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Effect of Supplemental Fat Source on Production, Immunity, Hepatic Gene Expression, and Metabolism of Periparturient Dai...

Permanent Link: http://ufdc.ufl.edu/UFE0022802/00001

Material Information

Title: Effect of Supplemental Fat Source on Production, Immunity, Hepatic Gene Expression, and Metabolism of Periparturient Dairy Cows
Physical Description: 1 online resource (297 p.)
Language: english
Creator: Amaral, Bruno
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: dairy, gene, immunity, metabolism
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: Experiments using periparturient Holstein cows were conducted to evaluate how supplemental fat sources enriched in specific fatty acids affected production, immunity, hepatic gene expression, and metabolism of periparturient dairy cows. In Experiment 1, fat supplements enriched with C18:1 (sunflower oil), Ca salt of trans C18:1, C18:2 (Ca salt of palm and soybean oils), or C18:3 (linseed oil) were fed (1.35 to 1.75% of dietary DM) in isolipid diets from 30 d before to 105 d post calving to 22 primiparous and 32 multiparous animals. Cows fed C18:3 tended to produce more 3.5% fat-corrected milk due to an improvement in concentration of milk fat compared to cows fed the C18:2 source. Supplementation with trans C18:1 increased trans C18:1 in plasma, milk fat, and liver fat. Supplementation with C18:2 increased C18:2 in plasma and milk fat. Supplementation with C18:3 increased C18:3 in plasma, milk fat, and liver fat. Animals fed C18:3 had greater plasma NEFA concentrations at wk 2 and 5 postpartum which were accompanied by upregulation of mRNA pyruvate carboxylase and phosphoenolpyruvate carboxykinase in the liver during this same time period. Concentrations of plasma IGF-1 and expression of hepatic IGFBP-3 mRNA increased at a faster rate postpartum for animals fed C18:2 or C18:3 compared to those fed cis or trans C18:1; this was accompanied by a faster rate of increase for plasma insulin of multiparous cows fed C18:2 or C18:3 sources. Primiparous cows supplementated with C18:3 had fewer neutrophils in the uterine flushing at 40 d postpartum. Trans C18:1 may have had immunostimulatory effects as evidenced by increasing concentrations of plasma acid soluble protein and haptoglobin of primiparous cows compared to those fed cis C18:1. In Experiment 2, fat supplements enriched with C18:2 (Ca salt of safflower oil) or C20:5 and C22:6 (Ca salt of palm and fish oils) were fed (1.5% of dietary DM) as well as a no-fat supplement control diet from 34 d before to 49 d post calving to 16 primiparous and 29 multiparous animals. Animals fed fish oil tended to consume less DM (% of body weight) and produce less milk fat compared to animals fed C18:2. Mean values for dry matter intake prepartum, milk yield, milk protein yield and concentration, body weight, body condition score, and plasma concentrations of glucose, nonesterified fatty acids, beta hydroxybutyrate, and prostaglandin F metabolite were unchanged across the 3 diets. Concentrations of plasma progesterone increased earlier in primiparous cows fed fish oil compared to safflower oil fed cows and return to first ovulation was improved by 6 day across parities. Consumption of fish oil appeared to have immunosuppressive effects. A greater proportion of the animals fed fish oil were diagnosed with a more severe case of metritis at 5 and 10 d postpartum, had lower blood concentrations of white blood cells and neutrophils and had circulating neutrophils that consumed fewer E. coli per neutrophil on -18, 0, 7, and 40 d postpartum. Primiparous cows fed fish oil had lower plasma concentrations of ceruloplasmin. In addition, animals fed fish oil had circulating lymphocytes that produced fewer cytokines when isolated and stimulated in vitro on 10, 20, and 30 d postpartum. On the other hand, the C18:2 fat source had immunostimulatory effects. Cows had a greater humoral response of IgG concentrations in serum postpartum to repeated ovalbumin injections, did not experience the decrease in concentration of blood neutrophils at 7 d postpartum that occurred in the other treatments, and multiparous cows had increased fibrinogen concentrations in plasma. Based upon greater plasma concentrations of acute phase proteins, primiparous cows were under greater stress from parturition and lactation compared to multiparous cows. In conclusion enrichement of the diet with specific fatty acids during the periparturient period were reflected in the incorporation of these fatty acids into different tissues. Omega-3 fatty acids attenuated immune responses compared to omega-6 supplementation and shortened return to first ovulation.
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 Bruno Amaral.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Staples, Charles R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022802:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022802/00001

Material Information

Title: Effect of Supplemental Fat Source on Production, Immunity, Hepatic Gene Expression, and Metabolism of Periparturient Dairy Cows
Physical Description: 1 online resource (297 p.)
Language: english
Creator: Amaral, Bruno
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: dairy, gene, immunity, metabolism
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: Experiments using periparturient Holstein cows were conducted to evaluate how supplemental fat sources enriched in specific fatty acids affected production, immunity, hepatic gene expression, and metabolism of periparturient dairy cows. In Experiment 1, fat supplements enriched with C18:1 (sunflower oil), Ca salt of trans C18:1, C18:2 (Ca salt of palm and soybean oils), or C18:3 (linseed oil) were fed (1.35 to 1.75% of dietary DM) in isolipid diets from 30 d before to 105 d post calving to 22 primiparous and 32 multiparous animals. Cows fed C18:3 tended to produce more 3.5% fat-corrected milk due to an improvement in concentration of milk fat compared to cows fed the C18:2 source. Supplementation with trans C18:1 increased trans C18:1 in plasma, milk fat, and liver fat. Supplementation with C18:2 increased C18:2 in plasma and milk fat. Supplementation with C18:3 increased C18:3 in plasma, milk fat, and liver fat. Animals fed C18:3 had greater plasma NEFA concentrations at wk 2 and 5 postpartum which were accompanied by upregulation of mRNA pyruvate carboxylase and phosphoenolpyruvate carboxykinase in the liver during this same time period. Concentrations of plasma IGF-1 and expression of hepatic IGFBP-3 mRNA increased at a faster rate postpartum for animals fed C18:2 or C18:3 compared to those fed cis or trans C18:1; this was accompanied by a faster rate of increase for plasma insulin of multiparous cows fed C18:2 or C18:3 sources. Primiparous cows supplementated with C18:3 had fewer neutrophils in the uterine flushing at 40 d postpartum. Trans C18:1 may have had immunostimulatory effects as evidenced by increasing concentrations of plasma acid soluble protein and haptoglobin of primiparous cows compared to those fed cis C18:1. In Experiment 2, fat supplements enriched with C18:2 (Ca salt of safflower oil) or C20:5 and C22:6 (Ca salt of palm and fish oils) were fed (1.5% of dietary DM) as well as a no-fat supplement control diet from 34 d before to 49 d post calving to 16 primiparous and 29 multiparous animals. Animals fed fish oil tended to consume less DM (% of body weight) and produce less milk fat compared to animals fed C18:2. Mean values for dry matter intake prepartum, milk yield, milk protein yield and concentration, body weight, body condition score, and plasma concentrations of glucose, nonesterified fatty acids, beta hydroxybutyrate, and prostaglandin F metabolite were unchanged across the 3 diets. Concentrations of plasma progesterone increased earlier in primiparous cows fed fish oil compared to safflower oil fed cows and return to first ovulation was improved by 6 day across parities. Consumption of fish oil appeared to have immunosuppressive effects. A greater proportion of the animals fed fish oil were diagnosed with a more severe case of metritis at 5 and 10 d postpartum, had lower blood concentrations of white blood cells and neutrophils and had circulating neutrophils that consumed fewer E. coli per neutrophil on -18, 0, 7, and 40 d postpartum. Primiparous cows fed fish oil had lower plasma concentrations of ceruloplasmin. In addition, animals fed fish oil had circulating lymphocytes that produced fewer cytokines when isolated and stimulated in vitro on 10, 20, and 30 d postpartum. On the other hand, the C18:2 fat source had immunostimulatory effects. Cows had a greater humoral response of IgG concentrations in serum postpartum to repeated ovalbumin injections, did not experience the decrease in concentration of blood neutrophils at 7 d postpartum that occurred in the other treatments, and multiparous cows had increased fibrinogen concentrations in plasma. Based upon greater plasma concentrations of acute phase proteins, primiparous cows were under greater stress from parturition and lactation compared to multiparous cows. In conclusion enrichement of the diet with specific fatty acids during the periparturient period were reflected in the incorporation of these fatty acids into different tissues. Omega-3 fatty acids attenuated immune responses compared to omega-6 supplementation and shortened return to first ovulation.
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 Bruno Amaral.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Staples, Charles R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022802:00001


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1 EFFECT OF SUPPLEMENTAL FAT SOURCE ON PRODUCTION, IMMUNITY, HEPATIC GENE EXPRESSION, AND METABOLISM OF PERIPARTURIENT DAIRY COWS By BRUNO CESAR DO AMARAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Bruno Cesar do Amaral

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3 To my wonderful and lovely wife Michelle and my beautiful littl e princess Natalie for being with me during this journey, for supporting me and for being the reason of my life. Dedico aos meus pais Joo e Maria da Penha pelos exemplos de vida, suporte e incenti vo que proporcionaram meu sucesso nesta etapa

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4 ACKNOWLEDGMENTS I thank m y advisor Dr. Charles Staples for allowing me to join his program and for supporting me to present my research at the American Dairy Science Association national meetings. I also want to extend my appreci ation to the committee members Dr. William Thatcher, for inspiring and implanting the pass ion for knowledge. Dr. Thatchers guidance was fundamental to my education; Dr. Lokenga Badi nga, for his help on the radioimmuno assays, for always being available to answer questions about my research a nd for the opportunity to use his lab for several analysis; Dr. Gbola Adesogan, fo r teaching the courses on advanced methods in nutrition technology and forage and quality evalua tion, for his guidance and friendship. I deeply appreciated Dr. Adesogans reco mmendation of not overloading my course work for the spring 2005 semester in order to balance my time with my family when Natalie was born. I will never forget this; and Dr. Carlos Risco for his valuable contributions and for al ways being willing to help. Thank go to Dr. Santos for sharing his deep knowledge in the field of nutrition and interpreting the data for my trials. Your cont ributions were essentia l for this dissertation. Thanks go to my wife Michelle for being so supportive during this journey and for making my PhD program so much easier. I also want to thank my brothers Alexandr e e Marco for cheering for me and being proud of the little things I did. They gave me power to work harder. Thanks go to my parents in law (Nergismar and Maria) for helping and being with us during my PhD program. I deeply appreciate that. Thanks go to my cumpadre and cumadre Manoel and Zaza for being with us during this journey. You are unforgettable. Thanks go to IFAS for supporting me with the Alumni Fellowship, to the Animal Science Graduate Student Association for giving me the opportunity to be a member of this group for 2

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5 years and learn how to work as a team, and to the Graduate Stude nt Council for giving financial assistance to present my research at the Amer ican Dairy Science Association annual meetings. Thanks go to Faith Cullens for being such a nice friend and my mommy when I first came to Gainesville. Your friendship is precious. Thanks go to Dr. Head for advising me on ho w to apply for my Ph.D. and for being a wonderful friend and a source of knowledge in several fields. Thanks to Joyce Hayen (Mommy) for all her help on the milk producti on data input, as well as her guidance, and suggestions in all my trials. Your suggestions were valuable. I also would like to thank Sergei Sennikov for helping me in my analysis and being a nice friend. Thanks to Werner Collante for his help on the radio immunoassays and mRNA analysis. His help and friendship facilitated my learning and made the long hours in the lab a fun time. Thanks go to Sam Kim for his valuable friends hip and for teaching me all the fatty acid analysis. Your friendship was wonderful. Thanks go to all the friends I made at the An imal Science Department and in Gainesville that direct or indirectly helped in research and staying in Gaines ville: Maria Padua (BP), Cristina Caldari-Torres, Carlos Alosilla (irmao), Jessi ca Belsito, Sergio Madrid, Marcio Liboni, Luciano and Aline Bonilla, Davi Brito, Reinaldo C ooke, Flavio, Luciano, Andre Pedroso (txutxu prexeca) and his family, Deans fam ily, Spinozas family and others. Thanks go to the all interns in Dr. Staples lab that helped with my research: Ocilon, Ozana, Katherine, and Jeniset. Thanks go to Mary Russel, David Armstrong, Johnnie Salvino, Jerry Langford, Larry Ooten, Stephanie Ooten, Thomas Orton, Thom as Oyster, Leon Sweat, Walt Thon, Robert Tillman, Kevin Turner, Jimmy Underwood in memmorium, Harley Wagenseller, Don

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6 Ferguson, Carrie Bradley, Carl Sapp (David ), Charles Anderson, Grady Byers (Pombaa), James Dunn, Ronald Miller, Sherry Hay, Leslie Henderson, Patty Best, Richard Braun, William Burgus, Patricia Cacace, Clinton Cathcart, Eric Diepersloot, Judith Fowler, Molly Gleason, Martina Griffin, Elese Guerrin, Brad Henderson, Jesse Hooten, James Klema, Brandy Kuba, Charlotte Kuczenska, Caffnioly Menese, Deborah Reed, Mose White, and Latasha Williams. Without all of you guys from the Dairy Research Unit, my degree would not have been possible. I want to thank Dr. Miles for making my stay at the University of Florida IFAS Animal Sciences Department funny and enjoyable. Ou r friendship will be remembered forever. Thank go to my Mom (Penha) for all the beau tiful messages she sent me during the Ph.D. program and gave me power to work even harder. Thank go to Sabrina Robinson for all your help and the high fives and Go Gators. I will miss that. Thanks also to Darlene Woodard in memmorium and Delores Bradshaw for warming my early mornings with a happy and friendly good morning and Go Gators cheer. I want to thank to Dr. Bates for all his help with the ELISA assays and for reading this dissertation and givi ng me his inputs. Thanks go to Sha Tao and Jacob Bubolz for helping me with Dr. Dahls experiment at the farm and allowing me more time to work on th e dissertation. You were a big part of this dissertation. Thank go to all cows that gently accepted to be part of this work. I deeply appreciated all you have done for me.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES .........................................................................................................................11LIST OF FIGURES .......................................................................................................................13LIST OF ABBREVIATIONS ........................................................................................................ 17ABSTRACT ...................................................................................................................... .............20 CHAP TER 1 INTRODUCTION .................................................................................................................. 232 LITERATURE REVIEW .......................................................................................................27Fatty Acids Nomenclature ...................................................................................................... 27Fatty Acid Metabolism ......................................................................................................... ..28Fatty Acid Sources ..................................................................................................................33Effect of supplemental fat on feed intake and production ...................................................... 34Effect of Supplemental Fat S ource on Fatty Acid Profile ......................................................37Milk .................................................................................................................................37Plasma ........................................................................................................................ ......41Liver ......................................................................................................................... .......43Caruncle ...........................................................................................................................44Effect of Supplemental Fat Sour ce on Hormones and Metabolites ........................................45Prostaglandin F2 .............................................................................................................45Progesterone .................................................................................................................. ..46Growth hormone and IGF-1 ............................................................................................47Glucose ....................................................................................................................... .....50Insulin ....................................................................................................................... .......51Nonesterified fatty acid ...................................................................................................52Effect of Supplemental Fat Source on Health and Immunity ................................................. 54Uterine Health .................................................................................................................54Production of Cytokines by Lymphocytes ...................................................................... 57Humoral Response ...........................................................................................................58Acute Phase Proteins .......................................................................................................59

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8 3 EFFECT OF DIETS ENRICHED IN DIFFER ENT FATTY ACIDS ON PLASMA, MILK, AND LIVER FATTY ACID PROFILE OF LACTATING HOLSTEIN COWS DURING SUMMER ..............................................................................................................67Abstract ...................................................................................................................... .............67Introduction .................................................................................................................. ...........68Material and Methods .............................................................................................................69Animals, Treatments, and Sampling ............................................................................... 69Sample Collection and Analysis ...................................................................................... 70Statistical Analysis .......................................................................................................... 73Results and Discussion ........................................................................................................ ...74DMI, Milk Production, and Milk Composition ............................................................... 74Fatty Acid Profile in Milk, Blood, and Liver .................................................................. 76Milk ..........................................................................................................................76Plasma ...................................................................................................................... 81Liver ......................................................................................................................... 84Conclusions .............................................................................................................................884 EFFECT OF DIETS ENRICHED IN DI FFER ENT FATTY ACIDS ON HORMONES, METABOLITES, ACUTE PHASE PROTEINS, AND HEPATIC GENE EXPRESSION OF LACTATING HOL STEIN COWS DURING SUMMER .................... 108Abstract ...................................................................................................................... ...........108Introduction .................................................................................................................. .........109Material and Methods ...........................................................................................................110Animals, Treatments, and Sampling ............................................................................. 110Sample Collection and Analysis .................................................................................... 111Statistical Analysis .......................................................................................................... ......115Results and Discussion ........................................................................................................ .116Metabolites and Hormones ............................................................................................ 116Glucose ................................................................................................................... 116Blood urea nitrogen ................................................................................................ 117NEFA ..................................................................................................................... 117Insulin ..................................................................................................................... 119Growth hormone and insulin -like growth factor-1 ................................................ 121Insulin-like growth factor / growth hormone ratio ................................................. 124Progesterone ........................................................................................................... 125Prostaglandin F2 metabolite ..................................................................................125Gene Expression ............................................................................................................126Pyruvate carboxylase and phos phoenol pyruvate carboxykinase .......................... 126Insulin-like growth factor family ........................................................................... 127Uterine Health ...............................................................................................................128Acute Phase Proteins .....................................................................................................129Conclusions ...........................................................................................................................131

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9 5 EFFECT OF OMEGA-3 AND OMEGA6 SUP PLEMENTATION ON ACUTE PHASE PROTEINS IN PLASMA OF PERIPARTURIENT DAIRY COWS .................... 170Abstract ...................................................................................................................... ...........170Introduction .................................................................................................................. .........171Material and Methods ...........................................................................................................172Animals, Treatments, and Sampling ............................................................................. 172Sample Analysis ............................................................................................................174Statistical Analysis ........................................................................................................ 175Results and Discussion ........................................................................................................ .175Conclusions ...........................................................................................................................1786 EFFECT OF OMEGA-3 AND OMEGA-6 SUP LLEMENTATION ON IMMUNITY AND PERFORMANCE OF PERIPA RTURIENT DAIRY COWS .................................... 187Abstract ...................................................................................................................... ...........187Introduction .................................................................................................................. .........188Material and Methods ...........................................................................................................190Animals, Treatments, and Sampling ............................................................................. 190Sample Collection and Analysis .................................................................................... 191Immune Status ...............................................................................................................195Neutrophil function ................................................................................................195Bovine peripheral blood mononuclear cell (PBMC) isolation and stimulation ..... 195Ovalbumin challenge .............................................................................................. 197Vaginoscopy ........................................................................................................... 198Uterine cytology ..................................................................................................... 199Statistical Analysis ........................................................................................................ 200Results and Discussion ........................................................................................................ .201DMI, Milk Production, and Milk Composition ............................................................. 202Fatty Acid Profile ..........................................................................................................204Milk ........................................................................................................................204Caruncle .................................................................................................................210Hormones and Metabolites ............................................................................................212Glucose ................................................................................................................... 212Blood urea nitrogen ................................................................................................ 213Beta hydroxy butyric acid ......................................................................................213Nonesterified fatty acid ..........................................................................................214Progesterone ........................................................................................................... 214Neutrophil Concentration a nd Function in Whole Blood .............................................. 216White Blood Cells in Whole Blood ............................................................................... 219Rectal Temperature .......................................................................................................219Uterine Flushing ............................................................................................................219Cytokines Produced By Lymphocytes .......................................................................... 220Humoral Response .........................................................................................................222Vaginoscopy .................................................................................................................. 223Conclusions ...........................................................................................................................225

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10 7 GENERAL DISCUSSION AND CONCLUSION ...............................................................265LIST OF REFERENCES .............................................................................................................274BIOGRAPHICAL SKETCH .......................................................................................................297

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11 LIST OF TABLES Table page 2-1 Common fatty acids ...........................................................................................................622-2 Major fatty acid compositi on of some fat sources. ............................................................633-1 Ingredient and chemical composition of TMR fed to Holstein cows during the prepartum period. ............................................................................................................. ..903-2 Ingredient and chemical composition of TMR fed to Holstein cows during the postpartum period. ............................................................................................................ .913-3 Fatty acid profile of the fat supplements. ...........................................................................923-4 Dry matter intake, milk yield, milk co mposition, feed efficiency, energy balance, postpartum body weight, and postpartum body condition score of Holstein cows. .......... 933-5 Effect of supplemental fat source on con centration of identified fatty acids of milk fat. ......................................................................................................................................953-6 Effect of supplemental fat source on concen tration of identified fatty acids of plasma. ... 973-7 Effect of supplemental fat source on liv er fatty acid profile on days 2, 14, and 28 postpartum..........................................................................................................................994-1 Concentration of plasma metabolites ............................................................................... 1324-2 Concentration of plasma hormones .................................................................................1334-3 Log of total neutrophil c ount in the uterine flusing and acute phase proteins. ................ 1344-4 Least squares means for hepatic PC, PEPCK, IGF-2, IGFBP-2, and IGFBP-3 mRNA abundance ..................................................................................................................... ...1356-1 Ingredient and chemical composition of TMR fed to Holstein cows during the prepartum period. ............................................................................................................. 2266-2 Ingredient and chemical composition of TMR fed to Holstein cows during the postpartum period. ........................................................................................................... 2276-3 Fatty acid profile of the fat supplements. .........................................................................2286-4 Dry matter intake, milk yield, milk composition, feed effi ciency, postpartum body weight, and postpartum body conditi on score of Holstein cows. .................................... 2296-5 Effect of supplemental fat source on con centration of identified fatty acids of milk fat. ....................................................................................................................................231

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12 6-6 Effect of supplemental fat source on c oncentration of identif ied fatty acids of caruncle. ..................................................................................................................... ......2326-7 Concentration of plasma metabolites ............................................................................... 2346-9 Uterine cytology of Holstein cows .................................................................................. 2366-10 Incidence and severity of metritis as measured by va ginoscopy scores at 5 and 10 DIM of Holstein cows. ..................................................................................................... 237

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13 LIST OF FIGURES Figure page 2-1 Structural formula of linoleic (omega-6 ) and lino lenic acid (omega-3) linoleic acid (numeric designation). .......................................................................................................642-2 Parent fatty acid and major metabolites w ithin each of the three omega fatty acid families .............................................................................................................................653-1 Least squares means for dry matter intake ....................................................................... 1013-2 Least squares means for dry matter intake as % of BW .................................................. 1023-3 Least squares means for milk yield ................................................................................ 1033-4 Least squares means for body weight .............................................................................. 1043-5 Least squares means for BCS. ......................................................................................... 1053-6 Least squares mean s for energy balance .......................................................................... 1063-7 Concentration of C18:2 in liver of Holstein cows. .......................................................... 1074-1 Least square means for plasma glucose. .......................................................................... 1364-2 Least square means for plasma BUN ............................................................................... 1374-3 Least square means for plasma NEFA ............................................................................. 1384-4 Polynomial regression curves (first order) of concentrations of plasma insulin .............1394-5 Polynomial regression curves (first order) of concentrations of plasma insulin ........... 1404-6 Polynomial regression curves (first order) of concentrations of plasma insulin .............1414-7 Polynomial regression curves (first order) of concentrations of plasma IGF-1 ............... 1424-8 Polynomial regression curves (first order) of concentrations of plasma IGF-1 from lactating Holstein cows ....................................................................................................1434-9 Polynomial regression curves (second order) of concentrations of plasma accumulated progesterone ................................................................................................ 1444-10 Least square means for days to first ovulation ................................................................. 1454-11 Polynomial regression curves (first order) of concentrations of plasma growth hormone ....................................................................................................................... ....146

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14 4-12 Polynomial regression curves (first order) of concentrations of plasm a growth hormone ....................................................................................................................... ....1474-13 Polynomial regression curves (first order) of concentrations of plasma growth hormone ....................................................................................................................... ....1484-14 Polynomial regression curves (seco nd order) of ratio of IGF-1/GH ............................... 1494-15 Polynomial regression curves (seco nd order) of ratio of IGF-1/GH ............................... 1504-16 Polynomial regression curves (seco nd order) of ratio of IGF-1/GH f .............................1514-17 Polynomial regression curves (second orde r) of concentrations of plasma PGFM ......... 1524-18 Effect of supplemental fat source on pyruvate carboxylase mRNA expression in liver of Holstein cows ......................................................................................................1534-19 Effect of supplemental fat source on pyruvate carboxylase mRNA expression in liver of Holstein cows. .....................................................................................................1544-20 Effect of supplemental fat source on phosphoenolpyruvate carboxykinase mRNA expression in liver of Holstein cows ................................................................................1554-21 Effect of supplemental fat sour ce on phosphoenolpyruvate carboxykinase mRNA expression in liver of Holstein cows ................................................................................1564-22 Effect of supplemental fat source on in sulin-like growth fact or-2 mRNA expression in liver of Holstein cows.. ................................................................................................1574-23 Effect of supplemental fat source on in sulin-like growth fact or-2 mRNA expression in liver of Holstein cows.. ................................................................................................1584-24 Effect of supplemental fat source on in sulin-like growth fact or-2 (IGF-2) mRNA expression in liver of Holstein cows ................................................................................1594-25 Effect of supplemental fat source on in sulin-like growth fact or binding protein-2 mRNA expression in liver of Holstein cows ................................................................... 1604-26 Effect of supplemental fat source on in sulin-like growth fact or binding protein-2 mRNA expression in liver of Holstein cows ................................................................... 1614-27 Effect of supplemental fat source on in sulin-like growth fact or binding protein-2 mRNA expression in liver of Holstein cows ................................................................... 1624-28 Effect of supplemental fat source on in sulin-like growth fact or binding protein-3 mRNA expression in liver of Holstein cows ................................................................... 1634-29 Concentrations of plasma haptoglobin ............................................................................164

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15 4-30 Concentrations of plasma haptoglobin .............................................................................1654-31 Mean concentration of plasma haptoglobin ..................................................................... 1664-32 Concentration of plas ma acid soluble protein .................................................................. 1674-33 Mean concentration of pl asma acid soluble protein ........................................................1684-34 Mean concentration of pl asma acid soluble protein ........................................................1695-1 Plasma concentrations of acute phase proteins of Holstein cows .................................... 1805-2 Effect of supplemental fat source on c oncentrations of prostaglandin F metabolite (PGFM) ........................................................................................................................ ....1815-3 Mean plasma concentrations of haptoglobin ...................................................................1825-4 Plasma concentrations of ceruloplasmin .......................................................................... 1835-5 Plasma concentration of acid soluble protein .................................................................. 1845-6 Plasma concentrations of acid soluble protein ................................................................. 1855-7 Plasma concentrations of fibrinogen ................................................................................1866-1 Least squares means for dry matter intake ....................................................................... 2386-2 Least squares means for milk yield .................................................................................. 2396-3 Least squares means for 3.5% fat corrected milk (FCM) ................................................ 2406-4 Least squares means for concentration of fat in m ilk of Holsteins cows .........................2416-5 Least squares mean s for energy balance .......................................................................... 2426-6 Least squares means for feed efficiency .......................................................................... 2436-7 Least squares means for body weight .............................................................................. 2446-8 Least squares means for body weight .............................................................................. 2456-9 Least squares means for body condition score ................................................................ 2466-10 Least squares means for con centration of plasma glucose .............................................. 2476-11 Least squares means for plasma glucose. ........................................................................ 2486-12 Least squares means for plasma BUN ............................................................................. 2496-13 Least squares means for plasma BHBA ........................................................................... 250

