28 Bt.18116.2.A1_at PAR P12 poly (ADP ribose) polymerase family, member 12 12.12 28.53 15.18 15.08 11.67 10.93 Bt.23171.2.S1_at PCBD1 pterin 4 alpha carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha 6186 4720 8324 5700 4442 6127 Bt.4718.1.S1_a t PCTP phosphatidylcholine transfer protein 3920 2300 2844 3065 2012 3886 Bt.3736.1.A1_at PDE4DIP phosphodiesterase 4D interacting protein (myomegalin) 7.75 7.75 11.94 6.94 7.38 8.22 Bt.444.1.S1_at PDE6C phosphodiesterase 6C, cGMP specific, cone, alpha p rime 496 206 137 568 661 321 Bt.6460.1.S1_at PDIA6 protein disulfide isomerase family A, member 6 5001 4358 5410 3275 3920 4674 Bt.11475.1.A1_at PDLIM5 PDZ and LIM domain 5 5.81 10.02 6.31 5.69 6.52 6.47 Bt.5916.1.S1_at PGCP plasma glutamate carboxypept idase 345 374 358 490 530 361 Bt.20281.2.S1_a_at PGM1 phosphoglucomutase 1 707 527 644 615 508 686 Bt.20281.3.S1_a_at PGM1 phosphoglucomutase 1 197 172 219 183 142 260 Bt.12820.1.S1_at PGRMC1 progesterone receptor membrane component 1 5596 3171 6074 596 7 6873 6656 Bt.15306.1.A1_at PHF3 PHD finger protein 3 1091 992 1198 1394 1528 1099 Bt.23955.1.A1_at PHOSPHO2 phosphatase, orphan 2 823 536 655 681 902 783 Bt.12864.1.S1_at PHPT1 phosphohistidine phosphatase 1 723 433 502 677 391 675 Bt.21680.2.S1_at P IR pirin (iron binding nuclear protein) 76.17 49.18 57.37 68.62 41.11 68.97 Bt.29432.1.A1_at PKHD1 similar to polycystic kidney and hepatic disease 1 (autosomal recessive) 27.26 51.65 51.16 19.39 29.92 30.47 Bt.13534.1.S1_at PLA2G16 phospholipase A2, gro up XVI 823 583 632 654 492 735 Bt.15713.2.S1_at PLEK pleckstrin 6.03 8.92 4.63 10.98 4.66 12.77 Bt.22283.1.S1_at PLEKHA2 pleckstrin homology domain containing, family A (phosphoinositide binding specific) member 2 351 486 351 328 389 388 Bt.29194.1.S1_a t PLIN4 similar to plasma membrane associated protein, S3 12 15.77 17.04 41.29 14.80 15.47 18.45

PAGE 400

400 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.28162.1.S1_at PLN phospholamban 111 114 97.93 86.08 222 155 Bt.15906.1.S1_at PLS3 plastin 3 1561 1430 1396 1312 2198 1828 Bt.12638.1.S1_at PML promyelocytic leukemia 26.73 57.34 34.26 28.75 24.86 25.20 Bt.23599.1.S1_at PON2 paraoxonase 2 1170 793 916 1077 1131 1205 Bt.6626.1.S1_at PPAP2A phosphatidic acid phosphatase type 2A 793 545 652 606 578 665 Bt.12803.1.S1_at PPARA peroxisome proliferator activated receptor alpha 77.00 98.10 64.16 80.09 44.00 89.23 Bt.9791.1.S1_at PPIF peptidylp rolyl isomerase F 1633 1666 1109 960 1014 1161 Bt.19839.1.A1_at Ppig peptidylprolyl isomerase G (cyclophilin G) 44.07 46.14 41.98 46.82 65.41 42.38 Bt.18634.1.A1_at PPM1K protein phosphatase, Mg2+/Mn2+ dependent, 1K 684 456 586 465 989 714 Bt.5319.1.S1_ at PRDX6 peroxiredoxin 6 1346 993 1390 1646 1293 1704 Bt.20145.1.S1_at PRELID1 PRELI domain containing 1 2029 1635 2245 1643 1345 2010 Bt.21189.1.S1_at PRKD2 protein kinase D2 192 237 238 206 116 170 Bt.6225.2.A1_at PRKD3 protein kinase D3 411 611 425 4 61 713 465 Bt.4404.1.A1_at PRSS2 protease, serine, 2 (trypsin 2) 4.87 4.87 4.51 135 4.87 4.51 Bt.13588.2.S1_at PSAT1 phosphoserine aminotransferase 1 33.41 15.87 49.01 22.87 21.42 45.88 Bt.13588.3.A1_at PSAT1 phosphoserine aminotransferase 1 108 40.63 1 27 76.79 50.96 166 Bt.9048.2.S1_a_at PSENEN presenilin enhancer 2 homolog (C. elegans) 584 377 508 488 420 509 Bt.12290.1.S1_at PSIP1 PC4 and SFRS1 interacting protein 1 997 988 916 1192 1956 938 Bt.20110.1.S1_at PSMF1 proteasome (prosome, macropain) in hibitor subunit 1 (PI31) 299 672 384 315 190 250 Bt.3715.1.S1_at PSMG4 proteasome (prosome, macropain) assembly chaperone 4 1074 589 851 1012 495 988 Bt.1645.1.S1_at PTGDS prostaglandin D2 synthase 21kDa (brain) 80.17 128 85.09 134 48.32 89.95 Bt.20261. 1.S1_at PTPN3 protein tyrosine phosphatase, non receptor type 3 30.06 48.18 53.85 59.45 61.03 47.50 Bt.24848.1.A1_at PTPRD protein tyrosine phosphatase, receptor type, D 57.63 54.58 103 94.80 174 79.25 Bt.21708.1.S1_at RAB4A RAB4A, member RAS oncogene fa mily 391 253 357 402 337 418 Bt.26308.2.A1_at RAD18 RAD18 homolog (S. cerevisiae) 7.65 7.65 7.15 8.22 14.93 6.70 Bt.8997.1.S1_at RANGAP1 Ran GTPase activating protein 1 104 412 161 192 61.07 84.54 Bt.8730.1.S1_at RAPGEF2 Rap guanine nucleotide exchange factor (GEF) 2 860 950 1232 769 684 797 Bt.22323.1.A1_a_at RASSF5 Ras association (RalGDS/AF 6) domain family member 5 350 315 454 392 253 327 Bt.22683.1.S1_at RBM10 RNA binding motif protein 10 189 312 244 188 164 155 Bt.17614.1.S1_at RBM25 RNA binding motif protein 25 52.73 102 87.44 60.50 107 61.42 Bt.27964.1.A1_at RCL1 RNA terminal phosphate cyclase like 1 3344 1769 2865 2794 1978 2998 Bt.20711.1.S1_at RDH16 retinol dehydrogenase 16 (all trans) 9295 7254 9157 6543 6134 7357 Bt.13743.1.A1_at RFK ri boflavin kinase 1134 1151 629 1124 1540 1135 Bt.20477.1.S1_at RFTN1 raftlin, lipid raft linker 1 52.21 20.49 23.74 53.05 27.33 28.75 Bt.6802.1.S1_at RGS5 regulator of G protein signaling 5 197 303 74.40 128 428 267

PAGE 401

401 Appendix A. Continued Treatment (Da m diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.28182.1.A1_at RGS5 regulator of G protein signaling 5 31.93 26.82 10.13 22.32 69.12 55.91 Bt.27940.1.A1_at RHBG Rh fam ily, B glycoprotein (gene/pseudogene) 70.50 37.81 100 35.93 36.70 73.26 Bt.24892.1.A1_at RIT1 Ras like without CAAX 1 427 422 273 366 598 375 Bt.6822.1.S1_at RNF150 similar to RING finger protein 150 14.66 22.33 19.97 15.64 18.31 16.33 Bt.10686.1.S1_at RNF170 ring finger protein 170 664 425 649 763 1048 742 Bt.920.1.S1_at RNF181 ring finger protein 181 78.12 93.55 81.91 94.58 76.12 180 Bt.28207.1.S1_at RNF19A ring finger protein 19A 401 442 386 531 740 419 Bt.15692.1.A1_at RNF19B ring finger protein 1 9B 61.63 88.13 51.45 103 44.99 65.76 Bt.6645.1.S1_at RNPC3 RNA binding region (RNP1, RRM) containing 3 339 290 343 393 530 278 Bt.28914.1.A1_at RP2 retinitis pigmentosa 2 (X linked recessive) 153 129 103 87.51 101 91.49 Bt.23317.1.S1_at RPL13 ribosomal protein L13 5393 2809 4534 5850 4506 5606 Bt.23548.1.S1_at RPL34 ribosomal protein L34 5877 4149 5191 5944 5568 6202 Bt.2822.1.S1_at RPL8 ribosomal protein L8 4798 3580 4545 4708 2963 4711 Bt.21268.1.S2_at RPS6KB1 ribosomal protein S6 kinase, 70kDa, pol ypeptide 1 365 257 355 441 516 355 Bt.1034.1.S1_at RPS8 ribosomal protein S8 18003 14263 15641 17448 14874 18099 Bt.4711.1.S1_at RPS9 ribosomal protein S9 2640 1838 2488 2499 1803 2637 Bt.5334.1.S1_at RPSA ribosomal protein SA 5533 4764 5548 4573 3737 5 316 Bt.22064.2.S1_at RSRC2 arginine/serine rich coiled coil 2 1061 1467 1277 1358 1681 973 Bt.196.1.S1_at S100A13 8KDa amlexanox binding protein 1032 354 425 821 465 875 Bt.17537.1.A1_at SAA4 serum amyloid A4, constitutive 1260 1042 1537 632 757 1174 B t.1552.1.S1_at SARS seryl tRNA synthetase 514 638 559 430 342 545 Bt.26302.1.A1_at SCML1 Sex comb on midleg like 1 (Drosophila) 23.15 16.89 21.50 17.62 62.56 28.09 Bt.11055.1.S1_at SDPR serum deprivation response 2274 2271 1801 2025 3705 2150 Bt.22483.1 .S1_at SEC31B SEC31 homolog B (S. cerevisiae) 148 176 123 182 148 130 Bt.27099.1.A1_at SEC62 SEC62 homolog (S. cerevisiae) 936 857 711 910 1325 736 Bt.28577.1.S1_at SENP6 SUMO1/sentrin specific peptidase 6 1468 1873 1454 1449 2146 1388 Bt.17451.2.A1_at SESTD1 SEC14 and spectrin domains 1; similar to SEC14 domain and spectrin repeat containing protein 1 (Huntingtin interacting protein like protein) (Protein Solo) 6.16 6.20 5.45 21.74 5.35 17.17 Bt.16234.2.S1_at SFRS18 splicing factor, arginine/serine ric h 18 52.98 73.69 115 92.25 154 71.87 Bt.16448.2.A1_at SFRS2IP splicing factor, arginine/serine rich 2, interacting protein 112 169 117 105 164 128 Bt.26408.1.A1_at SFRS2IP splicing factor, arginine/serine rich 2, interacting protein 1031 1201 1301 1296 1 915 1076 Bt.8206.1.S1_at SFRS7 splicing factor, arginine/serine rich 7, 35kDa 1328 2015 1730 1974 1598 1395 Bt.633.2.S1_a_at SFXN1 sideroflexin 1 285 295 1085 644 675 689 Bt.633.1.S1_at SFXN1 sideroflexin 1 429 434 1416 975 1004 824 Bt.27320.1.A1_at SG OL2 shugoshin like 2 (S. pombe) 99.46 108 104 106 206 85.05 Bt.5582.1.S1_at SH3BGR similar to SH3 domain binding glutamic acid rich protein (SH3BGR protein) 27.54 35.45 25.91 28.81 45.04 41.16 Bt.5220.1.S1_at SHBG sex hormone binding globulin 527 217 511 428 412 619

PAGE 402

402 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.7116.1.A1_at SIAE sialic acid acetylesterase 230 133 190 176 162 23 5 Bt.6038.1.S1_at SIGLEC1 sialic acid binding Ig like lectin 1, sialoadhesin 382 554 412 622 255 595 Bt.714.1.S1_at SIGMAR1 sigma non opioid intracellular receptor 1 89.59 60.87 74.67 72.40 63.46 89.73 Bt.23169.1.S1_at SIRPA signal regulatory protein al pha 277 402 340 204 175 303 Bt.16250.2.S1_at SLC10A1 solute carrier family 10 (sodium/bile acid cotransporter family), member 1 393 208 390 566 368 433 Bt.24007.1.A1_at SLC15A2 solute carrier family 15 (H+/peptide transporter), member 2 298 209 493 192 3 05 343 Bt.1207.1.S1_at SLC16A13 solute carrier family 16, member 13 (monocarboxylic acid transporter 13) 714 372 754 518 446 811 Bt.27443.1.S1_at SLC22A18 solute carrier family 22, member 18 136 75.40 134 106 113 134 Bt.3358.1.S1_at SLC25A1 solute carri er family 25 (mitochondrial carrier; citrate transporter), member 1 594 448 561 601 298 548 Bt.20520.1.S1_at SLC25A10 solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 979 565 748 830 627 776 Bt.11770.1.S1_at SLC25A20 solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 479 368 451 453 267 577 Bt.4880.1.S1_at SLC25A3 solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3 2977 2929 3104 2532 1797 2926 Bt.22577.2.S1_at SLC25A 33 solute carrier family 25, member 33 16.08 6.33 7.77 8.19 5.98 16.18 Bt.13332.1.S1_at SLC25A46 solute carrier family 25, member 46 913 1075 964 1271 1184 938 Bt.5083.1.S1_at SLC27A4 solute carrier family 27 (fatty acid transporter), member 4 112 104 11 6 63.46 27.03 108 Bt.28697.1.S1_at SLC31A1 solute carrier family 31 (copper transporters), member 1 6677 4474 6746 6669 6767 6431 Bt.8169.1.S1_at SLC39A6 solute carrier family 39 (zinc transporter), member 6 303 339 256 290 476 289 Bt.3195.1.S1_at SLC7A 9 solute carrier family 7 (cationic amino acid transporter, y+ system), member 9 101 27.02 75.47 55.28 82.00 70.47 Bt.15872.1.S1_at SLU7 SLU7 splicing factor homolog (S. cerevisiae) 386 323 223 288 859 440 Bt.27590.1.A1_at SMARCA4 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 89.68 164 143 93.75 79.51 87.00 Bt.22976.1.S1_at SMC4 structural maintenance of chromosomes 4 32.40 32.69 33.34 39.61 86.58 35.92 Bt.13336.1.A1_at SMC4 structural maintenance of c hromosomes 4 339 396 269 368 648 315 Bt.8491.1.S1_at SMOC2 SPARC related modular calcium binding 2 59.17 71.69 131 131 130 132 Bt.835.1.A1_at SNTB1 syntrophin, beta 1 (dystrophin associated protein A1, 59kDa, basic component 1) 166 139 128 190 218 151

PAGE 403

403 A ppendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.27468.1.A1_at SOAT2 sterol O acyltransferase 2 96.08 18.70 36.52 82.79 56.25 57.81 Bt.1736.1.A1_at SOCS1 suppressor of cytokine signaling 1 8.40 12.11 8.04 8.06 8.03 8.04 Bt.19339.3.A1_at SOCS6 similar to suppressor of cytokine signaling 6 302 327 183 308 405 250 Bt.2501.1.S1_at SOD2 superoxide dismutase 2, mitochondrial 611 360 597 532 754 519 Bt.24317.1.A1_at SOX6 SRY (sex determining region Y) box 6 95.50 106 130 80.38 138 103 Bt.27830.1.A1_at SP140 SP140 nuclear body protein 364 842 604 698 397 375 Bt.6289.1.S1_at SPTLC1 serine palmitoyltransferase, long chain base subunit 1 13 92 1319 1090 1409 1825 1250 Bt.11687.1.S1_a_at SRL sarcalumenin 4.52 16.90 4.52 4.52 4.52 4.52 Bt.13705.1.S1_at SSR2 signal sequence receptor, beta (translocon associated protein beta) 1983 1472 1924 1464 1077 1787 Bt.15037.1.S1_at ST3GAL1 ST3 beta gala ctoside alpha 2,3 sialyltransferase 1 556 646 537 512 248 445 Bt.11739.1.S1_a_at STAP2 signal transducing adaptor family member 2 1039 698 909 845 718 858 Bt.1920.2.S1_at STARD10 StAR related lipid transfer (START) domain containing 10 1280 734 826 1065 499 1076 Bt.24492.1.S1_at STAT2 signal transducer and activator of transcription 2, 113kDa 448 896 515 419 383 417 Bt.15334.2.A1_at STAT3 Signal transducer and activator of transcription 3 (acute phase response factor) 115 127 219 95.90 61.99 107 Bt.132 78.1.S1_at STEAP3 STEAP family member 3 408 351 407 441 270 437 Bt.28617.1.S1_at STOM Stomatin 1378 704 1083 1378 932 1513 Bt.27430.1.S1_at STRADB STE20 related kinase adaptor beta 237 136 150 209 197 196 Bt.7161.1.S1_at STRBP spermatid perinuclear RNA binding protein 285 194 275 240 230 263 Bt.3206.1.A1_at SUSD2 sushi domain containing 2 30.32 16.84 60.93 19.11 28.90 22.03 Bt.24249.1.S1_at SUV420H1 suppressor of variegation 4 20 homolog 1 (Drosophila) 53.00 56.11 69.72 85.08 111 57.87 Bt.8054.1.S1_at SYAP1 synapse associated protein 1, SAP47 homolog (Drosophila) 275 326 286 274 474 285 Bt.16614.1.A1_s_at SYNCRIP Synaptotagmin binding, cytoplasmic RNA interacting protein 229 349 227 346 282 186 Bt.20416.1.S1_at TAP1 transporter 1, ATP binding cassett e, sub family B (MDR/TAP) 145 334 191 202 171 140 Bt.4079.2.S1_a_at TARDBP TAR DNA binding protein 281 311 407 311 312 250 Bt.1987.1.S1_at TAX1BP3 Tax1 (human T cell leukemia virus type I) binding protein 3 1278 990 1298 967 891 1234 Bt.21764.1.S1_at TB C1D15 TBC1 domain family, member 15 217 199 148 210 404 285 Bt.21021.1.S1_at TBC1D7 TBC1 domain family, member 7 188 96.57 182 133 118 196 Bt.20229.1.S1_at TBRG4 transforming growth factor beta regulator 4 114 144 137 115 87.27 107 Bt.4053.1.S1_at TBXA2 R thromboxane A2 receptor 145 92.85 96.98 130 107 189 Bt.3026.1.A1_at TCEA3 transcription elongation factor A (SII), 3 81.41 32.84 45.07 90.09 38.01 56.55 Bt.5635.1.S1_at TCEAL1 transcription elongation factor A (SII) like 1 428 497 421 704 747 530 Bt.2 0091.1.S1_at TCF20 transcription factor 20 (AR1) 64.62 95.86 95.10 66.96 112 79.39

PAGE 404

404 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.25103.1.S1_at TDRD7 tudor domain containing 7 321 584 375 359 299 299 Bt.13834.1.S1_at TFRC transferrin receptor (p90, CD71) 3439 2637 4924 3246 4572 4708 Bt.6275.1.S1_at TGFBR1 transforming growth factor, beta receptor 1 476 419 288 543 618 402 Bt.4 619.1.S1_at TH1L TH1 like (Drosophila) 514 376 559 538 417 519 Bt.23605.2.S1_at THRA thyroid hormone receptor, alpha (erythroblastic leukemia viral (v erb a) oncogene homolog, avian) 198 176 394 161 178 209 Bt.10880.1.S1_at TIMM50 translocase of inner mi tochondrial membrane 50 homolog (S. cerevisiae) 138 108 113 117 70.76 135 Bt.22554.1.A1_at TK2 thymidine kinase 2, mitochondrial 116 171 175 171 122 116 Bt.22413.1.A1_at TLE4 Transducin like enhancer of split 4 (E(sp1) homolog, Drosophila) 87.59 122 128 97.08 110 96.48 Bt.13981.1.S1_at TM2D2 TM2 domain containing 2 847 692 685 1150 1196 673 Bt.20586.1.S1_a_at TM4SF5 transmembrane 4 L six family member 5 4265 2497 4507 3863 3462 3931 Bt.9567.1.S1_at TM7SF2 transmembrane 7 superfamily member 2 414 179 34 0 375 273 404 Bt.2416.1.S2_at TMBIM6 transmembrane BAX inhibitor motif containing 6 9072 6141 9467 9925 8946 9457 Bt.11176.2.S1_at TMEM14A transmembrane protein 14A 364 232 243 248 321 365 Bt.8039.1.S1_at TMEM170A transmembrane protein 170A 947 682 679 891 1093 645 Bt.26998.1.A1_s_at TNNC1 troponin C type 1 (slow) 4.57 94.99 4.57 4.57 4.57 4.57 Bt.6012.1.S1_at TNNC1 troponin C type 1 (slow) 4.51 33.57 4.51 4.67 4.54 4.54 Bt.9992.1.S1_at TNNC2 troponin C type 2 (fast) 6.58 101 6.77 6.55 6.52 6.55 Bt.1 2957.1.A1_at TNRC6B trinucleotide repeat containing 6B 751 970 938 885 502 736 Bt.21839.1.A1_at TOP1 Topoisomerase (DNA) I 1397 2081 1534 1591 1630 1255 Bt.19057.1.S1_at TOR1A torsin family 1, member A (torsin A) 97.29 32.48 44.88 41.19 47.25 83.48 Bt.8 42.1.A1_at TOR1AIP1 torsin A interacting protein 1 547 588 461 678 746 536 Bt.3487.1.S1_at TPI1 triosephosphate isomerase 1 1744 1287 1822 1263 908 1956 Bt.12477.2.S1_at TPM2 tropomyosin 2 (beta) 4.53 62.96 4.55 4.55 4.58 5.08 Bt.12477.1.S1_a_at TPM2 tr opomyosin 2 (beta) 124 228 82.25 110 89.41 219 Bt.17628.1.A1_at TRAK2 trafficking protein, kinesin binding 2 59.75 7.97 7.81 62.23 9.44 65.33 Bt.8235.1.S1_at TRAPPC5 trafficking protein particle complex 5 187 121 165 167 137 173 Bt.22980.1.S1_at TRIM21 tripartite motif containing 21 8.81 27.90 8.63 9.83 7.69 9.30 Bt.27071.1.S1_at TRIM38 tripartite motif containing 38 258 460 322 269 248 261 Bt.1529.2.A1_at TSG118 protein C16orf88 homolog 137 191 119 208 103 189 Bt.5444.1.S1_at TSPAN3 tetraspanin 3 236 0 1511 1841 2254 1796 2353 Bt.16052.2.A1_at TSPYL1 TSPY like 1 1125 1269 697 1330 1133 1114 Bt.460.1.S1_at TST thiosulfate sulfurtransferase (rhodanese) 3375 2078 2946 3039 2207 2978 Bt.20848.1.A1_at TTC36 tetratricopeptide repeat domain 36 2636 938 199 2 2670 1714 2921 Bt.21767.1.S1_at TTN titin 5.15 87.46 5.02 5.02 5.13 5.10 Bt.21767.1.S1_a_at TTN titin 7.33 325 8.82 10.04 10.30 10.02 Bt.5183.1.S1_at TUBA4A tubulin, alpha 4a 373 463 553 380 216 493 Bt.27119.1.A1_at TUBE1 tubulin, epsilon 1 268 281 2 19 241 442 280 Bt.2294.1.S1_a_at UBA7 ubiquitin like modifier activating enzyme 7 73.08 482 132 124 72.93 76.83 Bt.19006.2.A1_at UPB1 Ureidopropionase, beta 167 128 309 232 301 243

PAGE 405

405 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.17653.1.A1_at UPP2 uridine phosphorylase 2 1701 3367 969 2002 4761 2368 Bt.23164.1.S1_at UQCRC1 UQCRC1 protein 545 497 604 482 335 592 Bt.24095.1.A1_at USP1 Ubiquitin specific peptidase 1 568 742 559 605 921 582 Bt.21721.1.A1_at USP2 ubiquitin specific peptidase 2 4.74 6.27 4.52 17.26 4.74 4.75 Bt.14124.2.S1_at USP33 ubiquitin specific peptidase 33 118 142 104 161 242 120 Bt.17717.1.A1_at USPL1 ubiqui tin specific peptidase like 1 204 327 346 260 278 228 Bt.20427.2.S1_at UTP6 UTP6, small subunit (SSU) processome component, homolog (yeast) 56.55 236 234 136 131 133 Bt.25537.1.A1_at UXS1 UDP glucuronate decarboxylase 1 42.73 71.63 54.73 53.55 97.25 39.9 1 Bt.3549.1.A1_at VAMP4 vesicle associated membrane protein 4 178 176 123 173 273 144 Bt.24281.1.S1_at VAPA VAMP (vesicle associated membrane protein) associated protein A, 33kDa 976 796 844 1106 1047 1114 Bt.11270.2.S1_at VARS valyl tRNA synthetase 41. 86 61.05 59.62 41.51 23.90 54.54 Bt.282.1.S1_at VDAC1P5 voltage dependent anion channel 1 pseudogene 5 1278 1118 1117 1207 711 1303 Bt.28243.1.S1_a_at VNN1 vanin 1 2470 891 1744 2361 2126 2547 Bt.2170.1.A1_at VPS33A vacuolar protein sorting 33 homolog A (S. cerevisiae) 210 150 173 216 139 237 Bt.29587.1.S1_at WAC WW domain containing adaptor with coiled coil 113 115 135 164 205 127 Bt.20322.3.S1_a_at WDR18 WD repeat domain 18 75.31 51.90 104 49.18 51.90 70.11 Bt.5196.1.S1_at WDR55 WD repeat domain 55 643 676 754 611 497 702 Bt.28187.1.S1_at WEE1 WEE1 homolog (S. pombe) 120 126 79.14 195 125 130 Bt.26825.1.A1_at XRN2 5' 3' exoribonuclease 2 392 649 541 814 463 400 Bt.11237.1.S1_at YTHDC1 YTH domain containing 1 926 1284 1115 1134 1189 915 Bt.27876.1 .A1_at ZCCHC10 zinc finger, CCHC domain containing 10 17.59 28.98 13.44 17.77 17.15 17.26 Bt.12141.2.S1_a_at ZCCHC6 zinc finger, CCHC domain containing 6 310 621 375 316 403 294 Bt.23941.1.A1_at ZFP161 zinc finger protein 161 homolog (mouse) 240 306 283 214 325 278 Bt.3863.1.S1_at ZFP36 zinc finger protein 36, C3H type, homolog (mouse) 140 307 165 320 119 233 Bt.13489.1.S1_at ZMIZ1 zinc finger, MIZ type containing 1 95.10 186 109 126 124 148 Bt.12664.2.S1_at ZMYM5 Zinc finger, MYM type 5 96.90 82.83 10 1 141 212 90.81 Bt.17848.2.S1_at ZMYND8 zinc finger, MYND type containing 8 54.37 79.99 99.68 55.69 64.59 68.53 Bt.18023.1.S1_at ZNF322 zinc finger protein 322 271 237 285 335 399 264 Bt.10631.1.A1_at ZNF547 zinc finger protein 547 105 114 160 149 104 1 03 Bt.18479.1.A1_at ZNF608 zinc finger protein 608 142 211 174 121 123 178 Bt.1602.1.S1_at ZNF613 zinc finger protein 613 93.39 92.13 184 139 130 141 Bt.2186.1.S1_at ZNFX1 zinc finger, NFX1 type containing 1 243 1480 338 469 170 254 Bt.17229.1.A1_at ZN FX1 zinc finger, NFX1 type containing 1 4.62 7.64 4.63 5.27 4.63 4.62 Bt.7208.1.S1_at ZP2 zona pellucida glycoprotein 2 (sperm receptor) 46.64 15.48 204 24.62 51.46 22.87 Bt.29175.1.A1_at ZUFSP zinc finger with UFM1 specific peptidase domain 285 379 301 374 382 272 Bt.26650.1.S1_at ----16.66 9.37 17.83 17.81 43.39 22.64 Bt.841.1.S1_at --Transcribed locus 10.73 11.66 12.51 16.97 26.97 12.63 Bt.16425.1.A1_at --Transcribed locus 11.51 14.89 15.42 27.25 20.33 17.15