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16 6-14 Least squares means for plasma BHBA ........................................................................... 2516-15 Least squares means for plasma NEFA .......................................................................... 2526-16 Least squares means for plasma NEFA ........................................................................... 2536-17 Least squares means for acucumulated progesterone ...................................................... 2546-18 Least squares means for concentration of neutrophils in blood .......................................2556-19 Least squares means for per centage of neutrophils that carried out phagocytosis of E. coli in vitro in blood ........................................................................................................ .2566-20 Least squares means for median fluore scence intensity of ne utrophils in blood ............. 2576-21 Least squares means for oxidative burst of neutrophils in blood .....................................2586-22 Least squares means for concentration of white blood cells in blood ............................. 2596-23 Least squares means for rectal temperature ..................................................................... 2606-24 Least squares means for production of tumor necrosis factor alpha (TNF) produced by lymphocytes ................................................................................................................2616-25 Least squares means for pr oduction of interferon gamma (IFN) produced by lymphocytes ................................................................................................................... ..2626-26 Least squares means for pr oduction of interferon gamma (IFN) produced by lymphocytes ................................................................................................................... ..2636-27 Least squares means for production of immunoglobulin G ............................................. 264

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17 LIST OF ABBREVIATIONS ACC acetyl CoA carboxylase ACTH adrenocorticotropic hormone ADF acid detergent fiber ATGL adipose triglyceride lipase BCS body condition score BHBA beta-hydroxy butyric acid BUN blood urea nitrogen BW body weight Ca calcium CaVeg Ca salts of vegetabl e oil rich on linoleic acid CL corpus luteum CLA conjugated linoleic acid CO control CREB cAMP response element binding protein CV coefficient of variation DHA docohexanoic acid DHR dihydrorhodamine 123 DIM days in milk DM dry matter DMI dry matter intake ECM energy-corrected milk EFA essential fatty acid ELISA enzyme-linked immunosorbent assay EPA eicosapentaenoic acid

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18 FA fatty acid FAS fatty acid synthase FCM fat-corrected milk FPCM fat and protein-corrected milk GH growth hormone HCL hydrochloric acid HOSFO high oleic sunflower oil Hp haptoglobin HSL hormone sensitive lipase IGFBP insulin-like growth factor binding protein IGF insulin-like growth factor IgG immunoglobulin G IL interleukin INFinterferon gamma LPS lypopolysaccharide LSO linseed oil MUFA monounsaturated fatty acid NADPH nicotinamide adenine dinucleotide phosphate NDF neutral detergent fiber NEB negative energy balance NEFA non esterified fatty acid OVA ovalbumin PBMC peripheral blood mononuclear cell PBS phosphate buffer solution PC pyruvate carboxylase

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19 PEPCK phosphoenol pyruvate carboxykinase PG prostaglandin PGE2 prostaglandin E2 PGF2 prostaglandin F2 PGFM prostaglandin F2 metabolite PGH prostaglandin endoperoxide H PGHS prostaglandin e ndoperoxide H synthase PMA phorbol 12-myristate, 13-acetate PUFA polyunsaturated fatty acid RXR retinoid X receptor SBO soybean oil SCC somatic cell counts SREBP-1 sterol regulatory element-binding protein 1 TAI timed artificial insemination TG triglyceride TNFtumor necrosis factor alpha TRANS Ca salts of tr ans octadecenoic acid WBC white blood cells

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20 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF SUPPLEMENTAL FAT SOURCE ON PRODUCTION, IMMUNITY, HEPATIC GENE EXPRESSION, AND METABOLISM OF PERIPARTURIENT DAIRY COWS By Bruno Cesar do Amaral December 2008 Chair: Charles R. Staples Major: Animal Sciences Experiments using periparturient Holstein cows were conducted to evaluate how supplemental fat sources enriched in specific fa tty acids affected produc tion, immunity, hepatic gene expression, and metabolism of periparturient dairy cows. In Experiment 1, fat supplements enriched with C18:1 (sunflower oil), Ca salt of trans C18:1, C18:2 (Ca salt of palm and soybean oils), or C18:3 (linseed oil) were fed (1.35 to 1.75% of dietary DM) in isolipid diets from 30 d before to 105 d post calving to 22 primiparous and 32 multiparous animals. Cows fed C18:3 tended to produce more 3.5% fat-corrected milk due to an improvement in concentration of milk fat compared to cows fed the C18:2 source. Supplementation with trans C18:1 increased trans C18:1 in plasma, milk fat, and liver fat. Suppl ementation with C18:2 increased C18:2 in plasma and milk fat. Supplementation with C18:3 increased C18:3 in plasma, milk fat, and liver fat. Animals fed C18:3 had greater pl asma NEFA concentrations at wk 2 and 5 postpartum which were accompanied by upregulation of mRNA pyruvate carboxylase and phosphoenolpyruvate carboxykinase in the liver during th is same time period. Concentrations of plasma IGF-1 and expression of hepatic IGFBP-3 mRNA increased at a faster rate postpartum for animals fed C18:2 or C18:3 compared to those fed cis or trans C18:1; this was accompanied by a faster rate of increase for plasma insulin of multiparous cows fed C18:2 or C18:3 sources. Primiparous

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21 cows supplementated with C18:3 had fewer neutrophils in the uterine flushing at 40 d postpartum. Trans C18:1 may have had immunostimulator y effects as evidenced by increasing concentrations of plasma acid soluble protein a nd haptoglobin of primiparous cows compared to those fed cis C18:1. In Experiment 2, fat supplements enriched with C18:2 (Ca salt of saffl ower oil) or C20:5 and C22:6 (Ca salt of palm and fish oils) were fed (1.5% of dietary DM) as well as a no-fat supplement control diet from 34 d before to 49 d post calving to 16 primiparous and 29 multiparous animals. Animals fed fish oil te nded to consume less DM (% of body weight) and produce less milk fat compared to animals fe d C18:2. Mean values for dry matter intake prepartum, milk yield, milk protein yield a nd concentration, body weight, body condition score, and plasma concentrations of glucose, nonest erified fatty acids, beta hydroxybutyrate, and prostaglandin F metabolite were unchanged across the 3 diets. Concentrations of plasma progesterone increased earlier in primiparous cows fed fish oil compared to safflower oil fed cows and return to first ovulation was improved by 6 day across parities. C onsumption of fish oil appeared to have immunosuppressi ve effects. A greater proportion of the animals fed fish oil were diagnosed with a more severe case of me tritis at 5 and 10 d postpartum, had lower blood concentrations of white blood cells and neutr ophils and had circula ting neutrophils that consumed fewer E. coli per neutrophil on -18, 0, 7, and 40 d postpartum. Primiparous cows fed fish oil had lower plasma concentrations of cer uloplasmin. In addition, animals fed fish oil had circulating lymphocytes that produced fewer cytokines when is olated and stimulated in vitro on 10, 20, and 30 d postpartum. On the other hand, the C18:2 fat source had immunostimulatory effects. Cows had a greater humoral response of IgG concentrations in serum postpartum to repeated ovalbumin injections, did not experi ence the decrease in concentration of blood

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22 neutrophils at 7 d postpartum that occurred in the other treatments, and multiparous cows had increased fibrinogen concentrations in plasma Based upon greater plasma concentrations of acute phase proteins, primiparous cows were unde r greater stress from parturition and lactation compared to multiparous cows. In conclusion enrichement of the diet with specific fatty acids during the periparturient period were reflected in the incorporation of these fatty acids into different tissues. Omega-3 fatty acids attenuated immune responses compar ed to omega-6 supplementation and shortened return to first ovulation.

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23 CHAPTER 1 INTRODUCTION One of the most challenging periods in the life o f the amazing dairy cow is the transition from pregnancy to lactation. During this peri od the dairy cow undergoes dramatic physiological changes and most of the metabolic and infectious diseases (ketosis, hepatic lipidosis, displaced abomasum, hypocalcemia, retained fetal membranes, and mastitis) take place in part due to a suppression of the immune syst em (Goff and Horst, 1997). There is a marked reduction in dry matter intake as parturition approaches and the increase in intake in the early postpartum period is not sufficient to support milk production. As a consequence of the sharp peripartal decrease in DMI coupled with the progressive energy and nutrient demand by the growing fetus, fetal me mbranes, and the uter us as well as by the mammary gland to initiate lactogene sis, the transition da iry cow develops seve re negative energy balance (Goff and Horst, 1997; Jorritsma et al., 2003) that may reach -16 Mcal/d (Doepel et al., 2002). In an attempt to supply energy for milk production, the cow uses body reserves, mainly adipose tissue, as a source of energy to support milk production. As a result, a greater concentration of nonesterified fatty acids is found in plasma of dairy cows around parturition and impacts the transition into lactation. Lipid supplementation is an excellent nutritiona l tool to influence several functions in different species. For instance, profile of plasma fatty acids is affected by different sources of lipid supplemented to dairy cows. Petit (2003) reported that mid l actation cows (29 wk postpartum) fed flaxseeds had greater concentrations of C18:3 and C20:5 and a lower n-6/n-3 ratio in plasma compared to cows fed sunflower seeds; however, concentrations of C20:3 and C20:5 were not affected. Gonthi er et al. (2005) reported that cows fed flaxseeds had greater concentration of C18:3 in plasma compared to cows fed a control diet without flaxseeds. Loor et

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24 al. (2005a) reported that cows fed linseed oil ha d increased concentration of C18:3 in plasma compared to cows fed sunflower oil but concentr ations of C20:3 and C20:5 were not different. Ponter et al. (2006) also reported an increase in plasma concen tration of C18:3 of cows fed linseed oil. The change in the fatty acid profile of plasma will affect the pool of fatty acids reaching the mammary gland which in turn influences the milk fatty acid profile. Kalscheur et al. (1997) fed a high oleic acid sunflower oil and a partially hydrogenated ve getable shortening enriched in trans C18:1 to lactating dairy cows at 3.7% of dietary DM. Concentrations of cis C18:1in milk fat were 28.5% and 25.9% for cows fed sunflowe r oil and shortening, respectively. An increased concentration of C18:1 trans isomers in milk fat was reported when Holstein cows were supplemented with C18:1 trans isomers (Griinari et al., 1998; Selberg et al., 2004). Cows fed Megalac-R (rich in C18:2) at 2.5% of dietary DM had greater con centration of C18:2 in milk fat compared to cows fed a saturated fat supplem ent (3.7 vs. 2.6%; Harv atine and Allen, 2006b). Bilby et al. (2006) reported that la ctating Holstein cows fed a Ca sa lt mixture of palm and fish oil (1.9% of dietary DM) from 17 to 94 12 DIM ha d greater incorporati on of C20:5 and C22:6 into milk fat compared to cows fed whole cottonseeds. In addition to influencing plasma and milk fatty acid profile, lipid supplementation also influenced the incorporation of the specific fatty acid into several tissues such as the endometrium. Bilby et al. (2006c) re ported that cows fed a Ca salt mixture of palm and fish oil (1.9% of dietary DM) from 17 DIM until 94 12 DIM had greater incorporation of C20:5 (0.10 vs. <0.01%) and C22:6 (1.42 vs. 0.92%) in endometrium compared to cows fed whole cottonseeds. Similarly, Childs et al. (2008) fed cro ssbred beef primiparous cows increasing amounts of a partially rumen-protected fish oil at 0, 1.04, 2.08 or 4.15% of dietary DM and

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25 reported a linear increase in the concentration of C20:5 and C22:6 in endometrial tissue collected at slaughter. In addition, Mattos et al. (2004) reported a 7and 5-fold increase in the concentration of C20:5 and C22:6, re spectively in the carunc le of dairy cows fed fish oil at 2% of dietary DM during the last 3 wk prior to calving. Arachidonic acid (an omega-6 fatty acid) is the precursor for PGF2 synthesis. Fatty acids of the omega-3 family compete with the omega-6 fatty acids for elongases and desaturases in the tissues. By changing the fatty acid profile of the e ndometrium with more precursor it is plausible to expect an increase in PGF2 synthesis. Ewes infused with ei ther soybean oil (50% C18:2) or olive oil (16% C18:2) had greater serum PGF metabolite concentra tions than ewes infused with saline (Burke et al., 1996). In postpartum beef primiparous cows, infusion of lipid containing 20% of soybean oil through the jugular vein increased systemic concentrations of C18:2 and PGF metabolite after oxyto cin injection (Filley et al., 1999). In contrast, by incorporating more omega-3 fatty acids (C20:5 and C22:6) into the tissues, the precursors for PGF2 synthesis are reduced. Cows fed fish oil at 2% of dietary DM from 3 wk prepartum calving until parturition and at 1.8% of dietary DM during the postpar tum period had reduced concentration of PGF metabolite around parturition compared to cows fed olive oil at the same inclusion rate for the pre and postpartum period (Mattos et al., 2004). In bovine endometrial cells, incubation with arachidonic acid increased secretion of PGF2 whereas C20:5 was inhi bitory (Mattos et al., 2003). This illustrates the competition of precu rsors for processing by the prostaglandin H synthase (PGHS) enzymes involved in prostanoid synthesis. The reduced secretion of PGF2 observed in cells incubated with C20:5 is likely a result of a shift of the PGHS pathway from synthesis of prostanoids from th e 2 series to synthesis of pros tanoids of the 3 series. In the presence of C20:5, less of the arachidonic acid present will be converted to PGF2

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26 The PGF2 is an important eicosanoid that regulat es corpus luteum lifespan and might influence retention of fetal membranes and subse quent uterine health (Santos et al., 2008) and immunocompetence of the cow (Thatcher et al ., 2006) especially around parturition when the immune system is suppressed (Goff and Horst, 1997). The objective of this dissertati on was to evaluate how differe nt dietary fat sources will influence fatty acid profile of milk and tissue, production, immunity, hepatic gene expression, and metabolism of periparturient dairy cows.

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27 CHAPTER 2 LITERATURE REVIEW Fatty Acids Nomenclature Just as am ino acids are the individual units making up the class of nutrients called proteins, so fatty acids are the ma jor individual units of measure of what is broadly called lipids. Just as each amino acid has a distinct structur e and function in protein building, so each fatty acid (FA) has a distinct struct ure and possibly function in meta bolism (Staples et al., 2002). Fatty acids are carboxylic acids with a car boxyl group (COOH) at one end and a methyl (CH3) group at the other end (Voet and Vo et, 2004). The FA may contain no double bond (saturated) or contain one or more double bonds ( unsaturated). When it contains two or more double bonds, it is said to be a polyunsaturated FA (PUFA). Generally the double bonds are spaced in the more common FA at intervals of three carbons (Table 2.1). When counting carbons in FA, the carboxyl (COOH) carbon is always carbon number 1. Alternativ ely letters of the Greek alphabet are used to refer to specific carbons in a FA. For example, the carbon next to the number one (carboxyl) carbon is always called the alpha ( ) carbon which is followed by beta ( ) carbon, gamma ( ) carbon and so on. On the other side, the terminal methyl end (CH3) is always designated the omega ( the last letter in the Greek al phabet) carbon regardless of the length of the FA (See Figure 2.1). This omega system was originated by Holman (Holman, 1960b). The double bond location is designated by counting carbons beginning at the number 1 carboxyl carbon. For example, linoleic acid contai ns 2 double bonds located between carbons 9 and 10 and 12 and 13 while linolenic acid cont ains 3 double bonds located between carbons 9 and 10, 12 and 13, and 15 and 16 (Figure 2.1). In the literature, linoleic acid is referred to as C18:2, n-6 (C18:2 -6) and linolenic acid is referred to as C18:3, n-3 (C18:3 -3). The n refers

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28 to the location of the first double bond from the terminal methyl group (omega carbon) (Table 2.1). There are several omega families but the most important are -9, -6, and -3. Each family has a parent FA that is converted to ot her biologically-active acids within the same omega family (Figure 2.2). The only parent family that can be made by body tissues is the -9 FA, oleic acid. The -6 and -3 parent compounds (linoleic and linol enic acids) cannot be synthesized by body tissues and therefore must be in the diet. Th us, linoleic and linolenic acids are regarded as essential fatty acids (EFA) because they ar e required for normal function but cannot be synthesized by body tissues. Even th ough arachidonic acid is synthesi zed from linoleic acid, it is also considered by some researchers as an EFA. Fatty Acid Metabolism In 1929, Burr and Burr first described a c ondition characterized by eczem a, loss of weight, increased water consumption and poor repr oduction in rats kept on a fat-free diet. These symptoms could be reversed by the addition of ce rtain PUFA, essential FA (EFA), to the diet. Later on it was recognized that there were at le ast three major EFA: linoleic, linolenic, and arachidonic acid. Linoleic is the most common but not necessarily the most important of the three (Alfin-Slater and Aftergood, 1971). Linoleic (18:2 -6) and linolenic (18:3 -3) acids can undergo further desaturation and chain elongation to give two series of derivatives, the -6 and -3 series (Figure 2.2). The enzyme systems involved in these processes appear to have been lost in metazoan evolution even though linoleic acid is essential for vertebrate and invertebrate nutr ition (Alfin-Slater and Aftergood, 1971). The enzymes necessary to synthesize the EFA from nonessential FA are present only in plants (Gro ff et al., 1995). Competitive i nhibition occurs between the -6 and -

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29 3 series of PUFA and the balance of de rivatives is determined by the ratio, 18:2 -6 and 18:3 -3 in the diet. Regarding the metabolism of EFA, it is im portant to emphasize the difference between ruminant and monogastric species. In monogast ric species, the FA composition of body fat reflects the FA composition of the diet. Ho wever in ruminants, ingested PUFA are biohydrogenated to a large extent by ruminal micr oorganisms, resulting in a more saturated body fat (McDowell, 2000). In a recent review about the effects of dietary FA on incorportation of long chain FA and conjugated linoleic acid in la mb, beef and pork meat, Raes et al. (2004) reported that an increase in the omega-3 FA content of animal meats can be achieved by including fish oil/fish meal in the diet. Diets enriched in linolenic acid resulted in an increased concentration of linolenic acid, eicosapentaenoi c acid (EPA), and docosapentaenoic acid (DHA) in the meat. Increasing DHA content in meat was mainly achieved when fish oil/fish meal was included in the animal diet. In most studies, an increased -3 FA content in the intramuscular fat was accompanied with a decreased -6 FA deposition, mainly due to a lower dietary -6 FA supply among the treatments. This resulted in a more favorable -6/ -3 FA ratio in the meat while the PUFA/saturated FA ratio was less affected. A South Carolina study (Lundy et al., 2004) used four different oil supplements (2.45% soybean oil (SBO), 2.75% calcium salt of SBO, 2.75% amide of SBO or 2.75% of a mixture of the calcium salt and amide (80:20 wt/wt) of SBO). Supplements contained similar concentrations of C18:2 and C18:3 fatty acids (54.7 and 6.2, 52.2 and 4.6, 52.5 and 4.7, and 51.1 and 4.1 g/100 g of fat supplement respectively for the treatm ents cited above). Ruminal biohydrogenation of 18:2 -6 ranged from 92 to 95% and did not di ffer among diets when ruminal biohydrogenation was calculated based on the total grams of FA lost from the rumen (C18:2 consumed minus

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30 C18:2 in omasal flow) as a per centage of FA consumed. Losses of C18:2 and C18:3 from the mouth to the duodenum in ruminant species averaged from 80 to 92%, respectively in a data set containing more than 100 observations from published studies (Doreau and Chilliard, 1997). The EFA can be metabolized by mammalian tissues. In the omega-6 family, linoleic acid (C18:2) undergoes desaturation ( 6 insertion of double bond at carbon number 6) to form linolenic acid (C18:3 -6) (Figure 2.2). Then -linolenic acid (C18: 3 omega-6) undergoes elongation (addition of 2 car bons) to produce dihomo-linolenic acid (C20:3 omega-6; member of the eicosanoids). This FA is desaturated ( 5 desaturase) and forms arachidonic acid (20:4 -6) that undergoes elongation to form adrenic acid (C22:4) and th en another desaturation ( 4) step occurs to produce EPA (C22:5, omega-6). The adre nic acid is the precursor of C20:4 omega-6 and C22:5 omega-6 in rats (Sprencher, 1967). Form ation of these fatty acids from linoleic and arachidonic acid has been demonstrated in vivo (Mead, 1961; Davis and Coniglo, 1966). In the omega-3 family, -linolenic acid (C18:3) undergoes desaturation ( 6 desaturase) to produce C18:4 that elongates and forms C20:4 -3. This FA is desaturated ( -5) and forms EPA (C20:5). The fatty acids C20:4 -3 and C20:5 -3 are classified as eicosanoids. The EPA elongates and forms C22:5 (member of doc osanoids) that undergoes desaturation ( -4) and forms DHA (C22:6) (docosanoids) (Figure 2.2). The addition of DHA and EPA to a culture of mixed ruminal microorganisms from ruminally fi stulated Holstein cows caused a reduction in C18:1 and C18:2 biohydrogenation compared to cows not fed FA (AbuGhazaleh and Jenkins, 2004a). Indeed, DHA is the compound in fish oil that promotes vaccenic acid accumulation in mixed ruminal cultures when incubated with linoleic acid (AbuGhazaleh and Jenkins, 2004b). In the omega-9 family, oleic acid (C18:1) undergoes desaturations and elongations to form several fatty acids (Figure 2.2). Eicosatrieno ic acid (C20:3 -9) is important to the

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31 triene:tetraene ratio (C20:3 -9/C20:4 -6). In addition, the concentr ation of eicosatrienoic acid formed from oleic acid is mark edly reduced when the diet is supplemented with linoleic or linolenic acid. Holman (1960a), studying the ratio of trienoic-tetraenoic acids in tissu e lipids of rats fed EFA-deficient diets, proposed the use of the C20:3/C20:4 ratio as a measure of a linoleic acid deficiency. Later on, Mohrhauer and Holman ( 1963) reported that a trie ne to tetraene ratio of 0.4 or less indicated that the animal was ingesting adequa te amounts of linoleic acid. The ingestion of linoleic acid at 1% of the calories promoted EFA ad equacy as judged by the ratio of C20:3/C20:4 in rats (Mohrhauer and Holman, 1963), in guinea pigs (Reid et al., 1964), and in swine (Sewell and McDowelll, 1966). When inges tion of C18:2 is < 1% of the calories, the conversion of linoleic acid to ar achidonic acid is depr essed, and the synthesi s of eicosatrienoic acid from oleic acid and from palmitoleic acid (C16:1 omega-7) is increased (Alfin-Slater and Aftergood, 1971). Arachidonic acid is approximately three times more effective than linoleic acid in preventing deficiency sumptoms since arachidonic acid maintains the C20:3/C20:4 ratio below 0.4 when it was fed at 0.3% of calories. However, there are instances when the measurement ratio is inappropriate For example, when the forma tion of C20:3 is limited owing to inactivity of the necessa ry desaturase or when si gnificant amounts of dietary -3 PUFA are present in the diet (Sanders, 1988). The extent to which the parent EFA is conve rted to more unsaturated derivatives also depends on the affinity of the parent FA for the other metabolic pathways. Chain length and degree of unsaturation infl uences the rate of oxida tion of PUFA and affinity for acyl transferases. The rate of oxidation of FA decreases with increasing chain length. T hus, linoleic acid is oxidized at a faster rate than -linoleic or arachidonic acid. However C18:3 -3 is oxidized at a faster rate than C18:2. This may be because C18: 3 is not readily incorporated into phospholipids

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32 and C20:4 and C22:6 are avidly incorporated into phospholipids and are poor substrates for oxidation (Sanders, 1988). The greater activity of arachi donic acid as an EFA was also shown by Alfin-Slater and Kaneda (1962) in studies where the effectivenes s of arachidonic acid in reducing the elevated plasma and liver cholesterol conc entration of cholesterol fed-rats was more than twice that of linoleic acid. Dietary cholesterol also affects the transformation of linoleic acid to arachidonic acid. When cholesterol was fed to rats, a marked decrease in PUFA was observed in liver lipids with a virtual disappearance of arachidonic acid from serum (Kle in, 1959). Cholesterol in the diet evidently interferes with the enzymes involved in the co nversion of linoleic acid to arachidonic acid in aortic le cithin since an increase in linoleic acid and dihomo-linolenic acid (C20:3) (Alfin-Slater and Kaneda, 1962; Alfin-Slater and Aftergood, 1968) and a decrease in arachidonic acid (Morin, 1968) has been re ported when cholesterol was fed. Species differences exist in the capacity to c onvert the parent EFA to their longer-chain derivative. For example, cats are unable to synt hesize C20:4 in sufficien t quantities and have a requirement for C20:4 because they lack 6 desaturase and only can form C20:4 from C18:2 via an alternative pathway involving 8 desaturation of C20:2 omega-6 (MacDonald et al. 1984). Regarding humans, it has been argued that the activity of 6 desaturase may be low under certain conditions and that th is metabolic block may be overridden by consuming C18:3 (Manku et al., 1982). In a study of vegans whose di ets are devoid of EFA, Sanders et al. (1978) suggested that the conversion of C18:2 to C20:4 occurs readily in man but that of C18:3 into C22:6 may be limited by a slow rate of 4 desaturation. Indeed, the proportion of C22:6 in plasma phospholipids was not increased by additiona l C18:3 in the diet (Sanders and Younger,

PAGE 33

33 1981). Moreover, dietary C20:5 failed to incr ease the proportion of C22:6 in plasma and erythrocyte phospholipids in human s (Von Schacky and Weber, 1985). In addition to the species differences, ther e are sex differences in response to EFA deficiency and in EFA requirements. In the original study of Burr and Burr (1929), they observed that female rats retained more fat than did males when both were placed on fatdeficient diets. Later on Greenberg et al. (1950) revealed that th e male rat required much greater levels of linoleic acid than did the female to prevent deficiency symptoms. The minimum linoleic acid requirement of the female rat was es timated as 0.5% of dietary calories whereas that of the male rat was estimated as 1.3%. When studying the requirements of the female rat for linoleic and linolenic acids, Pudelkiewicz et al. ( 1968) reported that the FA of female rat tissues contained 1.3 1.6 times more PUFA than those of male rats. The dietary requirement for the hen was estimated at approximately 2% as C18: 2 for egg production but only 1% as C18:2 for hatchability of fertile eggs (Menge, 1968). Fatty Acid Sources The availability of fats for feeding to dairy cows is extensive and includes oilseeds, rendered fats such as tallow and yellow grease, ve getable oils, mixtures of animal and vegetable oils, marine oils, and protected fats (which ar e modified to reduce their metabolism by ruminal microorganisms). The major fatty acid composition of some fat sources is shown in Table 2.2. In summary, linoleic acid is found in a variet y of plant sources. Soybeans, cottonseeds and sunflower seeds as well as corn oil are particularly rich in linoleic acid and are good dietary sources. Linolenic acid is found in green leafy forages and linseed (also called flaxseed). Flaxseed oil has a high concentr ation of linolenic acid. Plant o ils containing PUFA may be protected partially to reduce rumen biohydrogenation in order to deliver more EFA to the small

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34 intestine. Fish meal and oil contains high c oncentrations of EPA and DHA and can provide a source of ruminally unde gradable protein. Effect of supplemental fat on feed intake and production In a review by Allen (2000), dry m atter intake (DMI) was affected often by the inclusion of fat in the diet and it was rela ted to the type of fat as well as the type and amount of forage used. The depression in intake was credited to ruminal fill due to inhibition of fiber digestion by fat, decreased palatability, and metabolic regulation of DMI by c holescystokinin on brain satiety centers, among others. However, no effect of lip id supplementation was detected when protected fats were fed at up to 5% of dietary DM (Moallem et al., 2000; Schroeder et al., 2003). Allred et al. (2006) reported that cows fed a Ca salt mixture of palm and fi sh oils at 2.7% of dietary DM had similar intake as cows not fed fat or cows fed extruded full-fat soyb eans at 5% of dietary DM. However, decreased DMI was reported when dairy cows were fed an unprotected mix of sunflower and fish oils at 4.5% of dietary DM (Shingfield et al., 2006) or when unprotected fish oil was infused ruminally compared to a ruminal infusion of a Ca salt mix of fish and palm oils at equal deliveries of fish oil of 145 g/d (Castaneda-Gutierrez et al., 2007). Andersen et al. (2008) reporte d no effect of feeding a hi ghly saturated fat (6.6% of dietary DM) or linseeds (16% of dietary DM) from 5 wk prepartum to calving on DMI of Danish Holstein dairy cows. When vegetable oils havi ng similar C18:2 but different C16:0, C18:1, and C18:3 proportions were fed to early lactating Hols tein cows at 2% of dietary DM, DMI was not different among treatment groups (Zheng et al., 2005) In addition, abomasal infusion of canola oil (high C18:1), soybean oil (high C18:2), or sunflower oil (high C18:2, low C18:3) did not affect DMI of Holstein cows in early lactation (Christensen et al. 1994). The PUFA can cause modifications in the ruminal environment and changes in the microbial population that result in decreased fi ber digestibility and a re duction in DMI (Doreau

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35 and Chilliard, 1997). Calcium salts of unsaturated fatty acids prevent rapi d modifications of the ruminal environment due to the slow release of unsaturated fatty acids (Fotouhi and Jenkins, 1992). As free unsaturated fatty acids are remove d from the free fatty acid pool, Ca salts of PUFA will further dissociate to maintain the balance between dissociated and undissociated unsaturated fatty acids, which confer ruminal inertness and minimize the fat effect on fiber digestion. Fat supplementation has had conflicting result s on milk yield and composition. Bharathan et al. (2008) reported that cows fed fish oil at 0.5% of dietary DM had lower (3.3 vs. 3.6%) milk fat concentration and yield of ECM (32.1 vs. 34.5 kg/d) compared to cows fed a control diet without fish oil. In contrast, Bu et al. (2007) reported that co ws supplemented with soybean oil or flaxseed oil at 4% of dietar y DM had greater milk yield without milk fat depression compared to cows fed a control diet without fat although co ncentration of milk fat was numerically lower for cows fed oil. The difference between the resu lts of Bharathan et al. (2008) and Bu et al. (2007) may have been due to the greater feed intake (25 vs. 16 kg/d) by cows used in the Bharathan et al. (2008) study. Cows supplemented with Ca salts of conjugated linoleic acid (CLA) or Trans monounsaturated fatty acids (M UFA) had similar milk and FCM production to that of cows not fed fat (Selberg et al., 2004). Dhiman et al. (2000) reported that cows fed linseed oil at 4.4% of dietary DM produced less 3.5% FC M (25.2 kg/d) compared to cows not fed fat (29.2 kg/d) or fed linseed oil at 2.2% of dietary DM (30.3 kg/d). C houinard et al. (1998) reported that the degree of unsaturation in the Ca salts made of canola o il (56% C18:1), soybean oil (55% C18:2) or linseed oil (51% C18:3) had a linear effect on FCM production with cows fed Ca salts of linseed oil producing more FCM (35.2 kg/d) comp ared to cows fed Ca salts of soybean oil (31.4 kg/d) or canola oil (30.1 kg/d).

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36 Different fat sources, quantities, and forms (protected vs. unprotected) influence ruminal fermentation by microorganisms differently which, in turn, affect milk composition distinctly. Trans C18:1 fatty acids produced during micr obial biohydrogenation (Pennington and Davis, 1975) or escaping from microbial biohydrogenation in the rumen and absorbed in the small intestine, can directly inhibit de novo synthesis of lipid in the mammary gland. Ahnadi et al. (2002) reported that mammary tissue from midlacta tion Holstein cows fed a diet of 3% protected fish oil had decreased mRNA abundance of lipogen ic enzymes such as acetyl CoA carboxylase ( ACC ), fatty acid synthase ( FAS), and stearoyl-Coa desaturase. Accordingly, Piperova et al. (2002) reported a reduction in FA synthesized de novo in mammary tissue from cows fed a milk fat-depressing diet characteri zed by increased formation of trans FA in the rumen and greater incorporation of trans FA into milk fat. In addition, the re duction in de novo s ynthesis of lipid in the mammary gland was consistent with a re duction in ACC and FAS activity and ACC mRNA relative abundance. Cows fed fish oil at an increasing rate of 0.33, 0.67, and 1.00% of dietary DM with soybeans to provide the balance of 2% added fat in the diet had lower milk fat compared to cows fed a control diet without fat supplement (Whitlock et al., 2006). Moate et al. (2008) reported a positive quadratic relationship between intake of fish oil fatty acids and production of total trans octadecenoic acids, with the maximum production of the trans isomers occurring with an intake of approximately 350 g/d of fish oil fatty acids. Abughazaleh et al. (2003b) reported that cows fed diets enriched in C18:2 (1% fish oil plus 4.3% sunflower seeds) tended to have lower ( P = 0.08) milk fat concentration (2.64 vs. 3.09%) compared to cows fed diets enriched in C18:3 (1% fish oil plus 5% flax seeds). They also reported that cows fed a supplemental C 18:1 source (sunflower seeds) tended to have a

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37 lower milk fat concentration (2.74%) compared to cows fed supplemental C18:3 or C18:0 (3.10%). Conflicting results on the effect of fat supplementation on DMI and milk yield are due to several differences among experiments such as propor tion of fat in the diet, protection of the fat, degree of saturation of the fatty acids, source of fat, stage of lact ation of the animal. Effect of Supplemental Fat Source on Fatty Acid Profile Milk Milk f at contains fatty acids derived from de novo synthesis by the mammary gland (C4:0 to C14:0 plus a portion of C16:0) and from mammary uptake of preformed fatty acids (a portion of C16:0 and all longer chain fatty acids). Source of fat supplement affects some of the short and medium chain fatty acids synthesized. Fat source, time of initiation of the s upplementation, inclusion rate, length of supplementation, and biohydrogenation extent will di fferentially affect the incorporation of the long chain fatty acids into milk fat of dairy cows. The PUFA are biohydrogenated by ruminal microorganisms to a great extent. Loor et al. (2004; 2005a) reported that the ruminal biohyd rogenation of C18:3 ranged from 93.2 to 97.1% when linseed oil was supplemented in the diet fro m 3 to 6% of dietary DM. Harvatine and Allen (2006a) reported that the extent of biohydrogenation of C18:2 in diets containing fat supplements differing in saturation varied from 84.5 to 86.6%. Even though most of the dietary C18:2 was likely biohydrogenated by ruminal microorganisms, some was escaping, being absorbed in the small intestine, and incorporated in milk fat. Lundy et al. (2004) reported that ruminal biohydrogenation of linolei c acid averaged 95% for unprotected soybean oil and 92% for the Ca sa lts of soybean oil leading to an additional 14 g/d of C18:2 delivered to the omasum. Harva tine and Allen (2006a) suppl emented Ca salts of

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38 unsaturated fatty acids and us ed a kinetic approach to es timate the extent of ruminal biohydrogenation in lactating cows They reported that protecti on of the 18-carbon PUFA from biohydrogenation was minimal in a commercial source of protected fat (Ca salts of fatty acids). Likewise in sheep, Fotouhi and Jenkins (1992) observed the extent of ruminal biohydrogenation of linoleic acid was 93% for free linoleic acid and 95% for Ca salts of linoleic acid and did not differ among treatments. Despite the extensive biohydrogenation of C18:2 in the rumen, sufficient quantities left the rumen to increa se the C18:2 concentr ation in milk fat. Kalscheur et al. (1997) fed a high oleic acid sunflower oi l and a partially hydrogenated vegetable shortening enriched in trans C18:1 to lactating dairy co ws at 3.7% of dietary DM. Concentration of cis C18:1in milk fat was 28.5% and 25.9% for cows fed sunflower oil and shortening respectively with a SEM of 1.0%. An increased concentration of C18:1 trans isomers in milk fat was reported when Holstein cows were supplemented with C18:1 trans isomers (Griinari et al., 1998; Selb erg et al., 2004) or infused abomasally with C18:1 trans isomers (Romo et al., 2000). Cows fed Megalac-R (2.5% of dietary DM) had greater concentration of C18:2 in milk fat compared to cows fed a sa turated fat supplement ( 3.7 vs. 2.6%) (Harvatine and Allen, 2006b). In contrast, Kell y et al. (1998) reported that cows fed sunflower oil (69.4% C18:2) had less C18:2 in milk fat compared to cows fed lins eed oil (LSO) (51.4% C18:3) but greater than cows fed peanut oil (51.5% cis -9 C18:1). Even though most of the dietary C18:2 was likely biohydrogenated by ruminal microorganisms, some was escaping, being absorbed in the small intestine, and incorporated in milk fa t. Mid lactation dairy co ws fed extruded soybeans at 2% of dietary DM had a greater concentration of C18: 2 in milk fat compared to cows fed fish oil at the same proportion of the diet (AbuGhazal eh et al., 2002). When lactating dairy ewes were supplemented with sunflower oil at a grea ter proportion (6% of diet ary DM), concentration

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39 of C18:2 in milk fat increased (3.76 vs. 2.87%) compared to ewes fed a control diet without fat (Hervs et al., 2008). Ponter et al (2006) reported that cows fed extruded linseed (2 kg/cow/day) had increased concentrations of C18:3 in milk fa t. Midlactation cows fe d linseed oil at 5.3% of dietary DM had greater concentrat ion of C18:3 in milk fat compar ed to cows fed sunflower oil (69.4% C18:2) (Kelly et al., 1998). Concentration of C20:5 and C22:6 in milk fa t are strongly and positively related to the intake of fish oil fatty acids (M oate et al., 2008). Bharatan et al (2008) reported a slight increase in concentration of C 20:5 and a tendency ( P = 0.07) for increasing C22:6 in milk fat of cows fed fish oil at 0.5% of dietary DM. Similarly, Shingfie ld et al. (2006) reported that cows fed fish oil at 1.5% of dietary DM had greater concentration of C20:5 and C22:6 in milk fat compared to cows fed a control diet. In contra st, Bilby et al. (2006c) reported onl y increased concentrations of C22:6 in milk fat of cows fed a Ca salt of pa lm and fish oils compared to cows fed whole cottonseed but no treatment effect on concentr ation of C20:5 in milk fat was detected. The cis -9, trans -11 CLA isomer is produced endogenously (Griinari et al., 2000) by a delta 9 desaturase in the mammary gland directly from the C18:1 trans -11 isomer (about 80% of milk fat cis -9, trans -11 CLA originates e ndogenously from C18:1 trans -11; Mosley et al., 2006). Researchers have reported that the C18:1 trans -11 isomer is the major trans isomer in ruminal fluid (Loor et al., 2005a) and in duodenal digest a (Piperova et al., 2002; Loor et al., 2004). Linoleic acid can be converted to trans -10 cis -12 CLA when ruminal ruminal pH is more acidic (Griinari et al., 1998; Bauman a nd Griinari, 2003; Loor et al, 2004). Harvatine and Allen (2006b) also reported an increase in trans -10, cis -12 CLA in milk fat of cows fed Megalac-R. AbuGhazaleh et al. (2002) reported no effect of fish oil supplementation on proportion of cis -9, trans -11 CLA in milk fat but concentration of trans -10, cis -12 CLA was increased

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40 compared to cows fed extruded soybeans. Bharathan et al. (2008) reported a 47% increase in total CLA concentration in milk fat of cows fed fish oil (0.5% of dietary DM) compared to cows not fed fish oil. Juchem et al. (2008) fed Ca salts of fish and palm oils or tallow at 0.95 and 0.90% of dietary DM, respectively, for the firs t 25 DIM and at 1.90 and 1.80% thereafter until 145 DIM. Cows supplemented with Ca salts of fi sh and palm oil had gr eater concentration of cis 9, trans -11 CLA in milk fat (0.76 vs. 0.53%) compared to cows fed tallow. Ruminal infusion of fish oil at 1.2% of dietary DM (276 g of menha den oil) increased 5 fold the concentration of cis 9, trans -11 CLA in milk fat compared to those fed a control diet without fat (Loor et al., 2005b). Lipid supplementation can be used to improve the quality of the human diet. The n6/n3 fatty acid ratio present in the diet of industrial societie s has increased as a re sult of the greater consumption of vegetable oils rich in n-6 fatty acids and a reduced consumption of fish and plant sources of n-3 fatty acids (Connor, 2000). This sh ift has been associated with coronary heart disease and other human ailments (Simopoulos, 2004) Reduction of the n6/n3 ratio in milk fat is a potential strategy to improve the quality of the human diet. Cows infused with linseed oil into the duode num (500 g/d) or fed linseed at 6.7% of dietary DM had a lower ratio of n6/n3 in milk fat compared to animals fed a mixture of linseed and fish oil or Ca salts of palm oil (Petit et al., 2002). Petit (2003) repor ted that the n6/n3 fatty acid ratio in milk fat also was reduced in mid to la te lactation Holstein cows that were fed a diet enriched in C18:3 (flaxseed suppl ementation) compared to cows fed a diet enriched in C18:2 (sunflower seed supplementation). In summary, despite the greater ruminal biohydrogenation of the fatty acids, lipid supplementation is an important nutritiona l tool to manipulate milk fatty acids.

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41 Plasma Phospholipids and cholestery l esters are th e major components of blood lipid and account for about 95% of the total lipids in plasma of ru minant animals. Lipids are transported in plasma in the form of lipoproteins for metabolism at various sites in the body. Plasma lipid composition collected from any given site in the body will be dependent on the extent of metabolism (Christie, 1981). Triglycerides and free fatty acids represent <5% and 1% of total plasma lipid, respectively (Christie, 1981). Po lyunsaturated fatty acids that escape ruminal biohydrogenation are preferentially converted to the plasma c holesteryl esters and phos pholipids (Christie, 1981). The latter two fractions are the most active meta bolically, supplying fatty acids to many other organs (e.g. mammary gland and adipose tissue). This appears to account for the comparatively low proportions of PUFA reaching the mammary gland and adipose tissue (Christie, 1981). Plasma cholesteryl esters and phospholipids have comparatively slow turnover, whereas triglyceride and free fatty acids fractions have a rapid turnover and supply fatty acids to other tissues such as the mammary gland and adipose ti ssue (Christie, 1981). Ther efore, the profile of fatty acids of plasma triglycerides represents th e profile of fatty acids available to the mammary gland. Although the most abundant single fatty acid ci rculating in plasma of lactating dairy cows is linoleic acid (up to 55% of total fatty acid), less than 1% of this is in the triglyceride form which is available for milk fat incorporation. The specific transfer of this fatty acid to the plasma phospholipids and cholesteryl este rs may be a mechanism for c onserving it for the essential functions elsewhere in the animal (Christie, 1981). Abomasal infusion of C18:1 trans fatty acids increased to a greater extent the concentration of C18:1 trans fatty acids in plasma compared to cows infused with high oleic sunflower oil (Gaynor et al., 1994).

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42 Interestingly, the proportion of PUFA in plasma lipid is about 9 fold greater than in milk fat and C18:2 makes up about 44% of total identifi ed FA in the plasma which is in the range reported by other researchers of up to 55% (C hristie, 1981; Petit, 2003; Harvatine and Allen, 2006b). Petit (2003) reported that mid lactation cows (29 wk postpartum) fed flaxseeds had greater concentrations of C18:3 and C20:5 and a lower n-6/n-3 rati o in plasma compared to cows fed sunflower seeds; however, concentrations of C20:3 and C20:5 were not affected. Gonthier et al. (2005) reported that cows fe d flaxseeds had a greater concen tration of C18:3 in plasma compared to cows fed a control diet without flax seeds. Loor et al. ( 2005a) reported that cows fed linseed oil had an increased concentration of C18:3 in plasma compared to cows fed sunflower oil but concentrations of C20:3 and C20:5 were not differe nt. Ponter et al. (2006) also reported an increase in plasma concentration of C18:3 of cows fed linseed oil. Interestingly, linseed oil supplementation reduced the plasma concentration of C22:5 and C22:6 compared to sunflower oil. Linseed oil shifted the proportion of unsaturated fatty acids to n-3 fatty acids at the expense of n-6, primarily C18:2. Zheng et al. (2005) reported that cows fed a control diet without supplemental fat had lower total lipid content in plas ma (1.2 mg/mL) compared to cows fed supplemental oils at 2.1% of dietary DM (cottonseed (2.8 mg/mL), soybean (2.9 mg/mL), or corn (2.8 mg/mL)). Childs et al. (2008) fed crossbred beef primiparous cows increasing amounts of a partially rumen protected fish oil at 0, 1.04, 2.08 and 4.15% of dietary DM and reported a linear increase in the concentration of C20:5 and C22:6 in plasma. Lipid supplementation affects plasma fatty acids moderately. The change in specific fatty acids in plasma will likely relate to the inclusion of the fat in the diet, protection of the fat, and stage of lactation among others.

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43 Liver Concentration of total lipid in liver ranges over tim e from 10 to 40% (DM basis) (Rouser et al., 1969; OKelly and Reich, 1975; Grum et al., 1996; Dann et al., 2005). Hepatic lipid composition in the early postpartum period can be altered by prepartum diets or by the extensive mobilization of body fat around partur ition (Drackley et al., 2001). Grum et al. (1996) reported that dietary fat supplementation during the nonlac tating period was associated with decreased accumulation of peripartum hepatic lipid. However, reduced DMI and loss of BCS in cows fed fat prepartum reduced the benefit of feeding s upplemental fat during the prepartum period. Later the same laboratory (Douglas et al., 2004) repor ted that feeding suppl emental fat during the nonlactating period did not affect peripartal lipid accumulation in liver and suggested that there was little clear benefit (or detriment) to peri partal health. The decr ease in peripartal concentrations of total hepatic lip id in the earlier study seemed unlik ely to be attributable directly to the supplemental fat used in that study. Howe ver, Grum et al., (1996) fed fat at 6.5% of the dietary DM whereas Douglas et al (2004) fed at 4% of the dietary DM. Recently Douglas et al. (2006) reported that cows fed supplemental fat at 4% of dietary DM during the faroff dry period (60 d before expected parturition) and at 3.6% of the dietary DM during the close-up dry period (2 wk before expected parturition) tended ( P < 0.10) to have lower accumulation of hepatic lipid than cows fed a control diet without fat. Five fatty acids combine to make up over 90% (D M basis) of the identified fatty acids in liver fat of lactating dairy cows: C16:0, C18: 0, C18:1, C18:2, and C20:4 (Rukkwamsuk et al., 1999; 2000; Moussavi et al., 2007a). Proportions of the various fa tty acids in the liver are influenced basically by the liver uptake of fatty acids from the circulating blood and to a lesser extent by their metabolism, i.e., de novo synthesis, desaturation and chain elongation of fatty

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44 acids within the liver (Sato el al., 2004). Synthesis (Emery et al., 1992) as well as desaturation (Bell, 1981; John et al., 1991) of fatty acids are limited in the ruminant liver. Fat supplementation influences the pool of fatty acids that are taken up by the liver. Cows fed fish meal at 5% of dietar y DM from 5 to 50 DIM had a great er concentration of C22:6 in liver samples taken at 21 DIM compared to cows not supplemented with fish meal but no differences among treatments were detected for hepa tic concentrations of C20:5 (Moussavi et al., 2007). Nevertheless, the n6/ n3 ratio in the liver was lowered fo r cows fed fish meal at 5% of dietary DM. Bilby et al. (2006c) reported that cows fed Ca salts of palm and fish oil at 1.9% of dietary DM from 17 DIM until 94 12 DIM had greater hepatic concentration of C20:5 and C22:6 than cows fed whole cottonseed. Concentration of fatty acids in the liver will likely be influenced by stage of lactation mainly due to mobilization of adipose tissue to support milk yield. However, lipid supplementation influences the uptake of fatty acids by the liver and the response of lipid supplementation depends on the proportion of fat in the diet, the protection of the fatty acid, and stage of lactation. More studies are needed to investigate the effects of lipid supplementation on the uptake of fatty acids by the liver. Caruncle The predominant f atty acid in the endometr ium of dairy (Bilby et al., 2006c) and beef cows (Burns et al., 2003) is C18:0, followed by cis -9 C18:1, C16:0, C18:2, and C20:4 Bilby et al. (2006c) reported th at cows fed a Ca salt mix of pa lm and fish oils had greater incorporation of C20:5 (0.10 vs. <0.01%) and C22:6 (1.42 vs. 0.92%) in the endometrium of lactating dairy cows. Similarly, Childs et al. (2008) fed crossbred beef primiparous cows increasing amounts of a partially rumen-protect ed fish oil at 0, 1.04, 2.08 and 4.15% of dietary DM and reported a linear increase in the concentration of C20:5 and C22:6 in endometrial tissue

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45 collected at slaughter. In additi on, Mattos et al. (2004) reported a 7 and 5 fold increase in the concentration of C20:5 and C22:6 in the caruncle of dairy cows fed fish oil at 2% of dietary DM during the last 3 wk prior to calving. Fish meal supplementation at 1.25, 2.5, or 5% of dietary DM or Ca salts of fish oil at 2.3% of dietar y DM increased endometrial composition of C20:5 and C22:6 as much as 3-fold compared to cont rol cows fed no fish pr oducts (Moussavi et al., 2007). Fish oil or meal supplementation increases the concentration of C20:5 and C22:6 in caruncle of dairy and beef cows. Effect of Supplemental Fat Source on Hormon es and Metabolites Prostaglandin F2 Lipid supplementation can affect hormones a nd metabolites directly or indirectly by changing the fatty acids in the membrane of tissues that synthesize hormones, by affecting animal metabolism or by affecting gene expression. Arachidonic acid is the precursor of PGF2 secreted by the endometrium. Fat sources enriched in omega-6 fatty acids likely increase pr ostaglandin concentrations in plasma. Infusions of lipids have shown positive effects on PGF Me tabolite (PGFM) concentrations in plasma. Ewes infused with either soybean oil (50% C18:2) or olive oil (16% C18:2) had greater serum PGFM concentrations than ewes infused with saline (Burke et al., 1996). In postpartum beef primiparous cows, infusion of lipid containi ng 20% soybean oil thr ough the jugular vein increased systemic concentrations of C18:2 a nd PGFM after oxytocin injection (Filley et al., 1999). In contrast, supplementation of lipids enri ched with omega-3 fatty acids has shown positive or negative effects on PGFM. Parturition is a proinflamatory process which is characterized by an incr eased secretion of PGF2 by the endometrium. In order to test the

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46 concept that fish oil fatty acids reduce PGF2 secretion by the endometrium using the natural challenge of parturition, Mattos et al. (2004) fed pregnant cows fish oil or olive oil at 2% of dietary DM from 3 wk relative to calving un til parturition and at 1.8% of dietary DM during the postpartum period. Cows fed fi sh oil had reduced concentra tion of PGFM in the first 2.5 DIM compared to cows fed olive oil. Fish m eal supplementation to dairy cows at 2.6, 5.2, or 7.8% of dietary DM reduced PGFM response after an oxytocin challenge (Mattos et al., 2002). Several others have reported reduced concen tration of PGFM when dairy cows were fed supplemental fish meal (Thatcher et al., 1997), whol e flaxseed (Petit et al., 2004), or fish oil (Petit et al., 2002). In contrast, Moussavi et al. (2007) reported that fish meal supplementation from 5 to 50 DIM did not affect PGFM after an oxytocin challenge ca rried out at 49 DIM. Similarly, Wamsley et al. (2005) observed that fish meal supplementation had no effect on the secretion of PGF2 in nonlactating primiparous cows having normal progesterone concentrations but decreased PGF2 in those having reduced progesterone concentration. Although in vitro studies have documented that -3 fatty acids C20:5 and C22:6 are very potent inhibors of PGFM secre tion by bovine endometrial cells, e ffects of lipid supplementation on PGFM secretion in vivo have had conflicting results main ly due to different methodologies used such as oxytocin challeng e during mid-lactation vs. partur ition as the natural challenge, infrequent blood sampling, low inclusion of fat in the diet, etc. More studies are needed to investigate the effects of lip id supplementation on PGFM. Progesterone Fat supplem entation to cattle has consisten tly increased plasma concentrations of cholesterol (Ryan et al., 1992; Ha wkins et al., 1995; Staples et al., 1998). Cholesterol is a precursor for the synthesis of progesterone by ov arian cells (Grummer and Carrol, 1991). Childs et al. (2008) reported that the overall mean concentration of pr ogesterone in plasma of beef

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47 primiparous cows fed a diet of 4.15% rumen-protect ed fish oil was greater during the 16 d of the estrous cycle than that of primiparous cows fe d the fish oil at 1.04% of dietary DM. Authors attributed this increase to increased concentratio n of plasma cholesterol and a larger CL on day 7 of the cycle. The hypercholestero lemia may increase CL steroidogene sis which in turn increases progesterone concentrations in plasma. Fat f eeding not only increased plasma progesterone concentration but also reduced progesterone clearance (Hawki ns et al., 1995). Staples and Thatcher (2005) summarized th e effects of fat supplementati on on the size of the dominant follicle and reported an average increase of 3.2 mm (23%) in the dominant follicle of cows fed supplemental fat compared to control cows not fed fat. A larger dominant follicle will form a larger CL which in turn synthe sizes more progesterone (Vasconcel os et al., 1999; Sartori et al., 2002). Other researchers (Bilby et al., 2006b, Moussavi et al., 2007) reported no effect of fish oil supplementation on plasma concentration of proges terone of dairy cows. It is important to emphasize that the differences in the response to fat supplementation is likely due to the proportion of fat in the diet, duration of fat feed ing, time of initiation of fat feeding, stage of lactation, or days of the estrous cycle, all of which could influen ce the uptake of the fatty acids by the tissues as well as their turnover. Lipid supplementation has shown to increase progesterone concentrations. However, more studies are needed to evaluate the effects of specific fatty acids on progesterone concentrations of dairy cows. Growth hormone and IGF-1 Nutrient partitioning for lactog enesis is mediated and sustai ned by alterations in the GHIGF axis. Under physiological conditions, pituitary-derived GH induces hepatic synthesis of IGF-1 via receptor-mediated signaling (Bichell et al., 1992) and consequently systemic IGF-1 negatively regulates GH production (Le Roith et al., 2001). Howeve r, in situations of high

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48 nutrient demand from the body such as after part urition, the state of NEB uncouples the GH-IGF axis in the liver (Thissen et al., 1994). This is associated with a reduction in total circulating IGF1 and elevated GH concentrations (Vanderhaar et el., 1995). Severe NEB reduces plasma concentrations of IGF-1 and hepa tic expression of IGFBP-3 compar ed to mild NEB (Fenwick et al., 2008). Almost all IGF secreted from the liver circulates as a bound complex and the majority of it (> 90%) is associated with IGFBP-3 (C lemmons, 1997). The IGFBP-3 is produced mainly in the liver and is the major transporter of IGF-1 in the peripheral circ ulation (Burger et al., 2005). The increase in IGFBP-3 mRNA expression in the liver prevented IGF-1 degradation and potentially increased availability to other tissues by providing a reservoir of IGF-1 (Boisclair et al., 2001). Li et al. (1999) reported that rats fed omega-3 fatty acids had greater concentrations of IGFBP-3 in plasma compared to rats fed omeg a-6 fat or a no-fat control diet. CastanhedaGutierrez et al., (2007) reported an increase in the concentrations of IGF-1 in plasma of cows fed CLA (7.1 g/d of each of the cis -9, trans -11 and trans -10, cis -12 isomers) compared to cows not fed fat but the mechanism by which CLA incr eases IGF-1 is unknown. During NEB in early lactation the liver is refractory to GH, resulting in low concentrations of circulating IGF-1, but greater insulin availability restores coupling of the GH-IGF-1 axis increa sing circulation of IGF1 (Butler et al., 2003). Castanheda-G utierrez et al. (2007) speculated that the effects of CLA to increase plasma IGF-1 in lactating cows ma y be mediated by subtle changes of hepatic sensitivity to insulin. Robinson et al. (2002) re ported that cows fed nonenzymatically browned full fat soybeans had greater concentrations of IGF-1 around the time of peak surge in LH compared to cows not supplemented with fat or those fed linseeds but ther e was no effect of fat source on insulin concentrations in plasma. Low circulating concentration of IGF-1 has been reported for cows fed prilled saturated fat (G rum et al., 1996; Beam and Butler, 1998) and young