PAGE 406

406 Appendix A. Continued Treatme nt (Dam diet Milk replacer) Affimetrix ID Gene Symbol Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.13815.1.S1_at --Transcribed locus 103 59.15 103 123 154 107 Bt.17034.1.A1_at ----4.73 4.65 4.73 18.47 4.52 4.86 Bt.24524.2.A1_at ----86.29 57.66 137 139 115 88.23 Bt.22188.1.S1_at ----457 421 945 678 769 482 Bt.17364.1.A1_at ----1029 428 829 2525 3215 1162 Bt.19906.1.A1_at ----18.89 8.97 14.98 29.04 39.94 18.61 Bt.16509.1.A1_at --Transcr ibed locus 144 118 219 413 302 207 Bt.16058.1.A1_at ----44.46 32.09 91.39 38.12 135 69.51 Bt.19120.1.A1_at ----47.81 40.36 48.95 42.94 93.84 83.78 Bt.6636.1.S1_at --Transcribed locus 18.13 59.09 7.92 8.46 8.64 8.11 Bt.19792.1.A1_at ----36 .89 95.64 36.34 35.04 39.15 28.17 Bt.28164.2.S1_at ----85.22 197 62.81 152 59.84 64.27 Bt.23735.1.A1_s_at ----5.02 22.02 10.90 4.87 10.31 11.26 Bt.15807.1.S1_at ----152 140 117 148 232 181 Bt.9785.1.S1_at ----74.59 60.19 67.60 60.70 114 84.10 Bt.24316.1.A1_at ----457 297 374 285 654 712 Bt.20501.1.S1_at ----7.25 6.90 7.51 6.97 10.81 27.43 Bt.19232.1.A1_at ----26.24 22.81 7.01 14.00 39.85 14.87 Bt.29581.1.A1_at ----4.77 5.29 15.84 4.97 5.56 4.93 Bt.22543.1.S1_at ---113 118 139 129 73.19 101 Bt.26926.1.S1_at ----57.61 61.11 134 63.84 57.91 59.95 Bt.18206.1.A1_at ----289 160 460 359 188 229 Bt.18861.1.A1_at ----303 143 278 449 245 224 Bt.6575.1.A1_at --Transcribed locus, strongly similar to NP_6634 76.1 [Mus musculus] 114 127 218 264 155 86.81 Bt.5771.1.S1_at --Transcribed locus 221 141 266 190 133 186 Bt.10130.1.S1_at --Transcribed locus 28.58 54.53 51.04 37.94 17.10 26.57 Bt.13308.1.S1_at --Transcribed locus 67.89 70.75 98.90 112 65.27 78. 63 Bt.28739.1.S1_at ----686 956 399 1353 237 893 Bt.6890.1.S1_at --Transcribed locus 3302 4919 2963 3959 2688 4242 Bt.28238.1.A1_at ----2814 1669 3155 1789 4600 2916 Bt.17263.1.S1_at ----514 402 567 285 544 428 Bt.22076.1.A1_at ----2 79 169 250 178 296 229 Bt.20592.1.S1_at ----65.36 31.50 66.84 36.22 66.60 49.91 Bt.13429.2.S1_at --Transcribed locus 68.33 28.75 79.35 41.39 73.70 55.60 Bt.15706.1.A1_at ----49.78 47.99 52.09 44.80 104 38.28 Bt.26416.1.A1_at ----117 71.20 90.14 107 321 77.13 Bt.3555.1.S1_at --Transcribed locus 71.43 47.21 52.21 74.13 46.12 60.80 Bt.23902.1.A1_at ----2879 2151 2262 2150 1734 2314 Bt.19118.1.A1_at ----552 360 319 470 460 634 Bt.22063.2.S1_at ----1688 985 1266 1718 1710 1680 Bt.12381.1.A1_at --Transcribed locus, moderately similar to NP_001026004.1 [Gallus gallus] 62.56 40.36 59.38 60.62 63.22 66.31 Bt.12360.1.S1_at ----334 240 234 303 301 353 Bt.1252.1.S1_at --Transcribed locus 413 285 395 504 348 449 Bt.20404.1 .S1_at ----374 237 240 358 294 286 Bt.23706.1.A1_at ----125 82.85 92.11 113 118 123 Bt.19284.1.A1_at ----185 377 292 201 222 222 Bt.11918.1.A1_at ----130 203 314 83.90 128 169 Bt.8920.1.S1_at --Transcribed locus 285 402 391 362 544 37 0 Bt.29960.1.S1_at --Transcribed locus 181 228 234 127 212 124 Bt.9098.1.A1_at --Transcribed locus 4.76 7.68 4.96 5.09 4.71 4.95 Bt.18873.1.A1_at ----77.18 364 133 121 63.16 77.74 Bt.29924.1.S1_at --Transcribed locus 1034 1645 1210 1261 1085 1102 Bt.10692.1.S1_at --CDNA clone IMAGE:8398549 243 365 318 300 343 248 Bt.16739.1.A1_at --Transcribed locus 1312 2385 6793 1848 2457 2075

PAGE 407

407 Appendix A. Continued Treatment (Dam diet Milk replacer) Affimetrix ID Gene Symbo l Gene Title CTL LLA CTL HLA SFA LLA SFA HLA EFA LLA EFA HLA Bt.7576.1.S1_at --Transcribed locus 23.44 31.94 23.56 24.86 27.80 23.24 Bt.26415.1.A1_at ----129 160 182 115 157 156 Bt.25832.1.S1_at ----36.53 46.31 42.28 41.34 80.74 32.84 Bt.262 32.2.A1_at ----28.82 62.09 31.86 31.65 28.36 29.81 Bt.19339.1.S1_at ----30.19 47.60 38.81 55.66 53.51 39.07 Bt.11791.2.S1_at --Transcribed locus 485 537 655 484 601 495 Bt.25196.1.A1_at ----100 167 120 153 147 151 Bt.2465.1.S1_at ----44.52 95.88 58.49 47.80 58.80 57.64 Bt.24940.1.A1_at ----53.13 335 177 158 277 177 Bt.19107.1.S1_at ----291 631 339 373 330 280 Bt.7349.1.S1_at --Transcribed locus 18.05 29.73 18.69 21.48 34.43 19.29 Bt.12854.1.S1_at --Transcribed locus 149 213 188 157 265 138 Bt.22335.1.S1_a_at ----741 1057 920 925 844 848 Bt.23306.1.S1_at ----448 699 526 556 638 596 Bt.25084.1.S1_at ----329 433 421 350 676 327 Bt.17073.1.S1_at ----122 142 120 80.25 70.20 163 Bt.16525.1.A1_at --Transc ribed locus 356 436 502 320 232 430 Bt.18914.1.S1_at ----311 385 345 273 274 381 Bt.10361.1.S1_at ----6.52 5.82 9.34 6.27 6.02 6.10 Bt.18847.1.A1_at ----14.55 36.38 28.42 17.59 9.14 25.33 Bt.13633.1.A1_at ----144 262 235 113 99.59 191 Bt.29324.1.S1_at ----48.08 51.61 44.40 55.15 95.49 35.23 Bt.21952.1.A1_at ----127 136 113 156 183 127 Bt.2962.1.S1_at ----108 121 94.43 150 342 111 Bt.15299.1.A1_at ----6.60 6.12 6.28 6.91 11.56 6.82 Bt.21957.1.S1_at ----103 109 120 162 210 112 Bt.29107.1.S1_at ----177 182 136 226 239 188 Bt.22044.1.S1_at ----173 180 107 182 305 155 Bt.17883.2.A1_at ----18.85 15.85 11.20 19.37 34.95 15.28 Bt.28101.1.S1_at ----118 102 118 108 180 105 Bt.22656.2.S1_at ----334 3 99 278 406 540 284 Bt.23900.1.A1_at ----259 280 221 270 396 260 Bt.17846.1.A1_at ----41.86 31.49 18.69 34.76 48.49 25.32 Bt.812.1.S1_at --Transcribed locus 215 235 131 204 248 185 Bt.14283.1.A1_at --Transcribed locus 123 116 109 118 232 116 Bt.8039.2.S1_a_at ----347 305 209 338 432 281 Bt.23992.1.A1_at ----148 331 109 319 203 124 Bt.25190.1.A1_at ----1023 1155 1092 1180 2108 1056 Bt.20666.1.S1_at ----96.51 111 84.56 122 182 88.08 Bt.16828.1.A1_at --Transcribed locus 16 .69 22.27 15.72 22.40 19.73 15.17 Bt.2765.1.S1_at ----222 244 199 267 334 226

PAGE 408

408 APPENDIX B DIFFERENTIALY EXPRES SED GENES FOR THE CO NTRAST OF FAT List of differential expressed genes in liver of Holstein males at 30 d of age. Effect of feeding fat pre partum (contrast FAT, control = reference). Calves born from dams fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Genes are ranked by adjusted P value in descendant order. Affimetrix ID Gene symbol Fold change Av. Exp. (SFA + EFA) Ave. Exp. Control Adjusted P value Regu lation Bt.26650.1.S1_at --1.89 23.63 12.49 1.84E 07 UP Bt.26769.1.S1_at GIMAP8 2.13 10.06 4.72 8.40E 07 UP Bt.7575.1.A1_at GPT2 1.72 290 168 3.68E 06 UP Bt.841.1.S1_at --1.47 16.40 11.18 3.79E 06 UP Bt.4404.1.A1_at PRSS2 2.21 10.76 4.87 1.01E 05 UP Bt.16425.1.A1_at --1.49 19.56 13.09 4.77E 05 UP Bt.1785.1.A1_at LOC532189 1.46 299 205 1.57E 04 UP Bt.6813.1.A1_at AKAP5 1.66 96.26 57.88 1.94E 04 UP Bt.6521.1.A1_at PARD6B 1.40 66.97 47.71 3.61E 04 UP Bt.13815.1.S1_at --1.54 120 77.94 4.01E 04 UP Bt.24779.2.S1_at CREM 1.59 8.26 5.20 4.01E 04 UP Bt.17034.1.A1_at --1.41 6.62 4.69 5.32E 04 UP Bt.18003.1.S1_at CUL3 1.53 14 .12 9.22 5.57E 04 UP Bt.19614.1.A1_at LIPC 1.42 3771 2661 6.01E 04 UP Bt.19006.2.A1_at UPB1 1.84 269 147 6.01E 04 UP Bt.17451.2.A1_at SESTD1 1.65 10.22 6.18 1.01E 03 UP Bt.24249.1.S1_at SUV420H1 1.44 78.58 54.53 2.11E 03 UP Bt.25663.1.A1_at CPNE8 1.62 176 108 2.22E 03 UP Bt.26538.1.S1_at LOC509420 1.70 31.44 18.45 2.51E 03 UP Bt.10686.1.S1_at RNF170 1.48 788 532 2.56E 03 UP Bt.10777.1.S1_at FOXP1 1.56 71.96 46.17 2.94E 03 UP Bt.24524.2.A1_at --1.67 118 70.54 3.65E 03 UP Bt.15691.1.S1_at KCNK5 1. 58 138 87.19 5.11E 03 UP Bt.22188.1.S1_at --1.59 698 439 5.60E 03 UP Bt.17364.1.A1_at --2.52 1672 664 8.42E 03 UP Bt.24848.1.A1_at PTPRD 1.92 108 56.08 1.35E 02 UP Bt.16234.2.S1_at SFRS18 1.67 104 62.48 1.36E 02 UP Bt.19906.1.A1_at --1.83 23.85 13.02 1.65E 02 UP Bt.12820.1.S1_at PGRMC1 1.51 6381 4213 1.73E 02 UP Bt.16509.1.A1_at --2.11 274 130 1.99E 02 UP Bt.16137.1.S1_at ALDH9A1 1.62 540 334 2.19E 02 UP Bt.27073.1.S1_at ACADL 1.48 928 629 2.84E 02 UP Bt.29879.1.S1_at KAT2B 1.45 84.48 58.0 7 2.92E 02 UP Bt.28278.1.S1_at ACE2 2.34 1080 463 3.00E 02 UP Bt.2587.2.S1_a_at FH 1.44 377 262 3.06E 02 UP Bt.16580.1.S1_at CD2AP 1.56 35.42 22.66 3.06E 02 UP Bt.633.2.S1_a_at SFXN1 2.60 755 290 3.06E 02 UP Bt.12745.1.A1_at ANTXR2 1.45 101 69.81 3.10 E 02 UP Bt.633.1.S1_at SFXN1 2.40 1034 431 3.24E 02 UP Bt.26302.1.A1_at SCML1 1.44 28.57 19.77 3.30E 02 UP Bt.8491.1.S1_at SMOC2 2.01 131 65.13 3.30E 02 UP Bt.16058.1.A1_at --2.00 75.65 37.77 3.48E 02 UP Bt.13834.1.S1_at TFRC 1.43 4307 3012 3.57E 02 UP Bt.25752.1.A1_at C7H5orf24 1.47 65.43 44.47 3.74E 02 UP

PAGE 409

409 Appendix B. Continued Affimetrix ID Gene symbol Fold change Av. Exp. (SFA + EFA) Ave. Exp. Control Adjusted P value Regu lation Bt.16250.2.S1_at SLC10A1 1.52 433 286 3.86E 02 UP Bt.1602.1. S1_at ZNF613 1.59 147 92.76 3.87E 02 UP Bt.16058.2.S1_at LOC100583040 2.17 48.14 22.14 4.04E 02 UP Bt.20514.1.S1_at ATG2B 1.44 347 241 4.06E 02 UP Bt.190.1.A1_at IGFBP1 1.79 50.90 28.51 4.20E 02 UP Bt.19544.1.A1_at ACSM2A 1.41 4937 3509 4.25E 02 UP Bt .20261.1.S1_at PTPN3 1.45 55.20 38.05 4.28E 02 UP Bt.7023.1.S1_at FAHD2A 1.44 657 457 4.37E 02 UP Bt.12579.1.A1_at GK5 1.94 2425 1247 4.37E 02 UP Bt.2905.1.S1_at NDRG2 1.45 2103 1451 4.40E 02 UP Bt.18440.2.S1_at LOC510382 1.47 8.83 5.99 4.64E 02 UP Bt .16276.1.A1_at ARSK 1.49 458 307 4.68E 02 UP Bt.19120.1.A1_at --1.45 63.76 43.93 4.71E 02 UP Bt.13777.2.S1_at GIMAP7 2.00 44.52 22.21 4.94E 02 UP Bt.12300.2.S1_at MYH2 11.29 4.53 51.17 4.61E 15 DOWN Bt.4922.1.S1_at MYL1 9.11 4.53 41.25 5.52E 15 DOWN Bt.8435.1.S1_at ACTA1 8.94 4.89 43.70 5.55E 13 DOWN Bt.26998.1.A1_s_at TNNC1 4.56 4.57 20.83 5.71E 13 DOWN Bt.1905.1.S1_at MYL2 4.87 4.57 22.25 5.71E 13 DOWN Bt.9992.1.S1_at TNNC2 3.91 6.60 25.78 3.20E 12 DOWN Bt.21767.1.S1_at TTN 4.19 5.07 21.22 3.20 E 12 DOWN Bt.12477.2.S1_at TPM2 3.61 4.68 16.90 3.36E 12 DOWN Bt.23696.1.A1_at LOC509457 3.00 13.69 41.08 3.90E 11 DOWN Bt.6012.1.S1_at TNNC1 2.69 4.57 12.31 8.24E 11 DOWN Bt.6620.1.S1_at MYH7 2.56 4.60 11.81 2.48E 10 DOWN Bt.20557.1.S1_at ACTN2 2.92 4.96 14.49 5.56E 09 DOWN Bt.11687.1.S1_a_at SRL 1.93 4.52 8.74 1.01E 08 DOWN Bt.8143.1.S1_at MX2 2.36 5.57 13.12 1.29E 07 DOWN Bt.22169.1.S1_at ENO3 2.19 9.82 21.49 2.44E 07 DOWN Bt.27463.1.A1_at HERC6 1.68 4.80 8.04 5.03E 07 DOWN Bt.4126.2.S1_at CYP4 A22 1.77 34.05 60.28 5.03E 07 DOWN Bt.10310.1.S1_at MYBPC1 1.52 4.71 7.15 5.20E 06 DOWN Bt.9655.2.S1_at LOC790332 3.04 30.57 92.93 5.25E 06 DOWN Bt.22199.1.S1_at DDIT4L 1.56 5.77 8.97 5.43E 06 DOWN Bt.9779.1.S1_at ISG12(B) 2.84 6.83 19.41 1.56E 05 DOWN Bt.6636.1.S1_at --3.95 8.28 32.73 1.75E 05 DOWN Bt.22065.1.S1_at LOC783920 1.59 4.87 7.72 2.09E 05 DOWN Bt.21461.1.S1_at LOC509034 2.51 5.98 15.03 4.04E 05 DOWN Bt.6972.1.S1_at KBTBD10 1.50 5.03 7.55 4.53E 05 DOWN Bt.21767.1.S1_a_at TTN 4.99 9.78 4 8.78 4.77E 05 DOWN Bt.4937.1.S1_at LOC505941 1.49 2326 3464 1.73E 03 DOWN Bt.4762.1.S1_at BOLA NC1 1.65 33.13 54.74 2.65E 03 DOWN Bt.12285.3.S1_a_at NMI 1.42 820 1164 3.07E 03 DOWN Bt.22980.1.S1_at TRIM21 1.78 8.83 15.68 3.07E 03 DOWN Bt.154.1.S1_at C CL8 1.46 12.67 18.44 4.66E 03 DOWN Bt.24492.1.S1_at STAT2 1.47 431 634 4.74E 03 DOWN Bt.28914.1.A1_at RP2 1.47 95.71 141 9.70E 03 DOWN Bt.19792.1.A1_at --1.73 34.42 59.40 1.05E 02 DOWN Bt.25957.1.S1_at MAVS 1.60 48.38 77.47 1.35E 02 DOWN Bt.22116.1. A1_at IL18BP 1.76 10.69 18.80 1.93E 02 DOWN Bt.18114.1.A1_at LOC100851000 1.52 42.78 64.88 1.96E 02 DOWN Bt.18415.1.A1_at FTSJD1 1.87 329 616 2.04E 02 DOWN Bt.15037.1.S1_at ST3GAL1 1.43 418 599 2.10E 02 DOWN

PAGE 410

410 Appendix B. Continued Affimetrix ID Gene sym bol Fold change Av. Exp. (SFA + EFA) Ave. Exp. Control Adjusted P value Regu lation Bt.5372.1.S1_at ICAM1 1.47 154 226 2.38E 02 DOWN Bt.20110.1.S1_at PSMF1 1.63 275 448 2.41E 02 DOWN Bt.8090.2.S1_at MYBBP1A 1.41 57.23 80.95 2.60E 02 DOWN Bt.6556.1. S1_at LOC504773 1.53 1522 2326 3.06E 02 DOWN Bt.18321.1.A1_at GNB4 1.84 159 293 3.24E 02 DOWN Bt.2186.1.S1_at ZNFX1 2.09 288 600 3.24E 02 DOWN Bt.8997.1.S1_at RANGAP1 1.84 112 207 3.42E 02 DOWN Bt.9791.1.S1_at PPIF 1.56 1058 1649 4.06E 02 DOWN Bt.2982 3.1.S1_x_at BOLA 2.33 20.25 47.12 4.28E 02 DOWN Bt.5768.1.S1_at IRF7 1.87 93.23 174 4.37E 02 DOWN Bt.5129.1.S1_a_at NNAT 1.87 21.31 39.77 4.96E 02 DOWN Bt.28164.2.S1_at --1.66 77.82 129 4.97E 02 DOWN

PAGE 411

411 APPENDIX C DIFFERENTIALY EXPRES SED GENES FOR TH E CONTRAST OF FATTY ACIDS List of differential expressed genes in liver of Holstein male at 30 d of age. Effect of feeding essential fatty acids prepartum (Contrast FA, reference = saturated fatty acid diet). Calves were born from dams fed diets supplemen ted with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Genes are ranked by adjusted P value in descendant order. Affimetrix ID Gene symbol Fold change Av. Exp. EFA Ave. Exp. SFA Adjusted P value Regu lation Bt.26150.1.A1_at L2HGDH 1.70 254 149 7.34E 05 UP Bt.18634.1.A1_at PPM1K 1.61 840 522 2.97E 04 UP Bt.20977.3.S1_at CCPG1 1.41 129 91.53 0.0034 UP Bt.24793.1.S1_at MN1 1.41 7.25 5.14 0.0092 UP Bt.26522.2.S1_at C1H3OR F34 1.44 57.92 40.21 0.0016 UP Bt.9140.1.S1_at GMNN 1.65 254 154 4.11E 04 UP Bt.15872.1.S1_at SLU7 2.43 614 253 8.40E 04 UP Bt.6397.2.S1_at HMGB2 1.70 1503 884 1.54E 03 UP Bt.3191.1.A1_at KLHL24 2.15 939 437 1.57E 03 UP Bt.1048.1.S1_at BORA 1.47 73.14 49.64 0.0384 UP Bt.24892.1.A1_at RIT1 1.50 473 316 3.42E 03 UP Bt.23735.1.A1_s_at --1.48 10.78 7.29 0.0296 UP Bt.27966.1.S1_at LOC532789 1.48 63.01 42.59 0.0077 UP Bt.22479.1.S1_at CPEB4 1.48 22.05 14.89 0.0291 UP Bt.21764.1.S1_at TBC1D15 1.93 339 176 3.72E 03 UP Bt.17653.1.A1_at UPP2 2.41 3358 1393 6.36E 03 UP Bt.3843.1.S1_at IGJ 1.51 989 655 6.36E 03 UP Bt.10777.1.S1_at FOXP1 1.51 88.54 58.49 0.0226 UP Bt.13743.1.A1_at RFK 1.57 1322 841 6.36E 03 UP Bt.15807.1.S1_at --1.56 205 131 7.33E 03 U P Bt.9785.1.S1_at --1.53 98.01 64.06 0.0052 UP Bt.24258.2.S1_at MAN1A1 1.57 841 536 8.27E 03 UP Bt.11751.1.A1_at KLHL23 1.57 83.88 53.54 0.0335 UP Bt.8903.1.S1_at C14H8ORF70 1.45 258 178 8.50E 03 UP Bt.20373.1.S1_at NRP1 1.46 1019 700 8.55E 03 UP B t.5582.1.S1_at SH3BGR 1.58 43.06 27.32 0.0214 UP Bt.16187.1.A1_at KBTBD6 2.42 330 136 8.92E 03 UP Bt.19274.1.A1_at C1QTNF7 1.59 7.42 4.65 0.0000 UP Bt.20267.1.S1_at GCLM 1.47 209 142 1.25E 02 UP Bt.24316.1.A1_at --2.09 682 327 1.25E 02 UP Bt.18540.1 .A1_at MGC165715 1.61 615 382 1.33E 02 UP Bt.20677.1.S1_at NSL1 1.62 70.65 43.64 0.0043 UP Bt.27889.1.S1_at DLD 1.62 81.34 50.06 0.0000 UP Bt.8829.1.S1_a_at IFT122 1.52 195 128 1.63E 02 UP Bt.19745.1.S1_at ELL2 1.61 528 327 1.97E 02 UP Bt.18928.1.A1_a t EIF4E3 1.70 266 156 2.39E 02 UP Bt.27119.1.A1_at TUBE1 1.53 351 230 2.45E 02 UP Bt.21433.1.S1_at MCM6 1.59 333 209 2.45E 02 UP Bt.1817.1.S1_at ETV1 1.72 33.94 19.79 0.0034 UP Bt.2.1.S1_at CDK1 1.72 32.97 19.16 0.0139 UP Bt.28764.1.A1_at LOC787057 1. 71 70.71 41.31 0.0053 UP Bt.9774.1.S1_a_at MGC165862 1.76 378 214 2.45E 02 UP Bt.26650.1.S1_at --1.76 31.34 17.82 0.0000 UP Bt.19519.1.S1_at HLTF 1.40 1608 1149 2.74E 02 UP

PAGE 412

412 Appendix C. Continued Affimetrix ID Gene symbol Fold change Av. Exp. EFA A ve. Exp. SFA Adjusted P value Regu lation Bt.18440.2.S1_at LOC510382 1.80 11.85 6.58 0.0140 UP Bt.17805.2.A1_at NUDT12 1.82 147 80.96 0.0079 UP Bt.2506.1.S1_at DKK3 1.85 76.89 41.47 0.0345 UP Bt.11772.2.S1_at LOC614339 1.86 116 62.29 0.0437 UP Bt. 18316.1.A1_at LOC513587 1.89 165 87.57 0.0468 UP Bt.26635.2.S1_at FZD1 1.89 134 71.06 0.0413 UP Bt.10007.1.A1_at CKAP2 1.92 106 55.40 0.0117 UP Bt.17517.1.S1_at MGC134574 1.41 539 383 3.23E 02 UP Bt.8135.1.S1_at LRAT 1.93 141 73.34 0.0251 UP Bt.19120. 1.A1_at --1.93 88.67 45.85 0.0052 UP Bt.26364.1.A1_at BTBD8 2.02 27.30 13.52 0.0001 UP Bt.28162.1.S1_at PLN 2.02 186 91.82 0.0090 UP Bt.14369.1.A1_at CYP39A1 1.68 176 105 4.04E 02 UP Bt.18792.1.S1_at DCTN6 2.10 55.91 26.67 0.0064 UP Bt.15906.1.S1_at PLS3 1.48 2005 1353 4.28E 02 UP Bt.11055.1.S1_at SDPR 1.48 2822 1910 4.29E 02 UP Bt.26302.1.A1_at SCML1 2.15 41.92 19.47 0.0008 UP Bt.22869.1.S2_at FABP5 2.26 25.09 11.08 0.0085 UP Bt.20501.1.S1_at --2.38 17.22 7.23 0.0052 UP Bt.9655.2.S1_at LOC790 332 2.40 47.41 19.72 0.0002 UP Bt.2999.1.A1_at LOC783843 1.44 149 103 4.37E 02 UP Bt.20399.1.S1_at HSD17B13 2.10 1029 490 4.44E 02 UP Bt.16382.1.A1_at CALCRL 1.76 338 192 4.82E 02 UP Bt.19232.1.A1_at --2.46 24.34 9.91 0.0429 UP Bt.23566.2.S1_at LOC7 85936 2.72 43.89 16.13 0.0296 UP Bt.28934.1.S1_at AREG 3.31 21.82 6.59 0.0139 UP Bt.6802.1.S1_at RGS5 3.46 338 97.59 0.0000 UP Bt.28182.1.A1_at RGS5 4.13 62.17 15.04 0.0090 UP Bt.26769.1.S1_at GIMAP8 4.29 20.83 4.86 0.0000 UP Bt.190.1.A1_at IGFBP1 4.7 0 110 23.48 0.0001 UP Bt.23696.1.A1_at LOC509457 9.00 41.06 4.56 0.0000 UP Bt.4404.1.A1_at PRSS2 5.26 4.69 24.68 3.77E 08 DOWN Bt.17415.3.A1_at ERRFI1 1.85 6.13 11.32 4.16E 06 DOWN Bt.17034.1.A1_at --1.99 4.69 9.34 1.32E 05 DOWN Bt.21721.1.A1_at USP 2 1.86 4.74 8.83 7.50E 05 DOWN Bt.29581.1.A1_at --1.69 5.24 8.87 3.37E 04 DOWN Bt.22543.1.S1_at --1.55 86.06 134 4.11E 04 DOWN Bt.26926.1.S1_at --1.57 58.92 92.33 7.02E 04 DOWN Bt.12957.1.A1_at TNRC6B 1.50 608 911 1.12E 03 DOWN Bt.8090.2.S1_at M YBBP1A 1.84 42.16 77.68 2.29E 03 DOWN Bt.5399.1.S2_at NADK 1.50 1191 1790 3.37E 03 DOWN Bt.5415.1.S1_at CCS 2.98 149 443 5.16E 03 DOWN Bt.6020.1.S1_at DNAJC11 1.52 107 161 6.36E 03 DOWN Bt.7963.1.S1_at EHD1 1.63 142 232 7.35E 03 DOWN Bt.3248.1.S1_at A LDH4A1 1.41 230 324 8.27E 03 DOWN Bt.2858.1.S1_at ABHD6 1.45 35.85 51.84 8.44E 03 DOWN Bt.21336.1.S1_a_at MAD2L2 1.66 88.39 147 8.44E 03 DOWN Bt.26825.1.A1_at XRN2 1.54 430 663 8.50E 03 DOWN Bt.11256.1.S1_at CNOT1 1.49 859 1283 8.55E 03 DOWN Bt.28245. 1.S1_at OSTBETA 1.90 656 1247 9.41E 03 DOWN Bt.11259.1.S1_at ISG12(A) 5.32 1394 7411 9.98E 03 DOWN Bt.4937.1.S1_at LOC505941 1.46 1925 2810 1.17E 02 DOWN Bt.18206.1.A1_at --1.96 208 406 1.17E 02 DOWN

PAGE 413

413 Appendix C. Continued Affimetrix ID Gene symbol Fo ld change Av. Exp. EFA Ave. Exp. SFA Adjusted P value Regu lation Bt.12304.1.S1_at ISG15 8.11 617 5004 1.39E 02 DOWN Bt.12980.3.S1_a_at CL43 1.69 6752 11431 1.40E 02 DOWN Bt.8997.1.S1_at RANGAP1 2.45 71.86 176 1.53E 02 DOWN Bt.15037.1.S1_at ST3G AL1 1.58 333 525 1.64E 02 DOWN Bt.154.1.S1_at CCL8 1.47 10.46 15.34 1.81E 02 DOWN Bt.20891.1.S1_at OAS2 3.83 615 2355 1.83E 02 DOWN Bt.3358.1.S1_at SLC25A1 1.44 404 581 1.97E 02 DOWN Bt.17223.1.S1_at IFI35 1.84 121 223 2.14E 02 DOWN Bt.20785.2.S1_at I FI44 3.06 280 855 2.39E 02 DOWN Bt.21181.1.S1_at FOXK2 1.46 61.30 89.57 2.45E 02 DOWN Bt.18861.1.A1_at --1.51 234 353 2.45E 02 DOWN Bt.3201.1.S1_at GRWD1 1.69 43.80 74.07 2.45E 02 DOWN Bt.17814.1.A1_at LOC100736585 1.45 687 998 2.51E 02 DOWN Bt.2232 3.1.A1_a_at RASSF5 1.47 288 422 2.51E 02 DOWN Bt.1332.1.S1_a_at COX10 1.43 74.97 107 2.65E 02 DOWN Bt.29194.1.S1_at PLIN4 1.46 16.89 24.72 2.78E 02 DOWN Bt.6575.1.A1_at --2.07 116 240 2.81E 02 DOWN Bt.27830.1.A1_at SP140 1.68 386 649 2.91E 02 DOWN B t.20490.1.S1_at CDC42EP4 1.68 1022 1720 2.96E 02 DOWN Bt.1730.1.A1_at ID1 2.33 589 1370 2.99E 02 DOWN Bt.7381.1.S1_at NPLOC4 1.41 103 145 3.02E 02 DOWN Bt.22554.1.A1_at TK2 1.45 119 173 3.34E 02 DOWN Bt.21189.1.S1_at PRKD2 1.57 141 222 3.47E 02 DOWN B t.5771.1.S1_at --1.43 157 224 3.50E 02 DOWN Bt.20785.1.A1_at IFI44 2.93 417 1220 3.60E 02 DOWN Bt.10130.1.S1_at --2.06 21.32 44.00 3.95E 02 DOWN Bt.6316.1.S1_at NR2F6 1.54 811 1251 4.04E 02 DOWN Bt.8436.1.S1_at IFI6 3.91 674 2633 4.11E 02 DOWN Bt. 3928.1.S1_at HNRNPAB 1.43 1225 1751 4.39E 02 DOWN Bt.13308.1.S1_at --1.47 71.64 105 4.70E 02 DOWN Bt.10631.1.A1_at ZNF547 1.49 104 154 4.82E 02 DOWN Bt.8078.1.S1_at ARPC4 1.50 43.17 64.75 4.82E 02 DOWN Bt.13184.1.S1_at LOC523126 6.27 14.65 91.91 4.82 E 02 DOWN Bt.15796.1.S1_at LOC508226 1.57 36.06 56.75 4.98E 02 DOWN

PAGE 414

414 APPENDIX D DIFFERENTIALY EXPRES SED GENES FOR THE CO NTRAST OF MILK REPLA CER List of differential expressed genes in liver of Holstein males at 30 d of age. Effect of feeding high linol eic acid (HLA) in milk replacer (contrast MR, reference = low linoleic acid (LLA) milk replacer). Genes are ranked by adjusted P value in descendant order. Affimetrix ID Gene symbol Fold change Av. Exp. HLA Ave. Exp. LLA Adjusted P value Regu lation Bt.23696.1.A1_at LOC509457 18.72 85.43 4.56 2.67E 16 UP Bt.4922.1.S1_at MYL1 4.37 19.77 4.52 1.37E 13 UP Bt.12300.2.S1_at MYH2 5.05 22.84 4.52 1.37E 13 UP Bt.26998.1.A1_s_at TNNC1 2.75 12.56 4.57 1.39E 11 UP Bt.8435.1.S1_at ACTA1 4.34 21.13 4.87 1.39 E 11 UP Bt.1905.1.S1_at MYL2 2.93 13.26 4.53 1.61E 11 UP Bt.12477.2.S1_at TPM2 2.49 11.33 4.55 9.93E 11 UP Bt.21767.1.S1_at TTN 2.56 13.08 5.10 1.11E 10 UP Bt.9992.1.S1_at TNNC2 2.46 16.30 6.62 3.36E 10 UP Bt.21798.1.S1_at GIMAP6 4.39 149 33.89 1.33E 09 UP Bt.6012.1.S1_at TNNC1 1.97 8.93 4.52 2.36E 09 UP Bt.6620.1.S1_at MYH7 1.90 8.68 4.57 5.71E 09 UP Bt.26769.1.S1_at GIMAP8 2.80 13.08 4.67 1.75E 08 UP Bt.4404.1.A1_at PRSS2 3.03 14.37 4.75 1.18E 07 UP Bt.20557.1.S1_at ACTN2 2.01 10.04 5.01 1.86E 0 7 UP Bt.11687.1.S1_a_at SRL 1.55 7.02 4.52 2.75E 07 UP Bt.15713.2.S1_at PLEK 2.13 10.77 5.07 3.01E 06 UP Bt.17451.2.A1_at SESTD1 2.34 13.23 5.65 4.35E 06 UP Bt.8143.1.S1_at MX2 1.82 10.01 5.49 5.83E 06 UP Bt.154.1.S1_at CCL8 1.99 20.26 10.18 9.25E 06 UP Bt.21721.1.A1_at USP2 1.72 8.01 4.67 1.39E 05 UP Bt.17034.1.A1_at --1.60 7.47 4.66 1.82E 05 UP Bt.22169.1.S1_at ENO3 1.55 15.89 10.23 2.68E 05 UP Bt.24793.1.S1_at MN1 1.60 8.18 5.10 9.43E 05 UP Bt.21767.1.S1_a_at TTN 3.66 31.97 8.73 1.42E 04 UP Bt.9779.1.S1_at ISG12(B) 2.00 13.70 6.83 3.66E 04 UP Bt.6449.1.S1_at FBLN5 1.50 125 83.35 5.08E 04 UP Bt.28739.1.S1_at --2.61 1049 402 1.10E 03 UP Bt.6038.1.S1_at SIGLEC1 1.72 590 343 2.23E 03 UP Bt.1529.2.A1_at TSG118 1.65 196 119 2.69E 03 UP Bt.49 37.1.S1_at LOC505941 1.43 3172 2224 3.38E 03 UP Bt.3863.1.S1_at ZFP36 2.03 284 140 3.97E 03 UP Bt.15692.1.A1_at RNF19B 1.61 84.18 52.25 4.05E 03 UP Bt.23912.1.A1_a_at CYP2E1 1.67 1720 1032 5.47E 03 UP Bt.9699.1.S1_at CYP26A1 2.05 3324 1624 7.15E 03 UP Bt.12803.1.S1_at PPARA 1.48 88.84 60.13 8.34E 03 UP Bt.22980.1.S1_at TRIM21 1.63 13.66 8.36 8.34E 03 UP Bt.27474.1.S1_at CLEC4F 4.43 105 23.64 8.56E 03 UP Bt.2186.1.S1_at ZNFX1 2.33 561 241 1.21E 02 UP Bt.4816.1.S1_at ANGPTL4 1.53 92.49 60.38 1.40E 02 UP Bt.1645.1.S1_at PTGDS 1.68 116 69.08 1.51E 02 UP Bt.12477.1.S1_a_at TPM2 1.82 176 97.08 1.79E 02 UP Bt.5768.1.S1_at IRF7 2.06 165 79.99 1.79E 02 UP Bt.28164.2.S1_at --1.82 124 68.42 1.97E 02 UP Bt.22498.2.S1_at HES4 2.12 22.58 10.65 1.97E 02 UP Bt.920.1.S1_at RNF181 1.49 117 78.68 2.35E 02 UP Bt.28623.1.S1_at FAT1 1.58 602 381 2.35E 02 UP

PAGE 415

415 Appendix D. Continued Affimetrix ID Gene symbol Fold change Av. Exp. HLA Ave. Exp. LLA Adjusted P value Regu lation Bt.6890.1.S1_at --1.46 4355 297 4 2.47E 02 UP Bt.8915.1.A1_at DHTKD1 1.56 134 85.54 2.63E 02 UP Bt.8997.1.S1_at RANGAP1 1.87 188 101 2.68E 02 UP Bt.29823.1.S1_x_at BOLA 2.32 40.90 17.61 3.16E 02 UP Bt.6141.1.S1_at DES 1.44 18.67 12.98 3.38E 02 UP Bt.367.1.S1_at OLR1 2.01 27.71 13.78 3.45E 02 UP Bt.17415.3.A1_at ERRFI1 1.57 5.97 9.35 8.09E 09 DOWN Bt.26650.1.S1_at --1.51 15.57 23.45 2.06E 08 DOWN Bt.7575.1.A1_at GPT2 1.62 190 308 3.91E 08 DOWN Bt.4126.2.S1_at CYP4A22 1.44 34.37 49.37 4.11E 08 DOWN Bt.29581.1.A1_at --1.48 5.06 7.49 1.26E 06 DOWN Bt.24779.2.S1_at CREM 1.58 5.64 8.89 2.34E 06 DOWN Bt.26538.1.S1_at LOC509420 1.67 20.36 34.04 1.28E 05 DOWN Bt.1817.1.S1_at ETV1 1.55 20.06 31.04 1.33E 05 DOWN Bt.20241.1.S1_at HAAO /// LOC786774 1.54 421 650 1.74E 05 DOWN Bt.9735 .1.S1_at APOM 1.60 837 1339 3.48E 05 DOWN Bt.28238.1.A1_at --1.67 2057 3444 3.52E 05 DOWN Bt.211.1.S1_at DNAJC3 1.52 994 1508 4.99E 05 DOWN Bt.26302.1.A1_at SCML1 1.55 20.30 31.46 7.58E 05 DOWN Bt.17263.1.S1_at --1.48 366 541 8.11E 05 DOWN Bt.2501 .1.S1_at SOD2 1.40 463 650 9.79E 05 DOWN Bt.3206.1.A1_at SUSD2 1.96 19.21 37.65 1.07E 04 DOWN Bt.11769.2.S1_at EID3 1.47 11.81 17.36 1.16E 04 DOWN Bt.18114.1.A1_at LOC100851000 1.50 40.11 60.24 1.47E 04 DOWN Bt.21101.1.A1_at ACMSD 2.29 179 410 1.62E 04 DOWN Bt.22076.1.A1_at --1.44 190 274 2.22E 04 DOWN Bt.20592.1.S1_at --1.72 38.47 66.26 2.68E 04 DOWN Bt.13429.2.S1_at --1.82 40.44 73.66 2.83E 04 DOWN Bt.3195.1.S1_at SLC7A9 1.81 47.22 85.36 2.99E 04 DOWN Bt.23905.1.A1_at ERRFI1 1.53 2969 4532 3.71E 04 DOWN Bt.15706.1.A1_at --1.49 43.50 64.61 3.89E 04 DOWN Bt.7208.1.S1_at ZP2 3.83 20.58 78.81 3.96E 04 DOWN Bt.12255.1.A1_at CYP2C19 1.47 21.61 31.84 4.84E 04 DOWN Bt.26416.1.A1_at --1.79 83.69 150 6.12E 04 DOWN Bt.24007.1.A1_at SLC15A2 1.4 8 239 355 7.21E 04 DOWN Bt.16058.1.A1_at --1.86 43.97 81.92 7.52E 04 DOWN Bt.18440.2.S1_at LOC510382 1.47 6.41 9.40 8.59E 04 DOWN

PAGE 416

416 APPENDIX E DIFFERENTIALY EXPRES SED FOR THE INTERACT ION FAT BY MILK REPL ACER List of differential expressed genes in li ver of Holstein males at 30 d of age. Effect of fat during prepartum and high linoleic acid in milk replacer during (Interaction of contrasts FAT by MR). Calves were fed a high or low linoleic acid milk replacer from 1 30 d of age and were born from dams fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Affimetrix ID Gene symbol Fold change Av. Exp. FAT HLA Ave. Exp. FAT LLA Adjusted P value Regu lation Bt.4126.2.S1_at CYP4A22 3.39 1.04 0.31 2.38E 07 UP Bt.26769.1.S1_at GIMAP8 4.30 4.42 1.03 1.49E 06 UP Bt.4404.1.A1_at PRSS2 5.26 5.07 0.96 6.40E 06 UP Bt.17451.2.A1_at SESTD1 3.55 3.12 0.88 1.22E 04 UP Bt.1978.3.S1_at LOC780933 6.86 3.39 0.49 2.91E 04 UP Bt.27964.1.A1_at RCL1 2.30 1.64 0.71 3.20E 04 UP Bt.17034.1.A1_at --2.08 2.04 0.98 3.21E 04 UP Bt.6774.2.S1_at MAP1LC3B 2.05 1.61 0.79 4.03E 04 UP Bt.3715.1.S1_at PSMG4 2.81 1.70 0.60 4.10E 04 UP Bt.28076.1.A1_at GSTO1 1.82 1.55 0.85 5.5 0E 04 UP Bt.23317.1.S1_at RPL13 2.43 2.04 0.84 6.99E 04 UP Bt.13530.1.S1_at DCI 1.83 1.65 0.90 8.58E 04 UP Bt.4449.1.S1_at AKR1A1 2.52 2.01 0.80 1.22E 03 UP Bt.3212.1.S1_at ISOC2 2.06 1.68 0.82 1.44E 03 UP Bt.9699.1.S1_at CYP26A1 6.34 1.77 0.28 1.51E 03 UP Bt.17628.1.A1_at TRAK2 55.66 8.00 0.14 1.70E 03 UP Bt.2965.1.A1_at LOC618434 2.45 1.71 0.70 1.82E 03 UP Bt.20477.1.S1_at RFTN1 3.91 1.91 0.49 1.86E 03 UP Bt.21464.2.S1_a_at GALT 3.46 2.23 0.65 1.90E 03 UP Bt.5129.1.S1_a_at NNAT 7.35 1.45 0.20 1. 92E 03 UP Bt.29268.1.S1_at GOLT1A 2.11 1.46 0.69 1.96E 03 UP Bt.9655.2.S1_at LOC790332 3.34 0.60 0.18 2.19E 03 UP Bt.7116.1.A1_at SIAE 2.00 1.53 0.76 2.37E 03 UP Bt.805.1.S1_at ADIPOR2 2.90 1.93 0.67 2.43E 03 UP Bt.9170.1.A1_at KIAA1147 2.91 1.82 0.62 2.46E 03 UP Bt.1667.1.S1_at CDC34 2.37 1.48 0.62 2.49E 03 UP Bt.21021.1.S1_at TBC1D7 2.16 1.67 0.78 2.77E 03 UP Bt.18861.1.A1_at --2.58 2.22 0.86 2.97E 03 UP Bt.10880.1.S1_at TIMM50 1.80 1.17 0.65 3.69E 03 UP Bt.10609.2.A1_at CYP20A1 2.15 1.54 0.72 3.69E 03 UP Bt.29398.1.S1_at LOC100582155 1.73 1.65 0.95 4.24E 03 UP Bt.27468.1.A1_at SOAT2 7.84 3.70 0.47 4.24E 03 UP Bt.13864.1.A1_at CDC26 1.65 1.37 0.83 4.60E 03 UP Bt.3857.1.S1_at ENDOG 2.57 1.66 0.64 4.60E 03 UP Bt.23912.1.A1_a_at CYP2E1 2.92 1 .44 0.49 4.60E 03 UP Bt.24881.1.S1_at LOC539690 2.72 1.80 0.66 4.65E 03 UP Bt.6334.1.A1_at DEGS1 1.49 1.13 0.76 4.78E 03 UP Bt.12240.1.A1_at GLYATL3 1.85 1.22 0.66 5.56E 03 UP Bt.24281.1.S1_at VAPA 1.45 1.39 0.96 5.61E 03 UP Bt.27443.1.S1_at SLC22A18 1.75 1.58 0.91 5.89E 03 UP Bt.12864.1.S1_at PHPT1 2.55 1.56 0.61 5.89E 03 UP Bt.2481.2.S1_at C23H6ORF105 2.69 1.89 0.70 5.89E 03 UP Bt.3555.1.S1_at --2.07 1.42 0.69 6.17E 03 UP

PAGE 417

417 Appendix E. Continued Affimetrix ID Gene symbol Fold change Av. Exp. F AT HLA Ave. Exp. FAT LLA Adjusted P value Regu lation Bt.6156.1.S1_at 3290025600 2.04 1.59 0.78 6.34E 03 UP Bt.6162.1.S1_at LOC613560 2.77 2.22 0.80 6.34E 03 UP Bt.23902.1.A1_at --1.51 1.04 0.69 6.40E 03 UP Bt.2049.1.S1_at LOC510634 3.09 1.67 0.5 4 6.40E 03 UP Bt.22577.2.S1_at SLC25A33 4.29 1.82 0.42 6.40E 03 UP Bt.3358.1.S1_at SLC25A1 1.86 1.28 0.69 6.42E 03 UP Bt.4475.1.S1_at NDUFS2 1.61 1.47 0.91 6.79E 03 UP Bt.2050.1.A1_at ACAA1 2.13 1.91 0.90 6.79E 03 UP Bt.26604.1.S1_at APLNR 3.01 1.66 0 .55 7.03E 03 UP Bt.5466.2.S1_a_at RPS4Y1 /// RPS4Y2 1.53 1.34 0.88 7.38E 03 UP Bt.17961.1.S1_at APOC4 1.71 1.46 0.86 7.40E 03 UP Bt.6143.1.S1_at LTA4H 2.01 1.40 0.69 7.40E 03 UP Bt.13381.1.S1_at CIDEC 1.46 1.41 0.97 7.40E 03 UP Bt.196.1.S1_at S100A13 5.55 2.39 0.43 7.69E 03 UP Bt.4053.1.S1_at TBXA2R 2.41 1.69 0.70 8.15E 03 UP Bt.2183.1.A1_at HEXB 2.16 1.52 0.71 8.57E 03 UP Bt.20329.2.S1_at ARL4D 2.32 1.80 0.77 9.42E 03 UP Bt.4336.1.S1_at CFD 3.13 2.59 0.83 9.42E 03 UP Bt.4503.1.S2_at MTCH2 1.57 1. 34 0.85 9.92E 03 UP Bt.22694.1.A1_at APOA5 2.48 2.16 0.87 9.92E 03 UP Bt.19064.1.A1_at BTD 2.17 1.41 0.65 1.02E 02 UP Bt.15530.1.S1_at LOC784762 /// RPL12 1.54 1.23 0.80 1.02E 02 UP Bt.4141.1.S1_at COPE 1.72 1.31 0.76 1.02E 02 UP Bt.4985.1.S1_at MRPL2 3 1.88 1.40 0.75 1.02E 02 UP Bt.25097.1.S1_at GMPS 1.60 1.48 0.93 1.08E 02 UP Bt.3248.1.S1_at ALDH4A1 1.70 1.43 0.84 1.08E 02 UP Bt.13534.1.S1_at PLA2G16 1.75 1.19 0.68 1.08E 02 UP Bt.21464.3.S1_a_at GALT 3.21 1.93 0.60 1.11E 02 UP Bt.19999.1.A1_at FI CD 6.52 3.42 0.52 1.20E 02 UP Bt.714.1.S1_at SIGMAR1 1.72 1.32 0.77 1.21E 02 UP Bt.18435.3.A1_at ANGEL1 2.09 1.36 0.65 1.21E 02 UP Bt.7161.1.S1_at STRBP 1.47 1.30 0.88 1.24E 02 UP Bt.19614.1.A1_at LIPC 1.57 1.78 1.13 1.24E 02 UP Bt.10797.2.S1_a_at LOC 615093 1.77 1.55 0.88 1.24E 02 UP Bt.13411.1.S1_at LRBA 1.45 1.51 1.04 1.29E 02 UP Bt.20252.2.S1_a_at GALK1 2.96 1.91 0.65 1.33E 02 UP Bt.8421.2.S1_at LOC100623159 1.91 1.84 0.96 1.41E 02 UP Bt.16001.1.S1_at CYP27A1 1.95 1.49 0.76 1.44E 02 UP Bt.19937 .1.S1_at LOC532189 1.49 1.40 0.94 1.48E 02 UP Bt.16250.2.S1_at SLC10A1 2.47 2.38 0.96 1.50E 02 UP Bt.13942.1.S1_at GLYCTK 3.33 2.29 0.69 1.50E 02 UP Bt.28697.1.S1_at SLC31A1 1.45 1.46 1.01 1.51E 02 UP Bt.20529.1.A1_at MBLAC1 1.75 1.52 0.87 1.54E 02 UP Bt.6521.1.A1_at PARD6B 1.49 1.71 1.15 1.56E 02 UP Bt.9735.1.S1_at APOM 2.10 1.71 0.81 1.56E 02 UP Bt.18323.1.A1_at LOC509506 2.84 1.91 0.67 1.56E 02 UP Bt.282.1.S1_at VDAC1P5 1.61 1.12 0.70 1.59E 02 UP Bt.1034.1.S1_at RPS8 1.47 1.25 0.85 1.62E 02 UP Bt.23548.1.S1_at RPL34 1.60 1.46 0.91 1.62E 02 UP Bt.20241.1.S1_at HAAO /// LOC786774 1.90 1.43 0.75 1.62E 02 UP

PAGE 418

418 Appendix E. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FAT HLA Ave. Exp. FAT LLA Adjusted P value Regu lation Bt.3865.3 .S1_a_at C25H16orf14 2.92 1.82 0.62 1.62E 02 UP Bt.11279.1.A1_at CLCN4 2.20 1.81 0.82 1.62E 02 UP Bt.19057.1.S1_at TOR1A 3.81 1.81 0.47 1.64E 02 UP Bt.1785.1.A1_at LOC532189 1.50 1.78 1.19 1.66E 02 UP Bt.2056.1.S1_at APEH 1.65 1.58 0.96 1.66E 02 UP Bt .21464.1.S1_at GALT 2.47 1.58 0.64 1.66E 02 UP Bt.1207.1.S1_at SLC16A13 2.15 1.74 0.81 1.70E 02 UP Bt.11135.1.S1_at MPV17 1.58 1.29 0.82 1.73E 02 UP Bt.27966.1.S1_at LOC532789 1.60 1.42 0.89 1.80E 02 UP Bt.4150.1.S1_at CTNNBL1 2.02 1.62 0.80 1.80E 02 U P Bt.27430.1.S1_at STRADB 2.06 1.49 0.73 1.80E 02 UP Bt.22170.1.S1_a_at AGPAT5 1.48 1.45 0.98 1.81E 02 UP Bt.2170.1.A1_at VPS33A 2.05 1.51 0.74 1.86E 02 UP Bt.4555.1.S1_at ETFB 1.89 1.54 0.82 1.87E 02 UP Bt.9567.1.S1_at TM7SF2 2.96 2.18 0.74 1.90E 02 UP Bt.5129.2.A1_at NNAT 7.05 1.48 0.21 1.91E 02 UP Bt.11178.1.S1_at GPC3 1.60 1.40 0.87 1.93E 02 UP Bt.27623.2.S1_a_at GRTP1 2.95 1.87 0.63 1.99E 02 UP Bt.11176.2.S1_at TMEM14A 1.69 1.29 0.77 2.00E 02 UP Bt.27036.1.S1_at CYP4F2 3.99 1.98 0.50 2.00E 02 UP Bt.9047.1.S1_at DDT 1.62 1.42 0.88 2.06E 02 UP Bt.20848.1.A1_at TTC36 4.25 2.98 0.70 2.06E 02 UP Bt.22590.1.S1_at AGPAT2 4.37 2.02 0.46 2.06E 02 UP Bt.19118.1.A1_at --2.19 1.52 0.69 2.08E 02 UP Bt.4619.1.S1_at TH1L 1.50 1.41 0.94 2.09E 02 UP Bt .13278.1.S1_at STEAP3 1.54 1.25 0.81 2.11E 02 UP Bt.21708.1.S1_at RAB4A 1.83 1.62 0.89 2.13E 02 UP Bt.18330.2.S1_at ASGR2 2.08 1.89 0.91 2.13E 02 UP Bt.22510.1.S1_at C11H2ORF7 2.47 1.42 0.57 2.13E 02 UP Bt.3026.1.A1_at TCEA3 4.27 2.17 0.51 2.13E 02 UP Bt.3999.1.S1_at NAGA 1.52 1.16 0.77 2.19E 02 UP Bt.19980.2.S1_at ApoN 2.03 1.72 0.84 2.19E 02 UP Bt.16832.1.A1_at DHDPSL 2.18 2.07 0.95 2.30E 02 UP Bt.643.1.S1_at LOC508666 2.37 2.15 0.90 2.30E 02 UP Bt.5319.1.S1_at PRDX6 1.69 1.69 1.00 2.36E 02 UP B t.20453.1.S1_at ABHD14A 2.12 1.48 0.70 2.36E 02 UP Bt.13324.4.S1_at IDH1 2.34 2.02 0.86 2.40E 02 UP Bt.26961.1.S1_at NUDT14 2.74 2.19 0.80 2.49E 02 UP Bt.24950.1.S1_at FBXL5 1.65 1.27 0.77 2.50E 02 UP Bt.20919.2.A1_at GNMT 4.50 2.69 0.60 2.50E 02 UP B t.2824.1.S1_at BLOC1S1 1.88 1.44 0.77 2.53E 02 UP Bt.5170.1.S1_at GRHPR 2.68 2.10 0.78 2.54E 02 UP Bt.22063.2.S1_at --1.98 1.73 0.87 2.56E 02 UP Bt.5193.1.S1_at ACP5 2.47 1.51 0.61 2.70E 02 UP Bt.23599.1.S1_at PON2 1.65 1.44 0.87 2.72E 02 UP Bt.1381 5.1.S1_at --1.59 1.95 1.23 2.90E 02 UP Bt.19664.1.A1_at C3H1ORF210 3.63 1.64 0.45 2.90E 02 UP Bt.23143.2.S1_at CSDE1 1.45 1.41 0.98 3.00E 02 UP Bt.5193.2.S1_a_at ACP5 2.36 1.51 0.64 3.00E 02 UP Bt.2415.1.S1_at ID2 1.44 0.92 0.64 3.03E 02 UP Bt.16496 .1.A1_at KNTC1 1.64 1.72 1.05 3.03E 02 UP Bt.15713.2.S1_at PLEK 1.73 1.33 0.77 3.03E 02 UP Bt.5412.1.S1_at BCKDHB 1.40 1.32 0.94 3.03E 02 UP

PAGE 419

419 Appendix E. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FAT HLA Ave. Exp. FAT LLA Adjusted P value Regu lation Bt.13251.1.S1_at MFNG 1.64 1.11 0.68 3.07E 02 UP Bt.4985.1.S1_a_at MRPL23 2.00 1.53 0.77 3.07E 02 UP Bt.2424.1.S1_at DPYD 1.45 1.57 1.08 3.16E 02 UP Bt.3195.1.S1_at SLC7A9 2.95 2.31 0.78 3.16E 02 UP Bt.5220.1.S1_at SHBG 2.72 2.37 0. 87 3.17E 02 UP Bt.5536.1.S1_at ITGB5 1.79 1.42 0.79 3.18E 02 UP Bt.17428.1.A1_at NHLRC3 2.21 1.74 0.79 3.18E 02 UP Bt.24205.1.A1_at FGB 1.87 1.08 0.58 3.20E 02 UP Bt.8617.1.S1_at CNRIP1 1.96 1.29 0.66 3.23E 02 UP Bt.20586.1.S1_a_at TM4SF5 1.68 1.56 0. 93 3.24E 02 UP Bt.2822.1.S1_at RPL8 1.72 1.32 0.76 3.29E 02 UP Bt.8235.1.S1_at TRAPPC5 1.75 1.41 0.80 3.39E 02 UP Bt.23572.1.S1_at CCNDBP1 2.46 1.35 0.55 3.39E 02 UP Bt.24001.1.A1_at LOC100433242 3.98 1.86 0.47 3.43E 02 UP Bt.20249.1.S1_a_at ABCD3 1.4 2 1.35 0.95 3.43E 02 UP Bt.5350.1.S1_at ETFA 1.61 1.34 0.83 3.43E 02 UP Bt.25088.1.A1_at GCSH 1.46 1.52 1.04 3.45E 02 UP Bt.5164.1.S1_at CA14 6.99 2.60 0.37 3.51E 02 UP Bt.4711.1.S1_at RPS9 1.74 1.40 0.80 3.54E 02 UP Bt.27073.1.S1_at ACADL 1.95 2.06 1 .06 3.58E 02 UP Bt.15705.1.S2_at DSTN 1.53 1.36 0.88 3.64E 02 UP Bt.2416.1.S2_at TMBIM6 1.56 1.58 1.01 3.64E 02 UP Bt.9310.1.S1_at C16orf5 1.78 1.23 0.69 3.64E 02 UP Bt.460.1.S1_at TST 1.92 1.45 0.76 3.76E 02 UP Bt.20520.1.S1_at SLC25A10 2.03 1.42 0.7 0 3.76E 02 UP Bt.1920.2.S1_at STARD10 2.90 1.46 0.50 3.76E 02 UP Bt.12381.1.A1_at --1.60 1.57 0.98 3.84E 02 UP Bt.26832.1.S1_at CANT1 5.11 1.87 0.37 3.86E 02 UP Bt.28617.1.S1_at STOM 2.81 2.05 0.73 3.95E 02 UP Bt.14213.1.A1_at CES2 1.63 1.50 0.92 4. 01E 02 UP Bt.12360.1.S1_at --1.72 1.36 0.79 4.01E 02 UP Bt.1252.1.S1_at --1.86 1.67 0.90 4.01E 02 UP Bt.9735.2.A1_at APOM 2.09 1.69 0.81 4.01E 02 UP Bt.13324.1.S1_a_at IDH1 2.54 2.19 0.86 4.01E 02 UP Bt.10371.1.S1_at LOC516241 2.12 1.21 0.57 4.03E 02 UP Bt.28243.1.S1_a_at VNN1 3.53 2.75 0.78 4.14E 02 UP Bt.8724.1.S1_at LOC100299281 2.99 2.36 0.79 4.14E 02 UP Bt.20404.1.S1_at --1.90 1.35 0.71 4.18E 02 UP Bt.17124.1.A1_s_at NUDT14 2.32 2.19 0.95 4.35E 02 UP Bt.20281.2.S1_a_at PGM1 1.52 1.23 0. 81 4.37E 02 UP Bt.28278.1.S1_at ACE2 4.06 4.70 1.16 4.40E 02 UP Bt.4718.1.S1_at PCTP 2.46 1.50 0.61 4.42E 02 UP Bt.23955.1.A1_at PHOSPHO2 1.46 1.36 0.93 4.50E 02 UP Bt.6177.1.S1_at ACOT8 4.38 2.64 0.60 4.54E 02 UP Bt.26953.1.A1_at MRPL36 1.68 1.32 0.7 9 4.59E 02 UP Bt.3300.1.S1_at LOC511523 2.34 1.71 0.73 4.63E 02 UP Bt.9048.2.S1_a_at PSENEN 1.67 1.32 0.79 4.65E 02 UP Bt.21721.1.A1_at USP2 1.48 1.44 0.98 4.65E 02 UP Bt.18037.2.A1_at ASPDH 2.36 1.58 0.67 4.65E 02 UP Bt.15997.1.S1_at P2RX4 2.92 2.08 0.71 4.65E 02 UP Bt.6626.1.S1_at PPAP2A 1.51 1.16 0.77 4.76E 02 UP Bt.21680.2.S1_at PIR 2.19 1.40 0.64 4.76E 02 UP Bt.6171.1.A1_at HIBADH 1.44 1.58 1.10 4.82E 02 UP