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49 primiparous cows fed a high linoleic acid (sunflowe r seeds) diet compared to animals not fed supplemental fat (Garcia et al., 2003). In contra st, fat supplementation di d not affect IGF-1 in plasma of lactating primiparous cows after 4 h of canola oil and oleamide feeding (DeLuca and Jenkins, 2000), or Holstein cows infused postrum inally from d 17 before expected calving date to d 21pospartum (Gagliostro et al., 1991). The discrepancy in the effects of lipid supplementation on IGF-1 concentration might be due to differences in the fat source, physiological state of the animal, and concentration of fat in the diet. More studies are needed to examine the relative import ance of these factors. Concentrations of IGF-1 in plasma may be a good indicator to monitor reproductive responsiveness to postpartum dietary treatments in high production dairy cows (Thatcher et al., 2006). If feeding PUFA increases IGF-1 concentr ation in plasma with increasing DIM early postpartum, this may stimulate estradiol secretion by the thecal and granulosa cells of the follicle and consequently promote cell proliferation and follicular growth. At timed AI (~ 79 DIM), the size of the dominant follicle was increased and CL volume was larger in cows fed PUFA compared to cows fed MUFA (Bilby et al., 2006 a). Bec-Villalobos et al. (2007) reported no differences in plasma concentrations of GH of cows fed partially hydrogenated fat compared to cows fed no supplemental fat. The lack of response of GH to fat supplementation has been reported in beef cattle fed 0 or 7.8% sunflower seeds (sampled every 28 d, Lammoglia et al., 2000), beef cattle fed 0 or 1.55 kg/d of safflower seeds (Bottger et al., 2002) and dairy cows fed diets of 0, 4.5, 9.0, 13.2, or 17.4% canola seeds (Khorasani et al., 1992). However, other researchers have shown an increase in GH concentration in plasma of late lactation cows infused with 1 kg/d of rapeseed oil into the abomasum (Gagliostro et al ., 1991) or mid lactation cows fed Energy Booster at 3% of dietary DM (Grum et al ., 1996). Feeding a mixture of Ca salts of palm

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50 oil and fish oil did not increas e plasma concentrations of GH of lactating dairy cows when injections of bST were not given (Bilby et al., 2006a). However when cows were injected with bST, cows fed the supplemental fat had a greater rise in plasma concentrations of GH compared to cows fed whole cottonseeds (Bilby et al., 2006a). Glucose Glucose concentrations in plasm a are not us ually affected by fat supplementation (Staples et al., 1998). Selberg et al. ( 2004) reported that supplementa tion of Ca salts of CLA or trans octadecenoic acid isomers did not a ffect plasma concentrations of glucose compared to cows fed no supplemental fat. Similarly, Moallem et al (2007) reported that supplementation with saturated or unsaturated fat did not affect concentration of pl asma glucose prepartum (65.4 vs. 66.1 mg/dL) or postpartum (59.7 vs. 59.7 mg/dL). Ho wever Andersen et al (2008) reported that cows fed whole linseeds (16% of dietary DM) prepartum had lowe r concentrations of plasma glucose postpartum compared to cows not fed fat or those fed a saturated fa t source. On the other hand, Moussavi et al. (2007a) report ed that cows fed fish meal at 5% of dietary DM or Ca salts of a mix of palm oil and fish oil at 2.3% of dietary DM from 5 to 50 DIM had greater concentration of glucose in plasma (57.6 and 57.3 mg/dL, respectively for fish meal and Ca salts of palm and fish oil) than cows fed fish meal at 2.5% of diet ary DM (51.1 mg/dL) or a control group not fed fat (53.4 mg/dL) but did not differ from cows fed fi sh meal at 1.25% of dietary DM (55.3 mg/dL). The authors attributed the incr ease in plasma glucose to a greater production of propionate in the rumen of cows fed fish o il as reported by others (W achira et al., 2000 and Fievez et al., 2003). Since propionate is the single most important s ubstrate for gluconeogenesis in ruminants ( Drackley et al., 2001), fish oil appare ntly shifts rum inal fermentation by decreasing methanogenesis that conserves energy and yields more propionate (Fievez et al., 2003). These

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51 effects seemed to be determined by the amount of the unique PUFA (i.e., EPA and DHA) present in fish oil products rather than simply by the total amount of PUFA fed. More studies are required to evaluate the eff ects of lipid supplementation on concentration of glucose in plasma of dairy cows. Insulin Concentration of plasma insulin usually refl ects energy in take. It in creases gradually as days postpartum increase and as DMI of dairy cows increase. Fat supplementation has had mixed results on circulating concentr ation of plasma insulin (Staples et al., 1998). In studies in which fat supplementation depressed plasma insulin (8 out of 17 studies re viewed by Staples et al., 1998), the diet and day differe nces were eliminated when energy balance was used as a covariate in the statistical mode l suggesting that insulin differe nces among diets were due to differences in EB. In rodents, feeding n-3 long chain PUFA (4.9% fish oil), as compared to a high fat diet, lowered concentrations of plasma insulin by sustaining glucose transporter protein GLUT4 receptors in the muscle, by preventing decreased expression of GLUT4 in adipose tissue, and by inhibiting both activity and expression of liver glucose-6-phosphatase that increased glucose uptake and metabolism (Delarue et al., 2004). Xiao et al. (2006) reported that different fatty acid profiles affected glucose-induced insulin secr etion in humans differently. Mashek et al. (2005) reported that cows infused intravenously with linseed oil had a lower insulin concentration in plasma compared with cows intravenously supplied with tallow. Cows fed whole linseed prepartum (16% of dietary DM) had reduced concentrations of plasma insulin prepartum compared to cows fed a saturated fat (6.6% of dietary DM) or no fat diet from 5 wk relative to calving until parturiti on (Andersen et al., 2008). In a ddition, feeding a Ca salt mixture of palm and fish oils reduced insulin concentratio n in plasma of dairy cows (Bilby et al., 2006c).

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52 Interpretation of eff ects of lipid supplementation on plas ma insulin should be cautions and EB should be included in th e model (Staples et al., 1998). Nonesterified fatty acid When the energy needed for m aintenance and la ctation is greater than the energy provided in the diet, the dairy cow will begin to mobilize he r body fat stores to lessen the energy deficit. Hormone sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from the TG in adipose tissue (Holm et al ., 2000). Hormone sensitive li pase is dephosphorylated and inactivated by insulin whereas an increment in the cAMP concentr ation and activation of protein kinase A by glucagon, epinephrine, and ACTH promote phosphorylation of HSL (Holm et al., 2000) which is activated when translocated from a cytosolic compartment to the surface of the lipid droplet (Egan et al., 1992; BrasaemLe et al., 2000). A second enzyme, adipose triglyceride lipase (ATGL), catalyzes the initial step in trig lyceride (TG) hydrolys is (Zimmermann et al., 2004). Thus, ATGL and HSL coordinately catabo lize stored TG in adip ose tissue of mammals (Zimmermann et al., 2004). Nonesterified fatty acids (NEFA) are released into the blood from adipose tissue and transported to hepatic and non-hepatic tissues. Gavino and Gavino (1992) studied the HSL-medi ated release of fatty acids from TG in cultured preadipocytes containing PUFA-enric hed triglyceride. They found that cultured preadipocytes challenged with 10 M of norepin ephrine tended to release more omega-6 and omega-3 PUFA than saturated fatty acids. Inde ed, crude preparations of HSL released C18:3 from the TG substrates twice as fast as cis -9 C18:1. Raclot et al. ( 2001) evaluated the fatty acid specificity of HSL in lipid emulsions and reported that HSL is slightly a ffected by the degree of unsaturation of the fatty acid in the TG. Thus, th is selectivity could affect the individual fatty acid supply from the tissues (Raclot, 2003).

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53 In a review of 50 treatment comparisons, Chill iard (1993) reported an average increase in concentration of pl asma NEFA of 41 M ( P < 0.005) over controls when supplemental fat was fed. Likewise, Drackley (1999) re ported an average increase in concentration of plasma NEFA of 81 M over controls when supplemental fat was fed after reviewing seven studies. This increase due to dietary fat supplementation is mu ch less than what is typically observed during the transition period when NEFA concentrations may increase up to 1 mM or more (Grummer, 1993). Selberg et al. (2004) reported that cows fe d a CLA supplement had greater concentrations of NEFA in plasma at wk 1 postpartum compared to cows fed trans fatty acids or a control diet without fat. However, Baumgard et al. (2000, 20 02) showed little or no effect of supplemental CLA on plasma NEFA concentrations. Moallem et al. (2007) reported no e ffect of saturated or unsaturated fat supplementation to lactating dairy cows on concen trations of NEFA in plasma (588 vs. 600 Eq/L). Fish meal or Ca salts of palm and fish oil supplemented to multiparous Holstein cows from 5 to 50 DIM did not affect concentration of NEFA in plasma (Moussavi et al., 2007). In contrast, Petit et al. (2007) reported that multiparous cows fed saturated fat at 1.7 and 3.5% of dietary DM pre and postpartum respec tively had greater concen tration of NEFA in plasma compared to multiparous cows fed w hole flaxseed at 3.3 and 11.0% of dietary DM during the pre and postpartum period, respectivel y, but diets had no effect on concentration of NEFA in plasma of primiparous cows (parity by treatment interaction). In summary, lipid supplementation increases NEFA. However, the effects fat sources enriched in different fatty acids on concentrations of NEFA in plasma of dairy cows merits further investigation.

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54 Effect of Supplemental Fat Sou rce on Health and Immunity Uterine Health Uterine function is often com promised by bact erial contamination of the uterine lumen after parturition. Pathogenic bacter ia frequently persist, causing ut erine disease, a key cause of infertility (Sheldon and Dobson, 2004) Bacteria can be cultured from samples collected from the uterine lumen of most dairy catt le in the first 2 wk after part urition in many situations. Although many cows eliminate these bacteria during the first 5 wk after parturition, persistence of bacterial infection causes uterine disease detectable by phys ical examination in 10 to 17% of animals (Le Blanc et al., 2002). The presence of pathogenic bacteria in the uterus causes inflammation, histological lesions of the endometrium, dela ys in uterine involution, and perturbs embryo survival (Sheldon et al., 2006). Thus uterine disease is associated with lower conception rates, increased intervals from calving to first service or conception, and more cattle culled for failure to conceive. During parturition, eicosanoids are produced in substantial quantities and play an important role in the regulation and control of parturition, and expulsi on of the placenta and uterine contents through opening of the cervix an d contractions of the uterus (Santos et al., 2008). Prostaglandin F2 is an important eicosanoid involved in the regulation of CL lifespan and likely influences retention of fe tal membranes and consequently uterine health. Arachidonic acid (C20:4, -6) is the precursor of the potent prostaglandin PGF2 The more C20:4 in the endometrial tissue available for ei cosanoid synthesis, the more PGF2 is likely to be secreted, which in turn may influence uterine health. Omega-3 fatty acids have been reported to suppress PGFM concentrations (Mattos et al., 2004; Petit et al., 2004) and reduce neutrophil function during the ea rly postpartum period (Thatcher et al., 2006). Recent studie s have confirmed the effects of in vivo exposure to PGs on

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55 the in vitro response of lymphocytes (Ramadan et al ., 1997; Lewis, 2003; Wulster-Radcliffe et al.,. 2003). The PGF2 has been reported to enhance immune function in vitro (Hoedemaker et al., 1992). In addition, PGF2 increased in vitro bactericidal activity of neutrophils from ovariectomized mares (Watson, 1988). Cox et al. (1995) reported that low numbers of neutrophils in vitro was due to clearance of apoptotic neutrophils by macrophage engulfmen t during inflammation which might influence endometrial repair (Kaitu'u-Lino et al., 2007) in the pospartum period. Seals et al. (2002) reported th at postpartum concentrati ons of plasma PGFM were inversely related to emergence of uterine infections; that is, postpartum cows with depressed PGFM concentrations were more likely to develop uterine infections. In addition, aberrant PGF2 and PGE2 production has been associated with reta ined placenta (Gross et al., 1987; Heuwieser et al., 1992), which in turn is associated w ith increased incidence of uterine infections. Omega-3 fatty acids are well known for being immunosuppressive (Calder, 1997; Pizato et al., 2006; Calder, 2007). Thatcher et al. (2006) reported that cows fed a rich source of C18:2 (28% C18:2) at 2% of dietary DM starting 4 wk prior to ca lving until 14 wk postpartum had greater concentration of PGFM in plasma and fewer health problems in the first 10 days postpartum compared to cows not fed fat prepartu m. The authors reported that the uterus and cells of the immune system had greater potentia l to secrete prostaglandins because of the increase in the supply of linoleic acid to tissues which likely enhanced postpartum uterine health and immunocompetence of the cow. More studies on the effect of lipid supplemention (starti ng on the dry period) on early postpartum uterine and how it will influence reproductive perfor mance later on during lactation merits further investigation.

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56 Neutrophils Calder et al. (1990) reported that macrophages enriched wi th C20:5 and C22:6 had lower phagocytic activity than what would be expected due to the degree of unsaturation of the fatty acid. Ballou and DePeters (2008) fe d 51 Jersey bull calves (5 1 d of age) milk replacers supplemented with 2% fatty acids having a 3:1 mix of corn and canola oils, a 1:1 mix of fish oil and the 3:1 mix of corn and canola oils, or fi sh oil only. Authors repo rted that fish oil supplementation had no effect on the ability of bl ood neutrophils from the calves to phagocytose E. coli. According to Calder (2007), studies th at investigate the num ber or proportion of phagocytes involved in engulfing the target material are not likely to detect an effect of PUFA because it is unlikely that such manipulation w ill completely stop phagocytes from engaging in the process of phagocytosis. However, PUFA mi ght affect the phagocyt ic activity, i.e. the amount of target material engulfed by thos e cells that are act ive (Calder, 2007). The production of reactive oxygen species by the action of NADPH oxidase of neutrophils is a critical mechanism to kill phagocytized bacteria, a process called oxidative burst. Supplementing rabbits with a high dose (5 g/kg /d) of fish oil decreased neutrophil oxidative burst by approximately 30%; however, a lowe r dose (0.22 g/kg/d) of fish oil had no influence on superoxide generation ( DAmbola et al., 1991). In contrast elderly m en supplemented with either a low (1.35 g/d), moderate (2.7 g/d), or high (4.05 g/d) dose of C20:5 had suppressed oxidative burst by neutrophils but no effect was detected in young men (Rees et al., 2006). Bartelt et al. (2008) reported th at healthy males, aged 18 to 40 years supplemented daily with capsules containing fish oil (166 mg of C20:5 a nd 119 mg of C22:6) for 8 wk had an immunestimulating effect on neutrophil oxi dative burst compared to s ubjects supplemented with olive oil. Discrepancies in the effects of the omega-3 fatty acids on immune function are likely due to

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57 the methodology used for measuring the immune status such as isolation of the neutrophils from whole blood prior to lipid in cubation vs. analysis in w hole blood, duration of lipid supplementation, age related effects, etc. More studies on the effect of lipid supplementation on ne utrophil concentration and function of periparturient dairy cows are necessary. Production of Cytokines by Lymphocytes One characteristic of inf lammatory responses is the great induction of diverse cytokines (Grinble, 1998). Cytokines are soluble proteins that are releas ed from immune cells (mainly monocytes and macrophages) in response to infec tion, injury, or foreign substances. Liberation of cytokines is indispensable for the initiation of the immune response and for the regulation of the multidirectional communication between the diffe rent cells involved (Seematter et al., 2004). The main pro-inflammatory cytokines are interle ukin-1 (IL-1b), interleuki n-6 (IL-6), interleukin2 (IL-2), interleukin-8 (IL-8), TNF, and IFN(Grinble, 1998), while the anti-inflammatory ones are IL-1ra, IL-4, IL-10, and IL-13 (Zhang and An, 2007). Dietary supplementation with EPA and DHA for 1 to 6 mo in humans diminished (Endres et al., 1989; Meydani et al. 1991; and Caughey et al., 1996) or di d not affect (Cooper et al., 1993, Kew et al., 2004) ex vivo production of TNFby peripheral blood mononuclear cells. Sierra et al. (2008) reported that lymphoc ytes from mice fed diets enriched in EPA and DHA produced less TNFcompared to mice fed a diet containi ng 53.8% C18:2. Lessard et al. (2004) fed flaxseed (5.9% of dietary DM), Ca salts of palm oil (2.7% of dietary DM) or micronized soybeans (9.4% of dietary DM) to Holstein cows from 6 wk prepartum until 6 wk postpartum. They reported that stimulated lymphocytes isolat ed from primiparous cows secreted more TNFcompared to those from multiparous cows but supplemental fat source had no effect on TNFsecretion. Lymphocytes isolated from physically stressed swimmers receiving 2.5 g of fish oil

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58 per day (0.9 g of EPA and 0.5 g of DHA) for 6 wk produced less TNFand IFNcompared to those receiving a placebo (mineral oil) (Andrade et al., 2008). Discrepancies among experiments could be due to difference between species, amount and source of n-3 PUFA added to diets, and physiological state of animals and humans. Differences in blood composition of omeg a-3 and omega-6 PUFA may influence production of mediators such as leukotrienes and prostaglandins which are known to help regulate cytokine production and consequently the response of im mune cells to stimuli. Fatty acids are precursors of prostaglandins. The omeg a-3 and omega -6 fatty acids are precursors of, respectively, the series 3 and series 2 prostaglandins (Yaqoob and Calder, 1995). Moreover, many effects mediated by PUFA on immune cells appear to be exerted in an eicosanoidindependent manner. The n-3 and n-6 PUFA may affect immune cell func tions by regulating the expression of key genes encoding for molecules involved in the signal transduction pathway such as nuclear transcription factorB and peroxisome proliferatoractivated receptors (Calder et al., 2002). There is limited data in the literature on the effects of lipid supplementation to periparturient dairy cows and production of cyto kines by lymphocytes. Mores studies are needed for a better understanding of the effects of lipid on immune func tion of periparturient dairy cows. Humoral Response The acquired immune response involves lym phocyt es and is highly specific to a certain antigen. Following activation, several days are needed for lymphoc ytes to become effective but the response persists af ter removal of the source of the init iating antigen. This persistence gives rise to immunological memory which is the ba sis for a stronger and more effective immune response to re-exposure to the same antigen. The humoral response d eals with extracellular

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59 pathogens through B lymphocytes which are characterized by their ability to produce immunoglobulins specific for an i ndividual antigen (Calder, 2007). Lessard et al. (2003) fed primiparous (n = 8) and multiparous (n = 22) Holstein cows whole flaxseed (10.4% of dietary DM), Megalac (3.8% of dietary DM), or micronized soybean (17.7% of dietary DM) from calving to 105 DIM. At insemination (between 60 and 72 DIM), cows were injected with ovalbumin and seru m samples were taken at 0, 10, 20, and 40 d post AI for analysis of immunoglobulin response to ova lbumin. Diet did not affect this humoral response. In another study to ev aluate the effect of fat feed ing during the prepartum period on humoral response, Lessard et al. (2004) inject ed cows with ovalbumin. They reported that multiparous cows fed micronized soybeans at 9.4% of dietary DM from 6 wk prior to calving until parturition had greater IgG concentration in colostrum compared to cows fed Megalac (2.7% of dietary DM) or flaxs eed (5.9% of dietary DM) but th ere was no effect of diet on antibody secretion against ovalbumin in serum. Da ta on the effect of different fat sources on humoral immunity of peripartur ient dairy cows are scarce. Acute Phase Proteins Acute phase proteins are produced in the liver in response to inflamma tion or stress and are released in the blood to help the immune syst em. Haptoglobin acts in plasm a as a scavenger molecule for free hemoglobin (Lim et al., 2000). Haptoglobin concentrati ons in plasma were increased in cows with fatty liver (Yoshino et al., 1992; Nakagawa et al., 1997; Petersen et al., 2004) or mastitis (Grnlund et al ., 2005; Eckersall et al., 2006; kerstedt et al., 2007). Bazinet et al. (2004) reported that pigs supplemented with omega-3 fatty acids had reduced haptoglobin concentrations in plasma compared to a control group fed a diet rich in omega-6. Haptoglobin was not affected by ener gy or starch concentrations in newly received feedlot calves

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60 (Berry et al., 2004). Diets en riched in omega-3, omega-6 or omega-9 did not affect plasma concentrations of haptoglobin in beef primiparous cows (Farran et al., 2008). Acid soluble protein (also called alpha 1-acid glycoprotei n) is an anti-inflammatory agent that controls inappropriate or extended activatio n of the immune system (Jafari et al., 2006) and inhibits prostaglandin E2 generation in plasma of rats (M atsumoto et al., 2007). Acid soluble protein is a minor acute phase protein constitu tively expressed by the liver, usually found in blood (Lecchi et al., 2008) and is increased with systemic inflammation (Hochepied et al., 2003). Acid soluble protein has a dual immunomodulatory effect in which it can activate monocytes and induce cytokine secretion or cause immunosuppression (Bennet a nd Schmid, 1980) in order to control immune status. Mouthiers et al. (2004) repor ted that alpha 1-acid glycopr otein gene was activated by retinoic acid through a DR-responsive element that involves retinoic X receptor (RXR). Since fatty acids are known to modulate gene expression through RXR nucl ear receptors, the effect of FA supplementation on acid soluble protein could be mediated via RXR. In addition, interleukins are modulators of the alpha 1-acid glycoprotein (Fourni er et al., 2000). Ceruloplasmin is a protein that binds copper and helps prevent oxidative damage to endothelial cells during inflammation (Uriu-Adams and Keen, 2005) Normal values in cattle range from 16.8 to 34.2 mg/dl (The Merck Veterinary Manual, 1997). Ceruloplasmin concentrations in plasma increased with increa sed bacterial contaminati on of the uterus during the first 2 wk postpartum compared to cows wi th low bacterial infec tion (Sheldon et al., 2003). Fibrinogen is a sticky, fibrous protein used to ma ke fibrin for blood clotting and tissue repair (Gentry, 2004). In addition, prostaglandins induce blood clotting. Normal values in cattle range from 100 to 600 mg/dl (The Merck Vete rinary Manual, 1997). One mechanism by which

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61 FA supplementation with omega-6 FA might incr ease fibrinogen concentrations in cows was likely due to up regulation of IL-6 (Meerarani et al., 2003) which is the major inducer of fibrinogen in hepatocytes (Albrecht et al., 2007). Studies on the effects of lipid supplementati on on acute phase proteins response in dairy cows are scarce and merits further investigation.

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62 Table 2-1. Common fatty acids (ada pted from Voet and Voet, 2004) Symbola Common Name Systematic name b Structure Saturated Fatty Acid 12:0 Lauric acid Dodecanoic acid CH3(CH2)10COOH 14:0 Myristic acid Tetradecanoic acid CH3(CH2)12COOH 16:0 Palmitic acid Hexadecanoic acid CH3(CH2)14COOH 18:0 Stearic acid Octadecanoic acid CH3(CH2)16COOH 20:0 Arachidic acid Eicosanoic acid CH3(CH2)18COOH 22:0 Behenic acid Docosanoic acid CH3(CH2)20COOH 24:0 Lignoceric acid Tetracosanoic acid CH3(CH2)22COOH Unsaturated Fatty Acid 16:1 n-7 Palmitoleic acid 9-hexadecanoic acid CH3(CH2)5CH=CH(CH2)7COOH 18:1 n-9 Oleic acid 9-octadecenoic acid CH3(CH2)7CH=CH(CH2)7COOH 18:2 n-6 Linoleic acid 9,12-octadecadienoic acid CH3(CH2)4(CH=CHCH2)2(CH2)6COOH 18:3 n-3 -Linolenic acid 9,12,15-octadecatrienoic acid CH3CH2(CH=CHCH2)3(CH2)6COOH 18:3 n-6 -Linolenic acid 6,9,12octadecatrienoic acid CH3(CH2)4(CH=CHCH2)3(CH2)3COOH 20:4 n-6 Arachidonic acid 5,8,11,14eicosatetraenoic acid CH3(CH2)4(CH=CHCH2)4(CH2)2COOH 20:5 n-3 EPA 5,8,11,14,17eicosapentaenoic acid CH3CH2(CH=CHCH2)5(CH2)2COOH 22:6 n-3 DHA 4,7,10,13,16,19docosahexenoic acid CH3CH2(CH=CHCH)6CH2COOH a Number of carbon atoms: number of double bounds. For unsaturated fatty acids, n is the number of carbon atoms, n-x is the double-bonded carbon atom and x is the number of that carbon atom counting from the methyl terminal ( ) end of the chain. b Numbers before the name indicate the double bound position in the carbon chain from the carboxyl end.

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63 Table 2-2. Major fatty acid com position of some fat sources. Fatty Acid C16:0 C18:0 C18:1 C18:2 C18:3 C20:5 C22:6 Tallow1 25 22 37 2 <1 ND2 ND Yellow grease1 17 10 44 17 1 ND ND Choice white grease3 24 11 48 12 1 ND ND Megalac4 44 5 39 9 <1 ND ND Flaxseed meal4 8 3 18 16 53 ND ND Flaxseed4 6 4 18 14 57 ND ND Soybean meal5 16 5 16 44 7 ND ND Fish meal5 22 5 7 ND <1 9 9 Energy booster 1006 28 51 8 1 <1 ND ND Megalac-R6 17 2 34 30 2 ND ND Linseed oil7 5 3 20 16 55 ND ND Safflower oil7 7 2 9 80 <1 ND ND Soybean oil7 8 3 24 58 8 ND ND Sunflower oil7 6 4 20 66 <1 ND ND Menhaden fish oil7 17 3 7 1 1 11 12 1 Oldick et al.,1997. 2 ND = not detected. 3 Onetti et al., 2001. 4 Petit et al., 2001. 5 Abu-Ghazaleh et al., 2001 6 Moallem et al., 2007. 7 Staples, 2006.

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64 H H H H H H H H H H H H H H H H CH3 C C C C C = C C C = C C C C C C C C COOH H H H H H H H H H H H H 2 3 4 5 1 7 8 9 10 6 11 13 14 15 16 12 17 18 Linoleic acid (Greek letter designation) H H H H H H H H H H H H H H H H CH3 C C C C C = C C C = C C C C C C C C COOH H H H H H H HH H H H H Linolenic acid (numeric designation) H H H H H H H H H H H H H H H H CH3 C C = C C C = C C C = C C C C C C C C COOH H H H H HH H H H H 18 2 345 1 789 10 6 11 13 14 15 16 12 17 Linolenic acid (Greek letters designation) H H H H H H H H H H H H H H H H CH3 C C = C C C = C C C = C C C C C C C C COOH H H H H H H H H H H Figure 2-1. Structural formula of linoleic (omega-6) and linolenic acid (omega-3) linoleic acid (numeric designation).

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65 Figure 2-2. Parent fatty acid and major metabolites within each of the three omega fatty acid families (partially adapted from Mattos et al., 2000).