PAGE 420

420 Appendix E. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FAT HLA Ave. Ex p. FAT LLA Adjusted P value Regu lation Bt.11078.2.S1_at AKR7A2 2.19 1.51 0.69 4.83E 02 UP Bt.2169.1.S1_at FUCA1 1.53 1.18 0.77 4.93E 02 UP Bt.11770.1.S1_at SLC25A20 1.92 1.39 0.72 4.93E 02 UP Bt.4126.1.A1_at CYP4A11 1.42 1.22 0.86 5.00E 02 UP Bt. 11739.1.S1_a_at STAP2 1.57 1.22 0.78 5.00E 02 UP Bt.15705.1.S1_at DSTN 1.67 1.16 0.69 5.00E 02 UP Bt.23706.1.A1_at --1.71 1.42 0.83 5.00E 02 UP Bt.6646.1.S1_at CTDSP1 2.22 1.25 0.56 5.00E 02 UP Bt.12300.2.S1_at MYH2 127.59 0.01 1.00 4.60E 15 DOWN Bt .4922.1.S1_at MYL1 83.33 0.01 1.00 5.47E 15 DOWN Bt.8435.1.S1_at ACTA1 85.98 0.01 1.04 4.43E 13 DOWN Bt.26998.1.A1_s_at TNNC1 20.79 0.05 1.00 5.73E 13 DOWN Bt.1905.1.S1_at MYL2 23.64 0.04 1.00 5.73E 13 DOWN Bt.12477.2.S1_at TPM2 13.18 0.08 1.01 3.13E 1 2 DOWN Bt.9992.1.S1_at TNNC2 15.59 0.06 1.01 3.13E 12 DOWN Bt.21767.1.S1_at TTN 17.03 0.06 0.99 3.13E 12 DOWN Bt.23696.1.A1_at LOC509457 9.01 0.11 1.00 3.88E 11 DOWN Bt.6012.1.S1_at TNNC1 7.31 0.14 1.00 7.87E 11 DOWN Bt.6620.1.S1_at MYH7 6.64 0.15 1.0 0 2.32E 10 DOWN Bt.20557.1.S1_at ACTN2 8.14 0.12 0.98 7.50E 09 DOWN Bt.11687.1.S1_a_at SRL 3.74 0.27 1.00 1.01E 08 DOWN Bt.27463.1.A1_at HERC6 3.39 0.32 1.10 7.12E 08 DOWN Bt.8143.1.S1_at MX2 5.96 0.17 1.04 7.12E 08 DOWN Bt.23735.1.A1_s_at --6.28 0. 34 2.11 2.63E 06 DOWN Bt.22199.1.S1_at DDIT4L 2.59 0.40 1.03 2.72E 06 DOWN Bt.22169.1.S1_at ENO3 3.54 0.24 0.86 3.04E 06 DOWN Bt.9779.1.S1_at ISG12(B) 10.37 0.11 1.13 4.71E 06 DOWN Bt.10310.1.S1_at MYBPC1 2.23 0.44 0.98 8.16E 06 DOWN Bt.6972.1.S1_at K BTBD10 2.59 0.41 1.07 8.16E 06 DOWN Bt.21767.1.S1_a_at TTN 42.06 0.03 1.30 9.64E 06 DOWN Bt.22065.1.S1_at LOC783920 2.35 0.41 0.97 5.52E 05 DOWN Bt.395.1.S1_at COX8B 1.88 0.53 1.00 7.81E 05 DOWN Bt.11199.1.S1_at MYOZ1 1.86 0.53 0.99 1.83E 04 DOWN Bt.1 7415.3.A1_at ERRFI1 1.86 1.02 1.89 2.46E 04 DOWN Bt.19284.1.A1_at --2.46 0.56 1.38 3.19E 04 DOWN Bt.17777.1.S1_at OPTN 2.69 0.50 1.34 3.21E 04 DOWN Bt.27891.1.S1_at LARS2 2.53 0.73 1.84 3.49E 04 DOWN Bt.16448.2.A1_at SFRS2IP 1.82 0.69 1.24 3.79E 04 D OWN Bt.11918.1.A1_at --2.62 0.59 1.54 3.79E 04 DOWN Bt.29581.1.A1_at --2.10 0.94 1.97 5.54E 04 DOWN Bt.20091.1.S1_at TCF20 2.10 0.76 1.60 5.93E 04 DOWN Bt.154.1.S1_at CCL8 2.56 0.43 1.10 6.21E 04 DOWN Bt.20427.2.S1_at UTP6 5.44 0.57 3.10 8.08E 04 DOWN Bt.12704.1.S1_at LOC514801 3.76 0.40 1.51 8.72E 04 DOWN Bt.19274.1.A1_at C1QTNF7 1.57 1.01 1.59 1.15E 03 DOWN Bt.28744.1.S1_at GBP4 10.23 0.22 2.27 1.33E 03 DOWN Bt.12285.3.S1_a_at NMI 2.14 0.48 1.03 1.48E 03 DOWN Bt.8920.1.S1_at --1.78 0.91 1. 62 1.68E 03 DOWN Bt.24361.1.S1_at ESF1 2.18 0.65 1.42 1.70E 03 DOWN Bt.12638.1.S1_at PML 2.33 0.47 1.09 1.86E 03 DOWN Bt.17229.1.A1_at ZNFX1 1.55 0.65 1.00 1.92E 03 DOWN Bt.28798.1.A1_at ANKRD22 1.97 0.53 1.04 1.96E 03 DOWN Bt.12760.1.S1_at INHBA 16.9 7 0.45 7.61 2.11E 03 DOWN

PAGE 421

421 Appendix E. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FAT HLA Ave. Exp. FAT LLA Adjusted P value Regu lation Bt.869.1.S1_at DPM1 1.70 0.72 1.23 2.31E 03 DOWN Bt.29823.1.S1_at BOLA 10.09 0.16 1.64 2.38E 03 DOWN Bt.26562.2.S1_at CCDC86 1.60 0.63 1.01 2.97E 03 DOWN Bt.27759.2.S1_at IDO1 2.63 0.49 1.30 2.97E 03 DOWN Bt.4937.1.S1_at LOC505941 2.05 0.47 0.96 3.33E 03 DOWN Bt.22116.1.A1_at IL18BP 3.98 0.28 1.13 3.33E 03 DOWN Bt.21798.1.S1_at GIMAP6 2.26 0.50 1.13 3.44E 03 DOWN Bt.18116.1.S1_at PARP12 2.27 0.64 1.44 4.24E 03 DOWN Bt.24492.1.S1_at STAT2 2.12 0.47 0.99 4.50E 03 DOWN Bt.21839.1.A1_at TOP1 1.67 0.68 1.13 4.60E 03 DOWN Bt.1817.1.S1_at ETV1 2.25 0.75 1.68 4.60E 03 DOWN Bt.22021.1.S1_at IFI16 5.2 8 0.37 1.94 4.60E 03 DOWN Bt.8436.1.S1_at IFI6 19.54 0.17 3.38 4.71E 03 DOWN Bt.29960.1.S1_at --2.24 0.55 1.23 4.74E 03 DOWN Bt.19792.1.A1_at --3.11 0.33 1.02 5.56E 03 DOWN Bt.27590.1.A1_at SMARCA4 2.15 0.55 1.19 5.89E 03 DOWN Bt.21981.3.S1_at ANT XR1 2.08 0.79 1.64 6.17E 03 DOWN Bt.27889.1.S1_at DLD 1.62 0.98 1.59 6.46E 03 DOWN Bt.11379.1.S1_at IFT52 2.06 0.66 1.37 6.69E 03 DOWN Bt.29823.1.S1_x_at BOLA 8.73 0.15 1.27 6.69E 03 DOWN Bt.22980.1.S1_at TRIM21 2.70 0.34 0.92 6.79E 03 DOWN Bt.13257.2 .A1_at LTV1 2.43 0.60 1.46 6.93E 03 DOWN Bt.18440.2.S1_at LOC510382 2.74 0.89 2.44 7.02E 03 DOWN Bt.14054.1.A1_at IFRD1 1.91 0.62 1.18 7.20E 03 DOWN Bt.12141.2.S1_a_at ZCCHC6 2.55 0.49 1.25 7.25E 03 DOWN Bt.9098.1.A1_at --1.55 0.65 1.01 7.40E 03 DOWN Bt.27143.1.A1_at ODF2L 1.70 0.80 1.36 7.56E 03 DOWN Bt.5197.1.S1_at G3BP1 1.80 0.67 1.21 7.64E 03 DOWN Bt.11475.1.A1_at PDLIM5 1.82 0.61 1.10 8.07E 03 DOWN Bt.15854.1.A1_at FUBP1 1.96 0.64 1.26 8.15E 03 DOWN Bt.28523.1.S1_at DTX3L 4.55 0.28 1.26 8.49 E 03 DOWN Bt.9391.2.S1_at BIRC3 1.58 0.87 1.37 8.57E 03 DOWN Bt.18873.1.A1_at --4.46 0.27 1.19 8.57E 03 DOWN Bt.8054.1.S1_at SYAP1 1.56 0.86 1.34 8.95E 03 DOWN Bt.24779.2.S1_at CREM 1.84 1.17 2.15 8.95E 03 DOWN Bt.22413.1.A1_at TLE4 1.71 0.79 1.36 1 .01E 02 DOWN Bt.12665.1.A1_at LOC531600 1.65 0.80 1.31 1.02E 02 DOWN Bt.14054.2.S1_at IFRD1 2.96 0.62 1.85 1.02E 02 DOWN Bt.11043.1.S1_a_at BCL2L12 1.45 0.71 1.03 1.05E 02 DOWN Bt.12300.1.S1_at MYH1 1.53 0.67 1.02 1.08E 02 DOWN Bt.29924.1.S1_at --1. 55 0.72 1.11 1.08E 02 DOWN Bt.24211.1.A1_at ASPN 1.92 0.68 1.31 1.20E 02 DOWN Bt.28764.1.A1_at LOC787057 2.07 0.79 1.64 1.20E 02 DOWN Bt.20416.1.S1_at TAP1 2.48 0.50 1.25 1.21E 02 DOWN Bt.17614.1.S1_at RBM25 3.07 0.60 1.84 1.21E 02 DOWN Bt.23941.1.A1_ at ZFP161 1.59 0.80 1.27 1.24E 02 DOWN Bt.26926.1.S1_at --1.51 1.01 1.53 1.27E 02 DOWN Bt.29432.1.A1_at PKHD1 3.05 0.47 1.44 1.28E 02 DOWN Bt.28577.1.S1_at SENP6 1.59 0.76 1.20 1.29E 02 DOWN Bt.15687.1.S1_at HERC4 1.83 0.80 1.46 1.33E 02 DOWN Bt.240 95.1.A1_at USP1 1.58 0.80 1.26 1.36E 02 DOWN Bt.2294.1.S1_a_at UBA7 6.61 0.20 1.34 1.44E 02 DOWN Bt.28139.1.S1_at LOC614107 3.54 0.32 1.12 1.50E 02 DOWN

PAGE 422

422 Appendix E. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FAT HLA Ave. Exp. FAT LLA Ad justed P value Regu lation Bt.10692.1.S1_at --1.82 0.75 1.36 1.50E 02 DOWN Bt.12663.1.S1_at KRT19 2.30 0.56 1.29 1.50E 02 DOWN Bt.20785.2.S1_at IFI44 5.37 0.28 1.49 1.50E 02 DOWN Bt.26364.1.A1_at BTBD8 1.74 0.80 1.40 1.54E 02 DOWN Bt.1927.1.S1_a t CRISPLD2 /// TIMM13 2.35 0.63 1.47 1.54E 02 DOWN Bt.27320.1.A1_at SGOL2 1.67 0.88 1.47 1.56E 02 DOWN Bt.13777.2.S1_at GIMAP7 4.90 0.91 4.44 1.56E 02 DOWN Bt.25471.1.S1_at ATXN3 2.33 0.74 1.73 1.56E 02 DOWN Bt.16739.1.A1_at --3.79 0.82 3.11 1.66E 02 DOWN Bt.7576.1.S1_at --1.45 0.75 1.09 1.69E 02 DOWN Bt.22683.1.S1_at RBM10 1.93 0.55 1.06 1.73E 02 DOWN Bt.25103.1.S1_at TDRD7 1.86 0.56 1.04 1.78E 02 DOWN Bt.20785.1.A1_at IFI44 5.49 0.26 1.44 1.78E 02 DOWN Bt.6636.1.S1_at --3.25 0.14 0.46 1.80E 02 DOWN Bt.26415.1.A1_at --1.57 0.84 1.31 1.91E 02 DOWN Bt.8053.1.S1_at ATAD1 1.57 0.68 1.07 1.99E 02 DOWN Bt.27589.1.A1_at DNAH12L /// LOC781795 1.71 0.71 1.22 1.99E 02 DOWN Bt.8206.1.S1_at SFRS7 1.52 0.82 1.25 2.00E 02 DOWN Bt.26408.1.A1_at SFRS2 IP 1.56 0.98 1.53 2.08E 02 DOWN Bt.2186.1.S1_at ZNFX1 4.23 0.23 0.99 2.08E 02 DOWN Bt.22064.2.S1_at RSRC2 1.76 0.78 1.38 2.13E 02 DOWN Bt.27830.1.A1_at SP140 2.21 0.61 1.34 2.26E 02 DOWN Bt.19620.1.A1_at IFI44 6.10 0.22 1.34 2.54E 02 DOWN Bt.27876.1.A 1_at ZCCHC10 1.43 0.60 0.86 2.75E 02 DOWN Bt.21565.1.S1_at IWS1 1.69 0.69 1.17 2.75E 02 DOWN Bt.4507.1.S1_at C4A 1.88 0.54 1.00 2.75E 02 DOWN Bt.22737.1.S1_at ERBB2IP 1.60 0.84 1.35 2.77E 02 DOWN Bt.16234.2.S1_at SFRS18 2.27 1.10 2.51 2.83E 02 DOWN Bt .9705.1.S1_at NKTR 1.52 0.86 1.31 3.00E 02 DOWN Bt.25832.1.S1_at --2.01 0.80 1.60 3.00E 02 DOWN Bt.26232.2.A1_at --2.11 0.49 1.04 3.03E 02 DOWN Bt.8997.1.S1_at RANGAP1 3.09 0.31 0.96 3.03E 02 DOWN Bt.6225.2.A1_at PRKD3 1.77 0.76 1.34 3.07E 02 DOWN Bt.19339.1.S1_at --1.54 0.98 1.51 3.13E 02 DOWN Bt.17777.3.S1_at OPTN 2.99 0.55 1.65 3.13E 02 DOWN Bt.15971.1.S1_at CCAR1 1.78 0.76 1.36 3.16E 02 DOWN Bt.11791.2.S1_at --1.42 0.91 1.29 3.17E 02 DOWN Bt.25196.1.A1_at --1.45 0.91 1.32 3.18E 02 DOW N Bt.21801.2.S1_at HNRNPL 1.50 0.79 1.19 3.18E 02 DOWN Bt.4079.2.S1_a_at TARDBP 1.41 0.90 1.27 3.19E 02 DOWN Bt.22869.1.S2_at FABP5 3.02 0.63 1.92 3.20E 02 DOWN Bt.13189.1.A1_at ORC4L 1.46 0.83 1.21 3.23E 02 DOWN Bt.17612.2.S1_at CFHR4 1.81 0.53 0.96 3.26E 02 DOWN Bt.6822.1.S1_at RNF150 1.82 0.72 1.30 3.29E 02 DOWN Bt.20270.1.S1_at MSL1 1.69 0.70 1.19 3.43E 02 DOWN Bt.24098.1.A1_at IFIH1 5.49 0.24 1.31 3.63E 02 DOWN Bt.8736.1.S1_at LOC520588 1.40 0.98 1.37 3.64E 02 DOWN Bt.28626.2.S1_at LOC521363 1.72 0.93 1.61 3.64E 02 DOWN Bt.1736.1.A1_at SOCS1 1.44 0.66 0.96 3.76E 02 DOWN Bt.5360.1.S1_a_at PAPOLA 1.89 0.81 1.52 3.85E 02 DOWN Bt.27403.1.S1_at LOC540987 1.49 0.95 1.41 3.98E 02 DOWN

PAGE 423

423 Appendix E. Continued Affimetrix ID Gene symbol Fold change Av Exp. FAT HLA Ave. Exp. FAT LLA Adjusted P value Regu lation Bt.17717.1.A1_at USPL1 2.04 0.75 1.52 3.98E 02 DOWN Bt.27071.1.S1_at TRIM38 1.90 0.58 1.09 3.99E 02 DOWN Bt.18116.2.A1_at PARP12 2.44 0.45 1.10 3.99E 02 DOWN Bt.16350.2.A1_s_at GBP5 1 .46 0.69 1.00 4.01E 02 DOWN Bt.2465.1.S1_at --2.41 0.55 1.32 4.01E 02 DOWN Bt.24940.1.A1_at --8.34 0.50 4.17 4.01E 02 DOWN Bt.24767.1.S1_at INTS3 1.67 0.87 1.45 4.05E 02 DOWN Bt.14464.1.A1_at GPHN 2.01 0.73 1.47 4.08E 02 DOWN Bt.24317.1.A1_at SOX6 1.64 0.86 1.40 4.14E 02 DOWN Bt.18045.1.S1_at MTPAP 1.76 0.80 1.41 4.18E 02 DOWN Bt.19107.1.S1_at --2.25 0.51 1.15 4.18E 02 DOWN Bt.27118.1.A1_at LOC510651 2.40 0.54 1.30 4.18E 02 DOWN Bt.17777.2.S1_at OPTN 2.71 0.62 1.68 4.18E 02 DOWN Bt.22283.1.S 1_at PLEKHA2 1.44 0.73 1.05 4.19E 02 DOWN Bt.4758.1.S1_at FABP3 1.86 0.59 1.10 4.19E 02 DOWN Bt.25537.1.A1_at UXS1 2.65 0.65 1.71 4.19E 02 DOWN Bt.22626.1.A1_at ANKRD12 1.92 0.70 1.34 4.26E 02 DOWN Bt.29194.1.S1_at PLIN4 1.65 0.97 1.60 4.30E 02 DOWN B t.5240.1.S1_at CTGF 2.54 0.65 1.66 4.30E 02 DOWN Bt.11259.1.S1_at ISG12(A) 7.05 0.25 1.73 4.30E 02 DOWN Bt.26804.1.S1_at LOC100847122 2.28 0.66 1.51 4.35E 02 DOWN Bt.17848.2.S1_at ZMYND8 1.91 0.77 1.48 4.37E 02 DOWN Bt.26892.1.S1_at NBN 1.73 0.69 1.20 4.46E 02 DOWN Bt.20110.1.S1_at PSMF1 2.16 0.42 0.90 4.59E 02 DOWN Bt.17432.1.S1_at ARL5B 1.42 0.84 1.20 4.60E 02 DOWN Bt.4898.1.S1_at BASP1 1.54 0.80 1.24 4.60E 02 DOWN Bt.7349.1.S1_at --2.05 0.68 1.41 4.60E 02 DOWN Bt.12854.1.S1_at --2.17 0.69 1. 50 4.63E 02 DOWN Bt.8323.1.S1_at DDX21 1.41 0.72 1.01 4.65E 02 DOWN Bt.22335.1.S1_a_at --1.42 0.84 1.19 4.65E 02 DOWN Bt.13489.1.S1_at ZMIZ1 1.67 0.73 1.22 4.65E 02 DOWN Bt.13777.1.S1_at GIMAP7 2.25 0.77 1.72 4.65E 02 DOWN Bt.18080.2.S1_at LOC787094 2.44 0.67 1.63 4.65E 02 DOWN Bt.6686.1.S1_at CASK 1.76 0.75 1.32 4.77E 02 DOWN Bt.25111.1.A1_at LOC508347 5.60 0.21 1.20 4.79E 02 DOWN Bt.23306.1.S1_at --1.57 0.82 1.29 4.90E 02 DOWN Bt.11237.1.S1_at YTHDC1 1.57 0.79 1.24 4.93E 02 DOWN Bt.25084.1.S 1_at --2.08 0.78 1.62 4.93E 02 DOWN

PAGE 424

424 APPENDIX F DIFFERENTIALY EXPRES SED FOR THE INTERACT ION FATTY ACID BY MI LK REPLACER List of differential expressed genes in liver of Holstein males at 30 d of age. Effect of feeding essential fatty acids prepartum a nd high linoleic acid in milk replacer (Interaction of contrasts FA by MR). Calves were fed a high or low linoleic acid milk replacer from 1 30 d of age and were born from dams fed diets supplemented with no fat (Control), saturated fatty acids (SFA), or essential fatty acids (EFA) starting at 8 wk before expected calving date. Affimetrix ID Gene symbol Fold change Av. Exp. FA by HLA Ave. Exp. FA by LLA Adjusted P value Regu lation Bt.23696.1.A1_at LOC509457 80.98 80.96 1.00 2.15E 13 UP Bt.26769 .1.S1_at GIMAP8 20.67 19.50 0.94 1.63E 08 UP Bt.17415.3.A1_at ERRFI1 3.64 1.03 0.28 3.19E 06 UP Bt.11918.1.A1_at --4.96 2.01 0.41 8.63E 05 UP Bt.9655.2.S1_at LOC790332 6.58 6.17 0.94 1.28E 04 UP Bt.27940.1.A1_at RHBG 5.58 2.04 0.37 1.47E 04 UP Bt.29 581.1.A1_at --2.83 0.99 0.35 4.21E 04 UP Bt.2858.1.S1_at ABHD6 2.71 1.14 0.42 1.01E 03 UP Bt.12910.1.S1_at OGDH 2.84 1.25 0.44 1.01E 03 UP Bt.17073.1.S1_at --3.46 2.03 0.58 1.37E 03 UP Bt.13381.1.S1_at CIDEC 1.91 1.92 1.00 1.46E 03 UP Bt.12508.1.S 1_at DCTPP1 3.14 1.70 0.54 1.46E 03 UP Bt.29194.1.S1_at PLIN4 3.33 1.25 0.37 1.46E 03 UP Bt.26926.1.S1_at --2.16 0.94 0.43 2.16E 03 UP Bt.3248.1.S1_at ALDH4A1 2.28 1.07 0.47 2.16E 03 UP Bt.27286.2.S1_at ECD 2.57 1.98 0.77 2.20E 03 UP Bt.11411.1.S1_a t CIAPIN1 2.81 1.32 0.47 2.30E 03 UP Bt.11270.2.S1_at VARS 3.28 1.31 0.40 2.82E 03 UP Bt.7413.1.S1_at GRN 1.90 1.19 0.63 3.14E 03 UP Bt.13376.1.S1_at DHRS1 3.15 1.56 0.49 5.54E 03 UP Bt.22533.1.S1_at ALDOA 2.80 1.75 0.62 5.69E 03 UP Bt.13641.1.S1_at G STZ1 1.85 1.14 0.62 5.86E 03 UP Bt.21467.1.S1_at COG4 2.27 1.44 0.63 6.34E 03 UP Bt.17537.1.A1_at SAA4 3.77 1.86 0.49 6.59E 03 UP Bt.6020.1.S1_at DNAJC11 2.15 0.97 0.45 8.76E 03 UP Bt.4643.1.S1_at LMAN2 1.76 1.21 0.69 8.81E 03 UP Bt.24793.1.S1_at MN1 1.97 1.98 1.00 8.81E 03 UP Bt.11256.1.S1_at CNOT1 2.18 0.99 0.45 8.81E 03 UP Bt.16525.1.A1_at --2.91 1.34 0.46 8.81E 03 UP Bt.1946.1.S1_at NSFL1C 1.89 1.11 0.58 9.06E 03 UP Bt.4141.1.S1_at COPE 1.95 1.25 0.64 9.19E 03 UP Bt.24662.1.S1_at AKT1S1 2.19 1.28 0.58 9.19E 03 UP Bt.26538.1.S1_at LOC509420 2.78 1.49 0.54 9.19E 03 UP Bt.12980.3.S1_a_at CL43 2.99 1.02 0.34 9.28E 03 UP Bt.21021.1.S1_at TBC1D7 2.28 1.48 0.65 9.99E 03 UP Bt.7237.2.S1_a_at HADHA 4.15 1.74 0.42 1.04E 02 UP Bt.10880.1.S1_at TIMM 50 1.83 1.15 0.63 1.06E 02 UP Bt.6556.1.S1_at LOC504773 3.18 1.37 0.43 1.10E 02 UP Bt.23735.1.A1_s_at --2.45 2.31 0.95 1.15E 02 UP Bt.28934.1.S1_at AREG 10.53 10.74 1.02 1.31E 02 UP Bt.5399.1.S2_at NADK 1.87 0.91 0.49 1.32E 02 UP Bt.4880.1.S1_at SLC 25A3 2.00 1.16 0.58 1.32E 02 UP

PAGE 425

425 Appendix F. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FA HLA Ave. Exp. FA LLA Adjusted P value Regu lation Bt.21216.1.S1_at CXorf56 2.15 1.26 0.59 1.32E 02 UP Bt.1983.1.S1_at EMR1 3.54 1.60 0.45 1.32 E 02 UP Bt.8775.1.S1_at AP1B1 1.62 1.05 0.65 1.32E 02 UP Bt.1753.1.S1_at ATP6V1E1 1.77 1.24 0.70 1.32E 02 UP Bt.805.1.S1_at ADIPOR2 2.68 1.46 0.54 1.32E 02 UP Bt.3736.1.A1_at PDE4DIP 1.92 1.18 0.62 1.34E 02 UP Bt.4937.1.S1_at LOC505941 2.03 0.98 0.48 1.36E 02 UP Bt.13588.2.S1_at PSAT1 4.59 2.01 0.44 1.64E 02 UP Bt.5096.1.S1_at CCT3 3.21 1.46 0.45 1.64E 02 UP Bt.25957.1.S1_at MAVS 2.64 1.35 0.51 1.69E 02 UP Bt.5083.1.S1_at SLC27A4 7.31 1.71 0.23 1.73E 02 UP Bt.8121.1.S1_x_at BOLA 2.58 1.42 0.55 1.8 4E 02 UP Bt.18914.1.S1_at --1.76 1.39 0.79 1.98E 02 UP Bt.13486.1.A1_at GLDC 2.36 1.08 0.46 2.06E 02 UP Bt.11279.1.A1_at CLCN4 2.58 1.55 0.60 2.06E 02 UP Bt.23366.1.S1_at CDIPT 2.68 1.42 0.53 2.07E 02 UP Bt.24007.1.A1_at SLC15A2 2.89 1.79 0.62 2.07E 02 UP Bt.23171.2.S1_at PCBD1 2.01 1.08 0.53 2.11E 02 UP Bt.1207.1.S1_at SLC16A13 2.64 1.57 0.59 2.14E 02 UP Bt.20997.1.S1_at C2H1orf144 3.65 1.18 0.32 2.18E 02 UP Bt.12030.2.S1_at ACTN4 2.08 1.23 0.59 2.26E 02 UP Bt.3023.1.S1_at NIT1 2.49 1.50 0.60 2 .26E 02 UP Bt.23169.1.S1_at SIRPA 2.89 1.49 0.51 2.26E 02 UP Bt.10387.1.S1_at ABCF1 2.91 1.42 0.49 2.26E 02 UP Bt.20265.1.A1_at ECD 1.85 1.54 0.83 2.28E 02 UP Bt.10361.1.S1_at --1.51 0.97 0.64 2.34E 02 UP Bt.20361.2.A1_at FBXL20 3.56 2.81 0.79 2.37E 02 UP Bt.8730.1.S1_at RAPGEF2 1.87 1.04 0.56 2.41E 02 UP Bt.5334.1.S1_at RPSA 1.73 1.16 0.67 2.53E 02 UP Bt.282.1.S1_at VDAC1P5 1.69 1.08 0.64 2.59E 02 UP Bt.28586.1.S1_at ERMP1 1.56 1.27 0.81 2.59E 02 UP Bt.1332.1.S1_a_at COX10 1.97 0.98 0.50 2.66E 02 UP Bt.26568.2.S1_a_at LOC531049 2.25 1.33 0.59 2.67E 02 UP Bt.13705.1.S1_at SSR2 2.18 1.22 0.56 2.70E 02 UP Bt.4902.1.S1_at CTSZ 1.90 1.52 0.80 2.78E 02 UP Bt.121.1.S1_at FRZB 3.29 2.58 0.78 2.78E 02 UP Bt.18847.1.A1_at --4.48 1.44 0.32 2.78E 02 UP Bt.8090.2.S1_at MYBBP1A 2.17 0.80 0.37 2.78E 02 UP Bt.19899.1.A1_at HGD 1.76 1.24 0.71 2.80E 02 UP Bt.653.1.S1_at NEK6 2.00 1.13 0.56 2.80E 02 UP Bt.8078.1.S1_at ARPC4 2.35 1.02 0.43 2.81E 02 UP Bt.20281.3.S1_a_at PGM1 2.19 1.42 0.65 2.89E 02 UP B t.27204.1.S1_at LPCAT3 6.25 1.42 0.23 2.99E 02 UP Bt.6460.1.S1_at PDIA6 1.97 1.43 0.72 3.04E 02 UP Bt.2580.1.S1_at GALM 2.05 1.58 0.77 3.04E 02 UP Bt.23164.1.S1_at UQCRC1 2.22 1.23 0.55 3.04E 02 UP Bt.1987.1.S1_at TAX1BP3 1.86 1.28 0.69 3.05E 02 UP Bt .5399.1.S1_at NADK 2.11 1.11 0.53 3.09E 02 UP Bt.12370.1.S1_at MLF2 3.83 1.41 0.37 3.27E 02 UP Bt.4475.1.S1_at NDUFS2 1.54 1.03 0.67 3.40E 02 UP Bt.5771.1.S1_at --1.96 0.98 0.50 3.40E 02 UP Bt.16137.1.S1_at ALDH9A1 2.74 1.35 0.49 3.40E 02 UP Bt.2459 7.1.S1_at GLG1 4.35 1.37 0.31 3.40E 02 UP