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66 Figure 2-3. Synthesis of the va rious prostaglandin (PG) series from fatty acid precursors (Adapted from Mattos et al., 2000). n-6 Family Linoleic acid, C18:2 -Linolenic acid, C18:3 Dihomo -linolenic acid, C20:3 Arachidonicacid, C20:4 n -3 Family -linolenic acid, C18:3 Stearidonic acid, C18:4 Eicosatetraenoic acid, C20:4 Eicosapentaenoic acid, C20:5 6 desaturase Elongase 5 desaturase PG -3 series PG -2 series PG -1 series PG H Synthas e PG H Synthas e PG H Synthas e

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67 CHAPTER 3 EFFECT OF DIETS ENRICHED IN DIFFERENT FAT TY ACIDS ON PLASMA, MILK, AND LIVER FATTY ACID PROFILE OF LACT ATING HOLSTEIN COWS DURING SUMMER Abstract The objective of the study was to evaluate how dietary fat sou rces enriched with oleic, trans -octadecenoic, linoleic, or linolenic acids aff ected plasma, liver, and milk fatty acid profiles of Holstein primiparous (n = 22) and multiparou s cows (n = 32) during the summer season. Fat supplements were the following: 1) high oleic sunflower oil (HOSFO Trisun, Humko Oil, 80% cis C18:1), 2) Ca salt of trans -octadecenoic acids (CaTRANSEn erG TR, Virtus Nutrition, 61% trans C18:1), 3) Ca salt of vegetable oils (CaVegMegalac-R, C hurch & Dwight Co, 29% C18:2), and 4) linseed oil (LSOArcher Daniels Midland, 55% C18:3 and 16% C18:2). Supplemental fats were fed at 1.35% of dietary DM beginning at 30 7 d prior to actual calving date. After calving, fats were fed at 1.5% (oils) and 1.75% (Ca salts) of dietary DM for 15 wk. Three blood samples collected on a Monday-We dnesday-Friday schedule between 21 and 28 DIM were analyzed for fatty acids using gas chromatography. Liver samples were taken via biopsy on 2, 14 2, and 28 2 DIM, immediately frozen in liquid nitrogen and kept at -80oC for fatty acid analysis. Milk without preservative was collected at 2 consecutive milkings at 7, 8, and 9 wk postpartum, composited based upon milk pr oduction, and frozen for fatty acid analysis. Feeding supplemental oleic acid increased the cis C18:1 content of plasma but not of liver fat. Oleic acid in milk fat of multiparous but not primiparous cows was increased by feeding supplemental oleic acid. Cows fed TRANS fats had greater concentrations of trans C18:1 isomers in plasma (1.5%), liver fat (1.4%), and milk fat (5.8%) compared to cows fed HOSFO. Concentrations of C18:2 in plasma (44.4%) and m ilk fat (4.5%) were greater in cows fed CaVeg compared to cows fed LSO (41.8 and 3.7% for plas ma and milk fat, respectively). Cows fed LSO had greater concentrations of C18:3 in plasma (4.9 vs. 2.3%), liver (1.4 vs. 1.0%) and milk (1.0

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68 vs. 0.6%) compared to cows fed CaVeg. Accord ingly, LSO supplementation increased C20:5 in plasma (0.7 vs. 0.5%), liver (0.9 vs. 0.5%), a nd milk (0.036 vs. 0.017%) compared to CaVeg-fed cows. Feeding dietary fats enriched with partic ular fatty acids in moderate amounts (1.5% of dietary DM) resulted in increased concentrations of those fatty acids in plasma, liver fat, and/or milk fat of dairy cows. Key Words: fatty acid, milk, liver, plasma Introduction Milk enriched in som e PUFA has demonstrat ed anticarcinogenic effects and a wide range of potential health benefits in biomedical studies with animal models (Pariza, 2004; Bhattacharya et al., 2006). Nutritional strategies to increase the healthy fatty acids in the milk can have a dramatic impact on human health. However, most PUFA consumed by dair y cows are biohydrogenated by ruminal microorganisms to more saturated fa tty acids before incorporation in to milk fat (Kalscheur et al., 1997). Thus, knowing the extent of transfer of diet ary PUFA to the small intestine in order to increase the incorporation of specific fatty acids into tissues will be beneficial to understand the effects of lipid supplementation on dairy cow ph ysiology (Petit et al., 2007), reproduction (Bilby et al., 2006a; Petit and Twagiramungu, 2006), an d immune function (Les sard et al., 2004). Ahnadi et al. (2002) reported th at midlactation Holstein cows fe d a diet of 3% gluataraldehydeprotected fish oil had decreased mRNA abundance of mammary lipogenic enzymes such as acetyl CoA carboxylase ( ACC ), fatty acid synthase ( FAS), and stearoyl-Coa desaturase. Flowers et al. (2008) evaluated the effect s of varying amounts of linseed o il in midlactation grazing dairy cows and reported a linear increase in the concentr ation of C18:3 in milk and a quadratic effect on milk fat of cows fed linseed oil compar ed to cows not supplemented with fat.

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69 However, comparison of various lipid supplem ents during the peripa rturient period on the fatty acid composition of plasma, liver, and milk of lactating Holstein cows during summer has not being evaluated to our knowledge. The objectiv e of the study was to evaluate the effects of diets enriched in oleic acid, trans -octadecenoic acid, linoleic acid, or linolenic acid on fatty acid composition of plasma, liver, and milk of lactating Holstein cows during summer. Material and Methods Animals, Treatments, and Sampling Experim ent was conducted at the University of Florida dairy research unit (Hague, FL) during the months of May through December 2004. All experimental animals were managed according to the guidelines approved by the University of Florida Animal Care and Use Committee. Periparturient Holstein primiparous (n = 22) and multiparous cows (n = 32) were sorted according to calving date, parity (primiparous or multiparous), BW, and milk production of the previous year for multiparous and then assigned to treatment at 30 7 d prior to their due date. Dietary supplemental treatments were the followi ng: 1) high oleic sunflowe r oil from genetically modified sunflower (HOSFO ; Trisun, Humko Oil, Memphis, TN ; 80% C18:1), 2) Ca salt of trans -octadecenoic acids (CaTRANS ; EnerG TR, Virtus Nutrition, Fairlawn, OH, 61% trans C18:1 isomers: 20.62% trans 6-8, 10.47% trans -9, 10.62% trans -10, 7.05% trans -11, and 8.73% trans -12), 3) Ca salt of vegetable oils ( CaVeg ; Megalac-R, Church & Dwight Co, Princeton, NJ; 36% C16:0 and 29% C18:2) and 4) linseed oil ( LSO ; Archer Daniels Midland, Redwing, MN, 55% C18:3 and 16% C18:2). Supplemental fats were fed at 1.35% of dietary DM during the prepartum period (Table 1). After calving, fats we re fed at 1.5% (oil sources) and 1.75% (Ca salt sources) of dietary DM for 15 wk to allow equa l concentration of dietary lipid (Table 2). Prepartum cows were housed in sod-based pens equipped with fans, sprinklers, and shaded Calan gates (American Calan Inc., Northwood, NH). Po stpartum cows were housed in a sand-bedded,

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70 free-stall barn equipped with fans, sprinklers, an d Calan gates. Intake of DM was measured daily. Cows were fed a TMR twice daily at 090 0 and 1300 h to allow 5 to 10% feed refusals daily. Corn silage was collected weekly and i mmediately dried for 1 h using a Koster (Koster Crop Tester, Inc., Strongsville, OH) to calculate the concentration of DM in order to maintain the formulated forage to concentrate ratio in the ration. Cows were milked thrice daily at 0100, 0900, and 1700 h. Cows were weighed and body condition scored (Edmonson et al., 1989) weekly after the 0900 h milking and before feed ing. Ethoxiquin (Fisher Scientific, Hampton, NH, USA) was used as an antioxidant by adding 0.32 g / kg of concentrate mix resulting in a dietary concentration of 0.015 and 0.020% for nonlact ating and lactating co ws, respectively (DM basis). Sample Collection and Analysis Representative sam ples of corn silage, bermudagrass hay, alfalfa hay, and concentrate mixes were collected on a weekly basis. Week ly samples were composited on a monthly basis and ground through a 1-mm Wiley mill screen (A. H. Thomas, Philadelphia, PA). Composited feed samples were analyzed for minerals and fat (acid hydrolysis for grain mixture containing CaVeg and LSO) composition (Dairy One, Ithaca, NY), NDF (Mer tens, 2002), ADF (AOAC, 1995), and CP using a macro elemental analyzer vario MAX CN (Elementar Analysensystene GmbH, Hanau, Germany). Blood (10 mL) was collected at 0700 h on Monday, Wednesday, and Friday from coccygeal vessels into sodium heparinized tubes (Vacutainer, B ecton Dickinson, Franklin Lakes, NJ) from calving until 47 3 DIM. Samples were put immediately on i ce until centrifuged at 2619 x g at 5C for 30 min (RC-3B refrigerated cen trifuge, H 600A rotor, Sorvall Instruments, Wilmington, DE). Plasma was separated and frozen at -20oC for subsequent fatty acid analyses.

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71 On 2, 14 2, and 28 2 DIM, liver samples were collect via biopsy, rinsed with sterile saline, snap-frozen in liquid N, and stored at -80oC until analyzed for fatty acids. Milk samples were collected weekly from 2 consecutive milkings using bronopol-B-14 as a preservative. Milk was measured for fat, true protein, and SCC by Southeast Milk lab (Belleview, FL) using a Bently 2000 NIR analyzer. Final concentrations of fat and protein were calculated after adjusting for milk production during those milk ings. Milk without preservative was collected at 2 consecutive milkings at 7, 8, and 9 wk postpartum, pooled based upon milk production, and frozen for fa tty acid analysis. To determine the fatty acid profile of milk fat, milk samples were composited (final volume of 45 mL) from wk 7, 8, and 9 postp artum according to milk production. Fat was isolated from milk by centrif ugation of thawed milk at 17,800 x g for 30 min at 8C. Fatty acids from about 325 mg of manually isolated fa t were extracted using a 3:2 (vol/vol) hexane/isopropanol solvent mixture (18 mL / g of fat). The extracted fatty acids were converted to methyl esters (Chouinard et al., 1999). Approximately 200 mg of the methyl esters were transferred into an acid-washed 15-mL glass tube to which 2 mL of hexane and 40 l of methyl acetate (Fisher Scientific, Hampton, NH, USA) we re added. The tube was vortexed until fat was dissolved. Forty l of sodium methylate solu tion (Fisher Scientific, Hampton, NH, USA) was added, the tube contents were vor texed, and allowed to react for 10 min at room temperature. Sixty l of oxalic acid solution (Fisher Scientific, Hampton, NH, USA) was added to terminate the reaction and the tube s were centrifuged at 2,000 x g for 5 min at 5oC. The top hexane layer containing the fatty acids in the methyl ester form was transferred to 2-mL crimp-top vials (Fisher Scientific, Hampton, NH, USA) for milk a nd liver analysis and 100 l crimp-snap vials (Fisher Scientific, Hampton, NH, USA) for plasma analysis..

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72 The fatty acid extraction and methylation pro cedures (Kramer et al., 1997) were the same for fat supplements, liver, and plasma samples. Fat supplements (approximately 200 mg), liver samples (approximately 200 mg of fresh weight), and plasma samples (3 blood samples collected during wk 4 of lactation were pooled to make up 1.5 mL) were freeze-dried for 24 h. One mg of internal standard (C19:0) was added in order to calculate total fa tty acid concentration. Lipid was extracted by adding 2 mL of sodium met hoxide (Acros, New Jersey, USA), vortexing, and incubating in a 50oC water bath for 10 min. After cooli ng for 5 min, 3 mL of 5% methanolic HCl (Fisher Scientific, Hampton, NH, USA) were added and the tubes vortexed. The tubes were incubated in an 80oC water bath for 10 min, removed from wa ter bath, and allowed to cool for 7 min. One mL of hexane and 7.5 mL of 6% K2CO3 were added. The tubes were vortexed and centrifuged at 194 x g for 5 min. The upper layer was transfer red into 10 mL glass tubes. The solvent was evaporated completely under N gas. He xane (100 l) was added in order to redisolve methylated fatty acids and the solution was transferred to the crimp-top vial. Fatty acid methyl esters were determined using a Varian CP-3800 gas chromatograph (Varian Inc., Palo Alto, CA) equipped with au to-sampler (Varian CP-8400), flame ionization detector, and a Varian capillary column (CP-S il 88, 100 m x 0.25 mm x 0.2 m ). The carrier gas was He, the split ratio was 10:1, and the injector a nd detector temperatures were maintained at 230oC and 250oC, respectively. One l of sample was injected via the auto-sampler into the column. The oven temperature was set initiall y 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 40 min. The peak was identified and calculated ba sed on the retention time and peak area of known standards.

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73 The desaturase index for cis -9 C16:1 and cis -9 C18:1 was defined as follows: [product of 9 desaturase] / [product of 9 desaturase + substrate of 9 desaturase]. For example, the cis-9 C16:1 desaturase index would be calculated as [ cis -9 C16:1] / [ cis -9 C16:1 + C16:0] (Kelsey et al., 2003). Statistical Analysis Measurem ents of daily DMI during the pre and postpartum periods, milk production, and milk composition were reduced to weekly means before statistical analyses were performed. Repeated measures data (DMI, milk producti on, milk fat, milk protein, BW, BCS, liver fatty acids) were analyzed using PROC MIXE D procedure of SAS according to the following model: Yijkl = + Fi + Pj + FPij +Ck (i j) +Wl + FWil + PWjl + FPWijl + Eijkl where Yijkl is the observation, is the overall mean, Fi is the fixed effect of dietary fat source (i = 1, 2, 3, and 4), Pj is the fixed effect of parity (j = 1 and 2), FPij is the interaction of fat source and parity, Ck (i j) is random effect of cow within fat source and parity (k = 1, 2, n), Wl is the fixed effect of week (l = 0, 1, 2, ), FWil is the interaction of fat source and week, PWjl is the interaction of parity and week, FPWijl is the three way interaction of fat source, parity and week, and Eijkl is the residual error. Data were tested to determine the structure of best fit, namely AR (1), ARH (1), CS, or CSH, as indicated by a lower Schw artz Baesian information criteri on value (Littell et al., 1996). Orthogonal contrasts used to detect treatment differences were the following: 1) HOSFO + CaTRANS vs. CaVeg + LSO, 2) HOSFO vs. CaTRANS, and 3) CaVeg vs. LSO. For liver samples collected at DIM not equally spaced (2, 14, and 28 DIM), the IML procedure of SAS was used to generate coefficients for testing of linear and quadratic day effects. After testing for

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74 the level of order that best fit up to quadratic, single degree of freedom contrasts of treatment by DIM were tested. Milk and plasma fatty acids were analy zed using PROC GLM of SAS. The model contained treatment, parity and treatment by parity interaction. The orthogonal contrasts mentioned above were also used to test for treatm ent effects. Differences were considered to be significant at P < 0.05 for all analyses. Results and Discussion Fat sources differed in FA profile, with HOSFO containing from 3 to 6 times more cis -9 C18:1 than the other sources, CaTRANS containing mainly C18:1 trans isomers (61%), CaVeg containing 2 to 14 times more C18:2 than the ot her sources, and LSO containing mainly C18:3 (55.2%) at a much greater concentrations than others (Table 3.3). DMI, Milk Production, and Milk Compositio n Orthogonal contrasts of treatment by parity in teractions were not significant for any dependent variable except for SCC (HOS FO vs. CaTRANS by parity interaction, P = 0.04; Table 3.4) so only main effects of treatment will be discussed. As expected, multiparous cows consumed more DM compared to primiparous cows (42 and 23% greater in the pre and postpartum periods, respectively. Table 3.4) although DMI expressed as a % of BW was not different betwee n parities. In addition, pattern of DMI (kg/d) over time did not differ among the dietary tr eatment groups (Figure 3.1). However, DMI expressed as % of BW appeared to increase for a longer period of time for animals fed LSO compared to cows fed CaTRANS (Figure 3.2; treatment by week interaction, P < 0.001). Andersen et al. (2008) reported no effect of feeding a highly satu rated fat (6.4% of dietary DM) or linseeds (16% of dietary DM) from 5 wk pr epartum to calving on DMI of Danish Holstein dairy cows. When vegetable oils having simila r C18:2 but different C16:0, C18:1, and C18:3

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75 proportions were fed to early la ctating Holstein cows at 2% of dietary DM, DMI was not different among treatment groups (Zheng et al., 2005) In addition, abomasal infusion of canola oil (high C18:1), soybean oil (high C18:2), or sunflower oil (high C18:2, low C18:3) did not affect DMI of Holstein cows in early lactation (Christensen et al. 1994). Yield of milk averaged 30.4 and 37.2 kg/d for primiparous and multiparous cows, respectively (Table 3.4). Fat source did not infl uence yield of milk, FPCM, or ECM over the 15wk postpartum period neither was pattern of milk yield over time influenced by diet (Figure 3.3). Cows fed LSO tended ( P = 0.08) to have a greater produc tion of FCM (33.2 vs. 29.6 kg/d) compared to cows fed CaVeg. Chouinard et al. (1 998) reported that the degr ee of unsaturation in the Ca salts made of canola oil (56% C18:1), soybean oil (55% C18:2) or linseed oil (51% C18:3) had a linear effect on FCM production with cows fed Ca salts of linseed oil producing more FCM (35.2 kg/d) compared to cows fed Ca salts of soybean oil (31.4 kg/d) or canola oil (30.1 kg/d). Dhiman et al. (2000) reported that cows fed linseed oil at 4.4% of dietary DM produced less 3.5% FCM (25.2 kg/d) compared to co ws not fed fat (29.2 kg/d) or fed linseed oil at 2.2% of dietary DM (30.3 kg/d). Bu et al. (2007) also reported that cows fed soybean oil (4% of dietary DM) or flaxseed oil (4% of dietar y DM) had similar ECM. Cows supplemented with Ca salts of CLA or Trans MUFA had similar m ilk and FCM production to that of cows not fed fat (Selberg et al., 2004). Although concentration (2.76%) a nd yield (0.90 kg) of milk pr otein were unchanged by fat source, cows fed LSO had greater ( P = 0.04) milk fat concentration (3.60 vs. 3.25%) and tended to have ( P = 0.09) greater yield of milk fat (1.16 vs. 0.99 kg/d) compared to cows fed CaVeg. Abughazaleh et al. (2003b) reported that cows fed diets enriched in C18:2 (1% fish oil plus 4.3% sunflower seeds) tended to have lower ( P = 0.08) milk fat concentration (2.64 vs.

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76 3.09%) compared to cows fed diets enriched in C18:3 (1% fish oil plus 5% flax seeds). Abughazaleh et al. (2003b) also reported that cows fed a supplemental C18:1 source (sunflower seeds) tended to have a lower milk fat c oncentration (2.74%) compared to cows fed supplemental C18:3 or C18:0 (3.10%). Although we did not compare HOSFO to LSO in the current study, a similar decline in milk fat concentration for cows fed HOSFO was observed (3.30 vs 3.60 %). This decrease in milk fat c oncentration due to CaVeg supplementation might have been due to an increase in the isomerization of C18:2 to trans -10, cis -12 C18:2 in the rumen which is responsible for milk fat depres sion (Bauman and Griinari, 2003; Mosley et al., 2002; Shingfield et al., 2006). The increased proportion of trans -10, cis -12 C18:2 in milk fat of cows fed CaVeg compared to cows fed LSO (Table 3.5) supports this speculation. Feed efficiency averaged 1.68 kg of 3.5% FCM per kg of DMI across the 15 wk postpartum period and did not differ among treatment groups. Multiparous cows lost BW in the first 6 wk postpartum and then plateaued whereas primiparous cows lost BW up to 4 wk and then plateaued (Figure 3.4). Multiparous cows were heavier (639 vs. 499 kg) than primiparous cows over the weeks of the study ( P < 0.001). All animals lost body condition until wk 5 postpartum a nd then plateaued. Treatments did not affect BW or BCS. Mean or pattern of energy balance did not differ among treatments. All animal groups were in negative energy balance for the first 3 wk postpartum and then in a positive energy balance until 15 wk postpartum (Figure 3.6). Fatty Acid Profile in Milk, Blood and Liver Milk The fatty acid profile of m ilk differed somewhat between parities. Compared to multiparous cows, the milk fat of primiparous cows contained a greater ( P < 0.02) proportion of

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77 trans C18:1 (4.4 vs. 3.6%) and cis -9, trans -11 C18:2 (1.2 vs. 1.0%). In addition, they had a greater ( P = 0.04) concentration of C18:0 (13.3 vs. 12.3%) and tended to have a lower ( P = 0.06) concentration of C18:2 (3.8 vs. 4.1%) compared to cows. Parity by treatment interactions for milk fa tty acids were predominantly nonsignificant (Table 3.5). A few notable exceptions are to be mentioned. The concentration of cis C18:1was increased in milk fat from cows (26.5 vs. 24.5% ) but an opposite response was detected in primiparous cows (24.3 vs. 27.1%) fed HOSFO vs. CaTRANS (parity by HOSFO vs. CaTRANS interaction, P = 0.03). Kalscheur et al. ( 1997) also fed a high oleic acid sunflower oil and a partially hydrogenated vegeta ble shortening enriched in trans C18:1 to lactating dairy cows but at 3.7% of dietary DM. Concentrations of cis C18:1in milk fat were 28.5% and 25.9% for cows fed sunflower oil and shortening, respectively wi th a SEM of 1.0% supporting our results with cows. Two additional parity by treatment interacti ons are to be mentioned. Compared to animals fed CaVeg, feeding LSO lowered ( P = 0.03) the concentration of C16:0 in milk fat of cows (32.0% vs. 28.3%) but not of primiparous cows (30.7 vs. 31.9%) but increased ( P = 0.03) the concentration of C18:0 in milk fat of cows (11.5 vs. 13.8%) but not of primiparous cows (13.9 vs. 13.1%). The multiparous animals in this stud y followed the expected response of milk fatty acids to fat supplementation source in that LSO is 94% C18 and 6% C16 whereas Ca Veg is 63% C18 and 36% C16 (Table 3.3). Milk contains fatty acids derived from de novo synthesis by the mammary gland (C4:0 to C14:0 plus a portion of C16:0) and from mammary uptake of pref ormed fatty acid (a portion of C16:0 and all longer chain fatty aci ds). Source of fat supplement a ffected some of the short and medium chain fatty acids. Cows fed HOSFO ha d greater concentrations of C6:0, C8:0, and C10:0 in milk fat ( P < 0.03) and tended ( P = 0.06) to have a greater concentration of C12:0

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78 compared to cows fed CaTRANS. This might be due to an effect of the trans C18:1 fatty acids on mammary gland activity. Trans C18:1 fatty acids pr oduced during microbial biohydrogenation (Pennington and Da vis, 1975) or escaping from microbial biohydrogenation in the rumen and absorbed in the sma ll intestine, can directly inhib it de novo synthesis of lipid in the mammary gland. Ahnadi et al. (2002) re ported that mammary tissue from midlactation Holstein cows fed a diet of 3% protected fish oil had decreased mRNA abundance of lipogenic enzymes such as acetyl CoA carboxylase ( ACC ), fatty acid synthase ( FAS), and stearoyl-Coa desaturase. Accordingly, Pipe rova et al. (2002) reported a re duction in FA synthesized de novo in mammary tissue from cows fed a milk fa t-depressing diet char acterized by increased formation of trans FA in the rumen and gr eater incorporation of trans FA into milk fat. In addition, the reduction in de novo synthesis of lipi d in the mammary gland was consistent with a reduction in ACC and FAS activity and ACC mRNA relative abundance. Feeding CaTRANS also increased (P = 0.02) cis -9 C16:1 in milk fat compared to HOSFO (1.10 vs. 0.97%). Cows fed supplemental MUFA (HOSFO + CaTRANS) had increased ( P < 0.001) C18:1 trans isomers in milk fat compared to cows fed supplementa l PUFA (CaVeg + LSO). This increase was due mainly to that of trans C18:1 feeding. Cows fed CaTRAN S had a greater concentration ( P = 0.01) of C18:1 trans isomers (5.6 vs. 4.0%) and cis -9, trans -11 C18:2 (1.22 vs. 0.98%) in milk fat compared to cows fed HOSFO. An increased concentration of C18:1 trans isomers in milk fat were reported when Holstein cows were supplemented with C18:1 trans isomers (Griinari et al., 1998; Selberg et al., 2004) or infused abomasally with C18:1 trans isomers (Romo et al., 2000). The cis -9, trans -11 CLA isomer is produced endogenously (Griinari et al., 2000) by a delta 9 desaturase in the mammary gl and directly from the C18:1 trans 11 isomer (about 80% of milk fat cis -9, trans -11 CLA originates endogenously from C18:1 trans -11) (Mosley et al., 2006). In the

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79 present experiment, we were unable to detect the specific trans isomers. However, researchers have reported that the C18:1 trans -11 isomer is the major trans isomer in ruminal fluid (Loor et al., 2005a) and in duodenal dige sta (Piperova et al., 2002; Loor et al., 2004). As expected, cows fed CaVeg had a greater ( P < 0.01) concentration of C18:2 in milk fat compared to cows fed LSO (4.4 vs. 3.6%). Harv atine and Allen (2006a) reported that the extent of biohydrogenation of C18:2 in diets containing fat supplements differing in saturation varied from 84.5 to 86.6%. Likewise, cows fed Mega lac-R (2.5% of dietary DM) had greater concentration of C18:2 in milk fat compared to cows fed a saturated fat supplement (3.7 vs. 2.6%) (Harvatine and Allen, 2006b). In contrast, Kelly et al. (1998) re ported that cows fed sunflower oil (69.4% C18:2) had less C18:2 in milk fat compared to cows fed LSO (51.4% C18:3) but greater than co ws fed peanut oil (51.5% cis -9 C18:1). Even though most of the dietary C18:2 was likely biohydrogenated by rumina l microorganisms, some was escaping, being absorbed in the small intestine, and incorporated in milk fat. The increase (P < 0.01) in CLA trans -10, cis -12 in milk fat of cows fed CaVeg co mpared to cows fed LSO (0.05 vs. 0.02%) support the finding of several rese archers (Griinari et al., 1998; Bauman and Griinari, 2003; Loor et al, 2004) who reported that C18:2 can be converted to trans -10, cis -12 CLA when ruminal ruminal pH is more acidic. Harvatine and Allen (2006b) also reported an increase in trans -10, cis -12 CLA in milk fat of cows fed Megalac-R. In contrast to our result, they also detected an increase in the cis -9, trans -11 CLA for cannulated cows fed Megalac-R. Expectedly, cows fed PUFA had greater concentrations of C18:3 (0.77 vs. 0.40%) and C20:5 (0.026 vs. 0.006%) in milk fat compared to cows fed the MUFA. Supplementation with LSO almost doubled the concentration of C18: 3 in milk fat compared to CaVeg (0.97 vs. 0.57%). Ponter et al. (2006) reported that cows fed extrude d linseed (2 kg/cow/day) had

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80 increased concentrations of C18:3 in milk fat. Midlactation cows fed linseed oil at 5.3% of dietary DM had greater concentrat ion of C18:3 in milk fat compar ed to cows fed sunflower oil (69.4% C18:2) (Kelly et al., 1998). Loor et al. (2004; 2005a ) reported that the ruminal biohydrogenation of C18:3 ranged from 93.2 to 97.1% when linseed oil was supplemented in the diet from 3 to 6% of dietary DM. Even t hough a great percentage of C18:3 is biohydrogenated in the rumen, LSO supplementation contributed to the incorporation of C18:3 into the milk. Linolenic acid can be desaturated by a 6 desaturase (a membrane bound, acyl-CoA desaturase) to C18:4 n-3. This fatty acid can be elongated to C20:4 n-3 and desaturated ( 5 desaturase) to C20:5 n-3 (Gurr et al., 2002). The two-fold increas e in the concentration of C20:5 in milk fat of cows fed LSO compared to cows fed CaVe g (0.036 vs. 0.017%) is likely explained by the desaturase and elongase activites on C18:3 in the mammary gland. The n6/n3 fatty acid ratio present in the diet of industrial societies has increased as a result of the greater consumption of vegetable oils rich in n-6 fatty acids and a reduced consumption of fish and plant sources of n-3 fatty acids (Connor 2000). This shift has been associated with coronary heart disease and other human ailm ents (Simopoulos, 2004). Reduction of the n6/n3 ratio in milk fat is a potential strategy to impr ove the quality of the hum an diet. The n6/n3 ratio in milk fat for cows fed LSO was half of that of cows fed CaVeg (5.0 vs. 9.8) as expected due to the increase in the concentration of C18:3 and C20:5 in the milk fat of cows fed LSO. Likewise, cows infused with linseed oil into the duodenum ( 500 g/d) or fed linseed at 6.7% of dietary DM had a lower ratio of n6/n3 in milk fat compared to animals fed a mixture of linseed and fish oil or Ca salts of palm oil (Petit et al., 2002). Petit (2003) reported that the n6/n3 fatty acid ratio in milk fat also was reduced in mid to late lactation Holstein cows that were fed a diet enriched in

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81 C18:3 (flaxseed supplementation) compared to co ws fed a diet enriched in C18:2 (sunflower seed supplementation). Plasma Phospholipids and cholestery l esters are th e major components of blood lipid and account for about 95% of the total lipids in plasma of ru minant animals. Lipids are transported in plasma in the form of lipoproteins for metabolism at various sites in the body. Plasma lipid composition collected from any site in the body will be dependent upon the extent of FA metabolism (Christie, 1981). Triglycerides and free fatty acids represent <5% and 1% of total plasma lipid, respectively (Christie, 1981). Po lyunsaturated fatty acids that escape ruminal biohydrogenation are preferentially incorporated into plasma cholesteryl esters and phospholipids (Christie, 1981). Plasma cholesteryl esters and phospholipids have comparatively slow turnover, while triglyceride and free fatty acids fractions have a rapid turnover and supply fatty acids to other tissues such as the mammary gland and adipose ti ssue (Christie, 1981). Ther efore, the profile of fatty acids of plasma triglycerides represents th e profile of fatty acids available to the mammary gland. Although the most abundant single fatty acid ci rculating in plasma of lactating dairy cows is linoleic acid (up to 55% of total fatty acid), less than 1% of this is in the triglyceride form which is available for milk fat incorporation. Th e specific transfer of th is acid to the plasma phospholipids and cholesteryl esters may be a mechanism for conserving it for essential functions elsewhere in the animal (Christie, 1981). When diets enriched in PUFA are fed to ru minants, those unsatu rated components that escape biohydrogenation in the rumen appear to be selectively taken up and esterified to the plasma phospholipids and choleste rol ester fractions as opposed to the triglycerides or unesterified fatty acids. The latter two fractions are the most active metabolically, supplying fatty acids to many other organs (e.g. mammary gland and adipose tissue). This appears to account for

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82 the comparatively low proportions of PUFA reaching the mammary gland and adipose tissue (Christie, 1981). For instance, Bilby et al. (2006c) repor ted an increase in the concentration of C20:5 and C22:6 in the endometrium, liver, a nd mammary tissue, but only C22:6 was increased in milk fat of cows fed fish oil compared to co ws fed whole cottonseed. Th is effect was only true for primiparous cows (1.70 vs. 0.99%) and not for cows (0.95 vs. 0.96%; parity by MUFA vs. PUFA interaction, P = 0.02). The 3 parity by orthogonal treatme nt contrasts were not significant for any identified FA in plasma in Table 3.6 with the exception of C18:1 trans and C20:0. Therefore, P values for these contrasts were not included in Table 3.6 but are discussed where considered relevant. Cows fed MUFA (HOSFO + CaTRANS) had greater ( P = 0.02) plasma concentrations of C18:1 trans isomers than cows fed PUFA (CaVeg + LSO) (1.32 vs. 0.97%; Table 3.6). This increase in the concentration of C18:1 trans fatty acids in plasma in animals fed MUFA vs. PUFA was primarily detected in primiparous cows (MUFA vs. PUFA by parity interaction, P = 0.02). Plasma concentrations of C18:1 trans fatty acids in primparous cows were greater when MUFA was fed instead of PUFA (1.70 vs. 0.99%) but multiparous cows had similar concentrations (0.95 vs. 0.96%). Abomasal infusion of C18:1 trans fatty acids increased to a greater extent the co ncentration of C18:1 trans fatty acids in plasma compared to cows infused with high oleic sunflower oil (G aynor et al., 1994). Supplementation with HOSFO increased ( P = 0.03) the concentration of cis -9 C18:1 in plasma compared to that of CaTRANS supplementation (12.3 vs. 10.7%). Gaynor et al (1994) reported a greater increase in cis -9 C18:1 in plasma of cows infused abomasally w ith high oleic sunflower oil compared to cows infused with C18:1 trans isomers.