PAGE 426

426 Appendix F. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FA HLA Ave. Exp. FA LLA Adjusted P value Regu lation Bt.9632.2.S1_at DMBT1 2.71 1.37 0.50 3.54E 02 UP Bt.15334.2.A1_at STAT3 3.95 1.12 0.28 3.54E 02 UP Bt.14207.1.S1_at GCAT 2.26 1.02 0.45 3.57E 02 UP Bt.15886.1.S1_at ACSL5 2.38 1.10 0.46 3.59E 02 UP Bt.12957.1.A1_at TNRC6B 1.55 0.83 0.54 3.69E 02 UP Bt.4431.1.S1_a_at ATP5B 1.56 1.26 0.81 3.69E 02 UP Bt.12586.1.A1_at LOC508439 1.71 1 .21 0.71 4.01E 02 UP Bt.13633.1.A1_at --4.00 1.70 0.42 4.07E 02 UP Bt.20207.1.A1_at ALG12 1.70 1.14 0.67 4.09E 02 UP Bt.227.3.A1_x_at GSTA1 1.96 1.38 0.70 4.09E 02 UP Bt.4604.1.S1_a_at ACSM1 1.97 1.08 0.55 4.09E 02 UP Bt.20145.1.S1_at PRELID1 2.04 1 .22 0.60 4.09E 02 UP Bt.2113.1.S1_at CNDP2 2.30 1.26 0.55 4.09E 02 UP Bt.11167.1.S1_at GLRX5 2.54 1.59 0.63 4.09E 02 UP Bt.2110.1.S1_at DPP3 2.99 1.29 0.43 4.09E 02 UP Bt.3487.1.S1_at TPI1 3.11 1.55 0.50 4.09E 02 UP Bt.23902.1.A1_at --1.40 1.08 0.77 4.15E 02 UP Bt.20229.1.S1_at TBRG4 1.47 0.93 0.64 4.25E 02 UP Bt.20711.1.S1_at RDH16 1.68 1.12 0.67 4.26E 02 UP Bt.20322.3.S1_a_at WDR18 2.87 1.43 0.50 4.27E 02 UP Bt.19922.1.S1_at HPD 3.45 1.98 0.58 4.27E 02 UP Bt.7915.1.S1_at MDH2 1.63 1.12 0.69 4. 35E 02 UP Bt.1059.3.S1_a_at ATP2A2 2.86 1.22 0.43 4.48E 02 UP Bt.23179.1.S1_at HSP90AA1 2.94 1.46 0.50 4.48E 02 UP Bt.227.2.A1_at GSTA1 2.01 1.07 0.53 4.50E 02 UP Bt.22783.1.S1_at ENO1 3.58 1.42 0.40 4.56E 02 UP Bt.17219.1.A1_at MPDU1 1.78 1.08 0.60 4 .56E 02 UP Bt.15691.1.S1_at KCNK5 2.12 1.08 0.51 4.64E 02 UP Bt.18479.1.A1_at ZNF608 2.09 1.48 0.71 4.71E 02 UP Bt.5183.1.S1_at TUBA4A 3.32 1.30 0.39 4.71E 02 UP Bt.5196.1.S1_at WDR55 1.74 1.15 0.66 4.72E 02 UP Bt.3811.1.S1_at MRPS18B 2.04 1.35 0.66 4 .72E 02 UP Bt.23605.2.S1_at THRA 2.87 1.30 0.45 4.74E 02 UP Bt.1552.1.S1_at SARS 2.07 1.27 0.61 4.75E 02 UP Bt.13588.3.A1_at PSAT1 5.39 2.16 0.40 4.88E 02 UP Bt.22543.1.S1_at --1.49 0.79 0.53 4.90E 02 UP Bt.9298.1.S1_at AARSD1 2.56 1.37 0.54 4.90E 0 2 UP Bt.4404.1.A1_at PRSS2 32.28 0.03 1.08 2.07E 08 DOWN Bt.841.1.S1_at --2.90 0.74 2.16 1.43E 05 DOWN Bt.19274.1.A1_at C1QTNF7 2.51 1.01 2.53 2.87E 05 DOWN Bt.17034.1.A1_at --3.63 0.26 0.96 3.05E 05 DOWN Bt.21721.1.A1_at USP2 3.82 0.27 1.05 4.09E 05 DOWN Bt.26364.1.A1_at BTBD8 4.13 0.99 4.10 8.63E 05 DOWN Bt.27889.1.S1_at DLD 2.30 1.07 2.47 1.53E 04 DOWN Bt.18003.1.S1_at CUL3 3.33 0.73 2.44 2.73E 04 DOWN Bt.26650.1.S1_at --1.91 1.27 2.43 7.94E 04 DOWN Bt.10084.1.S1_at CASP3 1.93 0.83 1.60 1 .01E 03 DOWN Bt.3549.1.A1_at VAMP4 2.67 0.83 2.23 1.37E 03 DOWN Bt.29324.1.S1_at --3.37 0.64 2.15 1.46E 03 DOWN Bt.18440.2.S1_at LOC510382 5.20 0.79 4.11 1.46E 03 DOWN Bt.20977.3.S1_at CCPG1 2.11 0.97 2.05 1.51E 03 DOWN Bt.26308.2.A1_at RAD18 2.56 0 .82 2.09 1.51E 03 DOWN Bt.18026.1.A1_at ERBB2IP 1.94 0.78 1.51 1.78E 03 DOWN

PAGE 427

427 Appendix F. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FA HLA Ave. Exp. FA LLA Adjusted P value Regu lation Bt.6645.1.S1_at RNPC3 2.18 0.71 1.55 1.78E 03 D OWN Bt.21952.1.A1_at --1.99 0.81 1.62 2.16E 03 DOWN Bt.9974.1.S1_at CCL3 5.53 0.38 2.11 2.29E 03 DOWN Bt.16425.1.A1_at --2.09 0.63 1.32 2.38E 03 DOWN Bt.27320.1.A1_at SGOL2 2.46 0.80 1.98 2.71E 03 DOWN Bt.2962.1.S1_at --4.90 0.74 3.62 3.80E 03 D OWN Bt.24892.1.A1_at RIT1 2.14 1.02 2.19 4.63E 03 DOWN Bt.8169.1.S1_at SLC39A6 1.86 1.00 1.86 4.68E 03 DOWN Bt.24249.1.S1_at SUV420H1 2.34 0.68 1.59 4.68E 03 DOWN Bt.22483.1.S1_at SEC31B 1.68 0.71 1.20 5.50E 03 DOWN Bt.9391.2.S1_at BIRC3 1.97 0.75 1.4 8 5.50E 03 DOWN Bt.12290.1.S1_at PSIP1 2.71 0.79 2.14 5.50E 03 DOWN Bt.29879.1.S1_at KAT2B 3.07 0.79 2.41 5.69E 03 DOWN Bt.29587.1.S1_at WAC 1.95 0.78 1.52 5.78E 03 DOWN Bt.17352.1.A1_at LOC785119 1.89 0.80 1.50 5.86E 03 DOWN Bt.20677.1.S1_at NSL1 2.4 4 1.04 2.53 5.86E 03 DOWN Bt.13743.1.A1_at RFK 2.42 1.01 2.45 6.09E 03 DOWN Bt.16789.1.A1_at C5H12orf11 1.98 0.89 1.77 6.33E 03 DOWN Bt.19575.1.S1_at HSPA14 1.73 0.66 1.15 6.59E 03 DOWN Bt.2424.1.S1_at DPYD 1.81 0.87 1.58 6.59E 03 DOWN Bt.17364.1.A1_a t --8.43 0.46 3.88 6.59E 03 DOWN Bt.26410.1.A1_at MTERF 2.26 0.82 1.85 7.53E 03 DOWN Bt.6802.1.S1_at RGS5 2.77 2.08 5.76 7.53E 03 DOWN Bt.20206.1.A1_at ATP11B 2.09 0.80 1.67 7.64E 03 DOWN Bt.9140.1.S1_at GMNN 1.96 1.17 2.31 7.64E 03 DOWN Bt.5692.1.S 1_at LOC100425208 2.59 0.63 1.62 7.87E 03 DOWN Bt.15299.1.A1_at --1.87 0.99 1.84 8.03E 03 DOWN Bt.24203.1.S1_at ANGPTL3 1.66 0.93 1.55 8.07E 03 DOWN Bt.22150.1.A1_at LZTFL1 1.77 0.97 1.72 8.39E 03 DOWN Bt.21957.1.S1_at --2.54 0.69 1.75 8.39E 03 DOW N Bt.29107.1.S1_at --2.11 0.83 1.76 8.76E 03 DOWN Bt.6275.1.S1_at TGFBR1 2.89 0.74 2.14 8.76E 03 DOWN Bt.22044.1.S1_at --3.33 0.85 2.84 8.76E 03 DOWN Bt.6397.2.S1_at HMGB2 2.22 1.14 2.54 8.81E 03 DOWN Bt.5129.1.S1_a_at NNAT 6.26 0.67 4.18 8.81E 03 DOWN Bt.25471.1.S1_at ATXN3 3.42 0.75 2.57 9.19E 03 DOWN Bt.18792.1.S1_at DCTN6 3.81 1.07 4.09 9.19E 03 DOWN Bt.16672.1.A1_at LOC698727 9.04 0.51 4.65 9.19E 03 DOWN Bt.24506.2.A1_at CHIC2 1.79 1.04 1.86 9.28E 03 DOWN Bt.19906.1.A1_at --4.16 0.64 2. 67 9.28E 03 DOWN Bt.22524.2.A1_at BBS5 1.89 0.89 1.69 9.62E 03 DOWN Bt.19339.1.S1_at --1.96 0.70 1.38 1.00E 02 DOWN Bt.28187.1.S1_at WEE1 2.37 0.67 1.58 1.04E 02 DOWN Bt.20758.1.S1_at LOC541014 1.96 0.76 1.49 1.04E 02 DOWN Bt.5542.2.S1_at NAP1L1 2.0 2 0.89 1.79 1.15E 02 DOWN Bt.7327.2.S1_a_at MGC133692 1.84 0.91 1.68 1.28E 02 DOWN Bt.16580.1.S1_at CD2AP 3.54 0.60 2.11 1.32E 02 DOWN Bt.26318.1.S1_a_at FAIM 4.91 0.62 3.05 1.32E 02 DOWN Bt.11751.1.A1_at KLHL23 2.78 0.94 2.61 1.32E 02 DOWN Bt.17883.2 .A1_at --3.96 0.79 3.12 1.34E 02 DOWN Bt.22730.1.S1_at FGFR1OP2 1.72 0.79 1.36 1.35E 02 DOWN Bt.26416.1.A1_at --4.93 0.72 3.56 1.56E 02 DOWN Bt.24361.1.S1_at ESF1 1.92 0.88 1.69 1.58E 02 DOWN

PAGE 428

428 Appendix F. Continued Affimetrix ID Gene symbol Fold cha nge Av. Exp. FA HLA Ave. Exp. FA LLA Adjusted P value Regu lation Bt.13768.1.S1_at DYNLT3 1.88 0.90 1.69 1.64E 02 DOWN Bt.5635.1.S1_at TCEAL1 2.36 0.75 1.77 1.64E 02 DOWN Bt.18220.1.A1_at CCDC112 3.74 0.74 2.77 1.64E 02 DOWN Bt.17653.1.A1_at UPP 2 4.15 1.18 4.91 1.64E 02 DOWN Bt.26408.1.A1_at SFRS2IP 1.77 0.83 1.47 1.81E 02 DOWN Bt.28101.1.S1_at --1.57 0.97 1.53 1.82E 02 DOWN Bt.6289.1.S1_at SPTLC1 1.89 0.89 1.67 1.82E 02 DOWN Bt.25471.2.A1_at ATXN3 2.50 0.85 2.13 1.82E 02 DOWN Bt.23178.1.S 2_at DCN 1.70 1.01 1.73 1.84E 02 DOWN Bt.11445.1.A1_at BCL10 2.68 0.85 2.27 1.84E 02 DOWN Bt.842.1.A1_at TOR1AIP1 2.05 0.79 1.62 1.98E 02 DOWN Bt.13981.1.S1_at TM2D2 2.99 0.59 1.75 2.01E 02 DOWN Bt.9774.1.S1_a_at MGC165862 3.10 1.00 3.10 2.05E 02 DOWN Bt.835.1.A1_at SNTB1 2.13 0.80 1.70 2.06E 02 DOWN Bt.22421.1.A1_at LOC530325 3.01 0.80 2.41 2.07E 02 DOWN Bt.4405.1.S1_s_at CCDC104 1.51 0.97 1.47 2.11E 02 DOWN Bt.27403.1.S1_at LOC540987 1.84 0.78 1.43 2.14E 02 DOWN Bt.27099.1.A1_at SEC62 2.30 0.81 1 .86 2.25E 02 DOWN Bt.2859.1.A1_at LOC540253 3.32 0.84 2.78 2.26E 02 DOWN Bt.19723.1.A1_at ACTR10 2.03 0.93 1.90 2.35E 02 DOWN Bt.19839.1.A1_at Ppig 1.72 0.91 1.56 2.35E 02 DOWN Bt.26150.1.A1_at L2HGDH 1.61 1.34 2.16 2.37E 02 DOWN Bt.6180.1.S1_at FRG1 2.15 0.96 2.05 2.37E 02 DOWN Bt.22656.2.S1_at --2.78 0.70 1.95 2.41E 02 DOWN Bt.367.1.S1_at OLR1 6.07 0.22 1.35 2.55E 02 DOWN Bt.18577.2.A1_at LOC472962 2.54 0.85 2.15 2.59E 02 DOWN Bt.3599.1.S1_at NPM1 1.52 0.93 1.40 2.59E 02 DOWN Bt.8054.1.S1_at S YAP1 1.59 1.04 1.66 2.59E 02 DOWN Bt.23900.1.A1_at --1.86 0.96 1.79 2.59E 02 DOWN Bt.26992.1.A1_at ADAM10 1.89 0.87 1.65 2.59E 02 DOWN Bt.17846.1.A1_at --3.56 0.73 2.59 2.59E 02 DOWN Bt.15685.1.A1_at MOSC2 1.57 0.87 1.36 2.66E 02 DOWN Bt.19212.1.S 1_at KLHL9 1.53 0.95 1.46 2.70E 02 DOWN Bt.29175.1.A1_at ZUFSP 1.74 0.73 1.27 2.70E 02 DOWN Bt.27187.1.S1_at MPHOSPH10 2.97 0.61 1.80 2.77E 02 DOWN Bt.13815.1.S1_at --1.72 0.87 1.50 2.78E 02 DOWN Bt.21268.1.S2_at RPS6KB1 1.81 0.80 1.45 2.78E 02 DOWN Bt.25832.1.S1_at --2.40 0.79 1.91 2.80E 02 DOWN Bt.8905.1.S1_at ITCH 2.02 0.70 1.41 2.85E 02 DOWN Bt.6341.1.S1_at DNAJC1 2.15 0.53 1.15 2.85E 02 DOWN Bt.22563.1.A1_s_at CSDE1 1.47 0.93 1.37 2.89E 02 DOWN Bt.9974.1.S1_a_at CCL3 2.89 0.48 1.39 2.90E 0 2 DOWN Bt.11233.1.S1_at LOC787143 /// TOP2B 1.47 0.87 1.27 2.92E 02 DOWN Bt.2048.1.S1_at AGPS 1.59 0.96 1.52 2.96E 02 DOWN Bt.812.1.S1_at --2.09 0.91 1.89 2.96E 02 DOWN Bt.19218.2.S1_at CNOT6 1.73 0.90 1.56 2.99E 02 DOWN Bt.21099.1.A1_at BRMS1L 1.75 1.02 1.78 2.99E 02 DOWN Bt.26828.1.S1_at CNTLN 5.22 0.62 3.21 2.99E 02 DOWN Bt.6899.1.S1_at LOC784769 1.77 0.80 1.41 3.01E 02 DOWN Bt.14124.2.S1_at USP33 3.13 0.75 2.33 3.01E 02 DOWN Bt.9069.1.S1_at ANKRD10 1.51 0.72 1.08 3.04E 02 DOWN Bt.24095.1.A1_ at USP1 1.71 0.96 1.65 3.04E 02 DOWN

PAGE 429

429 Appendix F. Continued Affimetrix ID Gene symbol Fold change Av. Exp. FA HLA Ave. Exp. FA LLA Adjusted P value Regu lation Bt.20932.1.S1_at NSA2 2.21 0.74 1.64 3.04E 02 DOWN Bt.19339.3.A1_at SOCS6 2.73 0.81 2.2 1 3.04E 02 DOWN Bt.16614.1.A1_s_at SYNCRIP 2.31 0.54 1.24 3.05E 02 DOWN Bt.18023.1.S1_at ZNF322 1.77 0.79 1.40 3.09E 02 DOWN Bt.19519.1.S1_at HLTF 1.86 1.03 1.91 3.09E 02 DOWN Bt.12664.2.S1_at ZMYM5 3.26 0.64 2.09 3.10E 02 DOWN Bt.19575.2.S1_at HSPA14 2.40 0.68 1.63 3.28E 02 DOWN Bt.14059.1.A1_at AUH 1.60 1.05 1.68 3.31E 02 DOWN Bt.22350.1.A1_at GMCL1 2.11 0.99 2.08 3.36E 02 DOWN Bt.17517.1.S1_at MGC134574 1.90 1.02 1.94 3.40E 02 DOWN Bt.14075.1.S1_at ARHGAP5 1.99 0.92 1.83 3.40E 02 DOWN Bt.9527.2 .S1_at KLF10 2.95 0.87 2.57 3.53E 02 DOWN Bt.27042.1.S1_at CENPC1 2.52 0.81 2.05 3.55E 02 DOWN Bt.27322.1.S1_at AP1AR 2.61 0.84 2.18 3.55E 02 DOWN Bt.1738.1.S1_at HIBCH 1.64 0.95 1.56 3.57E 02 DOWN Bt.23998.1.A1_a_at CUX2 4.19 0.59 2.49 3.59E 02 DOWN Bt.14129.1.S1_at LACTB2 1.64 0.99 1.62 3.65E 02 DOWN Bt.23960.1.S1_at CA5B 2.52 0.77 1.94 3.72E 02 DOWN Bt.3678.1.S1_at MKI67IP 1.65 0.76 1.25 3.83E 02 DOWN Bt.6993.2.A1_a_at NME7 1.92 0.99 1.90 3.83E 02 DOWN Bt.14283.1.A1_at --2.16 0.98 2.13 3.83E 0 2 DOWN Bt.8039.2.S1_a_at --2.48 0.83 2.06 3.83E 02 DOWN Bt.15306.1.A1_at PHF3 1.62 0.79 1.28 3.98E 02 DOWN Bt.28207.1.S1_at RNF19A 2.43 0.79 1.92 3.98E 02 DOWN Bt.15872.1.S1_at SLU7 2.52 1.53 3.86 3.98E 02 DOWN Bt.22064.2.S1_at RSRC2 1.84 0.72 1.32 4.00E 02 DOWN Bt.22976.1.S1_at SMC4 2.86 0.91 2.60 4.00E 02 DOWN Bt.28577.1.S1_at SENP6 1.54 0.96 1.48 4.01E 02 DOWN Bt.13556.1.S1_at CFH 2.98 0.52 1.55 4.05E 02 DOWN Bt.13332.1.S1_at SLC25A46 1.67 0.74 1.23 4.07E 02 DOWN Bt.16052.2.A1_at TSPYL1 1.94 0.84 1.63 4.07E 02 DOWN Bt.15706.1.A1_at --2.34 0.85 2.00 4.07E 02 DOWN Bt.27143.1.A1_at ODF2L 1.65 1.00 1.65 4.09E 02 DOWN Bt.21869.1.S1_at LOC537017 2.31 0.84 1.93 4.09E 02 DOWN Bt.18928.1.A1_at EIF4E3 2.43 1.09 2.65 4.09E 02 DOWN Bt.2 9506.1.S1_at CCDC82 3.21 0.66 2.13 4.09E 02 DOWN Bt.24749.1.S1_at LOC100430496 3.21 0.63 2.04 4.09E 02 DOWN Bt.23992.1.A1_at --4.78 0.39 1.87 4.09E 02 DOWN Bt.19232.1.A1_at --5.35 1.06 5.68 4.11E 02 DOWN Bt.13989.1.A1_at CAV2 1.99 1.00 1.99 4.15E 0 2 DOWN Bt.19994.1.S1_at LOC789597 1.94 0.87 1.69 4.25E 02 DOWN Bt.25190.1.A1_at --2.16 0.90 1.93 4.26E 02 DOWN Bt.18440.3.A1_at LOC510382 16.56 0.62 10.24 4.27E 02 DOWN Bt.24205.1.A1_at FGB 2.05 0.85 1.73 4.30E 02 DOWN Bt.20934.1.S1_at LOC100137763 4.04 0.94 3.80 4.32E 02 DOWN Bt.28945.1.A1_at LOC100440461 1.98 0.80 1.59 4.48E 02 DOWN Bt.20666.1.S1_at --2.98 0.72 2.15 4.48E 02 DOWN Bt.16828.1.A1_at --1.85 0.68 1.26 4.56E 02 DOWN Bt.13336.1.A1_at SMC4 2.82 0.86 2.42 4.56E 02 DOWN

PAGE 430

430 Appendix F Continued Affimetrix ID Gene symbol Fold change Av. Exp. FA HLA Ave. Exp. FA LLA Adjusted P value Regu lation Bt.5916.1.S1_at PGCP 2.01 0.74 1.48 4.61E 02 DOWN Bt.22676.1.A1_at GPN3 1.86 0.93 1.73 4.75E 02 DOWN Bt.16000.1.S1_at ENTPD4 2.02 0. 56 1.13 4.75E 02 DOWN Bt.444.1.S1_at PDE6C 8.50 0.57 4.81 4.75E 02 DOWN Bt.2765.1.S1_at --1.98 0.85 1.67 4.75E 02 DOWN Bt.5188.1.S1_at ABTB1 2.65 0.49 1.31 4.75E 02 DOWN Bt.22672.1.A1_at HPGD 3.34 0.93 3.11 4.75E 02 DOWN Bt.2899.1.S2_at FOS 4.65 0.3 4 1.57 4.75E 02 DOWN Bt.16276.1.A1_at ARSK 2.45 0.94 2.29 4.88E 02 DOWN Bt.22069.1.A1_at CCPG1 1.89 0.89 1.67 4.90E 02 DOWN Bt.2190.1.S1_at FUBP3 2.06 0.84 1.73 4.90E 02 DOWN Bt.8039.1.S1_at TMEM170A 2.22 0.72 1.61 4.91E 02 DOWN

PAGE 431

431 LIST OF REFERENCES Al, M. D. M., A. C. van Houwelingen, and G. Hornstra. 1997. Relation between birth order and the maternal and neonatal docosahexaenoic acid status. Eur. J. Clin. Nutr. 51:548 553. Allen, M. S. 2000. Effects of diet on short term regulation of feed intake by lactating dairy cattle. J. Dairy Sci. 83:1598 1624. Allen, M. S., and B. J.Bradford. 2012. Control of food intake by metabolism of fuels: a comparison across species. Proc. Nutr. Soc.71:401 409. Andersen M. H., D. Schrama, P. thor Straten, and J. C. Bec ker. 2006. Cytotoxic T cells. J Invest Dermatol. 126: 32 41. Antar, M. A., M. A. Ohlson, and M. O. Osborn. 1967. Fatty acid composition of different serum phospholipids in men and women. Eiochem. J. 105:117 119. Anthony, R. V., R. A. Bellows, R. E. Short, R. B. Staigmiller, C. C. Kaltenbach, and T. G. Dunn. 1986. Fetal growth of beef calves. I. Effect of prepartum dietary crude protein on birth weight, blood metabolites and steroid hormone concentrations. J. Anim. Sci. 62:1363 1374. Araujo, D. B., R. F. Coo ke, G. R. Hansen, C. R. Staples, and J. D. Arthington. 2010. Effects of rumen protected polyunsaturated fatty acid supplementation on performance and physiological responses of growing cattle following transportation and feedlot entry. J. Anim. Sci. 88:412 0 4132. Arens, R., K. Tesselaar, P. A. Baars, G. M. van Schijndel, J. Hendriks, S. T. Pals, P. Krimpenfort, J. Borst, M. H. van Oers, and R. A. van Lier. 2001. Constitutive CD27/CD70 interaction induces expansion of effector type T cells and results in IFN gamma mediated B cell depletion. Immunity 15:801 812. Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, and J. T. Eppig. 2000. Gene ontology: tool for the unification of biology. Nat. Gene t. 25:25 29. Association of Official Analytical Chemists. 2000. Official Methods of Analysis. 17th Ed. AOAC, Gaithersburg, MD, USA. Avila, C. D., E. J. DePeters, H. Perez Monti, S. J. Taylor, and R. A. Zinn. 2000. Influences of saturation ratio of suppleme ntal dietary fat on digestion and milk yield in dairy cows. J. Dairy Sci. 83:1505 1519. Badinga, L., R. J. Collier, W. W. Thatcher, C. J. Wilcox, H. H. Head, and F. W. Bazer. 1991. Ontogeny of hepatic bovine growth hormone receptors in cattle. J. Anim Sci. 69:1925 1934.

PAGE 432

432 Ballou, M. A., and E. J. DePeters. 2008. Supplementing milk replacer with omega 3 fatty acids from fish oil on immunocompetence and health of Jersey calves. J. Dairy Sci. 91:3488 3500. Ballou, M. A., R. C. Gomes, S. O. Juchem, and E. J. DeP eters. 2009. Effects of dietary supplemental fish oil during the peripartum period on blood metabolites and hepatic fatty acid compositions and total triacylglycerol concentrations of multiparous Holstein cows. J. Dairy Sci. 92:657 669. Banchero, G. E., G. Quintans, G. B. Martin, D. R. Lindsay, and J. T. Milton. 2004. Nutrition and colostrum production in sheep.1. Metabolic and hormonal responses to high energy supplement in the final stages of pregnancy. Reprod. Fertil. Dev. 16:633 643. Bancroft, T.A., 196 8. Topics in Intermediate Statistical Methods. Iowa State University Press, Ames, IA, USA. Banta, J. P., D. L. Lalman, F. N. Owens, C. R. Krehbiel, and R. P. Wettemann. 2006. Effects of interval feeding whole sunflower seeds during mid to late gestation on performance of beef cows and their progeny. J. Anim. Sci. 84:2410 2417. Banta, J. P., D. L. Lalman, F. N. Owens, C. R. Krehbiel, and R. P. Wettemann. 2011. Effects of prepartum supplementation of linoleic and mid oleic sunflower seed on cow performance, c ow reproduction, and calf performance from birth through slaughter, and effects on intake and digestion in steers. J. Anim. Sci. 89:3718 3727. Barker, D. 1997. Maternal nutrition, fetal nutrition, and disease in later life. Nutrition 13:807 813. Barker, D. J. P., C. N. Martyn, C. Osmond, C. N. Hales, and C. H. D. Fall. 1993. Growth in uterus and serum cholesterol concentrations in adult life. BMJ. 307:1524 1527. Barone, J., J. R. Hebert, and M. M. Reddy. 1989. Dietary fat and natural killer cell activity. A m. J. Clin. Nutr. 50:861 867. Bascom, S., R. E. James, M. L. McGilliard, and M. E. Van Amburgh. 2007. Influence of dietary fat and protein on body composition of Jersey bull calves. J. Dairy Sci. 90:5600 5609. Bauchart, D. 1993. Lipid absorption and transp ort in ruminants. J. Dairy Sci.76:3864 3881. Beam, A. L., J. E. Lombard, C. A. Kopral, L. P. Garber, A. L. Winter, J. A. Hicks, and J. L. Schlater. 2009. Prevalence of failure of passive transfer of immunity in newborn heifer calves and associated manageme nt practices on US dairy operations. J. Dairy Sci. 92:3973 3980.

PAGE 433

433 Bengoechea Alonso, M. T., and J. Ericsson. 2007. SREBP in signal transduction: cholesterol metabolism and beyond. Curr. Opin. Cell Biol. 19:215 222. Berger, A., D. M. Mutch, J. B. German, and M. A. Roberts. 2002. Dietary effects of arachidonate rich fungal oil and fish oil on murine hepatic and hippocampal gene expression. Lipids Health Dis. 1:2 23. Bernhagen, J., M. Bacher, T. Calandra, C. N. Metz, S. B. Doty, T. Donelly, and R. Bucala. 1996. An essential role for macrophage migration inhibitory factor in the tuberculin delayed type hypersensitivity reaction. J. Exp. Med. 183:277 282. Berr, F., A. Goetz, E. Schreiber, and G. Paumgartner. 1993. Effect of dietary n 3 versus n 6 polyunsaturated f atty acids on hepatic excretion of cholesterol in the hamster. J. Lipid Res. 34:1275 1284. Bieri, J.G., and E.L. Prival. 1966. Linoleic acid requirement of the chick. J. Nutrition 90:428 432. Bionaz, M., B. J. Thering, and J. J. Loor. 2012. Fine metabolic regulation in ruminants via nutrient/gene interactions: Saturated long chain fatty acids increase expression of genes involved in lipid metabolism and immune response partly 191. Bjermo, H., D. Iggman, J. Kull berg, I. Dahlman, L. Johansson, L. Perrson, J. Berglun, K. Pulkki, S. Basu, M. Uusitupa, M. Rudling P. Arner, T. Cedorholm, H. Ahlstrom, and U. Reserus. 2012. Effects of n 6 PUFAs compared with SFAs on liver fat, lipoproteins, and inflammation in abdominal obesity: a randomized controlled trial. Am J Clin Nutr. 95: 1 10. Black, A.C., 1999. Delayed type hypersensitivity: current theories with an historic perspective. Dermatol. Online J. 5:7 27. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipi d extraction and purification. Can. J. Biochem. Physiol. 37:911 917. Blum, J. W., and C. R. Baumrucker. 2008. Insulin like growth factors (IGFs), IGF binding proteins, and other endocrine factors in milk: role in the newborn. Pages 397 422 In: Bsze Z, edi tor. Bioactive components of milk. Advances in experimental medicine and biology, vol. 606. Springer. New York, USA. Bobe, G., J. W. Young, and D. C. Beitz. 2004. Pathology, etiology, prevention, and treatment of fatty liver in dairy cows. J. Dairy Sci. 8 7:3105 3124. Bordoni, A., M. Di Nunzio, F. Danesi, and P. L. Biagi. 2006. Polyunsaturated fatty acids: from diet to binding to PPARs and other nuclear receptors. Genes Nutr. 1:95 106.