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83 Interestingly, the proportion of PUFA in plasma lipid is about 9-fold greater than in milk fat and C18:2 makes up about 44% of total identifi ed fatty acid in the plasma which is in the range reported by other researchers of up to 55% (Christie, 1981; Petit, 2003; Harvatine and Allen, 2006b). In the present experiment, cows fed LSO had a lesser concentration of C18:2 (41.8 vs. 44.4%), greater concentrations of the n-3 fatty acids (C18:3 (4.9 vs. 2.3%), C20:3 (0.06 vs. 0.04%), C20:5 (0.71 vs. 0.46%), and C22:5 (0.65 vs. 0.53%)), and lower n-6/n-3 ratio (7.5 vs. 15.0) in plasma compared to cows fed CaVeg. Si milarly, Petit (2003) reported that mid lactating cows (29 wk postpartum) fed flaxseeds had grea ter concentrations of C18:3 and C20:5 and a lower n-6/n-3 ratio in plasma compared to cows fed sunflower seeds; howev er, concentrations of C20:3 and C20:5 were not affecte d. Gonthier et al. (2005) reported that co ws fed flaxseeds had greater concentration of C18:3 in plasma compared to cows fed a c ontrol diet without flaxseeds. Loor et al. (2005a) reported that cows fed linse ed oil had increased con centration of C18:3 in plasma compared to cows fed sunflower oil bu t concentrations of C 20:3 and C20:5 were not different. Ponter et al. (2006) also reported an increase in plas ma concentration of C18:3 of cows fed linseed oil. Interestingly, linseed oi l supplementation reduced the plasma concentration of C22:5 and C22:6 compared to sunflower oil. Linseed oil shifted the proportion of unsaturated fatty acids to n-3 fatty acids at the expense of n-6, primarily C18:2. Total lipid content of plasma (1.34, 1.31, 1.36, and 1.25 mg/mL for HOSFO, CaTRANS, CaVeg, and LSO respectively) was lower than e xpected for lactating dairy cows (range from 2.24 to 4.84 mg/mL as reviewed by Christie, 1981). Zh eng et al. (2005) report ed that cows fed a control diet without supplement al fat had lower total lipid co ntent in plasma (1.2 mg/mL) compared to cows fed supplemental oils at 2.1% of dietary DM (cottonseed (2.8 mg/mL), soybean (2.9 mg/mL), or corn (2.8 mg/mL)). Th e findings of Noble and OKelly (1974) that

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84 exposure of cattle to high temperature produce a steady decline in the plasma total lipid concentration support the lower lipid content of plas ma detected in this experiment in cows fed different sources of fat supplements during the summer months in Flor ida. The lower proportion of fat in the diet in the present experiment ( 1.5% of dietary DM) also likely explains the lower lipid content in plasma of co ws fed different fat sources. Liver Concentration of total lipid in liver ranged ove r tim e from 15 to 31% (DM basis) with a mean of 21.2%. This is in the lower range re ported by other researcher s (Rouser et al., 1969; OKelly and Reich, 1974; Grum et al., 1996; Dann et al., 2005). Grum et al. (1996) reported that dietary fat supplementation during the nonlacta ting period was associated with decreased accumulation of peripartum hepatic lipid. However, reduced DMI and loss of BCS in cows fed fat prepartum confounded the eff ect of feeding supplemental fat during the prepartum period. Later the same laboratory (Douglas et al., 2004) re ported that feeding supplemental fat during the nonlactating period did not affect peripartal lipid accumulation in liver and suggested that there was little clear benefit or detriment to peripartal health. The decrease in peripartal concentrations of total lipid seemed unlikely to be attributable di rectly to the supplemental fat used in that study. However, Grum et al., (1996) fed fat at 6.5 % of the dietary DM whereas Douglas et al. (2004) fed at 4% of the dietary DM. Recently D ouglas et al. (2006) reported that cows fed supplemental fat at 4% of dietary DM during th e faroff dry period and at 3.6% of the dietary DM during the closeup dry period tended ( P < 0.10) to have lower accumulation of hepatic lipid than cows fed a control diet without fat. Five fatty acids combined to make up over 90% (DM basis) of the identified fatty acids in liver fat. The average proportion of each of these fatty acids in the current study was the following: 29.0% C16:0, 17.4% C18:0, 21.7% C18:1, 12.3% C18:2, and 9.9% C20:4. These

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85 proportions were similar to thos e reported for lactating dairy cows by Rukkwamsuk et al. (1999; 2000), and Moussavi et al. (2007). A ll other fatty acids identified were present at < 3.5% of liver DM. Hepatic lipid composition in the early postpar tum period can be alte red by prepartum diets or by the extensive mobilization of body fat around parturition (Drackley et al., 2001). Concentration of fat in liver peaked at 14 DIM, being 19.0, 24.7, and 19.7% (DM basis) at 2, 14, and 28 DIM (effect of DIM, P < 0.01). This peak was likely due to the greater uptake of NEFA mobilized from adipose tissue during the early pos tpartum weeks when energy balance is most negative. The pattern of the individual fatty acid s detected in liver over DIM supports this idea. The fatty acids that are most common in adipose tissue (C16:0 and C18:1 cis ) were at their lowest concentration (P = 0.001) at 28 DIM; namely 29.1, 31.8, and 26.1% (SE = 1.0%) for C16:0 and 22.7, 23.2, and 19.3% (SE = 0.7%) for C18: 1 at 2, 14, and 28 DIM, respectively. The fatty acids that would be absorbed in greater amounts postpartum from increasing intake of DM were in greater concentration ( P = 0.001) at 28 DIM; namely 17.5, 15.2, and 19.5% (SE = 0.8%) for C18:0 and 11.9, 11.9, and 13.2% (SE = 0.3%) for C18:2 at 2, 14, and 28 DIM, respectively. In addition, the proportion of C20: 4, which is synthesized from C18:2, was greatest at 28 DIM as well ( P = 0.001), being 9.9, 8.6, and 11.2% (SE = 0.6%) at 2, 14, and 28 DIM, respectively. Although making up a much smaller pr oportion of the fatty acids in liver, the concentration of all other individual fatty acids also changed or tended to change over DIM, with the exceptions of C20:0 and C20:3, indicating that th e liver has an extensive turnover of fatty acids during the first 28 d postpartum. Collectively, the proporti on of MUFA was lower (24.3, 25.2 and 21.8%) and that of PUFA was greater at 28 DIM (25.6, 23. 9, and 28.8% for 2, 14, and 28 DIM, respectively; P < 0.001). The proportion of saturated fatty acids remained fairly constant at 50% across DIM.

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86 Across DIM, multiparous cows had more fat ( P = 0.02) in their liver than primiparous cows (25.8 vs. 16.6% of DM). This was likely due to greater mobilization of fat from adipose to the liver by multiparous cows due to their greater production of m ilk and more negative energy status. The average concentration of C16:0 f its this explanation, being 31.7% for multiparous cows and 26.3% for primiparous cows (SE = 1.3%; parity, P < 0.01) although average concentration of liver C18:1 was not different between parities (21.8 and 21.7% for multiparous and primiparous cows, respectively). Likewise concentration of C18:2 was not different between multiparous and primiparous cows (12.8 vs. 11.9%, SE = 0.4%). Average concentration of C20:4 was lower in liver fat of multiparous compared to primiparous cows (8.5 vs. 11.3%; SE = 0.7%) suggesting that multiparous cows were le ss efficient in converting C18:2 to C20:4 or that multiparous cows were utilizing a greater proportion of C20:4 in the synthesis of prostaglandins than were primiparous cows. A lternatively, the additional intake of C18:3 by cows fed LSO may have suppressed the conversion of C20:4 to PGF2 because liver concentration of C20:4 was great er at 2 DIM (Table 3.7). Par ity by DIM interaction was not significant for any of these major fatty acids. The only liver fatty acids for which a significant ( P < 0.05) treatment contrast by parity interaction or a treatment contrast by parity by day interaction was detected were those found in small concentrations (< 1.85% of tissue DM). The interaction of CaVeg vs. LSO by parity was significant ( P = 0.03) for C17:0 and C20:3. Also the MU FA vs. PUFA by parity interaction was significant ( P = 0.05) for trans C18:1. The liver fat of primiparous cows contained more trans C18:1 when fed unsaturated fat supplements of HOSFO and CaTRANS compared to those fed the PUFA fat supplements of CaVeg and LSO (1.41 vs. 0.99%) but the liver fat of multiparous cows was unchanged when fed these diets ( 0.93 vs. 1.00%). A significant interaction of

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87 treatment by parity by DIM was detected only for C15:0, trans C18:1, trans -10, cis -12 C18:2, and C22:6. Therefore columns of probability valu es for these 2-way and 3-way interactions are not included in Table 3.7. Proportions of the various fatty acids in the liver are influenced basically by the liver uptake of fatty acids from the ci rculating blood and to a lesser ex tent by their metabolism, i. e., de novo synthesis, desaturation and chain elongation of fatty acids within the liver (Sato el al., 2004). Generally, synthesis (Emery et al., 1992) as well as desaturation (Bell, 1981; John et al., 1991) of fatty acid is limited in the ruminant liver. Feeding fat sources enriched in certain fatty acids did result in incr eased concentration of those fatty acids in liver fat (Table 3.7). Feeding CaTRANS increased the trans C18:1 content of liver fat compared to the f eeding of HOSFO (1.38 vs. 0.96%; P = 0.01). Feeding LSO increased the C18:3, C20:5, and C22:6 contents of liver fa t compared to the feeding of CaVeg (1.38 vs. 0.97%, 0.87 vs. 0.44%, and 0.20 vs.0.07%, respectively, P 0.01). As a result of these shifts in n-3 fatty acids, the n-3 to n-6 ratio was lower for cows fed LSO compar ed to those fed CaVeg (5.3 vs. 8.0; P < 0.001). Cows fed fish meal at 5% of dietary DM from 5 to 50 DIM had greater concentration of C22:6 in liver samples taken at 21 DIM compared to cows not supplemented with fish meal but no differences among treatment were detected for hepatic concentrations of C20:5 (Moussavi et al., 2007). Ne vertheless, the n6/n3 ra tio in the liver was lowered also for cows fed fish meal at 5% of di etary DM (Moussavi et al., 2007). Feeding a fat enriched in C18:1 did not increase C18:1 in liver fat. Feeding a Ca salt enriched in C18: 2 resulted in a greater concentration of C18:2 in liver fat at 14 DI M compared to feeding LSO (13.3 vs. 10.6%; Figure 3.7; CaVeg vs. LSO by quadr atic DIM interaction, P < 0.001). This shift may have been due more to effects of LSO on liver lipid profiles than of CaVeg. This is explained in more detail in

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88 the following paragraph. Dietar y fat source did not influence tota l lipid content of the liver. This may be due to the considerable variation in the quantity of lipid in liver tissue among cows (Kinsella and Butler, 1970). The CV was 47, 70, and 45%, respectively for 2, 14, and 28 DIM (Table 3.7). Effect of fat source on liver was influenced in some cases by DIM. Cows fed LSO experienced a larger in crease in liver fat from 2 to 14 DI M (15.9 vs. 28.1%) compared to cows fed CaVeg (18.5 vs. 19.7%; CaVeg vs. LSO by quadratic DIM interaction, P = 0.06). This increase in total liver lipid was likely due to a greater mobilization of fatty acids from adipose tissue to liver in cows fed LSO. This is suppor ted by the fact that the proportion of C16:0 in liver fat had a greater increase from 2 to 14 DIM when animals were fed LSO (27.2 vs. 34.4%) compared to those fed CaVeg (30.3 vs. 30.2%; CaVeg vs. LSO by quadratic DIM interaction, P < 0.01). Likewise the proportion of cis C18:1 in liver fat had a gr eater increase from 2 to 14 DIM when animals were fed LSO (21.2 vs. 24.8%) compared to those fed CaVeg (23.2 vs. 22.0%; CaVeg vs. LSO by quadratic DIM interaction, P < 0.01). As a resu lt of these changes, the proportion of 2 other major fatty acids resp onded by decreasing in co ncentration at 14 DIM compared to 2 DIM as a matter of dilution. The proportion of C18:0 in liver fat decreased from 2 to 14 DIM when animals were fed LSO (18.9 vs. 12.7%) compared to those fed CaVeg (16.6 vs. 16.2%; CaVeg vs. LSO by qua dratic DIM interaction, P < 0.01). Likewise the proportion of C20:4 in liver fat decreased from 2 to 14 DIM when animals were fed LSO (11.3 vs. 6.9%) compared to those fed CaVeg (9.3 vs. 9.0%; CaVeg vs. LSO by quadratic DIM interaction, P < 0.01). Conclusions Supplem enting periparturient dairy cows with diets enriched in oleic, trans -octadecenoic, linoleic, or linolenic acids affected the fatty acid composition of plasma, liver, and milk

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89 postpartum. Trans -octadecenoic supplementation increased C18:1 trans fatty acids in plasma, liver, and milk. Diet enriched in linoleic acid increased this fatty acid in milk fat but not in plasma or liver fat. Enrichment of linolenic acid in the diet resulted in greater incorporation of C18:3 and C20:5 in plasma, liver, and milk fat but C22:6 was detected in increased concentration only in liver. LSO supplementation likely suppressed the conversion of C20:4 to PGF2 early postpartum because liver concentration of C20:4 was greater at 2 DIM. Feeding supplemental oleic acid did increase the C18:1 c ontent of plasma but not of liver fat. Oleic acid in milk fat of multiparous but not primiparous cows was in creased by feeding supplemental oleic acid. Supplementation of fats enriched in different fatty acids influenced the preferential incorporation of specific fatty acids into plasma, liver, and milk fat without compromising cow performance.

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90 Table 3-1. Ingredient and chemical compositi on of TMR fed to Holstein cows during the prepartum period. Treatments1 HOSFO CaTRANS CaVeg LSO Ingredient (% of dietary DM) Corn silage 45.0 45.0 45.0 45.0 Bermudagrass hay 15.0 15.0 15.0 15.0 Ground corn 14.5 14.5 14.5 14.5 Citrus pulp 5.2 5.2 5.2 5.2 Soybean meal 12.5 12.5 12.5 12.5 Trace mineralized salt 2 0.1 0.1 0.1 0.1 Mineral and Vitamin premix3 6.5 6.5 6.5 6.5 Ethoxiquin4 0.015 0.015 0.015 0.015 High oleic sunflower oil 1.35 CaTRANS 1.35 ... ... CaVeg 1.35 Linseed oil ... 1.35 Component NEL, Mcal/kg of DM 1.57 1.56 1.56 1.57 CP, % of DM 14.9 14.8 15.2 14.9 NDF, % of DM 38.9 38.7 38.5 38.9 ADF, % of DM 20.8 20.8 21.0 21.2 Ether extract, % DM 4.6 4.2 4.1 4.3 Ca, % of DM 1.91 1.93 2.06 1.80 P, % of DM 0.33 0.32 0.33 0.33 Mg, % of DM 0.33 0.31 0.35 0.32 K, % of DM 1.41 1.38 1.40 1.42 Na, % of DM 0.19 0.18 0.20 0.19 S, % of DM 0.42 0.42 0.42 0.42 Cl, % of DM 0.76 0.78 0.80 0.63 Fe, mg/kg of DM 328 307 316 312 Zn, mg/kg of DM 50 46 55 47 Cu, mg/kg of DM 19 19 22 20 Mn, mg/kg of DM 40 39 42 42 1 HOSFO = high oleic sunflower oil (Trisun, Hum ko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn, OH) ; CaVeg = Megalac-R (Church & Dwight Co, Princet on, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN); 2 Trace mineralized salt cont ained minimum concentrations of 40% Na, 55% Cl, 0.25% Mn, 0.2% Fe, 0.033% Cu, 0.007% I, 0.005% Zn, and 0.0025% Co (DM basis). 3 Mineral and vitamin premix contained 22.8% CP, 22.9% Ca, 0.20% P, 0.2% K, 2.8% Mg, 0.7% Na, 2.4% S, 8% Cl, 147 mg/kg of Mn, 27 mg/kg of Fe, 112 mg/kg of Cu, 95 mg/kg of Zn, 7 mg/kg of Se, 8 mg/kg of I, 11 mg/kg of Co, 268,130 IU of vitamin A/kg, 40,000 IU of vitamin D/kg, and 1129 IU of vitamin E/kg (DM basis). 4 Fisher Scientific, Hampton, NH, USA.

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91 Table 3-2. Ingredient and chemical compositi on of TMR fed to Holstein cows during the postpartum period. Treatments1 HOSFO CaTRANS CaVeg LSO Ingredient (% of dietary DM) Corn silage 37.5 37.5 37.5 37.5 Alfalfa hay 10.0 10.0 10.0 10.0 Cottonseed hulls 2.4 2.4 2.4 2.4 Ground corn 21.9 21.6 21.6 21.9 Citrus pulp 5.1 5.1 5.1 5.1 Soybean meal 9.6 9.6 9.6 9.6 Soyplus2 6.9 6.9 6.9 6.9 Mineral and vitamin mix3 4.7 4.7 4.7 4.7 Biophos4 0.4 0.4 0.4 0.4 Ethoxiquin5 0.015 0.015 0.015 0.015 High oleic sunflower oil 1.50 1.50 CaTRANS 1.75 ... ... CaVeg 1.75 Linseed oil ... 1.50 Component NEL, Mcal/kg of DM 1.68 1.68 1.68 1.68 CP, % of DM 17.0 17.0 17.2 16.7 NDF, % of DM 31.7 32.0 32.4 33.7 ADF, % of DM 18.7 18.9 18.9 18.9 Ether extract, % DM 4.3 5.1 4.8 4.4 Ca, % of DM 1.17 1.26 1.16 1.03 P, % of DM 0.49 0.47 0.45 0.46 Mg, % of DM 0.29 0.28 0.27 0.27 K, % of DM 1.50 1.48 1.49 1.48 Na, % of DM 0.47 0.45 0.40 0.40 S, % of DM 0.24 0.22 0.23 0.23 Cl, % of DM 0.42 0.35 0.41 0.43 Fe, mg/kg of DM 276 281 274 272 Zn, mg/kg of DM 118 107 105 105 Cu, mg/kg of DM 19 19 17 17 Mn, mg/kg of DM 83 89 67 69 1 HOSFO = high oleic sunflower oil (Trisun, Hum ko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn, OH) ; CaVeg = Megalac-R (Church & Dwight Co, Princet on, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN). 2 West Central Soy, Ralston, IA. 3 Mineral and vitamin mix containe d 26.4% CP, 10.2% Ca, 0.90% P, 3.1% Mg, 1.5 % S, 5.1% K, 8.6 % Na, 11698 mg/kg of Zn, 512 mg/kg of Cu, 339 mg/kg of Fe, 2231 mg/kg of Mn, 31 mg/kg of Co, 26 mg/kg of I, 7.9 mg/kg of Se, 147,756 IU of vitamin A/kg, and 787 IU of vitamin E/kg (DM basis). 4 IMC-Agrico, Bannockburn, IL. 5 Fisher Scientific, Hampton, NH, USA.

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92 Table 3-3. Fatty acid profile of the fat supplements (% of identified fatty acids). Fat supplements1 HOSFO CaTRANS CaVeg LSO C14:0 ND20.3 0.9 ND C16:0 4.6 15.0 36.3 6.0 cis -9 C16:1 ND 0.1 ND ND C17:0 ND 0.1 0.1 ND C18:0 2.7 8.3 3.9 3.2 C18:1 trans family ND 61.0 0.6 ND cis -9 C18:1 78.8 13.0 26.1 19.6 C18:2 n-6 13.7 2.0 28.5 16.0 CLA ND ND ND ND cis -9, trans -11 ND 0.2 0.5 ND trans -10, cis -12 ND ND 0.1 ND C18:3 n-3 0.2 ND 3.0 55.2 1 HOSFO = high oleic sunflower oil (Trisun, Humko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn, OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN). 2 ND = not detected.

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93Table 3-4. Dry matter intake, milk yield, milk composition, feed efficiency, energy balance, postpartum body weight, and postp artum body condition score of Holstein cows fed diets supplemented with high oleic sunf lower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed o il (LSO) from 4 wk prepartum to 15 wk postpartum. Treatments1 Orthogonal contrasts 2, P = Measure HOSFO CaTRANS CaVeg LSO SE Parity Week Treatment*Week A B C D E F P3 M4 P M P M P M Dry matter intake (kg/d) Prepartum 7.00 10.55 7.57 10.76 7.07 10.35 8.07 10.76 0.66 <0.001 <0.001 0.10 0.85 0.55 0.31 0.69 0.78 0.66 Postpartum 15.46 17.69 14.00 18.45 14.18 17.43 14.77 18.45 0.90 <0.001 <0.001 0.60 0.76 0.69 0.39 0.92 0.21 0.82 Dry matter intake (% BW) Postpartum 2.96 2.86 2.83 2.91 3.01 2.88 3.11 3.03 0.17 0.64 <0.001 <0.001 0.34 0.82 0.48 0.70 0.57 0.89 Milk yield, kg/d 32.35 35.58 29.65 38.55 27.81 34.81 29.53 36.68 2.05 <0.001 <0.001 0.92 0.30 0.89 0.40 0.79 0.15 0.69 3.5% FCM, kg/d5 30.64 34.58 26.96 36.54 26.77 32.49 29.94 36.41 1.90 <0.001 <0.001 0.51 0.57 0.64 0.08 0.80 0.13 0.85 3.5% FPCM6 (kg/d) 29.90 33.59 26.54 36.25 26.72 31.79 28.89 35.81 1.76 <0.001 <0.001 0.860 0.54 0.84 0.10 0.78 0.09 0.61 ECM7 (kg/d) 30.22 33.95 26.83 36.64 27.01 32.13 29.21 36.20 1.78 <0.001 <0.001 0.60 0.54 0.84 0.10 0.78 0.09 0.61 Milk fat, % 3.22 3.36 3.02 3.26 3.35 3.15 3.65 3.55 0.16 0.97 <0.001 0.07 0.16 0.34 0.04 0.31 0.95 0.98 Milk protein, % 2.75 2.76 2.79 2.76 2.70 2.80 2.76 2.80 0.08 0.58 <0.001 1.00 0.98 0.83 0.75 0.51 0.81 0.76 Milk fat yield, kg/d 1.03 1.18 0.87 1.22 0.91 1.08 1.06 1.27 0.07 <0.001 0.25 0.71 0.97 0.62 0.09 0.56 0.20 0.34 Milk protein yield, kg/d 0.88 0.97 0.81 1.06 0.77 0.95 0.80 1.03 0.05 <0.001 <0.001 0.93 0.37 0.67 0.63 0.78 0.13 0.22 Milk SCC, x 1000/mL 116 310 307 88 148 178 65 101 21 0.82 <0.001 0.64 0.27 0.79 0.22 0.56 0.04 0.82 Feed eficiency 8 1.68 1.68 1.65 1.71 1.69 1.60 1.73 1.72 0.11 0.91 <0.001 0.69 0.88 0.97 0.48 0.64 0.81 0.70 Energy balance9 (Mcal/d) 3.78 4.68 3.60 4.49 4.20 5.18 4.32 5.48 1.25 0.28 <0.001 0.33 0.46 0.88 0.87 0.92 1.00 0.95 BW, kg 533 640 500 650 479 632 482 635 22 <0.001 <0.001 0.93 0.13 0.61 0.89 0.44 0.32 1.00 BCS 3.14 3.11 3.37 3.13 2.86 2.98 2.94 3.19 0.16 0.82 <0.001 0.11 0.08 0.42 0.37 0.14 0.49 0.70 1 HOSFO = high oleic sunflower oil (Trisun, Humko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn, OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN). 2 Orthogonal contrast of means were the following: A = MUFA (HOSFO + TRANS) vs. Poly (CaVeg + LSO), B = HOSFO vs. TRANS, C = CaV eg vs. LSO, D = contrast A by parity interaction, E = contrast B by parity interaction, and F = contrast C by parity interaction. 3 Primiparous cows. 4 Multiparous cows.

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945 3.5% FCM = (0.4324*milk yield) + (16.216*milk fat yield). 6 3.5% Fat and protein corrected milk = (12.82 kg of fat) + (7.13 kg of protein) + (0.323 kg of milk). 7Energy corrected milk = (0.327 milk kg) + (12.95 kg of fat) + (7.20 kg of protein). Tyrrel and Reid, 1965. 8 Feed efficiency = kg of 3.5% FCM / kg of DMI. 9 Energy balance = net energy of intake (net energy of maintenance + net energy of lactation). NRC, 2001.

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95Table 3-5. Effect of supplemental fat source on concentration of identified fatty acids of milk fat. Fatty acid Treatments1 HOSFO CaTRANS CaVeg LSO S.E. Parity Contrast2 ------% of identified fatty acids-----A B C D E F C4:0 3.18 4.32 3.88 4.43 0.54 <0.001 0.46 0.14 0.50 0.52 0.03 0.21 C6:0 1.11 0.92 0.90 1.03 0.06 0.15 0.40 0.02 0.15 0.60 0.51 0.59 C8:0 0.61 0.48 0.49 0.57 0.04 0.34 0.70 0.02 0.15 0.63 0.69 0.97 C10:0 1.29 1.00 1.03 1.16 0.09 0.28 0.57 0.02 0.31 0.36 0.87 0.75 C12:0 1.51 1.25 1.26 1.37 0.10 0.32 0.51 0.06 0.45 0.23 0.99 0.60 C14:0 11.95 10.92 10.54 11.23 0.54 0.99 0.32 0.18 0.40 0.12 0.81 0.35 C15:0 0.33 0.34 0.34 0.33 0.02 0.21 0.83 0.76 0.64 0.08 0.20 0.59 C16:0 30.13 30.12 31.32 30.05 0.73 0.15 0.45 0.99 0.24 0.99 0.70 0.03 cis -9 C16:1 0.97 1.10 0.97 0.94 0.04 <0.001 0.07 0.02 0.68 0.29 0.16 0.59 C17:0 0.69 0.73 0.72 0.70 0.02 <0.001 0.87 0.09 0.52 0.81 0.01 0.18 C18:0 12.87 12.33 12.66 13.47 0.47 0.04 0.33 0.41 0.25 0.77 0.75 0.03 C18:1 trans family 3.96 5.59 3.50 3.07 0.36 0.02 <0.001 <0.001 0.41 0.14 0.12 0.95 cis -9 C18:1 25.92 24.95 26.02 25.58 1.10 0.82 0.74 0.52 0.79 0.03 0.91 0.15 C18:2 n-6 3.73 3.97 4.43 3.65 0.15 0.06 0.23 0.26 0.01 0.50 0.47 0.55 CLA cis -9, trans -11 0.98 1.22 0.99 1.12 0.06 0.01 0.49 0.01 0.16 0.49 0.98 0.86 trans -10, cis -12 0.03 0.03 0.05 0.02 0.01 0.62 0.28 0.72 0.01 0.20 0.63 0.97 C18:3 n-3 0.44 0.46 0.57 0.97 0.03 0.71 <0.001 0.65 <0.001 0.37 0.62 <0.01 C20:4 n-6 0.27 0.23 0.26 0.24 0.02 0.82 0.93 0.09 0.44 0.18 0.59 0.67 C20:5 n-3 0.008 0.004 0.017 0.036 0.006 0.27 0.01 0.65 0.04 0.24 0.82 0.90 C22:6 n-3 0.000 0.001 0.001 0.001 0.001 0.15 0.65 0.18 0.84 0.19 0.91 0.84 SAT3 63.7 62.4 63.2 64.3 1.3 0.40 0.60 0.49 0.54 0.22 0.64 0.17 MUFA4 30.8 31.6 30.5 29.6 1.2 0.31 0.33 0.63 0.62 0.13 0.55 0.20 PUFA5 5.5 5.9 6.3 6.1 0.2 0.55 0.03 0.15 0.39 0.33 0.60 0.35 PUFA/MUFA 0.18 0.19 0.21 0.21 0.01 0.18 0.01 0.62 0.84 0.04 0.32 0.56 n6/n3 ratio6 10.8 11.5 9.8 5.0 0.3 0.28 <0.001 0.09 >0.001 0.82 0.91 0.01 Desaturase index7 cis -9 C16:1 0.03 0.03 0.03 0.03 0.01 0.05 0.14 0.15 0.76 0.45 0.52 0.27 cis -9 C18:1 0.67 0.67 0.67 0.65 0.01 0.13 0.61 0.86 0.24 0.05 0.96 0.71

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96 1 HOSFO = high oleic sunflower oil (Trisun, Humko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn, OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN). 2 Orthogonal contrasts: A) HOSFO + CaTRANS vs. CaVeg + LSO; B) HOSFO vs. CaTRANS, C) CaVeg versus LSO, D) Contrast A by parity in teraction, E) Contrast B by parity interaction, and F) Contrast C by parity interaction. 3 SAT = C4:0 + C6:0 + C8:0 + C10:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0; 4 MUFA = cis -9 C16:1 + C18:1 trans family + c is -9 C18:1. 5 PUFA = C18:2 + C18:3 + cis -9, trans -11 CLA + trans -10, cis -12 CLA + C20:4 + C20:5 + C22:6. 6 n-6/n-3 ratio = (C18:2 + cis -9, trans11 CLA + trans -10, cis -12 CLA + C20:4) / (C18:3 + C20:5 + C22:6). 7 Desaturase indexes are ratios of the 9-desaturase product divided by the sum of the 9-desaturase product and substrate. For example, the desaturase index for cis -9 C16:1 would be ( cis -9 C16:1)/( cis -9 C16:1 + C16:0).

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97Table 3-6. Effect of supplemental fat source on co ncentration of identified fatty acids of plasma. Treatments1 S.E. Parity Contrast2 Fatty acid HOSFO CaTRANS CaVeg LSO ------------% of identified fatty acid-------A B C C14:0 0.71 0.68 0.7 0.67 0.04 0.58 0.77 0.56 0.59 C15:0 0.38 0.37 0.36 0.35 0.02 0.08 0.32 0.94 0.81 C16:0 16.6 16.2 17.2 16.4 0.4 0.19 0.36 0.47 0.21 cis -9 C16:1 0.66 0.71 0.65 0.71 0.03 <0.001 0.84 0.26 0.21 C17:0 0.55 0.6 0.58 0.59 0.02 0.01 0.57 0.21 0.84 C18:0 15.5 15.0 15.2 15.7 0.3 0.67 0.39 0.20 0.29 C18:1 trans family 1.15 1.51 0.90 1.05 0.14 0.01 0.02 0.07 0.47 cis -9 C18:1 12.3 10.7 11.6 11.2 0.5 0.09 0.79 0.03 0.67 C18:2 n-6 43.4 45.3 44.4 41.8 0.9 0.04 0.16 0.12 0.05 C18:3 n-3 2.37 2.46 2.32 4.89 0.17 0.11 <0.001 0.69 <0.001 cis -9, trans -11 CLA 0.13 0.13 0.16 0.14 0.01 0.01 0.01 0.73 0.22 CLA trans -10, cis -12 0.04 0.04 0.03 0.05 0.01 <0.001 0.97 0.78 0.11 C20:3 0.06 0.05 0.04 0.06 0.01 <0.001 0.58 0.07 0.02 C20:4 n-6 4.89 5.11 4.72 4.83 0.25 0.13 0.37 0.52 0.77 C20:5 n-3 0.53 0.53 0.46 0.71 0.05 0.14 0.28 0.97 0.01 C22:5 n-3 0.55 0.55 0.53 0.65 0.03 <0.001 0.16 0.99 0.01 C22:6 n-3 0.08 0.06 0.06 0.08 0.01 0.01 0.51 0.29 0.28 SAT3 33.8 32.9 34.2 33.8 0.5 0.20 0.18 0.15 0.60 MUFA4 14.2 12.9 13.1 13.0 0.5 0.01 0.35 0.10 0.87 PUFA5 52.0 54.2 52.7 53.2 0.9 0.34 0.86 0.08 0.70 PUFA/MUFA 3.71 4.27 4.21 4.28 0.25 0.24 0.32 0.11 0.85 n6/n3 ratio6 13.8 14.0 15.0 7.5 0.5 <0.001 <0.001 0.69 <0.001 Desaturase index7 di161 0.04 0.04 0.04 0.04 0.01 0.01 0.56 0.24 0.14 di181 0.44 0.42 0.43 0.41 0.01 0.10 0.54 0.17 0.51 Total lipid (mg/mL) 1.34 1.31 1.36 1.25 0.08 0.01 0.83 0.78 0.38

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981 HOSFO = high oleic sunflower oil (Trisun, Hum ko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairla wn, OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (A rcher Daniels Midla nd, Redwing, MN). 2 Orthogonal contrasts: A) HOSFO + CaTRANS vs. CaVeg + LS O; B) HOSFO vs. CaTRANS, C) CaVeg versus LSO. 3 SAT = C14:0 + C15:0 + C16:0 + C17:0 + C18:0; 4 MUFA = cis -9 C16:1 + C18:1 trans family + cis -9 C18:1. 5 PUFA = C18:2 + C18:3 + cis -9, trans -11 CLA + CLA trans -10, cis -12 + C20:4 + C20:5 +C22:5 + C22:6. 6 n-6/n-3 ratio = (C18:2 + cis -9, trans -11 CLA + CLA trans -10, cis -12+ C20:4) / (C18:3 + C20:5 +C22:5 + C22:6). 7 Desaturase indexes are ratios of the 9-desaturase product divided by the sum of the 9-desaturase product and substrate. Fo r example, the desaturase index for cis -9 C16:1 would be ( cis -9 C16:1)/( cis -9 C16:1 + C16:0).

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99Table 3-7. Effect of supplemental fat source on liver fatty acid prof ile on days 2, 14, and 28 postpartum. Treatments1 (Trt) Effects Contrast2 Fatty acid HOSFO CaTRANS CaVeg LSO S.E. Parity Day Trt x day Trt x parity x day A B C DIM of biopsy 2 14 28 2 14 28 2 14 28 2 14 28 ---------------------------------------------% of identified fatty acid-----------------------------------------C14:0 2.47 2.79 2.47 2.68 3.21 2.52 2.52 2.64 2.45 2.38 3.40 2.62 0.32 0.001 <0.001 0.37 0.26 0.94 0.55 0.52 C15:0 0.15 0.18 0.17 0.16 0.20 0.23 0.14 0.16 0.21 0.16 0.20 0.21 0.02 0.19 <0.001 0.40 0.02 0.98 0.20 0.38 C16:0 28.74 30.83 24.69 30.33 31.66 25.26 30.30 30.24 27.34 27.16 34.44 26.97 2.06 0.01 <0.001 0.06 0.17 0.65 0.68 0.93 cis -9 C16:1 0.73 0.87 1.05 0.97 1.31 1.14 0.83 0.92 1.13 0.76 1.08 0.91 0.13 0.52 0.01 0.52 0.52 0.49 0.07 0.78 C17:0 0.73 0.80 0.93 0.69 0.69 0.84 0.70 0.68 0.80 0.65 0.62 0.83 0.06 0.02 <0.001 0.82 0.37 0.27 0.31 0.76 C18:0 17.80 16.17 20.74 16.77 15.74 19.39 16.61 16.24 18.54 18.89 12.68 19.34 1.66 0.04 0.001 0.01 0.21 0.62 0.63 0.94 C18:1 trans family 0.78 0.83 1.27 0.98 1.23 1.94 0.81 1.11 1.06 0.79 0.97 1.24 0.17 0.05 <0.001 0.46 0.04 0.14 0.01 0.96 cis -9 C18:1 23.28 23.10 19.08 23.31 21.96 18.28 23.12 22.40 20.21 21.03 25.22 19.81 1.44 0.96 <0.001 0.08 0.46 0.70 0.70 0.95 C18:2 n-6 12.16 11.65 13.17 11.47 11.92 13.77 12.25 13.29 13.63 11.90 10.61 12.38 0.69 0.20 <0.001 0.01 0.23 0.99 0.94 0.12 cis -9, trans 11 CLA 0.47 0.41 0.35 0.47 0.42 0.37 0.51 0.49 0.50 0.50 0.49 0.42 0.04 0.01 0.001 0.55 0.90 0.06 0.85 0.51 CLA trans 10, cis -12 0.02 0.01 0.03 0.00 0.02 0.01 0.02 0.01 0.02 0.01 0.00 0.00 0.01 0.50 0.67 0.53 0.40 0.32 0.31 0.23 C20:0 0.02 0.04 0.02 0.04 0.03 0.02 0.02 0.02 0.03 0.02 0.03 0.05 0.01 0.26 0.84 0.35 0.51 1.00 0.81 0.36 C18:3 n-6 0.99 0.82 0.76 0.99 0.86 0.87 1.02 0.96 0.93 1.34 1.36 1.44 0.07 0.05 0.10 0.40 0.27 <0.001 0.45 <0.001 C20:3 0.04 0.05 0.04 0.05 0.03 0.07 0.02 0.03 0.03 0.08 0.05 0.09 0.02 0.01 0.38 0.80 0.09 0.68 0.77 0.02 C20:4 n-6 9.83 9.33 12.06 9.07 9.02 11.99 9.30 9.05 10.68 11.29 6.90 9.99 1.23 0.01 0.001 0.04 0.17 0.53 0.80 0.86 C20:5 n-3 0.40 0.40 0.89 0.52 0.51 0.86 0.35 0.41 0.56 0.80 0.57 1.23 0.12 0.06 0.001 0.25 0.38 0.50 0.56 0.01 C22:5 n-3 1.45 1.32 1.76 1.41 1.35 1.95 1.37 1.47 1.58 1.95 1.27 2.09 0.19 0.001 0.001 0.01 0.24 0.63 0.79 0.23 C22:6 n-3 0.05 0.11 0.10 0.10 0.08 0.19 0.08 0.06 0.07 0.29 0.09 0.22 0.04 0.001 0.02 0.01 0.01 0.38 0.44 0.01 MUFA3 24.76 24.88 21.32 25.29 24.39 21.38 21.77 24.41 22.40 22.58 27.28 21.97 1.51 0.86 <0.001 0.08 0.59 0.85 0.98 0.97 PUFA4 25.41 24.24 29.18 24.03 24.08 30.10 24.94 25.83 28.04 28.15 21.34 28.01 2.00 0.08 <0.001 0.01 0.13 0.94 0.98 0.88 PUFA/MUFA 1.11 1.02 1.46 1.03 1.12 1.59 1.07 1.14 1.45 1.32 0.83 1.49 0.17 0.44 <0.001 0.01 0.23 0.95 0.80 0.97 n6/n3 ratio5 7.73 8.18 7.63 7.11 7.74 6.93 8.01 8.12 8.00 5.67 5.54 4.80 0.42 0.001 0.03 0.63 0.09 0.02 0.22 <0.001 Desaturase index6 cis -9 C16:1 0.02 0.03 0.04 0.03 0.04 0.04 0.03 0.03 0.04 0.03 0.03 0.03 0.01 0.02 <0.001 0.94 0.65 0.41 0.04 0.86 cis -9 C18:1 0.57 0.60 0.49 0.58 0.58 0.49 0.58 0.58 0.53 0.53 0.66 0.51 0.04 0.19 <0.0001 0.24 0.68 0.64 0.99 0.90 Total lipid (% dry weight) 19.20 20.22 14.66 22.48 30.88 21.39 18.48 19.71 20.98 15.94 28.16 21.88 4.52 0.02 0.01 0.20 0.76 0.77 0.27 0.68

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100 1 HOSFO = high oleic sunflower oil (Trisun, Humko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn, OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN). 2 Ortogonal contrasts: A) HOSFO + CaTRANS vs. CaVeg + LSO; B) HOSFO vs. CaTRANS, C) CaVeg versus LSO. 3 MUFA = cis -9 C16:1 + C18:1 trans family + cis -9 C18:1. 4 PUFA = C18:2 + C18:3 + cis -9, trans -11 CLA + trans -10, cis -12 CLA + C20:4 + C20:5 +C22:5 + C22:6. 5 n-6/n-3 ratio = (C18:2 + cis -9, trans -11 CLA + trans -10, cis -12 CLA + C20:4) / (C18:3 + C20:5 + C22:5 + C22:6). 6 Desaturase indexes are ratio of the 9-desaturase product divided by the sum of the 9-desaturase product and substrate. For example, the desaturase index for cis -9 C16:1 would be ( cis -9 C16:1)/(cis -9 C16:1 + C16:0).

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101 Figure 3-1. Least squares means for dry matter intake of primiparous (A) and multiparous (B) Holsteins cows fed diets supplemented w ith high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Parities differed (P <0.001).

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102 Figure 3-2. Least squares means for dry matter inta ke as % of BW of Ho lsteins cows fed diets supplemented with high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oil (CaVeg) or linse ed oil (LSO) from 4 wk prepartum to 15 wk postpartum. There was a treatment by week interaction (P < 0.01). Cows fed CaTRAN had less DMI at we ek 13 compared to cows fed LSO (P < 0.001).

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103 Figure 3-3. Least squares means for milk yield of primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postp artum. Parities differed (P < 0.001).

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104 Figure 3-4. Least squares means for body wei ght of primiparous (A ) and multiparous (B) Holstein cows fed diets supplemented w ith high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Parities differed (P < 0.001).

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105 Figure 3-5. Least squares means for BCS of Holstein cows fed diets supplemented with high oleic sunflower oil (HO SFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable o il (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Treatments did not differ.

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106 Figure 3-6. Least squares means for energy balance of Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oi l (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Treatments did not differ.

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107 Figure 3-7. Concentration of C18: 2 in liver of Holstein cows fed diets supplemented with high oleic sunflower oil (HO SFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable o il (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. The asterisk at day 14 indicates that co ws fed CaVeg had a greater concentration of C18:2 in liver compared to cows fed LSO (CaVeg vs. LSO by DIM quadratic interaction; P < 0.001).

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108 CHAPTER 4 EFFECT OF DIETS ENRICHED IN DI FFER ENT FATTY ACIDS ON HORMONES, METABOLITES, ACUTE PHASE PROTEINS AND HEPATIC GENE EXPRESSION OF LACTATING HOLSTEIN COWS DURING SUMMER Abstract The objective of the study was to evalua te how dietary fat sources of oleic, trans octadec enoic, linoleic, or linolenic acids affect ed hormones, metabolites, acute phase proteins, and hepatic gene expression of Holstein primipar ous cows (n = 22) and cows (n = 32) during the summer season. Fat supplements were the follow ing: 1) high oleic sunflower oil (HOSFO; Trisun, Humko Oil, 80% C18:1), 2) Ca salt of trans -octadecenoic acids (CaTRANS; EnerG TR, Virtus Nutrition, 61% trans C18:1), 3) Ca salt of vegetable oils (CaVeg; Megalac-R, Church & Dwight Co, 30% C18:2), and 4) li nseed oil (LSO; Archer Daniel s Midland, 56% C18:3 and 16% C18:2). Supplemental fats were fed at 1.35% of dietary DM beginning at 29 d prior to expected calving date. After calving, fats we re fed at 1.5% (oils) and 1.75% (Ca salts) of dietary DM for 15 wk. Blood samples were taken thrice weekly during 7 wk for measurement of insulin, bST, IGF-1, haptoglobin, acid soluble protein. Weekly samples from calving until 7 wk postpartum were analyzed for NEFA, glucose, BUN, and BHBA. Primiparous cows fed PUFA tended to have greater concentration of plasma NEFA and lower concentrations of insulin than primiparous cows fed MUFA. Primiparous cows fed MUFA had a faster decline in GH concentrations in plasma compared to those had PUFA whereas no treat ment differences were detected among multiparous cows. Concentrations of IGF-1 increased at a faster rate for animals fed PUFA compared to those fed MUFA. Cows fe d CaVeg had greater concentrations of glucose in plasma at wk 1 and 2 compar ed to cows fed LSO. However, hepatic mRNA expression of the gluconeogenic enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxykinase, were upregulated in cows fed LSO compared to co ws fed CaVeg. Primiparous cows fed CaTRANS

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109 had greater concentration of hapt oglobin and acid soluble protein compared to primiparous cows fed HOSFO suggesting a proinf lammatory effect. Dietary fa tty acids altered hormones, metabolites, acute phase proteins, and gene expr ession of lactating dairy cows differently. Key Words : fatty acid, acute phase protein, hormone, gene expression Introduction Fat supplem entation is a common nutritional tool used by dair y nutritionists to increase the energy density of the diet and to influen ce reproduction (Staples et al., 1998; Bilby et al. 2006c; Petit and Twagiramungu, 2006, Moussavi et al., 2007b), metabolism (Petit et al., 2007; Andersen et al., 2008), milk yield (Bu et al., 2007 ), milk composition (Bilby et al., 2006c, Huang et al., 2008), and health of dair y cows (Lessard et al. 2004; Rodr iguez-Sallaberry et al., 2007). Inconsistent effects of lipid supplementa tion on performance of dairy cows may be partially explained by differences in the profile of specific fatty acids fed, by the form in which the fat was fed (oil or Ca salt form), the rate of lipid inclusion in the diet, and the time of initiation of fat feeding, among others. The eff ects of dietary PUFA on plasma hormones and metabolites have been inconsistent and the studie s have not included all the same measurements. Researchers have reported that fat supplementa tion increased (Robinson et al., 2002), decreased (Grum et al., 1996; Beam and Butler, 1998; Garcia et al., 2003) or had no effect (Gagliostro et al., 1991; DeLuca and Jenkins, 2000) on IGF-1 concen tration in plasma. Bec-Villalobos et al. (2007) reported no difference in GH concentrations in plasma of cows fed partially hydrogenated fat compared to cows fed no supplemental fat. The lack of response of fat supplementation on GH has been reported in beef (Lammoglia et al ., 2000; Bottger et al., 2002) and dairy cows (Khorasani et al., 1992). However, other researcher s reported an increase in GH concentration in plasma of cows fed different s ources of fat (Gagliostro et al., 1991; Grum et al., 1996; Bilby et al., 2006b).

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110 Mashek et al. (2005) reported that cows infused intravenously with linseed oil had lower insulin concentrations in plasma compared with cows intravenous ly supplied with tallow. Cows fed a 16% linseed diet prepartum had reduced concentrations of plas ma insulin prepartum compared to cows fed a highly saturated fat or control diet from 5 wk before calving until parturition (Andersen et al., 2008) In addition, a Ca salt of palm oil and fish oil supplement reduced insulin concentra tion in plasma of dairy cows (Bilby et al., 2006b). Glucose concentrations in plasma have been affected by dietary fat supplementation (Moallem et al., 2007; Moussa vi et al., 2007a; Andersen et al., 2008). However, studies examining the effects of fat supplementation on the hepatic mRNA of gluconeogenic enzymes of periparturient dairy cows are scarce. Lipids are important modulator s of immune function (Calder, 2007). However, little is known about the effects of source of fatty aci d supplementation on secr etion of acute phase proteins by the liver of pe riparturient dairy cows. The objective of this study was to evaluate the effects of dietar y fat sources rich in oleic, trans-octadecenoic, linoleic, or linolenic acids on hormones, se lected metabolites, acute phase proteins, and hepatic gene expression of pe riparturient Holstein cows during summer. Material and Methods Animals, Treatments, and Sampling Experim ent was conducted at the University of Florida dairy research unit (Hague, FL) during the months of May through December 2004. All experimental animals were managed according to the guidelines approved by the University of Florida Animal Care and Use Committee. Periparturient Holstein primiparous (n = 22) and multiparous (n = 32) cows were assigned to treatment at 30 7 d prior to their calvi ng date. Dietary supplemental treatments were the following: 1) high oleic sunflower oil fr om genetically modified sunflower ( HOSFO ; Trisun,

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111 Humko Oil, Memphis, TN; 80% C18:1), 2) Ca salt of trans -octadecenoic acids ( CaTRANS ; EnerG TR, Virtus Nutrition, Fairlawn, OH, 61% trans C18:1 isomers), 3) Ca salt of vegetable oils ( CaVeg ; Megalac-R, Church & Dwight Co, Prince ton, NJ; 36% C16:0 and 29% C18:2), and 4) linseed oil (LSO ; Archer Daniels Midland, Redwing, MN, 55% C18:3 and 16% C18:2). Supplemental fats were fed at 1.35% of dietary DM during the prepartum period (refer to Table 3.1). After calving, fats were fe d at 1.5% (oil sources) and 1.75% (Ca salt sources) of dietary DM for 15 wk to allow equal concentration of di etary lipid (refer to Table 3.2 in the previous chapter). Calculated calving date, parity (primiparous or multiparous), BW and milk production of the previous year for multiparous cows we re similar among treatment groups. Prepartum cows were housed in sod-based pens equipped wi th fans, sprinklers, and shaded Calan gates (American Calan Inc., Northwood, NH). Postpart um cows were housed in a sand-bedded, freestall barn equipped with fans, sp rinklers, and Calan gates. Inta ke of DM was measured daily. Cows were fed TMR twice daily at 0900 and 1300 h to allow 5 to 10% feed refusals daily. Corn silage was collected weekly and immediately dr ied for 1 h using a Koster (Koster Crop Tester, Inc., Strongsville, OH) to calculate the concentration of DM in or der to maintain the formulated forage to concentrate ratio in the ration. Cows were milked thrice daily at 0100, 0900, and 1700 h. Cows were weighed and body condition scored (Edmonson et al., 1989) weekly after the 0900 h milking and before feeding. Ethoxiquin (Fisher Scientific, Hampton, NH, USA) was used as an antioxidant by adding 0.33 g / kg of concentrate mix resulting in a dietary concentration of 0.015 and 0.020% for nonlactating a nd lactating cows, resp ectively (DM basis). Sample Collection and Analysis Blood (10 mL) was collected at 0700 h on Monday, W ednesday, and Friday from coccygeal vessels into sodium heparinized tubes (Vacutainer, B ecton Dickinson, Franklin Lakes, NJ) from calving until 47 3 DIM. Samples were put immediately on i ce until centrifuged at

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112 2619 x g at 5oC for 30 min (RC-3B refrigerated centrif uge, H 600A rotor, Sorvall Instruments, Wilmington, DE). Plasma was separated and frozen at -20oC for subsequent metabolite and hormone analyses. Plasma concentrations of NEFA (NEFA-C k it; Wako Fine Chemical Industries USA, Inc., Dallas, TX; as modified by (Johnson, 1993) were determined once weekly for 7 wk. A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to determine weekly concentrations of plasma BUN (a m odification of Coulombe and Favreau, 1963 and Marsh et al., 1965) and plasma glucose (a modification of Gochman and Schmitz, 1972). A double antibody radioimmunoassay (RIA) was used to determine plasma concentrations of insulin (Badinga et al., 1991; Ma lven et al., 1987), growth hormone ( GH) (Badinga et al., 1991), and IGF-1 (Badinga et al., 1991) on every plas ma sample collected. The sensitivity of the assays were 1 ng/mL, 1 ng/mL, and 14 pg/mL for insulin, somato tropin, and IGF-1, respectively. The intraand interassay CV were 9.9% and 3.7%, 6.6% and 11.6%, and 3.1% and 7.5%, respectively for insulin, GH, and IGF-1. Concen trations of progesteron e were determined on every plasma sample collected using Coat-A-Count Kit (DPC Diagnostic Products Inc., Los Angeles, CA) solid phase 125I RIA. The sensitivity of the assay was 0.1 ng/mL and the intraand interassay CV were 4.9 and 7.8%, respectively. A polyethylene glycol RIA procedure described by Meyer et al. (1995) was used to analyze for the concentration of 15-keto-13,14-dihydroprostaglandin F2 metabolite ( PGFM ) in each plasma sample collected during the first 10 DIM. Sensitivity of the assay was 31.2 pg/mL and the intraand interassay CV were 8.1 and 4.3%, respectively. Haptoglobin concentrations of plasma were determined by measuring haptoglobin/hemoglobin complexing (Makimura a nd Suzuki, 1982). Acid soluble protein was

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113 extracted from plasma with 0.6 M perchloric ac id and analyzed with the bicinchoninic acid kit (Sigma-Aldrich, Saint Louis, MO). On 2, 14 2, and 28 2 DIM, liver samples were collect via biopsy, rinsed with sterile saline, snap-frozen in liquid N, and stored at -80oC until analyzed for mRNA abundance. Total cellular RNA was isolated from liver (approximately 250 mg, wet basis) by pipeting 3 mL of TriZol (Invitrogen, Carlsbad, CA) into a 15-mL sterile tube contai ning the liver sample. The sample was blended using a homogenizer (PowerGen 700, Fisher Scientific, Hampton, NH, USA) coupled with a generator sawtooth (7 mm x 195 mm) (Fisher Scientific, Hampton, NH, USA) and incubated for 5 min at room temperature with 200 l of chloroform. Tubes were centrifuged at 12,000 x g for 15 min at 4o C. The aqueous upper layer was transferred to a 1.5mL microfuge tube, 500 l of isopropanol added, and the tubes mixed by inversion and incubated at room temperature for 10 min. The supernatant was decanted and 1 mL of 75% ethanol was added to the RNA pellet. The tubes were centrifuged at 12,000 x g for 3 min at 4o C. Supernatant was decanted and the remaining ethanol evaporated at room temperature for 5 min. The RNA pellet was suspended in 25 l of sterile water and the RNA concentration was quantified. Ten g of RNA was fractionated in 1% agarose-formaldehyde gel and blotted to a BioTrans 0.2 micron nylon membrane (MP Biomedical, Irvine, CA) by capillary action. The RNA was cross-linked to the membrane by UV irradiation and baked at 80C for 1 h. Two sets of BioTrans nylon membranes were generated from the same extracted RNA sample in order to probe three genes in each set. The first set of RNA filters were hybridized consecutively with random primer-labeled pyruvate carboxylase (PC), IGF Binding Protein-II ( IGFBP-II), and phosphoenolpyruvate carboxykinase (PEPCK) cDNA and the second set of RNA filters were hybridized with random primer labeled IGF-1, IGFBP-III and IGF-II cDNA probes. After