PAGE 434

434 Bottger, J. D., B. W. Hess, B. M. Alexander, D. L. Hixon, L. F. Woodard, R. N. Funston, D. M. Hallford, and G. E. Moss. 2002. Effects of supplementation with high linoleic or oleic cracked safflower seeds on postpartum reproduction and calf performance of primiparous beef heifers. J. Anim. Sci. 80:2023 2030. Bourre, J. M., M. Piciotti, O. Dumont, G. Pascal, and G. Durand. 1990. Dietary linoleic acid and polyunsaturated fatty acids in rat brain and other organs. Minimal requirements of linoleic acid. Lipids 25: 465 472. Bouwens, M., O. van de Rest, N. Dellschaft, M. G. Bromhaar, L.C. de Groot, J. M. Geleijnse, M. Muller, and L. A. Afman. 2009. Fish oil supplementation induces antiinflammatory gene expression profiles in human blood mononuclear cells. Am J Clin. Nutr. 90:415 424. Bradford, B. J., K. J. Harvatine, and M. S. Allen. 2008. Dietary unsaturated fatty acids increase plasma glucagon like peptide 1 and cholecystokinin and may decrease premeal ghrelin in lactating dairy cows. J. Dairy Sci. 91:1443 1450. Brun Hansen, H. C., A. H. Kampen, and A. Lund. 2006. Hematological value s in fifteen calves during the first six months of life. Vet. Clin. Pathol. 35:182 187. Bueno, A. A., L. M. Oyama, C. S. M. Motoyama, C. R. S. Biz, V. L. Silveira, E. B. Ribeiro, and C. M. O. do Nascimento. 2010. Long chain saturated fatty acids increase h aptoglobin gene expression in C57BL/6J mice adipose tissue and 3T3 L1 cells. Eur. J. Nutr. 49:235 241. Buhler, C., H. Hammon, G. L. Rossi, and J. W. Blum. 1998. Small intestinal morphology in eight day old calves fed colostrum for different durations or on ly milk replacer and treated with long R3 insulin like growth factor I and growth hormone. J. Anim.Sci. 76:758 765. Burdge, G. C., and P. C. Calder. 2005. Conver linolenic acid to longer chain polyunsaturated fatty acids in human adults. Reprod. Nutr. Dev. 45:581 597. Burr, G. O., and M. M. Burr. 1929. A new deficiency disease produced by the rigid exclusion of fat from the diet. J. Biol. Chem. 82:345 36 7. Burr, G. O., and M. M. Burr. 1930. On the the nature and role of the fatty acids essential in nutrition. J. Biol. Chem. 86:587 621. Burr, G.O., M.M. Burr and E.S. Miller. 1932. On the fatty acids essential in nutrition. III. J. Biol. Chem. 97:1 9. Calda ri Torres, C. 2009. Effects of long chain fatty acids on production, metabolism and immunity of Holstein cows. PhD. Thesis. University of Florida, Gainesville.

PAGE 435

435 Caldari Torres, C., A. L. Lock, C .R. Staples, and L. Badinga. 2011. Performance, metabolic, and endocrine responses of periparturient Holstein cows fed 3 sources of fat. J Dairy Sci. 94:1500 1510. Calder, P. C. 2006. Polyunsaturated fatty acids and inflammation. Prostaglandins Leukot. Essent. Fatty Acids 75:197 202. Calder, P. C. 2008. The relations hip between the fatty acid composition of immune cells and their function. Prostaglandins Leukot Essent Fatty Acids. 79:101 8. Calder, P. C. 2012. Mechanisms of action of (n 3) fatty acids. J. Nutr. 142:592S 599S. Calder, P. C., and R. J. Deckelbaum. 2011. Harmful, harmless or helpful? The n 6 fatty acid debate goes on. Curr Opin Clin Nutr Metab Care. 14:113 124. Calder, P. C., J. A. Bond, D. J. Harvey, S. Gordon, and E. A. Newsholme. 1990. Uptake and incorporation of saturated and unsaturated fatty acids i nto macrophage lipids and their effect upon macrophage adhesion and phagocytosis. Biochem. J. 269:807 814. Calloway, C. D., J. W. Tyler, R. K. Tessman, and D. Hostetler. 2002. Comparison of refractometers and test endpoints in the measurement of serum prot ein concentration to assess passive transfer status in calves. J. Am. Vet. Med. Assoc. 221:1605 1608. Camandola, S., G. Leonarduzzi, T. Musso, L. Varesio, and R. Carini. 1996. Nuclear id. Biochem. Biophys. Res. Commun. 229:643 647. Campbell, F. M., M. J. Gordon, and A. K. Dutta Roy. 1994. Plasma membrane fatty acid binding protein (FABPpm) from the sheep placenta. Biochim. Biophys. Acta 1214:187 192. Campbell, J. M., L. E. Russell, J. D Crenshaw, E. M. Weaver, S. Godden, J. D. Quigley, J. Coverdale, and H. Tyler. 2007. Impact of irradiation and immunoglobulin G concentration on absorption of protein and immunoglobulin G in calves fed colostrums replacer. J. Dairy Sci. 90:5726 5731. Capp er, J. L., R. G. Wilkinson, A. M. Mackenzie, and L. A. Sinclair. 2006. polyunsaturated fatty acid supplementation during pregnancy alters neonatal behavior in sheep. J. Nutr. 136:397 403. Carbone, A., P. J. Psaltis, A. J. Nelson, R. Metcalf, J. D. Richards on, M. Weightman, A. Thomas, J. W. Finnie, G. D. Young, and S. G. Worthley. 2012. Dietary Omega 3 supplementation exacerbates left ventricular dysfunction in an ovine model of anthracycline induced cardiotoxicity. J. Cardiac. Fail.18:502 511.

PAGE 436

436 Caroprese, M ., A. Marzano, G. Entrican, S. Wattegedera, M. Albenzio, and A. Sevi. 2009. Immune response of cows fed polyunsaturated fatty acids under high ambient temperatures. J. Dairy Sci. 92:2796 2803. Carstens, G. E., D. E. Johnson, M. D. Holland, and K. G. Odde. 1987. Effects of prepartum protein nutrition and birth weight on basal metabolism in bovine neonates. J. Anim. Sci. 65:745 751. Cave, M., I. Deaciuc, C. Mendez, Z. Song, S. Joshi Barve, S. Barve, and C. McClain. 2007. Nonalcoholic fatty liver disease: pred isposing factors and the role of nutrition. J. Nutr. Biochem. 18:184 195. Cha J. Y. and J. J. Repa 2007 The liver X receptor (LXR) and hepatic lipogenesis. The carbohydrate response element binding protein is a target gene of LXR. J. Biol. Chem. 28 2 : 743 751 Chan, J. K., B. E. McDonald, J. M. Gerrard, V. M. Bruce, B. J. Weaver, and B. J. Holub. 1993. Effect of dietary alpha linolenic acid and its ratio to linoleic acid on platelet and plasma fatty acids and thrombogenesis. Lipids 28:811 817. Ch apkin, R. S., S. D. Somers, L. Schumaker, and K. L. Erickson. 1988. Fatty acid composition of macrophage phospholipids in mice fed fish or borage oil. Lipids 23:380 383. Chase, C. C. L., D. J. Hurley, and A. J. Reber. 2008. Neonatal immune development in t he calf and its impact on vaccine response. Vet. Clin. North Am. Food Anim. Pract. 24:87 104. Chechi, K., and S. Cheema. 2006. Maternal diet rich in saturated fats has deleterious effects on the plasma lipids of mice. Exp. Clin. Cardol. 11:129 135. Chen, J ., and X. Liu. 2009. The role of interferon gamma in regulation of CD4+ T cells and its clinical implications. Cell Immunol. 254:85 90. Chen, W., and J. Y. Chiang. 2003. Regulation of human sterol 27 hydroxylase gene (CYP27A1) by bile acids and hepatocyte nuclear factor 4 alpha (HNF4alpha). Gene 313:71 82. Christie, W. W. 1989. Gas Chromatography and Lipids: A Practical Guide. The Oily Press, Ayr, Scotland. Clarke, S. D. 2001. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechani sm to improve the metabolic syndrome. J. Nutr. 131:1129 1132. Clarke, S. D., D. R. Romsos, and G. A. Leveille. 1977. Influence of dietary fatty acids on liver and adipose tissue lipogenesis and on liver metabolites in meal fed rats. J. Nutr. 107:1277 1287.

PAGE 437

437 Corrigan, M. E., C. R. Wyatt, E. R. Low, B. E. Depenbusch, M. J. Quinn, F. P. Wang, J. J. Higgins, and J. S. Drouillard. 2009. Effect of melegestrol acetate on L selectin integrin expression of polymorphonuclear leukocytes from heifers challenged wit h lipopolysaccharide. Intern. J. Appl. Res. Vet. Med. 7:169 180. Costello, M., B. A. Fiedel, and H. Gewurz. 1979. Inhibition of platelet aggregation by native and desialised alpha 1 acid glycoprotein. Nature 281:677 678. Cray, C., J. Zaias, and N. H. Altm an. 2009. Acute phase response in animals: a review. Comp. Med. 59:517 5226. Crooks, S. W., and R. A. Stockley. 1998. Leukotriene B4. Int. J. Biochem. Cell Biol. 30:173 178. Cunningham, H. M., and J. K. Loosli. 1954. The effect of fat free diets on young d airy calves with observations on metabolic fecal fat and digestion coefficients for lard and hydrogenated coconut oil. J. Dairy Sci 37:453. Czernichow, S., D. Thomas, and E. Bruckert. 2010. n 6 Fatty acids and cardiovascular health: a review of the evidenc e for dietary intake recommendations. Br J Nutr. 104:788 796. da Silva, D. G., P. R. L. Silva, P. C. da Silva, and J. J. Fagliari. 2011. Serum protein concentrations, including acute phase proteins, in calves experimentally infected with Salmonella Dublin. Pesq. Vet. Bras. 31:551 554. Daniels, K. M., S. R. Hill, K. F. Knowlton, R. E. James, M. L. McGilliard, and R. M. Akers. 2008. Effects of milk replacer composition on selected blood metabolites and hormones in preweaned Holstein heifers. J. Dairy Sci. 91: 2628 2640. Das, U. N. 2003. Can perinatal supplementation of long chain polyunsaturated fatty acids prevent diabetes mellitus?. Eur. J. Clin. Nutr. 57:218 226. Deignan, T., A. Alwan, J. Kelly, J. McNair, T. Warren, and C. OFarrelly. 2000. Serum haptoglobi n: an objective indicator of experimentally induced Salmonella infection in calves. Res. Vet. Sci. 69:153 158. Delves, P. J., and I. M. Roitt. 2000. The immune system, First of two parts. N. Engl. J. Med. 343:37 49. DeNise, S. K., J. D. Robison, G. H. Stot t, and D. V. Armstrong. 1989. Effects of passive immunity on subsequent production in dairy heifers. J. Dairy Sci. 72:552 554. Dentin R., J. P. Pegorier, F. Benhamed, F. Foufelle, and P. Ferre. 2004. Hepatic glucokinase is required for the synergistic acti on of ChREBP and SREBP 1c on glycolytic and lipogenic gene expression. J. Biol. Chem. 279:20314 20326.

PAGE 438

438 Dentin, R., F. Benhamed, J. P. Pgorier, F. Foufelle, B. Viollet, and S. Vaulont. 2005. Polyunsaturated fatty acids suppress glycolytic and lipogenic gen es through the inhibition of ChREBP nuclear protein translocation. J. Clin. Invest. 115:2843 2854. Dervishi, E., M. Joy, J. Alvarez Rodriguez, M. Serrano and J. H. Calvo. 2012. The forage type (grazing versus hay pasture) fed to ewes and the lamb sex affec t fatty acid profile and lipogenic gene expression in the longissimus muscle of suckling lambs. J. Anim. Sci. 90:54 66. Detilleux, J. C., M. E. Kehrli, J. R. Stabel, A. E. Freeman, and F. H. Kelley. 1995. Study of immunological dysfunction in periparturien t Holstein cattle selected for high and average milk production. Vet. Immunol. Immunopathol. 44:251 267. Di Nunzio, M., D. van Deursen, A. J. M. Verhoeven, and A. Bordoni. 2010. N 3 and n 6 polyunsaturated fatty acids suppress sterol regulatory element bin ding protein activity and increase flow of non esterified cholesterol in HepG2 cells. Br. J. Nutr. 103:161 167. Dietz, R. E., J. B. Hall, W. D. Whittier, F. Elvinger, and D. E. Eversole. 2003. Effects of feeding supplemental fat to beef cows on cold tolera nce in newborn calves. J. Anim. Sci. 81:885 894. Diwakar, B. T., B. R. Lokesh, and K. A. Naidu. 2011. Modulatory effect of a linolenic acid rich garden cress (Lepidium sativum L.) seed oil on inflammatory mediators in adult albino rats. Br. J. Nutr.106:530 539. Donkin, S. S., and L. E. Armentano. 1995. Insulin and glucagon regulation of gluconeogenesis in preruminating and ruminating bovine. J. Anim. Sci. 73:546 551. Donovan, G. A., L. R. Dohoo, D. M. Montgomery, and F. L. Bennet. 1998. Associations between passive immunity and morbidity and mortality in dairy heifers in Florida, USA. Prev. Vet. Med. 34:41 46. Douglas, G. N., T. R. Overton, H. G. Bateman, and J. K. Drackley. 2004. Peripartal metabolism and production of Holstein cows fed diets supplemented w ith fat during the dry period. J. Dairy Sci. 87:4210 4220. Drackley, J. K. 2005. Interorgan lipid and fatty acid metabolism in growing ruminants. Pages 323 350 in: Biology of metabolism in growing animals. Burrin DG, Mersmann HJ, editors. Elsevier Ltd. Dr ackley, J. K. 2008. Calf nutrition from birth to breeding. Vet. Clin. Food Anim. 24:55 86.

PAGE 439

439 Drackley, J. K., and J. B. Andersen. 2006. Splanchnic metabolism of long chain fatty acids in ruminants. Pages 199 224 in Ruminant Physiology: Digestion, Metabolism and Impact of Nutrition on Gene Expression, Immunology and Stress. K. Sejrsen, T. Hvelplund, and M. O. Nielsen, ed. Wageningen Academic Publishers. Wageningen, the Netherlands. Drake, A. J., B. R. Walker, and J. R. Seckl. 2005. Intergenerational consequen ces of fetal programming by in uterus exposure to glucocorticoids in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288:R34 38. Drake, A.J., and B. R. Walker. 2004. The intergenerational effects of fetal programming: non genomic mechanisms for the inh eritance of low birth weight and cardiovascular risk. J. Endocrinol. 180:1 16. Dubois, V., S. Breton, M. Linder, J. Fanni, and M. Parmentier. 2007. Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. Eur. J. Lipid Sci. Tech nol. 109:710 732. Ducheix, S., J. M. A. Lobaccaro, P. G. Martin, and H. Guillou. 2011. Liver X receptor: an oxysterol sensor and a major player in the control of lipogenesis. Chem. Phys. Lipids 164:500 514. Duske, K., H. M. Hammon, A. K. Langhof, O. Bellma nn, B. Losand, K. Nurnberg, G. Nurnberg, H. Sauerwein, H. M. Seyfert, and C. C. Metges. 2009. Metabolism and lactation performance in dairy cows fed a diet containing rumen protected fat during the last twelve weeks of gestation. J. Dairy Sci. 92:1670 1684 Duval, C., M. Muller, and S. Kersten. 2007. PPARalpha and dyslipidemia. Biochim. Biophys. Acta 1771:961 971. Dwyer, C. M., A. B. Lawrence, S. C. Bishop, and M. Lewis. 2003. Ewe lamb bonding behaviours at birth are affected by maternal undernutrition in p regnancy. Br. J. Nutr. 89:123 136. Eastridge, M. 2002. Effects of feeding fats on rumen fermentation and milk composition. Pages 47 57 in Proc. 37th Annual Pacific Northwset animal nutrition conference. Vancouver, Canada. Elanco. 1996. Body condition scor ing in dairy cattle. Elanco Animal Health Bull. AI 8478. Elanco Animal Health, Greenfield, IN. Elmes, M., P. Tew, Z. Cheng, S. E. Kirkup, D. R. E. Abayasekara, P. C. Calder, M. A. Hanson, D. C. Wathes, and G. C. Burdge. 2004. The effect of dietary suppleme ntation with linoleic acid to late gestation ewes on the fatty acid composition of maternal and fetal plasma and tissues and the synthetic capacity of the placenta for 2 series prostaglandins. Biochim. Biophys. Acta 1686:139 147.

PAGE 440

440 Encinias, H. B., A. M. Enc inias, J. J. Spickler, B. Kreft, and M. L. Bauer. 2001. Effects of prepartum high linoleic safflower seed supplementation for gestating cows on performance of cows and calves. Pages 3 7 in Proc. of 5th International Safflower Conference. Montana, USA. Enci nias, H. B., G. P. Lardy, A. M. Encinias, and M. L. Bauer. 2004. High linoleic acid safflower seed supplementation for gestating ewes: effects on ewe performance, lamb survival, and brown fat stores. J. Anim. Sci. 82:3654 3661. Enke, U., L. Seyfarth, E. Sc hleussner, and U. R. Markert. 2008. Impact of PUFA on early immune and fetal development. Br. J. Nutr. 100:1158 1168. Espinoza, J. L., J. A. Ramirez Godinez, J. A. Jimenez, and A. Flores. 1995. Effects of calcium soaps of fatty acids on postpartum reproduc tive activity in beef cows and growth of calves. J. Anim. Sci. 73:2888 2892. Etherton, T. D., and D. E. Bauman. 1998. Biology of somatotropin in growth and lactation of domestic animals. Physiol. Rev. 78:745 761. Fang, C. X., F. Dong, D. P. Thomas, H. Ma, L. He, and J. Ren. 2008. Hypertrophic cardiomyopathy in high fat diet induced obesity: role of suppression of forkhead transcription factor and atrophy gene transcription. Am. J. Physiol. Heart Circ. Physiol. 295:H1206 H1215. FAO (Food and Agriculture Orga nization of the United Nations). 2010. Fats and fatty acids in human nutrition Report of an expert consultation. FAO, Rome, Italy. Farran, T. B., C. D. Reinhardt, D. A. Blasi, J. E. Minton, T. H. Elsasser, J. J. Higgins, and J. S. Drouillard. 2008. Source of dietary lipid may modify the immune response in stressed feeder cattle. J. Anim. Sci. 86:1382 1394. Ferezou Viala, J., A. F. Roy, C. Serougne, D. Gripois, M. Parquet, V. Bailleux, A. Gertler, B. Delplanque, J. Djiane, M. Riottot, and M. Taouis. 2007. Lo ng term consequences of maternal high fat feeding on hypothalamic leptin sensitivity and diet induced obesity in the offspring. Am. J. Regul. Integr. Comp. Physiol. 293:R1056 R1062. Fernandez, M. L., and K. L. West. 2005. Mechanisms by which dietary fatty acids modulate plasma lipids. J. Nutr. 135:2075 2078. Fielitz, J., M. S. Kim, J. M. Shelton, S. Latif, J. A. Spencer, D. J. Glass, J. A. Richardson, R. Bassel Duby, and E. N. Olson. 2007. Myosin accumulation and striated muscle myopathy result from the los s of muscle RING finger 1 and 3. J. Clin. Invest. 117:2486 2495.

PAGE 441

441 Flag, J., P. Grka, Z. M. Kowalski, U. Kaczor, P. Pietrzak, and R. Zabielski. 2011. Insulin like growth factors 1 and 2 (IGF 1 and IGF 2) mRNA levels in relation to the gastrointestinal trac t (GIT) development in newborn calves. Pol. J. Vet. Sci.14:605 613. Foldager, J., and K. Sejrsen. 1987. Mammary gland development and milk production in dairy cows in relation to feeding and hormone manipulation during rearing. Page 102 in Research in Catt le Production,Danish Status and Perspectives. Landhusholdningsselskabet, Frederiksberg, Denmark. Foote, M. R., B. J. Nonnecke, D. C. Beitz, and W. R. Waters. 2007. High growth rate fails to enhance adaptive immune responses of neonatal calves and is associ ated with reduced lymphocyte viability. J. Dairy Sci. 90:404 417. Forman, B. M., C. Chen, and R. M. Evans.1997. Hypolipodemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator activated 4317. Fowden, A. L., D. A. Giussani, and A. J. Forhead. 2006. Intrauterine programming of physiological systems: causes and consequences. Physiology 21:29 37. Fritsche, K. L. 2008. Too much linoleic acid promotes inflammation Prostaglandins Leukot. Essent. Fatty Acids 79:173 175. Fritsche, K. l., C. Feng, and J. Berg. 1997. Dietary fish oil enhances circulating interferon c in mice during listeriosis without altering in vitro production of this cytokine. J. Interferon Cytokine Res.17:271 277. Funston, R. N., D. M. Larson, and K. A Vonnahme. 2010. Effects of maternal nutrition on conceptus growth and offspring performance: Implications for beef cattle production. J. Anim. Sci. 88:E205 E215. Furman Fratczak K., A. Rzasa, T. Stefaniak. 2011. The influence of colostral immunoglobulin J. Dairy Sci. 94:5536 5543. Ganheim, C., S. Alenius, and K. Persson Waller. 2007. Acute phase proteins as indicators of calf herd health. Vet J. 173:645 651. Gao, M., and M. Karin. 2005. Re gulating the regulators: control of protein ubiquitination and ubiquitin like modifications by extracellular stimuli. Mol. Cell 19:581 593. Garry, F.B., R. Adams, M.B. Cattell, and R.P. Dinsmore. 1996. Comparison of passive immunoglobulin transfer to dairy calves fed colostrum or commercially available colostral supplement products. JAVMA 1:107 110.

PAGE 442

442 Gentleman, R. C., V. J. Carey, D. M. Bates, B. Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, and J. Gentry. 2004. Bioconductor: open software d evelopment for computational biology and bioinformatics. Genome Biol. 5:R80.1 R80.16. Georgiev, I. P. 2008a. Differences in chemical composition between cow colostrum and milk. Bulgarian J. Vet. Med. 11:3 12. Georgiev, I. P. 2008b. Effect of colostrum insu lin like growth factors on growth and development of neonatal calves. Bulgarian J. Vet. Med 11:75 88. Georgiev, I. P., T. M. Georgieva, M. Pfaffl, H. M. Hammon, and J.W. Blum. 2003. Insulin like growth factor and insulin receptors in intestinal mucosa of n eonatal calves. J. Endocrinol. 176:121 132. Gibson, R. A., B. Muhlhausler, and M. Makrides. 2011. Conversion of linoleic acid and alpha linolenic acid to long chain polyunsaturated fatty acids (LCPUFAs), with a focus on pregnancy, lactation and the first 2 years of life. Matern. Child Nutr. 7:17 26. Gicquel, C., A. El Osta, and Y. Le Bouc. 2008. Epigenetic regulation and fetal programming. Best Pract. Res. Clin. Endocrinol. Metab. 22:1 16. Glickman, M. H., and A. Ciechanover. 2002. The ubiquitin proteasome proteolytic pathway: destruction for the sake of construction. Physiol. Rev. 82:373 428. Gochman, N., and J. M. Schmitz. 1972. Application of a new peroxide indicator reaction to the specific automated determination of glucose with glucose oxidase. Clin. C hem. 18:943 950. Godden, S. M., D. M. Haines, and D. Hagman. 2009b. Improving passive transfer of immunoglobulins in calves. II: Interaction between feeding method and volume of colostrum fed. J. Dairy Sci. 92:1758 1764. Goebl, N. A., C. M. Babbey, A. Datt a Mannan, D. R. Witcher, V. J. Wroblewski, K. W. Dunn. 2008. Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells. Mol. Biol. Cell. 19:5490 5505. Gorjao, R., M. F. Cury Boaventura, T. M. de Lima, and R. Curi. 2007. Reg ulation of human lymphocyte proliferation by fatty acids. Cell Biochem. Funct. 25:305 315. Goyens, P. L., M. E. Spilker, P. L. Zock, M. B. Katan, and R. P. Mensink. 2006. Conversion of alpha linolenic acid in humans is influenced by the absolute amount of alpha linolenic acid and linoleic acid in the diet and not by their ratio. Am. J. Clin. Nutr. 84:44 53.

PAGE 443

443 Graulet, B., D. Gruffat, D. Durand, and D. Bauchart. 2004. Small intestine and liver microsomal triacylglycerol transfer protein in the bovine and rat: effects of dietary coconut oil. J. Dairy Sci. 87:3858 3868. Graulet, B., D. Gruffat Mounty, D. Durand, and D. Bauchart. 2000. Effects of milk diets containing beef tallow or coconut oil on the fatty acid metabolism of liver slices from preruminant calves. Br.J. Nutr. 84:309 318. Greco L. F., M. Garcia, M. G. Favoretto, R. S. Marsola, L. T. Martins, R. S. Bisinotto, E. S. Ribeiro, F. S. Lima, W. W. Thatcher, C. R. Staples, and J. E. P. Santos. 2010. Fatty acid supplementation to periparturient dairy cows fe d diets containing low basal concentrations of fatty acids. J. Dairy Sci. E Suppl. 1: 393. Greenberg, S. M., C. E. Calbert, E. E. Savage, and H. J. Deuel. 1950. The effect of fat level of the diet on general nutrition. J. Nutr. 41: 473 486. Gruffat Mouty, D., B. Graulet, D. Durand, M. E. Samson Bouma, and D. Bauchart. 2001. Effects of dietary coconut oil on apolipoprotein B synthesis and VLDL secretion by calf liver slices. Br.J. Nutr. 86:13 19. Gruffat Mouty, D., B. Graulet, D. Durand, M. E. Samson Bouma, and D. Bauchart. 1999. Apolipoprotein B production and very low density lipoprotein secretion by calf liver slices. J. Biochem. 126:188 193. Grummer, R. R. 2008. Nutritional and management strategies for the prevention of fatty liver in dairy cattle. Vet. J. 176:10 20. Guilloteau, P, R. Zabielski, and J.W. Blum. 2009. Gastrointestinal tract and digestion in the young ruminant: ontogenesis, adaptations, consequences and manipulation. J Physiol Pharmacol. 60:37 46. Gunstone, F. D. 1996. Fatty acids Nomenclatu re, structure, isolation and structure determination, biosynthesis and chemical synthesis. In: Fatty Acid and Lipid Chemistry, chapter 1. Blackie Academic & Professional, Chapman & Hall, London, UK. Guo, L., H. Fang, and J. Collins. 2006a. Differential gen e expression in mouse primary hepatocytes exposed to the peroxisome proliferator BMC Bioinformatics 7:S18. Guo, L., L. Zhang L, and Y. Sun. 2006b. Differences in hepatotoxicity and gene expression profiles by anti diabetic PPA R gamma agonists on rat primary hepatocytes and human HepG2 cells. Mol. Divers 10:349 360. Gurr, M. I., J. L. Harwood, and K. N. Frayn. 2002. Lipids: definition, isolation, separation, and detection. Pages 1 65 in Lipid Biochemistry: An introduction, ed. B lackwell Science Ltd, Oxford, UK.

PAGE 444

444 Hagiwara, K., M. Domi, and J. Ando. 2008. Bovine colostral CD8 positive cells are potent IFN gamma producing cells. Vet. Immunol. Immunopathol. 124:93 98. Hammon, H. M., I. A. Zanker, and J. W. Blum. 2000. Delayed colostr um feeding affects IGF I and insulin plasma concentrations inneonatal calves. J. Dairy Sci. 83:85 92. Hara, A., and N. S. Radin. 1978. Lipid extraction of tissues with a low toxicity solvent. Anal. Biochem. 90:420 426. Harhaj, E. W, and V. W. Dixit. 2012. Regulation of NF kB by deubiquitinases. Immunol. Rev. 246:107 124. Harmon, S. D., X. Fang, T. L. Kaduce, S. Hu, V. Raj Gopal, J. R. Falck, and A. A. Spector. 2006. Oxygenation of omega 3 fatty acids by human cytochrome P450 4F3B: Effect on 20 hydroxyeicosa tetraenoic acid production. Prostaglandins Leukot. Essent. Fatty Acids 75:169 177. Harris, W.S. 2006. The omega 6/omega 3 ratio and cardiovascular disease risk: uses and abuses. Curr. Atheroscler. Rep. 8:453 459. Hartsock, A., and W. J. Nelson. 2008. Adhe rens and tight junctions: Structure, function and connections to the actin cytoskeleton. Biochim. Biophys. Acta 1778:660 669. Hashemi, M., M. J. Zamiri, and M. Safdarian. 2008. Effects of nutritional level during late pregnancy on colostral production and blood immunoglobulin levels in Karakul ewes and their lambs. Small Rumin. Res. 75: 204 209. Hayashi, H., S. Muruyama, M. Fukuoka, T. Kosakai, K. Nakajima, T. Onaga, and S. Kato. 2012. Fatty acid binding protein expression in the gastrointestinal tract of calves and cows. Japanese Animal Science Journal. doi: 10.1111/j.1740 0929.2012.01038.x. Hebert, J. R., J. Barone, M. M. Reddy, and J. Y. Backlund. 1990. Natural killer cell activity in a longitudinal dietary fat intervention trial. Clin. Immunol. Immunop athol. 54:103 116. Heegaard, P. M., D. L.Godson, M. J. Toussaint, K. Toornehooj, L.E. Larsen, B. Viuff, and L. Roonsholt. 2000. The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respir atory syncytial virus. Vet. Immunol. Immunopathol. 77:151 159. Heinrichs, A. J. and J. A. Elizondo Salazar. 2009. Reducing failure of passive immunoglobulin transfer in dairy calves. Rev. Med. Vet. 160:436 440.

PAGE 445

445 Hernndez, A., J. A. Yager, B. N. Wilkie, K. E. Leslie, and B. A. Mallard. 2005. Evaluation of bovine cutaneous delayed type hypersensitivity to various test antigens and a mitogen using several adjuvants. Vet. Immunol. Immunopathol. 104:45 58. Hertz, R., J. Magenheim, I. Berman, and J. Bar Tana. 19 98. Fatty acyl CoA thioesters are ligands of hepatic nuclear receptor 4 alpha. Nature 392:512 516. Hess, B. W. 2003. Supplementing fat to the cow herd. Pages 156 165 in Proc. Range Beef Cow Symp. XVIII, Mitchell. Nebraska Printworks, Scottsbluff, NE. Hidir oglou, M., T. R. Batra, and M. Ivan. 1995. Effects of supplemental vitamins E and C on the immune responses of calves. J. Dairy Sci. 78:1578 1583. Hill, E. G., E. L. Warmanen, C. L. Silbernick and R. T. Holman 1961 Essential fatty acid nutrition in swine. I. Linoleate requirement estimated from triene:tetraene ratio of tissue lipids. J. Nutrition, 74: 335 341 Hill, J. O., J. C. Peters, L. L. Swift, D. Yang, T. Sharp, N. Abumrad, and H. L. Greene. 1990. Changes in blood lipids during six days of overfeeding with medium or long chain triglycerides. J. Lipid Res. 31:407 416. Hill, T. M., H. G. Bateman II, J. M. Aldrich, and R. L. Schlotterbeck. 2009. Effects of changing the essential and functional fatty acid intake of dairy calves. J. Dairy Sci. 92:670 676. Hi ll, T. M., M. J. VanderHaar, L. M. Sordillo, D. R. Catherman, H. G. Bateman II, and R. L. Schlotterbeck. 2011. Fatty acid intake alters growth and immunity in milk fed calves. J. Dairy Sci. 94:3936 3948. Acid glycoprotein: an acute phase protein with inflammatory and immunomodulating properties. Cytokine Growth Factor Rev. 14:25 34. Hocquette, J. F., and D. Bauchart. 1999. Intestinal absorption, blood t ransport and hepatic and muscle metabolism of fatty acids in preruminant and ruminant animals. Reprod. Nutr. Dev.39:27 48. Hollmann, D., and D. K. Beede. 2012. Comparison of effects of dietary coconut oil and animal fat blend on lactational performance of Holstein cows fed a high starch diet. J. Dairy Sci. 95:1484 1499. Holman, R. T., L. Smythe, and S. Johnson. 1979. Effect of sex and age on fatty acid composition of human serum lipids. Am. J. Clin. Nutr. 32:2390 2399. Horton, J. D., Y. Bashmakov, I. Shimo mura, and H. Shimano. 1998. Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice. Proc. Natl. Acad. Sci. USA. 95:5987 5992.

PAGE 446

446 Hostetler, H. A., A. D. Petrescu, A. B. Kier, and F.Schroeder. 2005. Peroxisome proliferator activated receptor alpha interacts with high affinity and is conformationally responsive to endogenous ligands. J. Biol. Chem. 280:18667 18682. Hotchkiss R. S, K. C. Chang, P. E. Swanson, K. W. Tinsley, J. J. Hui, P. Kendler, S. Xanthoudakis, S. Roy, C. Ba ck, E. Grimm, R. Aspitotis, Y. Han, D. W. Nicholson, And I. E. Karl. 2000. Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat. Immunol. 1:496 501. Howell, G., X. Deng, C. Yellaturu, E. A. Park, H. G. Wilcox, and R. Raghow 2009. N 3 polyunsaturated fatty acids suppress insulin induced SREBP 1c transcription via reduced transactivating capacity if LXR. Biochim. Biophys. Acta 1791:1190 1196. Huang, D. W., B. T. Sherman, and R. A. Lempicki. 2009. Systematic and integrative an alysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 4:44 57. Hulbert, L. E., C. J. Cobb, J. A. Carroll, and M. A. Ballou. 2011. Effects of changing milk replacer feedings from twice to once daily on Holstein calf innate immune r esponses before and after weaning. J. Dairy Sci. 94:2557 2565. Husnjak, K., and I. Dikic. 2012. Ubiquitin binding proteins: decoders of ubiquitin mediated cellular functions. Annu. Rev. Biochem. 81, 291 322. Huuskonen, A., H. Khalili, J. Kiljala, E. Joki Tokola, and J. Nousiainen. 2005. Effects of vegetable fats versus lard in milk replacers on feed intake, digestibility, and growth in finnish Ayrshire bull calves. J. Dairy Sci. 88:3575 3581. Hwang, M. N., C. H. Min, H. S. Kim, H. Lee, K. A. Yoon, and S. Y Park. 2007. The nuclear localization of SOCS6 requires the N terminal region and negatively regulates Stat3 protein levels. Biochem. Biophys. Res. Commun. 360:333 338. Igarashi, M., K. Ma, L. Chang, J. M. Bell, and S. I. Rapoport. 2 deprivation for 15 weeks up regulates elongase and desaturase expression in rat liver but not brain. J. Lipid Res. 48:2463 2470. Inagaki, T., P. Dutchak, G. Zhao, X. Ding, L. Gautron, V. Parameswara, Y. Li, R. Goetz, M. Mohammadi, and V. Esser. 2007. Endocrine regulation of the fasting mediated induction of fibroblast growth factor 21. Cell Metab. 5:415 425. Innis SM. Essential fatty acids in growth and development. 1991. Prog Lipid Res.30:39 103. Israel, E.J., S. Tay lor, Z. Wu, E. Mizoguchi, R.S. Blumberg, A. Bhan, and N.E. Simister. 1997. Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells. Immunology 92:69 74.

PAGE 447

447 Jacobson, N.L., C. Y. Cannon, and B. H. Thomas. 1949. Filled milks for dairy calves. I. Soybean oil versus milk fat. J. Dairy Sci. 32:429 434. Jaeschke H. 2006. Mechanisms of liver injury. II. Mechanisms of neutrophilinduced liver cell injury during hepatic ischemia reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol. 290: G1083 G1088, Jain, N. C., M. J. Paape, and R. H. Miller. 1991. Use of flow cytometry for determination of differential leukocyte counts in bovine blood. Am. J. Vet. Res. 52:630 636. Jambrenghi, A.C., G. Paglialonga, A. G noni, F. Zanotti, F. Giannico, G. Vonghia, and G.V. Gnoni. 2007. Changes in lipid composition and lipogenic enzyme activities in liver of lambs fed omega 6 polyunsaturated fatty acids. Comparative Biochemistry and Physiology, Part B. 147: 498 503. Jaster, E. H. 2005. Evaluation of quality, quantity, and timing of colostrum feeding on immunoglobulin G1 absorption in Jersey calves. J. Dairy Sci. 88:296 302. Jenkins, J. K., G. Griffith, and I. K. G. Kramer. 1988. Plasma lipoproteins in neonatal, preruminant, and weaned calf. Dairy Sci. 71:3003 3012. Jenkins, K. J. 1988. Factors affecting poor performance and scours in preruminant calves fed corn oil. J. Dairy Sci. 71:3013 3020. Jenkins, K. J., and J. K. G. Kramer. 1986. Influence of low linoleic and linolenic acids in milk replacer on calf performance and lipids in blood plasma, heart, and liver. J. Dairy Sci. 69:1374 1386. Jenkins, K. J., and J. K. G. Kramer. 1990. Effects of dietary corn oil and fish oil concentrate on lipid composition of calf tissues. J. Da iry Sci. 73:2940 2951. Jenkins, K. J., J. K. G. Kramer, F. D. Sauer, and D. B. Emmons. 1985. Influence of triglycerides and free fatty acids in milk replacers on calf performance, blood plasma, and adipose lipids. J. Dairy Sci. 68:669 680. Jenkins, K. J., J. K. Kramer, and D. B. Emmons. 1986. Effect of lipids in milk replacers on calf performance and lipids in blood plasma, liver, and perirenal fat. J. Dairy Sci. 69:447 459. Johnson, M. M., and J. P. Peters. 1993. Techincal note: An improved method to quant ify nonesterified fatty acids in bovine plasma. J. Anim. Sci. 71:753 756. Johnston, J. B., H. Oueliet, L. M. Podust, and P. R. Ortiz de Montellano. 2011. Structural control of cytochrome P450 catalyzed x hydroxylation. Arch. Biochem. Biophys. 507:86 94. Jones, E. A., and T. A. Waltman. 1972. The mechanism of intestinal uptake and transcellular transport of IgG in the neonatal rat. J. Clin. Invest. 51:2916.

PAGE 448

448 Jump, D. B. 2002. The biochemistry of n 3 polyunsaturated fatty acids. J. Biol. Chem. 277:8755 8758. Jump, D. B., S. D. Clarke, A. Thelen, and M. Liimatta, M. 1994. Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids. J. Lipid Res. 35:1076 1084. Kadowaki, T., and T. Yamauchi. 2005. Adiponectin and adiponectin receptors. Endocr. Rev. 26:439 451. Kadowaki, T., T. Yamauchi, N. Kubota, K. Hara, K. Ueki, and K. Tobe. 2006. Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. J. Clin. Invest. 116:1784 1792. Kampen, A. H. ,I. Olsen, T. Tollersrud, A. K. Storset, and A. Lund. 2006. Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life. Vet. Immunol. Immunopathol. 113, 53 63. Kanehisa, M., and S. Goto. 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28:27 30. Karibe, A., L. S. Tobacman, J. Strand, C. Butters, N. Back, L. L. Bachinski, A. E. Arai, A. Ortiz, R. Roberts, E. Himsher, and L. Fananapazir. 2001. Hypertrophic tropomyo sin mutation (V95A) is associated with mild cardiac phenotype, abnormal calcium binding to troponin, abnormal myosin cycling, and poor prognosis. Circulation 103:65 71. Karsten, S., G. Schafer, and P. Schauder. 1994. Cytokine production and DNA synthesis b y human peripheral lymphocytes in response to palmitic, stearic, oleic, and linoleic acid. J. Cell. Physiol. 161:15 22. Kehoe, S. I., and A. J. Heinrichs. 2007. Bovine colostrum nutrient composition. CAB. Reviews: Perspectives in Agriculture, Veterinary Sc ience, Nutrition and Natural Resources 2: No. 029:1 10. Kelley, D. S., L. B. Branch, and J. M. Lacono. 1989. Nutritional modulation of human immune status. Nutr. Res. 9:965 975. Kelley, D. S., R. M. Dougherty, L. B. Branch, P. C. Taylor, and J. M. Lacono.1 992. Concentration of dietary n 6 polyunsaturated fatty acids and human immune status. Clin. Immunol. Immunopathol. 62:240 244. Kelsey, J.A., B.A. Corl, R.J. Collier, and D.E. Bauman. 2003. The effect of breed, parity and stage of lactation on conjugated l inoleic acid (CLA) in milk fat from dairy cows. J. Dairy Sci. 86:2588 2597. Kennedy, G. C., and J. Mitra. 1963. Body weight and food intake as initiating factors for puberty in the rat. J. Physiol. 166:408 416.

PAGE 449

449 Kersten, S. 2008. Peroxisome proliferator act ivated receptors and lipoprotein metabolism. PPAR Res. 2008:132960. Khaidakov, M., S. Mitra, B. Y. Kang, X. Wang, S. Kadlubar, G. Novelli, V. Raj, M. Winters, W. C. Carter, and J. L. Mehta. 2011. Oxidized LDL receptor 1 (OLR1) as a possible link between ob esity, dyslipidemia and cancer. PLoS ONE. 6:e20277. Kim, H. J., M. Miyazaki, and J. M. Ntambi. 2002. Dietary cholesterol opposes PUFA mediated repression of the stearoyl CoA desaturase 1 gene by SREBP 1 independent mechanism. J. Lipid Res. 43:1750 57. Kind t, T., J. Richard, A. Goldsby, A. Barbara, and A. Oldstone. 2007. Kuby Immunology. 6 th edition. W.H. Freeman and Company. NY. Klaus, G. G. B., A. Bennett, and E. W. Jones. 1969. A quantitative study of the transfer of colostral immunoglobulins to the newb orn calf. Immunology 16: 293 299. Klemens, C. M., D. R. Berman, and E. L. Mozurkewich. 2011. The effect of perinatal omega 3 fatty acid supplementation on inflammatory markers and allergic diseases: a systematic review. BJOG. 118: 916 25. Knowles, T. G., J E. Edwards, K. J. Bazeley, S. N. Brown, A. Butterworth, and P. D Warriss. 2000. Changes in the blood biochemical and haematological profile of neonatal calves with age. Vet. Rec. 147:593 598. Koletzko, B., E. Larque, and H. Demmelmair. 2007. Placental t ransfer of long chain polyunsaturated fatty acids (LC PUFA). J. Perinat. Med. 35:S5 S11. Kramer, J. K. G., C. Cruz Hernandez, and J. Q. Zhou. 2001. Conjugated linoleic acids and octadecenoic acids: Analysis by GC. Eur. J. Lipid Sci. Technol. 103:600 609. Kramer, M. S. 1987. Determinants of low birth weight: methodological assessment and meta analysis. Bull World Health Organ. 65:663 737. Laarman, A. H., A. L. Ruiz Sanchez, T. Sugino, L. L. Guan, and M. Oba. 2012. Effects of feeding a calf starter on molecu lar adaptations in the ruminal epithelium and liver of Holstein dairy calves. J. Dairy Sci. 95:2585 2594. Lake, S. L., E. J. Scholljegerdes, D. M. Hallford, G. E. Moss, D. C. Rule, and B. W. Hess. 2006a. Body condition score at parturition and postpartum s upplemental fat effects on metabolite and hormone concentrations of beef cows and their suckling calves. J. Anim. Sci. 84:1038 1047. Lake, S. L., E. J. Scholljegerdes, R. L. Atkinson, V. Nayigihugu, S. I. Paisley, D. C. Rule, G. E. Moss, T. J. Robinson, an d B. W. Hess. 2005. Body condition score at parturition and postpartum supplemental fat effects on cow and calf performance. J. Anim. Sci. 83:2908 2917.

PAGE 450

450 Lake, S. L., E. J. Scholljegerdes, T. R. Weston, D. C. Rule, and B. W. Hess. 2006b. Postpartum suppleme ntal fat, but not maternal body condition score at parturition, affects plasma and adipose tissue fatty acid profiles of suckling beef calves. J. Anim. Sci. 84:1811 1819. Lake, S. L., E. J. Scholljegerdes, W. T. Small, L. Belden, S. I. Paisley, D. C. Rule and B. W. Hess. 2006c. Immune response and serum immunoglobulin G concentrations in beef calves suckling cows of differing body condition score at parturition and supplemented with high linoleate or high oleate safflower seeds. J. Anim. Sci. 84:997 1003. Lam, F. W., A. R. Burns, C. W. Smith, and R. E. Rumbaut. 2011. Platelets enhance neutrophil transendothelial migration via P selectin glycoprotein ligand 1. Am. J. Physiol. Heart Circ. Physiol. 300:H468 H475. Lambert, M.R., N.L. Jacobson, R.S. Allen and J .H. Zaletel. 1954. Lipid deficiency in the calf. J. Nutr. 52:259 272. Lammoglia, M. A., R. A. Bellows, E. E. Grings, and J. W. Bergman. 1999. Effects of prepartum supplementary fat and muscle hypertrophy genotype on cold tolerance in newborn calves. J. Ani m. Sci. 77:2227 2233. Lands, W. E., A. Morris, and B. Libelt.1990. Quantitative effects of dietary polyunsaturated fats on the composition of fatty acids in rat tissues. Lipids 25:505 516. Lawrence, T., D. A. Willoughby, and D. W. Gilroy. 2002. Anti inflam matory lipid mediators and insights into the resolution of inflammation. Nat. Rev. Immunol. 2, 787 795 Lawson, C., and S. Wolf. 2009. ICAM 1 signaling in endothelial cells. Pharmacol. Rep. 61:22 32. Lee, R. S., S. B. Tikunova, K. P. Kline, H. G. Zot, J. E. Hasbun, N. V. Minh, D. R. Swartz, J. A. Rall, and J. P. Davis. 2010. Effect of Ca2+ binding properties on the rate of skeletal muscle force redevelopment. Am. J. Physiol. Cell Physiol. 299:C1091 C1099. Leiber F, R. Hochstrasser, H.R. Wettstein, and M. Kre uzer. 2011. Feeding transition cows with oilseeds: effects on fatty acid composition of adipose tissue, colostrum and milk. Livest Sci 138:1 12. Lengqvist, J., A. Mata de Urquiza, A C. Bergman, T. M. Willson, J. Sjvall, T. Perlmann, and W. J. Griffiths. 2 004. Polyunsaturated fatty acids including docosahexaenoic and arachidonic acid bind to the retinoid X receptor alpha ligand binding domain. Mol. Cell. Proteomics 3:692 703.

PAGE 451

451 Leplaix Charlat, L., D. Bauchart, D. Durand, P. M. Laplaud, and M. J. Chapman. 199 6. Plasma lipoproteins in preruminant calves fed diets containing tallow or soybean oil with and without cholesterol. J. of Dairy Sci. 79:1267 1277. Lessard, M., N. Gagnon, D. L. Godson, and H. V. Petit. 2004. Influence of parturition and diets enriched in n 3 or n 6 polyunsaturated fatty acids on immune response of dairy cows during the transition period. J. Dairy Sci. 87:2197 2210. Levy, O. 2007. Innate immunity of the newborn: basic mechanisms and clinical correlates. Nature Rev.Immunol. 7:379 390. Lewis G. S., M. C. Wulster Radcliffe, and J. H. Herbeinc. 2008. Fatty acid profiles, growth, and immune responses of neonatal lambs fed milk replacer and supplemented with fish oil or safflower oil. Small Rumin. Res. 79:167 173. Ley, K., C. Laudanna, M. I. Cybu lsky, and S. Nourshargh. 2007. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Rev. Immunol. 7:678 689. Li, T., and J. Y. Chiang. 2009. Regulation of bile acid and cholesterol metabolism by PPARs. PPAR Res. 2009:501739. Liou, Y. A., D. J. King, D. Zibrik, and S. M. Innis. 2007. Decreasing linoleic acid with constant alpha linolenic acid in dietary fats increases (n 3) eicosapentaenoic acid in plasma phospholipids in healthy men. J. Nutr. 137:945 952. Litherland, N. B., R L. Wallace, and M. Bionaz. 2010. Effects of peroxisome proliferator activated receptor in weaned dairy calves. J. Dairy Sci. 93:2404 2418. Littel, R.C., G. A. Milliken, W. W. Stroup, and R D. Wolfinger. 1996. SAS System for Mixed Models, SAS Institute, Cary, NC. Liu, W.M., F. X. Shi, L. Z. Lu, C. Zhang, Y. L. Liu, J. Zhang, Z. R. Tao, J. D. Shen, G. Q. Li, and D. Q. Wang. 2011. Effects of linoleic acid and eicosapentaenoic acid on cell pro liferation and lipid metabolism gene expression in primary duck hepatocytes. Mol. Cell Biochem. 352:19 24. Lofthouse, S. A., A. E. Andrews, A. D. Nash, and V. M. Bowles. 1995. Humoral and cellular responses induced by intradermally administered cytokine a nd conventional adjuvants. Vaccine 13:1131 1137. Lundy, F. P., E. Block, W. C. Bridges, Jr., J. A. Bertrand, and T. C. Jenkins. 2004. Ruminal biohydrogenation in Holstein cows fed soybean fatty acids as amides or calcium salts. J. Dairy Sci. 87:1038 1046.

PAGE 452

452 Lynch, E. M., B. Earley, M. McGee, and S. Doyle. 2010. Effect of abrupt weaning at housing on leukocyte distribution, functional activity of neutrophils, and acute phase protein response of beef calves. BMC Vet. Res. 6:39 47. Magalhes, V. J. A., F. Susca, F. S. Lima, A. F. Branco, I. Yoon, and J. E. P. Santos. 2008. Effect of feeding yeast culture on performance, health, and immunocompetence of dairy calves. J. Dairy Sci. 91:1497 1509. Makimura, S., and N. Suzuki. 1982. Quantitative determination of bovine serum haptoglobin and its elevation in some inflammatory diseases. Jpn. J. Vet. Sci. 44:15 21. Mallard, B. A., L. C. Wagter, M. J. Ireland, and J.C.M. Dekkers. 1997. Effects of growth hormone, insulin like growth factor I, and cortisol on periparturient a ntibody response profiles of dairy cattle. Vet. Immunol. Immunopathol. 60:61 68. Manku, M. S., D. F. Horrobln, Y.S. Huang, and N. Morse. 1983. Fatty acids in plasma and red cell membranes in normal humans. Lipids. 18:906 918. Marodi L. 2002. Down regulati on of Th1 responses in human neonates. Clin Exp Immunol. 128:1 2. Marrapodi, M., and J. Y. Chiang. 2000. Peroxisome proliferator activated receptor alpha (PPAR transcription. J. Lipid Res. 41:514 520. Marsh, W. H., B. Fingerhut, and H. Miller. 1965. Autom ated and manual direct methods for the determination of blood urea. Clin. Chem. 11:624 627. Mashek, D. G, S. J. Bertics, and R. R. Grummer. 2002. Metabolic fate of long chain unsaturated fatty acids and their effects on palmitic acid metabolism and glucone ogenesis in bovine hepatocytes. J. Dairy Sci. 85:2283 2289. Mashek, D. G., and R. R. Grummer. 2003. Effects of long chain fatty acids on lipid and glucose metabolism in monolayer cultures of bovine hepatocytes. J. Dairy Sci. 86:2390 2396. Mashek, D. G., an d R. R. Grummer. 2004. Effects of conjugated linoleic acid isomers on lipid metabolism and gluconeogenesis in monolayer cultures of bovine hepatocytes. J. Dairy Sci. 87:67 72. McMaster, C. R., and T. R. Jackson. 2004. Phospholipid synthesis in mammalian c ells. Pages 5 30 in: Lipid Metabolism and Membrane Biogenesis, G. Daum (Ed.), Springer Verlag, Berlin, Heidelberg, Germany. Mehta, J. L., J. Chen, P. L. Hermonat, F. Romeo, and G. Novelly. 2006. Lectin like, oxidized low density lipoprotein receptor 1 (LOX 1): a critical player in the development of atherosclerosis and related disorders. Cardiovasc Res 69:36 45.

PAGE 453

453 Metzig M, D. Nickles, C. Falschlehner, J. Lehmann Koch, B. K. Straub, W. Roth, and M. Boutros. 2011. An RNAi screen identifies USP2 as a factor req uired for TNF alpha induced NF kappaB signaling. Int. J. Cancer. 129: 607 618. Michalski, M. C., N. Leconte, V. Briard Bion, J. Fauquant, J. L. Maubois, and H. Gouddranche. 2006. Microfiltration of raw whole milk to select fractions with different fat glo bule size distributions: Process optimization and analysis. J. Dairy Sci. 89:3778 3790. Mierlita, D.; I. Padeanu, C. Maerescu, I. Chereji, E. Halma, and F. Lup. 2011. Comparative study regarding the fatty acids profile in sheep milk related to the breed an d parity. 10:221 232 Mills, J. K., D. A. Ross, and M. E. Van Amburgh. 2010. The effects of feeding medium chain triglycerides on the growth, insulin res ponsiveness, and body composition of Holstein calves from birth to 85 kg of body weight. J. Dairy Sci. 93:4262 4273. Mizota, T., C. Fujita Kambara, N. Matsuya, S. Hamasaki, T. Fukudome, and H. Goto. 2009. Effect of dietary fatty acid composition on Th1/Th2 polarization in lymphocytes. J. Parenter. Enteral Nutr. 33:390 396. Moallem, U., A. Arieli, and H. Lehrer. 2007. Effects of prepartum propylene glycol or fats differing in fat acid profiles on feed intake, production, and plasma metabolites in dairy cows. J. Dairy Sci. 90:3846 3856. Moallem, U., and M. Zachut 2012. Short communication: The effects of supplementation of various n 3 fatty acids to late pregnant dairy cows on plasma fatty acid composition of the newborn calves. J. Dairy Sci. 95:4055 4058. M oallem, U., D. Werner, H. Lehrer, M. Zachut, L. Livshitz, S. Yakoby, and A. Shamay. 2010. Long term effects of ad libitum whole milk prior to weaning and prepubertal protein supplementation on skeletal growth rate and first lactation milk production. J. Da iry Sci. 93:2639 2650. Moonsie Shageer, S., and D. N. Mowat. 1993. Effect of level of supplemental chromium on performance, serum constituents, and immune status of stressed feeder calves. J. Anim. Sci. 71:232 238. Moreno, T., A. Varela, B. Oliete, J. A. C arballo, L. Snchez, and L. Montserrat. 2006. Nutritional characteristics of veal from weaned and unweaned calves: Discriminatory ability of the fat profile. Meat Science. 73:209 217. Morin, D. E., G. C. McCoy, and W. L. Hurley. 1997. Effects of quality, q uantity, and timing of colostrum feeding and addition of a dried colostrum supplement on immunoglobulin G1 absorption in Holstein bull calves. J. Dairy Sci. 80:747 753.

PAGE 454

454 Morrison, S. J., H. C. F. Wicks, R. J. Fallon, J. Twigge, L. E. R. Dawson, A. R. G. Wyl ie, and A. F. Carson. 2009. Effects of feeding level and protein content of milk replacer on the performance of dairy herd replacements. Animal 3:1570 1579. Motojima, K., P. Passilly, J. M. Peters. 1998. Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator activated receptor alpha and gamma activators in a tissue and inducer specific manner. J. Biol. Chem. 273:16710 16714. Murley, W. R., N. L. Jacobson, G. H. Wise, and R. S. Allen. 1949. Filled milks for dairy calves II. Comparative effects of various types of soybean oils and of butter oil on health, growth and certain blood constituents. J. Dairy Sci. 32:609 619. Naeye, R. L., W. A. Blanc, and C. Paul. 1973. Effects of maternal nutrition on the human fetus. Pediatr ics 52:494 503. Nakajima, K. T., Y. Kodaira, H. Ichioka, H. Nitta, S. Nakagawa, H. Yamamoto, H. Chikakiyo, and H. Ohtani. 1982. A new method of serum mucoprotein (acid soluble glycoproteins) assay using Coomassie brilliant blue G 250. Kit&w Kagaluc (Japane se Journal of Clinical Chemistry), 3, 214 221 (in Japanese with English summary) Nelson, D. L., and M. M. Cox. 2008. Oxidative phosphorylation and photophosphorylation. Chapter 19 in Lehninger Principles of Biochemistry, 5th ed., Freeman and Company, New Y ork, NY. Nikkari, T., P. Luukkainen, P. Pietinen, and P. Buska. 1995. Fatty acid composition of serum lipid fractions in relation to gender and quality of dietary fat. Ann. Med. 27:491 498. Niu, Y D., W. Xie, and W X. Qin. 2011. Molecular mechanism for the involvement of nuclear receptor FXR inHBV associated hepatocellular carcinoma. Acta Pharmaceutica Sinica 1:73 79. polyunsaturated fatty acid in the diet of the ewe and the essen tial fatty acid status of the neonatal lamb. J. Nutr. 108:1868 1876. Noble, R. C., M. L. Crouchman, D. M. Jenkinson, and J. H. Moore. 1975. Relationship between lipids in plasma and skin secretions of neonatal calf with particular reference to linoleic aci d. Lipids 10:128 133. Node, K., Y. Huo, X. Ruan, B. Yang, M. Spiecker, K. Ley, D. C. Zeldin, and J. K. Liao. 1999. Anti inflammatory properties of cytochrome P450 epoxygenase derived eicosanoids. Science 285:1276 1279.

PAGE 455

455 Novak, E. M., R. A. Dyer, and S. M. I nnis. 2008. High dietary omega 6 fatty acids contribute to reduced docosahexaenoic acid in the developing brain and inhibit secondary neurite growth. Brain Res. 1237:136 145. Novak, K. N., E. Davis, C. A. Wehnes, D. R. Shields, J. A. Coalson, A. H. Smith, T. G. Rehberger. 2012. Effect of supplementation with an electrolyte containing a Bacillus based direct fed microbial on immune development in dairy calves. Res. Vet. Sci. 92:427 434. and immune status of calves. Pol. J. Vet. Sci. 15:77 82. NRC. 1995. Nutrient Requirements for l aboratory animals. 4th rev. ed. Natl. Acad. Sci., Washington, DC. NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, DC. Nuernberg, K., D. Dannenberger, K. Ender, and G. Nuernberg. 2007. Comparison of different met hylation methods for the analysis of conjugated linoleic acid isomers by silver ion HPLC in beef lipids. J. Agric. Food Chem. 55:598 602. Lai. 1996. Ruminant pregastric lipases: experimental evidence of their potential as industrial catalysts in food technology. Colloids Surf. B: Biointerf. 7:189 205. Chapter 1 in: Foods Lipids: Chem istry, Nutrition and Biotechnology Marcel Dekker. Inc., New York, USA. Oda, S., H. Satoh, T. Matsunga, T. Kuhara, Y. Shoji, A. Nihei, M. Ohta, and Y. Sasaki. 1989. Insulin like growth factor I, GH, insulin, and glucagon concentrations in bovine colostrum a nd in plasma of dairy cows and neonatal calves around parturition. Comp. Biochem. Physiol. 94:805 812. Onetti, S. G., R. D. Shaver, M. A. McGuire, D. L. Palmquist, and R. R. Grummer. 2002. Effect of supplemental tallow on performance of dairy cows fed diet s with different corn silage:alfalfa silage ratios. J. Dairy Sci. 85:632 641. Osellame, L. D., T. S. Blacker, and M. R. Duchen. 2012. Cellular and molecular mechanisms of mitochondrial function. Best Pract. Res. Clin. Endocrinol. Metab. doi:10.1016/j.beem. 2012.05.003. Osgerby, J. C., D. C. Wathes, D. Howard, and T. S. Gadd. 2002. The effect of maternal under nutrition on ovine fetal growth. J. Endocrinol.173:131 141.

PAGE 456

456 Owczarek Lipska, M., P. Plattet, L. Zipperle, C. Drogemuller, H. Posthaus, G. Dolf, and M. H. Braunschweig. 2011. A nonsense mutation in the optic atrophy 3 gene (OPA3) causes dilated cardiomyopathy in Red Holstein cattle. Genomics 97:51 57. Ozanne, S. E., and C. N. Hales. 2002. Early programming of glucose insulin metabolism. Trends Endocrinol. Metab. 13:368 373. Palm, N.W., and R. Medzhitov. 2009. Pattern recognition receptors and control of adaptive immunity. Immunol. Rev. 227, 221 233. Palmquist, D. L. 2009. Omega 3 fatty acids in metabolism, health, and nutrition and for modified animal prod uct foods. Prof. Anim. Sci. 25:207 249. Pang, W. Y., B. Earley, T. Sweeney, S. Pirani, V. Gath, and M. A. Crowe. 2009. Effects of banding or burdizzo castration of bulls on neutrophil phagocytosis and respiratory burst, CD62 L expression, and serum interle ukin 8 concentration. J. Anim. Sci. 87:3187 3195. Pauletti, P., A. R. Bagaldo, L. Kindlein, and R. Machado. 2007. Insulin like growth factor I, passive immunity transfer, and stereological characteristics of small intestine of newborn calves. Anim. Sci. J. 78:631 638. Pawar, A., D. Botolin, D. J. Mangelsdorf, and D. B. Jump. 2003. The role of LXR in the fatty acid regulation of hepatic gene expression. J. Biol. Chem. 278:40736 40743. PPAR Res. 2010:572405. doi: 10.1155/2010/572405. Pennacchio L. A., and E. M. Rubin. 2003. Apolipoprotein A5, a newly identified gene that affects plasma triglyceride levels in humans and mice. Arterioscler. Thromb. Vasc. Biol. 23:529 534. Perdomo, M. C. 2011. Dietary strategies to modulate performance, health, and immune responses in Holstein calves. PhD. Thesis. University of Florida, Gainesville. Petit, H. V., and R. Berthia ume. 2006. Effect of feeding different sources of fat during gestation and lactation on reproduction of beef cows and calf performance. Can. J. Anim. Sci. 86:235 243. Petit, H. V., M. F. Palin, and L. Doepel. 2007. Hepatic lipid metabolism in transition d airy cows fed flaxseed. J. Dairy Sci. 90:4780 4792. Pettitt, D. J., W. C. Knowler, P. H. Bennett, K. A. Aleck, and H. R. Baird 1987. Obesity in offspring of diabetic Pima Indian women despite normal birth weight. Diabetes Care 10:76 80.

PAGE 457

457 Piantoni, P., K. M. Daniels, R. E. Everts, S. L. Rodriguez Zas, H. A. Lewin, W. L. Hurley, R. M. Akers, and J. J. Loor. 2012. Level of nutrient intake affects mammary gland gene expression profiles in preweaned Holstein heifers. J. Dairy Sci. 95: 2550 2561. Piot, C., J. F. H ocquette, J. H. Veerkamp, D. Durand, and D. Bauchart. 1999. Effects of dietary coconut oil on fatty acid oxidation capacity of the liver, the heart and skeletal muscles in the preruminant calf. Br. J. Nutr. 82:299 308. Postic, C., R. Dentin, P. D. Denechau d, and J. Girard. 2007. ChREBP, a transcriptional regulator of glucose and lipid metabolism. Annu. Rev. Nutr. 27:179 192. Pownall, H. J., and J. A. Hamilton. 2003. Energy translocation across cell membranes and membrane models. Acta Physiol. Scand. 178:357 365. Probst, S., E. Oechslin, P. Schuler, M. Greutmann, P. Boye, W. Knirsch, F. Berger, L. Thierfelder, R. Jenni, and S. Klaassen. 2011. Sarcomere gene mutations in isolated left ventricular noncompaction cardiomyopathy do not predict clinical phenotype. Circ. Cardiovasc. Genet. 4:367 374. Pudelkewicz, C., J. Seufert, and R. T. Holman 1968. Requirement of the female rat for linoleic and linolenic acids. J. Nutr. 94: 138 46. Quigley, J. D. III, and J. J. Drewry. 1998. Nutrient and immunity transfer from cow to calf pre and postcalving. J. Dairy Sci., 81:2779 2790. Quigley, J. D., III, J. J. Drewry, and K. R. Martin. 1998. Estimation of plasma volume in Holstein and Jersey calves. J. Dairy Sci. 81:1308 1312. Quigley, J. D., III, K. R. Martin, H. H. Dowlen, L. B. Wallis, and K. Lamar. 1994. Immunoglobulin concentration, specific gravity, and nitrogen fractions of colostrum from Jersey cattle. J. Dairy. Sci. 77:264 269. Quigley, J. D., III, T. A. Wolfe, and T. H. Elsasser. 2006. Effects of additional milk rep lacer feeding on calf health, growth, and selected blood metabolites in calves. J. Dairy Sci. 89:207 216. Rabiee, A. R., K. Breinhild, W. Scott H. M. Golder E. Block and I. J. Lean. 2012. Effect of fat additions to diets of dairy cattle on milk produ ction and components: A meta analysis and meta regression. J. Dairy Sci. 95:3225 3247. Radcliff, R. P., M. J. VandeHaar, L. T. Chapin, T. E. Pilbeam, D. K. Beede, E. P. Stanisiewski, and H. A. Tucker. 2000. Effects of diet and injection of bovine somatotro pin on prepubertal growth and first lactation milk yields of Holstein cows. J. Dairy. Sci. 83:23 29.

PAGE 458

4 58 Raeth Knight, M., H. Chester Jones, S. Hayes, J. Linn, R. Larson, D. Ziegler, B. Ziegler, and N. Broadwater. 2009. Impact of conventional or intensive mil k replacer programs on Holstein heifer performance through six months of age and during first lactation. J. Dairy Sci. 92:799 809. Rajaraman, V., B. J. Nonnecke, and R. L. Horst. 1997. Effects of replacement of native fat in colostrum and milk with coconut oil on fat soluble vitamins and immune function in calves. J. Dairy Sci. 81:2380 2390. Rakhshandehroo, M., B. Knoch, M. Muller, and S. Kersten. 2010. Peroxisome proliferator activated receptor alpha target genes. PPAR Res. 2010:612089. doi:10.1155/2010/61 2089. Rakhshandehroo, M., G. Hooiveld, M. Miuller, and S. Kersten. 2009. Comparative and human. PLoS One 4:e6796. doi:10.1371/journal.pone.0006796. Ramsden, C. E, J. R. Hibbeln, S. F. Majchrzak, and J. M. Davis. 2010. n 6 fatty ac id specific and mixed polyunsaturate dietary interventions have different effects on CHD risk: a meta analysis of randomised controlled trials. Br. J. Nutr. 104:1586 1600. Rapoport, S. I., J. S. Rao, and M. Igarashi. 2007. Brain metabolism of nutritionall y essential polyunsaturated fatty acids depends on both the diet and the liver. Prostaglandins Leukotr. Ess. Fatty Acids 77:251 261. Rath, T., T. T. Kuo, K. Baker, S. W. Qiao, K. Kobayashi, M. Yoshida, D. Roopenian, E. Fiebiger, W. I. Lencer and R. S. Blum berg. 2012. The immunologic Functions of the neonatal Fc receptor for IgG. J. Clin. Immunol. 9. DOI 10.1007/s10875 012 9768 y. Reddy, J. K., and M. S. Rao. 2006. Lipid Metabolism and Liver Inflammation. II. Fatty liver disease and fatty acid oxidation. Am J. Physiol. Gastrointest. Liver Physiol. 290:G852 G858. Reid, M. E., J. G. Bieri, P. A. Plack and E. L. Andrews 1964 Nutritional studies with the guinea pig. X. Determination of the lino leic acid requirement. J. Nutr., 82: 401 408. Renatus, M., S. G. P arrado, A. D'Arcy, U. Eidhoff, and B. Gerhartz. 2006. Structural basis of ubiquitin recognition by the deubiquitinating protease USP2. Structure 14:1293 1302. Reveneau, C., C. V. D. M. Ribeiro, M. L. Eastridge, and J. L. Firkins. 2012. Interaction of unsa turated fat or coconut oil with monensin in lactating dairy cows fed 12 times daily. I. Fatty acid flow to the omasum and milk fatty acid profile. J. Dairy Sci. 95:2061 2069.

PAGE 459

459 Richard, M. J., J. W. Stewart, T. R. Heeg, K. D. Wiggers, and N. L. Jacobson. 198 0. Blood plasma lipoprotein and tissue cholesterol of calves fed soybean oil, com oil, vegetable shortening or tallow. Atherosclerosis 37:513 520. Robison, J. D., G. H. Stott, and S. K. DeNise. 1988. Effects of passive immunity on growth and survival in th e dairy heifer. J. Dairy Sci. 71:1283 1287. Rodrigues, H. G., M. A. R. Vinolo, and J. Magdalon. 2010. Dietary free oleic and linoleic acid enhances neutrophil function and modulates the inflammatory response in rats. Lipids 45:809 819. Roffler, B., A. Fh, S. Sauter, H. M. Hammon, G. Brem & J. B. Blum, 2003. Intestinal morphology, epithelial cell proliferation, and absorptive capacity in neonatal calves fed milk borne insulin like growth factor I or a colostrum extract. J. Dairy Sci. 86:1797 1806. Sampath H., and J. M. Ntambi. 2005. Polyunsaturated fatty acid regulation of genes of lipid metabolism. Annu. Rev. Nutr. 25:317 340. Sanchez Madrid, F., and M. A. del Pozo. 1999. Leukocyte polarization in cell migration and immune interactions. Embo J. 18:501 11 Sanders, T.A.B. 1988. Essential and trans fatty acids in nutrition. Nutr. Res. Reviews 1, 57 78. Santschi, D.E., H.R. Wettstein, F. Leiber, A.K.M. Witschi, and K. Kreuzer. 2009. Colostrum and milk fatty acids of dairy cows as influenced by extruded linse ed supplementation during the transition period. Can. J. Anim. Sci. 89:383 392. SAS Institute. 2009. SAS/STAT 9.2 User's Guide. SAS Inst., Inc., Cary, NC. Sasaki, M., C. L. Davis, and B. L. Larson. 1976. Production and turnover of IgG1 and IgG2 immunoglobu lins in the bovine around parturition. J. Dairy Sci. 59: 2046 2055. Sato K, Y. Cho, S. Tachibana, T. Chiba, W. J. Schneider, and Y. Akiba. 2005. Impairment of VLDL secretion by medium chain fatty acids in chicken primary hepatocytes is affected by the chai n length. J Nutr 135:1636 1641 Sato, H. 1994. Plasma ketone levels in neonatal calves fed medium chain triglycerides in milk. J. Vet. Med. Sci. 56:781 782. Savas, U., M. H. Hsu, and E. F. Johnson. 2003. Differential regulation of human CYP4A genes by perox isome proliferators and dexamethasone. Arch. Biochem. Biophys. 409:212 220.

PAGE 460

460 Schagger, H., and K. Pfeiffer. 2001. The ratio of oxidative phosphorylation complexes I V in bovine heart mitochondria and the composition of respiratory chain supercomplexes. J. B iol. Chem. 276:37861 37867. Schmitz, G., and J. Ecker. 2008. The opposing effects of n 3 and n 6 fatty acids. Prog. Lipid Res. 47:147 155. Schroeder, H. W., and L. Cavacini. 2010. Structure and function of immunoglobulins. J. Allergy Clin. Immunol. 125:S4 1 52. Scrimgeour, C. 2005. Chemistry of fatty acids. Chapter 1 in: Bailey's Industrial Oil and Fat Products, Edible Oil and Fat Products: General Applications. Y. H. Hui, ed. John Wiley and Sons, Inc., New York, NY, USA. Sekiya, M., N. Yahagi, T. Matsuzaka Y. Najima, M. Nakakuki, and R. Nagai. 2003. Polyunsaturated fatty acids ameliorate hepatic steatosis in obese mice by SREBP 1 suppression. Hepatology 38:1529 1539. Semple, R. K., A. Sleigh, P. R. Murgatroyd, C. A. Adams, L. Bluck, S. Jackson, A. Vottero, D. Kanabar, V. Charlton Menys, P. Durrington, M. A. Soos, T. Adrian Carpenter, D. J. Lomas, and E. K. Cochran. 2009. Post receptor insulin resistance contributes to human dyslipidemia and hepatic steatosis. J. Clin. Invest. 119:315 322. Sewalem, A., F. Mi glior, G. J. Kistemaker, P. Sullivan, and B. J. Van Doormaal. 2008. Relationship between reproduction traits and functional longevity in Canadian dairy cattle. J. Dairy Sci. 91:1660 1668. Shamay, A., D. Werner, U. Moallem, H. Barash, and I. Bruckental. 200 5. Effect of nursing management and skeletal size at weaning on puberty, skeletal growth rate, and milk production during first lactation of dairy heifers. J. Dairy Sci. 88:1460 1469. Shembade, N., N. S. Harhaj, K. Parvatiyar, N. G. Copeland, N. A. Jenkins L. E. Matesic, and E. W. Harhaj. 2008. The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin editing enzyme A20. Nat. Immunol. 9:254 262. Silvestre, F. T., T. S. Carvalho, P. C. Crawford, J. E. Santos, C. R. Staples, T. Jenkins, and W. W. Thatcher. 2011. Effects of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: II. Neutrophil fatty acids and function, and acute phase proteins. J. Dairy Sci. 94:2285 2301. Simon, S. I., Y. Hu, D. Vestweber, and C. W. Smith. 2000. Neutrophil tethering on E 2 integrin binding to ICAM 1 through a mitogen activated protein kinase signal transduction pathway. J. Immunol. 164:4348 4358.

PAGE 461

461 Singh, K., R. A. Erdman, K. M. Swanson, A. J. Molenaar, N. J. Maqbool, and T. T. Wheeler. 2010. Epigenetic regulation of milk production in dairy cows. J. Mammary Gland Biol. Neoplasia 15:101 112. Siracusa, M. C., Perrigoue J. G., Comeau M. R., and D. Artis 2010. New paradigms in basophil development, regulation and function. Immunol. Cell Biol. 88,:275 284 Smith, J. M., M. E. Van Amburgh, M. C. Diaz, M. C. Lucy, and D. E. Bauman. 2002. Effect of nutrient intake on the development of the somatotropic axis and its responsiveness to GH in Holstein bull calves. J. Anim. Sci. 80:1528 1537. Smits, E., C. Burvenich and R. Heyneman. 1997. Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood. Vet. Immunol. Immunopathol. 56:259 269. Smyth, G. K. 2005. Limma: linear models for microa rray data. Pages 397 420 in: Bioinformatics and Computational Biology Solutions using R and Bioconductor. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, W. Huber (eds), Springer. New York, USA. Smyth, S. S., R. P. Mcever, A. S. Weyrich, C. N. Morrell, M. R. Hoffman, G. M. Arepally, P. A. French, H. L Dauerman, and R. C. Becker. 2009. Platelet functions beyond hemostasis. J. Thromb. Haemost. 7:1759 1766. Soares, M. C. 1986. Effect of dietary protected lipids on the essential fatty acid status of the newbo rn kid. J. Nutr. 116:1473 1479. Soberon, F., E. Raffrenato, R. W. Everett, and M. E. Van Amburgh. 2012. Pre weaning milk replacer intake and effects on long term productivity of Dairy calves. J. Dairy Sci. 95: 783 793. Sokolovic, M., A. Sokolovic, and D. W ehkamp. 2008. The transcriptomic signature of fasting murine liver. BMC Genomics 9:528. doi:10.1186/1471 2164 9 528. Sparks A. L., J.G. Kirkpatrick, C.S. Chamberlain, D. Waldner, and L.J. Spicer. 2003. Insulin like growth factor I and its binding proteins in colostrum compared to measures in serum of Holstein neonates. J. Dairy Sci. 6:2022 2029. Stanley, J. C., R. L. Elsom, P. C. Calder, B. A. Griffin, W. S. Harris, S. A. Jebb, J. A. Lovegrove, C. S. Moore, R. A. Riemersma, and T. A. B. Sanders. 2007. UK Fo od Standards Agency Workshop Report: the effects of the dietary n 6:n 3 fatty acid ratio on cardiovascular health. Br J Nutr 98:1305 1310. Stanton, T. L., J. C. Whittier, T. W. Geary, C. V. Kimberling, and A. B. Johnson. 2000. Effects of trace mineral supp lementation on cow calf performance, reproduction and immune function. Prof. Anim. Sci. 16:121 127.

PAGE 462

462 Stoeckman, A. K., L. Ma, and H. C. Towle. 2004. Mlx is the functional heteromeric partner of the carbohydrate response element binding protein in glucose re gulation of lipogenic enzyme genes. J. Biol. Chem. 279:15662 15669. Storch, J., and L. McDermott. 2009. Structural and functional analysis of fatty acid binding proteins. J. Lipid Res. 50:S126 S131. Stott, G. H., D. B. Marx, B. E. Menefee, and G. T. Nighte ngale. 1979a. Colostral immunoglobulin transfer in calves. I. Period of absorption. J. Dairy Sci. 62:1632 1638. Stott, G. H., D. B. Marx, B. E. Menefee, and G. T. Nightengale. 1979c. Colostral immunoglobulin transfer in calves. III. Amount of absorption. J. Dairy Sci. 62:1902 1907. Stott, G. H., D. B. Marx, B. E. Menefee, and G. T. Nightengate. 1979b. Colostral immunoglobulin transfer in calves. II. The rate of absorption. J. Dairy Sci. 62:1766 1773. Sun, Y., J. Zhang, L. Lu, S. S. Chen, M. T. Quinn, and K T. Weber. 2002. Aldosterone induced inflammation in the rat heart : role of oxidative stress. Am. J. Pathol. 161:1773 1781. Swift, U., J. O. Hill, I. C. Peters, and H. L. Greene. 1990. Medium chain fatty acids: evidence for incorporation into chylomicron triglycerides in humans. Am. I. Clin. Nutr. 52:834 836. Tajsharghi, H. 2008. Thick and thin filament gene mutations in striated muscle diseases. Int. J. Mol. Sci. 9:1259 1275. Takahashi, R., K. Okumura, T. Asai, T. Hirai, H. Murakami, R. Murakami, Y. Numa guchi, H. Matsui, M. Ito, and T. Murohara. 2005. Dietary fish oil attenuates cardiac hypertrophy in lipotoxic cardiomyopathy due to systemic carnitine deficiency. Cardiovasc. Res. 68:213 223. Talloen, W., D. A. Clevert, S. Hochreiter, D. Amaratunga, L. Bij nens, S. Kass, and H. W. Gohlmann. 2007. I/NI calls for the exclusion of non informative genes: a highly effective filtering tool for microarray data. Bioinformatics 23:2897 2902. Thanasak, J., K. E. Mller, S. J. Dieleman, A. Hoek, J. P. Noordhuizen, and V. P. Rutten. 2005. Effects of polyunsaturated fatty acids on the proliferation of mitogen stimulated bovine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol.104: 289 295. Thanasak, J., V. P. M. G. Rutten, J. Th. Schonewille, A. Hoek, A. C. B eynen, J. P. T. M. Noordhuizen, and K. E. Mller. 2004. Effect of dietary n 6 polyunsaturated fatty acid supplement on distinct immune functions of goats. J Vet Med A Physiol Pathol Clin Med. 51:1 9.

PAGE 463

463 Theurer, M. L., E. Block, W. K. Sanchez, and M. A. McGui re. 2009. Calcium salts of polyunsaturated fatty acids deliver more essential fatty acids to the lactating dairy cow. J. Dairy Sci. 92:2051 2056. Thoden, J. B., D. J. Timson, R. J. Reece, and H. M. Holden. 2004. Molecular structure of human galactose mutar otase. J. Biol. Chem. 279:23431 23437. Thome, M. 2004. CARMA1, BCL 10 and MALT1 in lymphocyte development and activation. Nature Rev. Immunol. 4:348 359. Thomson, A. W., and P. A. Knolle. 2010. Antigen presenting cell function in the tolerogenic liver envi ronment. Nat. Rev. Immunol. 10:753 766. Tilg, H., and A. R. Moschen. 2006. Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat. Rev. Immunol. 6:772 783. Tinoco, J., R. Babcock, I. Hincenbergs, B. Medwadowski, and P. Miljanich. 1978. Linolenic acid deficiency: changes in fatty acid patterns in female and male rats raised on a linolenic acid deficient diet for two generations. Lipids. 13:6 17. Tinoco, J., R. Babcock, I. Hincenbergs, B. Medwadowski, P. Miljanich, and M. A. William s. 1979. Linolenic acid deficiency. Lipids. 14:166 173. Toussant, M. J., M. D. Wilson, and S. D. Clarke. 1981. Coordinate suppression of liver acetyl coA carboxylase and fatty acid synthetase by polyunsaturated fat. J. Nutr. 111:146 153. Tsiplakou, E., K. C. Mountzouris, and G. Zervas. 2006. The effect of breed stage of lactation and parity on sheep milk fat CLA content under the same feeding practices. Liv. Sci. 105:162 167. Tyler, J. W., D. D. Hancock, S. M. Parish, D. E. Rea, T. E. Besser, S. G. Sanders, and L. K. Wilson. 1996. Evaluation of 3 assays for failure of passive transfer in calves. J. Vet. Intern. Med. 10:304 307. Ueki, K., T. Kondo, Y. H. Tseng, and C. R. Kahn. 2004. Central role of suppressors of cytokine signaling proteins in hepatic steato sis, insulin resistance, and the metabolic syndrome in the mouse, Proc. Natl. Acad. Sci. U.S.A. 101:10422 10427. Ulberth, F., and F. Schrammel. 1995. Accurate quantitation of shortchain, medium chain, and long chain fatty acid methyl esters by split inject ion capillary gas chromatography. J. Chromatogr. A. 704:455 463. Van Gool, C. J. A. W., C. Thijs, and P. C. Dagnelie. 2004. Determinants of neonatal IgE level: parity, maternal age, birth season and perinatal essential fatty acid status in infants of atopi c mothers. Allergy: 59:961 968.

PAGE 464

464 Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber and non starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74: 3568 3597. Villeneuve, P., M. Pin a, and J. Graille. 1996. Determination of pregastric lipase specificity in young ruminants. Chem. Phys. Lipids. 83:161 168. Vyas, D., B. B. Teter, and R. A. Erdman. 2012. Milk fat responses to dietary supplementation of short and medium chain fatty acids i n lactating dairy cows. J. Dairy Sci. 95:5194 5202. Wagter, L .C., B. A. Mallard, B. N. Wilkie, K. E. Leslie,P J. Boettcher, and J. C. Dekkers. 2003. The relationship between milk production and antibody response to ovalbumin during the peripartum period. J. Dairy Sci. 86:169 173. Wallace, F. A., E. A. Miles, C. Evans, T. E. Stock, P. Yaqoob, and P. C. Calder. 2001. Dietary fatty acids influence the production of Th1 but not Th2 type cytokines. J. Leukoc. Biol. 69:449 457. Wanders, R. J. A., S. Ferdinandus se, P. Brites, and S. Kemp. 2010. Peroxisomes, lipid metabolism and lipotoxicity. Biochim. Biophys. Acta 1801:272 280. Wanders, R. J., and H. R. Waterham. 2006. Biochemistry of mammalian peroxisomes revisited. Annu. Rev. Biochem. 75:295 332. Weaver, D. M., J. W. Tyler, D. C. VanMetre, D. E. Hostetler, and G. M. Barrington. 2000. Passive transfer of colostral immunoglobulins in calves. J. Vet. Intern. Med. 14:569 577. Weaver, K. L., P. Ivester, M. C. Seeds, L. D. Case, J. Arm, and F. H. Chilton. 2009. Effect of dietary fatty acids on inflammatory gene expression in healthy humans. J. Biol. Chem. 284:15400 15407. Weber, P. S. D., S. A. Madsen, G. W. Smith, J. J. Ireland, and J. L. Burton. 2001. Pre translational regulation of neutrophil L selectin in glucocort icoid challenged cattle. Vet. Immunol. Immunopathol. 83:213 240. Weickert, M. O., and A. F. H. Pfeiffer. 2006. Signaling mechanisms linking hepatic glucose and lipid metabolism. Diabetologia 49:1732 1741. West, I. C. 2000. Radicals and oxidative stress in diabetes. Diabet. Med. 17:171 180. Whelan, J. 2008. The health implications of changing linoleic acid intakes. Prostaglandins Leukot. Essent. Fatty Acids 79:165 167. White, H. M., S. L. Koser, and S. S. Do untranslated region variant expression during transition to lactation and feed restriction in dairy cows. J. Anim. Sci.89:1881 1892.

PAGE 465

465 White, H. M., S. L. Koser, and S. S. Donkin. 2011b. Differential regulation of pyruvate carboxylase promoters by fatty acids and peroxisome proliferator activated receptor 3436. Wolfrum, C., C. M. Borrmann, T. Borchers, and F. Spener. 2001. Fatty acids and hypolipidemic drugs regulate peroxisome proli ferator activated receptors alpha and gamma mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc. Natl. Acad. Sci. U.S.A. 985:2323 2328. Woolums A. R. 2010. Immune development of the ruminant neonate. In: 2010 Penn State Dairy Cattle Nutrition Workshop; 2010; Grantville,PA, USA; 2010. p. 1 5. Wrenn, T. R., J. R. Weyant, C. H. Gordon, H. K. Goering, L. P. Dryden, and J. Bitman. 1973. Growth, plasma lipids and fatty acid composition of veal calves fed polyun saturated fats. J. Anim. Sci. 37:1419 1427. Wright, P. 1987. Enzyme immunoassay: observations on aspects of quality control. Vet. Immunol. Immunopathol. 17. 441 452. Wu, Z., R. A. Irizarry, R. Gentleman, F. Martinez Murillo, and F. Spencer. 2004. A model b ased background adjustment for oligonucleotide expression arrays. J. Am. Stat. Assoc. 99:909 917. Yahagi, N., H. Shimano, A. H. Hasty, M. Amemiya Kudo, H. Okazaki, Y. Tamura, Y. Iizuka, F. Shionoiri, K. Ohashi, J I. Osuga, K. Harada, T. Gotoda, R. Nagai, S Ishibashi, and N. Yamada. 1999. A crucial role of sterol regulatory element binding protein 1 in the regulation of lipogenic gene expression by polyunsaturated fatty acids. J. Biol. Chem. 274:35840 35844. Yamad, K., and T. Noguchi. 1999. Nutrient and hor monal regulation of pyruvate kinase gene expression. Biochem J 337:1 11. Yamashita, H., M. Takenoshita, M. Sakurai, R. K. Bruick, W. J. Henzel, W. Shillinglaw, D. Arnot, and K. Uyeda. 2001. A glucose responsive transcription factor that regulates carbohydr ate metabolism in the liver. Proc. Natl. Acad. Sci. U.S.A. 98:9116 9121. Yaqoob, P., and P. C. Calder. 2007. Fatty acids and immune function: new insights into mechanisms. Br. J. Nutr. 98:S41 S45. Yaqoob, P., H. S. Pala, M. Cortina Borja, E. A. Newsholme, and P. C. Calder. 2000. Encapsulated fish oil enriched in a tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions. Eur J. Clin. Invest. 30:260 274.

PAGE 466

466 Yoshikawa, T., H. Shimano, N. Yahagi, M. A memiya Kudo, and T. Matsuzaka. 2003. Cross talk between peroxisome proliferator activated receptor (PPAR) alpha and liver X receptor (LXR) in nutritional regulation of fatty acid metabolism. I. PPARs suppress sterol regulatory element binding protein 1c pr omoter through inhibition of LXR signaling. Mol. Endocrinol. 17:1240 1254. Yoshikawa, T., H. Shimano, N. Yahagi, T. Ide, and M. Amemiya Kudo. 2002. Polyunsaturated fatty acids suppress sterol regulatory element binding protein 1c promoter activity by inhib ition of liver X receptor (LXR) binding to LXR response elements. J. Biol. Chem. 277:1705 1711. Zhao, A., J. Yu, J. L. Lew, L. Huang, S. D. Wright, and J. Cui. 2004. Polyunsaturated fatty acids are FXR ligands and differentially regulate expression of FXR targets. DNA Cell Biol. 23:519 526. Zhu, P., Y. Y Goh, H. F. A. Chin, S. Kersten, and N. S. Tan. 2012. Angiopoietin like 4: a decade of research. Biosci. Rep. 32:211 219. Zipfel, P. F. 2009. Complement and immune defense: from innate immunity to human dis eases. Immunol. Lett. 126:1 7.

PAGE 467

467 BIOGRAPHICAL SKETCH Miriam Garcia Orellana earned her bachelor degree in animal s ciences at Universidad Nacional Agraria la Molina in December 1997 and her engineer degree in animal sciences in December 1999 From August 19 99 to September 2003, Miriam to the highlands of Peru to work as an animal produ ction consultant assisting low income farmers. In 2004, Miriam was awarded a scholarship from Consejo Nacional de Ciencia y Tecnologia in Peru and returned to Universidad Nacional Agraria la Molina, where she got a master in ruminant nutrition after defend Effect of feeding two diets with different nutritive value on milk production and composition and metabolic Miriam worked with Dr. Carlos A. Gomez at Universi dad Nacional Agraria la Molina as an associated researcher where she gained enormous experience in dairy cattle nutrition. In fall 2008, she was awarded the University of Florida CALS Alumn i Graduate Award to pursue her PhD in animal s ciences under the gui dance of Dr. Charles R. Staples. During her program Miriam has lead two projects and participated in several others, she also has served as teaching assistant and invited instructor. She is impassioned for teaching and research, after graduating; she is pl anning to pursue a career that would allow her to stay on the same path.