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114 hybridization, RNA filters were washed twice for 20 min in 30 mL of 2 x saline sodium citrate and 0.1% SDS at 50C, followed by one 15-min wash in 0.1x saline sodium citrate, and 0.1% SDS at 50C. The filters were blotted dry and exposed to x-ray films for 24 h (IGFBP-II), 48 h (PEPCK, IGF-1, and IGF-2), and 72 h (PC and IGBP-III) at C. Hybridization signals for each target gene were quantifie d by densitometric analysis. At 40 2 DIM, a single assessment of ut erine cytology was conducted. Cows were flushed using a 53.3 cm silicon Fo ley catheter (i.e., 18 Fr and 5 cc) The vulva was cleaned with chlorhexidine diacetate (Nolvasan, Fort Dodge, WI) and dried with a paper towel. The catheter was introduced through the cervix in to the previously pregnant ut erine horn. The air balloon was placed approximately 1 cm past the bifurcation of the uterine horn and inflated with air to a volume consistent with the size of the uterine horn. Sterile saline (20 mL of 0.9%) was infused into the uterine horn and aspirated back using a syringe with a Foley connector. The aspirated solution was placed into a steril e 50-mL conical tube and vortexed. A 50-l aliquot of mixed flush was placed into a bullet tube and mixed with 50 l of a trypan blue solution (0.4%) for 1 min. A 10 l sample of the solution was placed in each side of the hemacytometer in order to count total white blood cells (WBC) and to determ ine cell viability using a magnification of 40x. Cells not stained were considered live and cells stained with trypa n blue were considered dead. Concentration of WBC was determined by coun ting 5 squares (i.e., each square = 0.2 0.2 mm2) from each side of the hemocytometer in the la rge middle square using a magnification of 40x. Concentration of WBC wa s calculated as follows: WBC (number / mL) = total WBC count in 10 squares/2 50,000 dilution factor 2. Proportion of cell viability was calculated as follows: cell viability (%) = (number of WBC not stained (via ble) / number of staine d and not stained WBC) 100. After determination of total and viable WBC, 20 l of mixed flush was pipetted onto a

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115 glass slide and smeared (2 slides per flush). Smear was air-dried and stained using the DiffQuick (Fisher Diagnostics, Middletown, VA) st ain. Slides were examined for WBC and neutrophil numbers at magnificat ion of 40x. Number of tota l WBC as well as number of neutrophils were counted and per cent of neutrophils calculated as follows: % neutrophils = total number of neutrophils / total number of WBC 100. The percentage of neutrophils was used to estimate the number of ne utrophils per mL of flush solution using the hemacytometer results as follows: number of neutrophils / mL of flush = total WBC count / mL (hemacytometer) % neutrophils. Statistical Analysis Repeated measures d ata (plasma concentra tions of NEFA, BUN, and glucose, liver mRNA expression for PC, PEPCK, IGF-1, IGF-II, IGFBP-II, and IGFBP-III) were analyzed using the PROC MIXED procedure of SAS according to the following model: Yijkl = + Fi + Pj + FPij +Ck (i j) +Wl + FWil + PWjl + FPWijl + Eijkl where Yijkl is the observation, is the overall mean, Fi is the fixed effect of dietary fat source (i = 1, 2, 3, and 4), Pj is the fixed effect of parity (j = 1 and 2), FPij is the interaction of fat source and parity, Ck (i j) is random effect of cow within fat source and parity (k = 1, 2, n), Wl is the fixed effect of week (l = 0, 1, 2, ), FWil is the interaction of fat source and week, PWjl is the interaction of parity and week, FPWijl is the three way interaction of fat source, parity and week, and Eijkl is the residual error. Data were tested to determine the structure of best fit, namely AR (1), ARH (1), CS, or CSH, as indicated by a lower Schw artz Baesian information criteri on value (Littell et al., 1996). Orthogonal contrasts used to detect treatment differences were the following: 1) HOSFO + CaTRANS vs. CaVeg + LSO, 2) HOSFO vs. CaTRANS, and 3) CaVeg vs. LSO. For liver samples collected at DIM not equally spaced (2, 14, and 28 DIM), the IML procedure of SAS

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116 was used to generate coefficients for testing of linear and quadratic day effects. After testing for the level of order that best fit up to cubic, single degree of fr eedom contrasts of treatments by week were tested. Data that did not have a common DIM at the time of collection (plasma concentrations of insulin, IGF-1, GH, and progesterone) were mode led using the CS structure as a polynomial function of time using regression an alysis and coefficients were obt ained to plot the curves after the level of order (linear, quadratic cubic, or quartic) that best fit the data was determined. Homogeneity of regression was performed to dete rmine if the curves differed for each of the orthogonal contrasts cited above. Acute phase proteins and log of total ne utrophil counts were analyzed using Proc Glimmix of SAS since the data did not have a normal distribution. Results and Discussion Metabolites and Hormones Glucose Ani mals fed LSO had ( P < 0.05) lower concentrations of gl ucose in plasma at weeks 1 and 2 postpartum compared to CaVeg-fed cows (Figure 4.1). Petit et al. (2007) reported that cows fed saturated fat at 1.7% of dietary DM starting 6 wk prior to calving ha d lower plasma glucose concentrations at weeks 1, 2, and 4 postpartum comp ared to cows fed a control diet without fat supplement or a diet supplemented with flaxseed at 3.3% of dietary DM. The authors attributed the hypoglycemia to the lower DMI and more negativ e energy balance of the cows fed saturated fat. In the present study, ther e were no treatment differences fo r either DMI or energy balance (Chapter 3) but concentration of plasma NEFA were greater at wk 2 for cows fed LSO. Consistent with our data, Ande rsen et al. (2008) reported that cows fed a high linseed diet prepartum had lower concentration of glucose in plasma compared to cows fed a highly saturated

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117 fat or control diet. Conversely, Mo ussavi et a. (2007a) found that cows fed Ca salts of fish oil at 2.3 % of dietary DM had greater glucose concentr ations in plasma compared to cows fed a control diet or a diet of 2.5% (dietary DM) menhaden fish meal. In another study (Moallem et al., 2007), plasma concentrations of glucose did not differ among co ws fed Megalac-R, saturated fat, or a control diet. Mashek and Grummer (2003) reported that gl uconeogenesis was reduced in hepatocytes incubated with C22:6 compared to other LCFA. In the present study, co ws fed LSO had greater hepatic concentration of C22:6 co mpared to cows fed CaVeg and thus may explain the decrease in plasma glucose concentrations. Blood urea nitrogen Plasm a concentrations of BUN were high at calving likely due to the aminoacid break down from the uterus, declined until wk 2 and th en gradually increased until wk 7 in primiparous and multiparous cows (Figure 4.2). Plasma c oncentrations of BUN did not differ among treatments as reported by others feeding multiple fat sources (Petit et al., 2007). NEFA When the energy needed for m aintenance and la ctation is greater than the energy provided in the diet, the dairy cow will begin to mobilize he r body fat stores to lessen the energy deficit. Hormone sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from the TG in adipose tissue (Holm et al ., 2000). Hormone sensitive li pase is dephosphorylated and inactivated by insulin whereas an increment in the cAMP concentr ation and activation of protein kinase A by glucagon, epinephrine, and ACTH to promote phosphorylation and activation of HSL (Holm et al., 2000) which is translocated fro m a cytosolic compartmen t to the surface of the lipid droplet (Egan et al., 1992; BrasaemLe et al., 2000). A second enzyme, adipose triglyceride lipase (ATGL), catalyzes the initial step in trig lyceride (TG) hydrolys is (Zimmermann et al.,

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118 2004). Thus, ATGL and HSL coordinately catabo lize stored TG in adip ose tissue of mammals (Zimmermann et al., 2004). Nonesterified fatty acids (NEFA) are released into the blood from adipose tissue and transported to hepatic and non-hepatic tissues. Animals fed LSO had a greater concentration of NEFA in plasma at wk 2 ( P = 0.02) and 5 ( P = 0.08) postpartum compared to animals fed CaVeg (treatment by DIM interactio n) (Figure 4.3). Because animals at wk 2 appeared to have similar intakes of DM, produc tion of milk and loss of BW, it is difficult to explain this difference in NEFA values. In addition, primiparous cows fed PUFA (CaVeg or LSO) tended to have greater mean concentratio n of plasma NEFA than primiparous cows fed MUFA (HOSFO or CaTRANS) (611 vs. 436 mEq/L) whereas cows fed these supplements did not differ (564 vs. 522 mEq/L; Table 4.1) (parit y by [HOSFO + CaTRANS] vs. [CaVeg + LSO] interaction, P = 0.08). Others have reported that liver lipid accumulation occu rs in the presence of increased plasma concentra tion of NEFA at calving (Skaar et al., 1989; Vazquez-Anon et al., 1994). The concentrations of plasma NEFA in the present study were not high enough to cause hepatic lipid accumulation (Drackley, 2001). Selberg et al. (2004) reported that cows fe d a CLA supplement had greater concentrations of NEFA in plasma at wk 1 postpartum compared to cows fed trans fatty acids or a control diet without fat. However, Baumgard et al. (2000, 20 02) showed little or no effect of supplemental CLA on plasma NEFA concentrations. Moallem et al. (2007) reported that cows fed 215 g/d of Megalac-R starting at 256 d of pregnancy throug h 110 d postpartum had greater concentration of NEFA in plasma postpartum compared to cows not fed fat but did not differ from cows fed a saturated fat source. In contrast, Petit et al. (2007) reported that multiparous cows fed a saturated fatty acid source starting prepartum had a greater concentration of NEFA in plasma at wk 1, 2, and 4 compared to cows supplemented with flaxs eed or not supplemented with fat but there was

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119 no effect of fat source on NEFA of primiparous cows. DeFrain et al. (2005) evaluated the effect of feeding propionate with and without Ca salts of fatty acids on hormone and metabolites of periparturient dairy cows. Cows supplemente d with 178 g/d of propionate and 154 g of LCFA had lesser concentration of NEFA in plasma comp ared to cows fed 120 g/ d of propionate and 93 g of LCFA (623 vs. 875 Eq/L). Andersen et al. (2008) reported that feed ing linseed at 1.5% of dietary DM increased the prepartum concentratio ns of plasma NEFA compared to cows fed a low fat control diet but did not differ from co ws fed a highly saturated fat diet. However, postpartum concentrations of NEFA in plasma did not differ among treatments. Fat supplementation influences the fatty acid profile of adipose tissue in dairy cows (Chilliard et al., 1991), beef cattl e (Ducket et al., 1993; Garcia et al., 2003; Gillis et al., 2004) and sheep (Bolte et al., 2002; Cooper et al., 2004). We specu late that fat supplementation starting prepartum changed the fatty acid profil e of the adipose tissue in such way that primiparous cows fed PUFA had a higher proporti on of PUFA on the TG in the adipose tissue which in turn were preferentially hydrolyzed by the HSL and increased the NEFA concentration in plasma compared to primiparous cows fed MUFA. Insulin Concentration of plasma insulin usually refl ects energy in take. It in creases gradually as days postpartum increase and as DMI of dairy cows increase. Fat supplementation has had mixed results on circulating concentr ation of plasma insulin (Staples et al., 1998). In studies in which fat supplementation depressed plasma insulin (8 out of 17 studies re viewed by Staples et al., 1998), the diet and day differe nces were eliminated when energy balance was used as a covariate in the statistical mode l suggesting that insulin differe nces among diets were due to differences in EB. In the present study, ther e was no difference in mean EB among treatments (Chapter 3) but the dietary treatment effect wa s significant for plasma insulin concentrations.

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120 Primiparous cows fed PUFA tended to have lowe r mean concentrations of insulin in plasma compared to primiparous cows fed MUFA (0.44 vs. 0.51 ng/mL) whereas multiparous cows experienced the opposite effect (0.47 vs. 0.41 ng/mL ; MUFA vs. PUFA by parity interaction (P = 0.06) (Table 4.2). The lower concentration of insulin in plasma probably stimulated HSL and thus TG hydrolysis in the adipos e tissue leading to increased NE FA concentrations in plasma. This was most evident in primiparous co ws. Gavino and Gavino (1992) studied the HSLmediated release of fatty acids from TG in cultured preadipocytes containing PUFA-enriched triglyceride. They found that cu ltured preadipocytes challenged with 10 M of norepinephrine tended to release more omega-6 and omega-3 PUFA than saturated fatty acids. Indeed, crude preparations of HSL released C18:3 from the TG substrates twice as fast as cis -9 C18:1. Raclot et al. (2001) evaluated the fatty acid specificity of HSL in lipid emulsions and reported that HSL is slightly affected by the degree of unsaturation of the fatty acid in the TG. Thus, this selectivity could affect the individual fa tty acid supply from the tissues (Raclot, 2003). Feeding n-3 long chain PUFA, as compared to a high fat diet, lowered concentrations of plasma insulin in rodents by sustaining glucose transporter protein GLUT4 receptors in the muscle, by preventing decreased expression of GLUT4 in adipose tissue, and by inhibiting both activity and expression of liver glucose-6-phosphatase that increased glucose uptake and metabolism (Delarue et al., 2004). Xiao et al. (2006) reporte d that different fatty acid profiles affected glucose-induced insulin secretion in humans differently. Mash ek et al. (2005) reporte d that cows infused intravenously with linseed oil had a lower insuli n concentration in plasma compared with cows intravenously supplied with tallow. Cows fed lin seed prepartum had reduced concentrations of insulin prepartum compared to cows fed a satura ted fat or no fat diet from 5 wk relative to calving until parturition (Andersen et al., 2008). In addition, fi sh oil supplementation reduced

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121 insulin concentration in plasma of dairy cows (Bilby et al., 2006c). Multiparous cows fed the MUFA supplements experienced a slower increase in plasma concentrations of insulin compared to those fed the PUFA supplements whereas the increase over time for primiparous cows did not differ (MUFA vs. PUFA by parity by DIM interaction, P = 0.01; Figure 4.5) This interaction was due mostly to the effect of feeding CaTRANS. Multiparous cows fed the CaTRANS supplement experienced a slower in crease in plasma concentrations of insulin compared to those fed HOSFO whereas the increase over time for primiparous cows did not differ (HOSFO vs. CaTRANS by parity by DIM interaction, P = 0.03; Figure 4.6). Growth hormone and insulin-like grow th factor-1 Nutrient partitioning for lactog enesis is mediated and sustai ned by alterations in the GHIGF axis. Under physiological conditions, pituitary-derived GH induces hepatic synthesis of IGF-1 via receptor-mediated signaling (Bichell et al., 1992) and consequently systemic IGF-1 negatively regulates GH production (Le Roith et al., 2001). Howeve r, in situations of high nutrient demand from the body such as after part urition, the state of NEB uncouples the GH-IGF axis in the liver (Thissen et al., 1994). This is associated with a reduction in total circulating IGF1 and elevated GH concentrations (Vanderhaar et el., 1995). Severe NEB reduces plasma concentrations of IGF-1 and hepa tic expression of IGFBP-3 compar ed to mild NEB (Fenwick et al., 2008). Plasma concentrations of IGF-1 increased at a faster rate postpartum in animals fed PUFA compared to those fed MUFA (PUFA vs. MUFA by DIM interaction, P < 0.05; Figure 4.7 and 4.8) regardless of similar NEB among treatments Almost all IGF secreted from the liver circulates as a bound complex and the majority of it (> 90%) is associated with IGFBP-3 (Clemmons, 1997). Cows fed PUFA experienced a rise in the expression of hepatic IGFBP-3 mRNA overtime (Figure 4.28) compared to that of cows fed MUFA which remained unchanged.

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122 This would be expected based upon IGF-1 results. IGFBP-3 is produced mainly in the liver and is the major transporter of IGF-1 in the peripheral circulation (B urger et al., 2005). The increase in IGFBP-3 mRNA expression in the liver pr evented IGF-1 degradat ion and potentially increased availability to other tissues by providing a reservoir of IGF-1 (Boisclair et al., 2001). Li et al. (1999) reported that ra ts fed omega-3 fatty acids had gr eater concentrations of IGFBP-3 in plasma compared to rats fed omega-6 fat or a no-fat control diet. Ca stanheda-Gutierrez et al., (2007) reported an increase in the concentrations of IGF-1 in plasma of cows fed CLA (7.1 g/d of each of the cis -9, trans -11 and trans -10, cis -12 isomers) compared to cows not fed fat but the mechanism by which CLA increases IGF-1 is unknow n. During NEB in early lactation the liver is refractory to GH, resulting in low concentr ations of circulating IG F-1, but greater insulin availability restores coupling of the GH-IGF-1 axis increasing circulation of IGF-1 (Butler et al., 2003). In the present stu dy, mean concentrations of insulin in plasma tended to be greater for multiparous cows fed PUFA compared to co ws fed MUFA but the opposite was true for primiparous cows (Figure 4.2 and 4.3). Castanheda-G utierrez et al. (2007) speculated that the increase in plasma IGF-1 due to feeding CLA to lactating cows may be mediated by subtle changes on hepatic sensitivity to insulin. This is in agreement with the effects of supplementing PUFA to multiparous cows in the present experime nt. Robinson et al. (2002) reported that cows fed nonenzymatically browned full-fat soybeans ha d greater concentratio ns of IGF-1 around the time of peak surge in LH compared to cows not supplemented with fat or those fed linseeds but there was no effect of fat source on insulin concentrations in plas ma. Low circulating concentration of IGF-1 has been reported for co ws fed prilled saturated fat (Grum et al., 1996; Beam and Butler, 1998) and young primiparous cows fed a high linoleic acid (sunflower seeds) diet compared to animals not fed supplementa l fat (Garcia et al., 2003). In contrast, fat

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123 supplementation did not affect IGF-1 in plasma of lactating Jersey cows fed a mixture of canola oil and oleamide (DeLuca and Jenkins, 2000), or Holstein cows infused postruminally with rapeseed oil (1.0 to 1.1 kg/d) from d 17 befo re expected calving date to d 21pospartum (Gagliostro et al., 1991). The discrepancy in the effects of lipid supplementation on IGF-1 concentration might be due to differences in the fat source, physiological state of the animal, and concentration of fat in the diet. Concentrations of IGF-1 in plasma may be a good indicator of repr oductive responsiveness to postpartum dietary treatments in high produc tion dairy cows (Thatche r et al., 2006). If feeding PUFA increases IGF-1 c oncentration in plasma with in creasing DIM early post partum as observed in the current study, this may st imulate estradiol secr etion by the thecal and granulosa cells of the follicle and consequently promote cell proliferation and follicular growth. At TAI (~ 79 DIM) the size of the dominant foll icle was increased in cows fed PUFA compared to cows fed MUFA in a previously published po rtion of the present study (Bilby et al., 2006a). Subsequently, CL volume was larger in cows fe d PUFA (Bilby et al., 200 6a). Prior to AI, accumulated concentrations of progesterone between animals fed PUFA and those fed MUFA did not differ (Figure 4.9). Primiparous cows fed HOSFO had a faster rise in plasma progesterone than those fed TRANS (SFO vs. TRANS by parity by DIM interaction; P < 0.01) (Figure 4.9). However, there was no treatment effect on day to fist ovulation, ranging from 20.8 to 26.6 DIM (Figure 4.10). Mean concentrations of GH ranged from 5.9 to 8.3 ng/mL of plasma (Table 4.2) and did not differ among treatments nor was treatment by pa rity interaction signif icant. However the patterns of plasma concentrations of GH over DIM were affected by diet. Multiparous cows fed CaVeg had greater concentrations of plasma GH dur ing the first 4 wk after calving compared to

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124 cows fed LSO whereas the GH concentrations of primiparous cows fed CaVeg were not different soon after calving but were more persistent over DIM (Figure 4.11 and 4.12). Bec-Villalobos et al. (2007) reported no differences in plasma concentrations of GH of lactating cows fed partially hydrogenated fat compared to cows fed no supplemen tal fat. The lack of response of GH to fat supplementation has been reported in beef cattle fed 0 or 7.8% sunflower seeds and sampled every 28 d (Lammoglia et al., 2000), beef cattle fe d 0 or 1.55 kg/d of safflower seeds (Bottger et al., 2002) and dairy cows fed diets of 0, 4.5, 9.0, 13.2, or 17.4% canola seeds (Khorasani et al., 1992). However, other researchers have shown an increase in GH concentration in plasma of late lactation cows infused with 1 kg/d of rapeseed oil into the abomasum (Gagliostro et al., 1991) or mid lactation cows fed Energy Booster at 3% of dietary DM (Grum et al., 1996). Feeding a mixture of Ca salts of palm oil and fi sh oil did not increase pl asma concentrations of GH of lactating dairy cows when injections of bST were not given (Bilby et al., 2006a) which agrees with data in the current study. However when cows were injected with bST, cows fed the supplemental fat had a greater rise in plasma concentrations of GH compared to cows fed whole cottonseeds (Bilby et al., 2006a) and IGF-1 was less indicating on un-coupling response due to fish oil. Insulin-like growth factor / growth hormone ratio The inability of GH to s timulate hepatic IGF-1 production during periods of NEB is termed GH resistance (Donaghy and Baxter, 1 996) or uncoupling of the GH-IGF axis. The ratio of IGF-1:GH indicates how coupled the GH-IG F axis is. A lower ratio indicates a greater degree of uncoupling. Multiparous cows fed PUFA had a greater IGF-1:GH ratio over time than those fed MUFA (PUFA vs. MUFA by parity by DIM interaction, P < 0.01) (Figure 4.14 and 4.15) whereas the response over time was not diffe ren for primiparous cows. These results are in agreement with the insulin data (Figure 4.2 and 4.3). Butler et al. (2003) reported that early

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125 lactation cows subjected to a hyperinsulinemia-euglycemic clamp had a linear increase in the concentrations of IGF-1 in plasma compared to cows infused with saline. The increase in IGF-1 in plasma was associated with an increase in hepatic GHR 1A, which indi cated that insulin can be an important metabolic signal for the GH-IGF ax is. The effect of insulin was likely mediated directly by increasing IGF-1 gene expression or indirectly by stim ulating an increase in hepatic GHR expression which in turn allows GH to act through its receptor in order to mediate IGF-1 secretion and keep the GH-IGF axis coupled. Primip arous cows fed LSO had a faster increase in the IGF-1/GH ratio compared to primiparous co ws fed CaVeg but no differences were detected among multiparous cows (CaVeg vs. LSO by parity by DIM interaction, P <0.05) (Figure 4. 16) Progesterone Concentration of accumulated p rogresterone increas ed at a faster rate in primiparous cows fed HOSFO compared to primiparous cows fe d CaTRANS (HOSFO vs. CaTRANS by parity by DIM interaction (Figure 4.9, P < 0.05) but did not differ between multiparous cows. Usually fat supplementation increases plasma concentrations of cholesterol (Ryan et al., 1992; Hawkins et al., 1995) and cholesterol is a precursor for the synthesis of progesterone by ovarian cells (Grummer and Carrol, 1991). The mechanism by which primiparous cows fed HOSFO had greater concentrations of accumulated progest erone in plasma compared to CaTRANS fedprimiparous cows is unknown and merits further investigation, although da y to first ovulation was not affected (Figure 4.10). Prostaglandin F2 metabolite The patterns over time of concentrations of plasma PGFM of multiparous cows were not different among treatments (Figure 4.17). Howe ver primiparous cows fed the CaTRANS fat source had greater concentrations initially and then decreased at a faster rate over the next 7 d compared to those fed HOSFO (CaTRANS vs. HOSFO by parity by DIM interaction, P < 0.05;

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126 Figure 4.17). Rodriguez-Sallaberry et al. (2007) fed Ca salts of trans fatty acids at 1.8% of dietary DM or saturated fat at 1.5% of dietary DM from approximately 28 d before calculated calving date and continued through 21 DIM. They reported that multiparous cows fed Ca salts of trans fatty acids had greater conc entration of PGFM from calvi ng through 7 DIM compared to cows fed saturated fat but no differences between treatments were detected in primiparous cows. Authors suggested that the response in cows was related to a greater susc eptibility of multiparous cows to hypocalcemia (Goff and Horst, 1997) which likely affected signaling mechanisms within the cell and altered an imals ability to respond to hormonal or dietary treatment. The fact that we detected the difference among primiparous cows th at are much less susceptible to hypocalcemia than multiparous cows suggests that hypocalcem ia likely did not account for the treatment differences. However, the impact of dietary octa decaenoic acids on PGFM of periparturient dairy animals is consistent. Gene Expression Pyruvate carboxylase and phospho enol pyruvate carboxykinase Liver of cows fed LSO had upregulated m R NA of gluconeogenic enzymes (PC, Figure 4.18; and PEPCK, Figure 4.20) compared to cows fed CaVeg (treatment by day linear; P < 0.05). Accordingly, Rozance et al. (2007) reported an upregulation of PEPCK in hypoglycemic fetal livers of sheep compared to control fetal liver po ssibly through peroxisome proliferator-activated receptorcoactivator-1 (PGC1 ) and cAMP response element binding protein (CREB). Thus, soon after the upregulation of th e PC and PEPCK (after 2 wk postpartum), concentrations of blood glucose did not differ among treat ments. Selberg et al. (2004) reported that liver of cows fed supplemental trans -C18:1 monoenes had increased PC mR NA expression at 2 and 28 but not at 14 DIM compared to cows fed CLA supplement or a control diet without fat. In contrast to PC, hepatic PEPCK mRNA transcripts were upregulated in cows fed trans -C18:1 monoenes only

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127 at 14 DIM. Despite differences in the expressi on of the gluconeogenic enzymes in the liver, plasma glucose concentrations were not increas ed maybe due to the differences between the peripheral processing of glucose and the enzy matic activity of the gluconeogenic enzymes (Goodridge, 1987). As reported by others (Greenfield et al., 2000; Williams et al., 2006, Pershing et al., 2002; Loor et al ., 2006), PC was elevated at calving and decreased overtime during the postpartum period (Figure 4.19) wher eas changes in PEPCK mRNA are delayed until after calving when DMI has increased (Figure 4.21). The influence of different fat sources on gluconeogenic enzyme expression in the liver merits further investigation. The difference in the glucose concentrations in plasma might be due to an influence of the fat supplements on the expression of gluconeogenic enzymes in the liver since this metabolic pathway is the major contributor of glucose to the ruminant, as well as differences in enzyme activity, the availability of gluc oneogenic substrates to the liver, or less use of the glucose for milk production. Insulin-like growth factor family Hepatic m RNA expression of IGF-2 was upre gulated on 14 DIM compared to 2 and 28 DIM (Figure 4.22). Hepatic conten t of IGF-2 mRNA did not change over DIM in cows fed LSO but was maximal at 14 DIM in cows fed Ca Veg (LSO vs. CaVeg by DIM interaction; P = 0.01) (Figure 4.23). In addition, the liver of primipar ous cows had greater expression of IGF-2 mRNA compared to those of multiparous cows (Figure 4.24). Expression of IGFBP-2 increa sed from 2 to 14 DIM but de creased at 28 DIM (Figure 4.25), with the decrease being greater for liver of cows fed MUFA compared to the cows fed PUFA (MUFA vs. PUFA by DI M quadratic interaction, P = 0.02) (Figure 4.26). Liver of primiparous cows had greater expression of IG FBP-2 mRNA compared to those of multiparous cows (Figure 4.27)

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128 Hepatic expression of IGFBP-3 was only di fferent at 28 DIM with cows fed PUFA expressing greater IGFBP-3 than those fed MU FA (PUFA vs. MUFA by DIM linear interaction, P = 0.01) (Figure 4.28). Uterine Health Uterine function is often com pro mised in cattle by bacterial contamination of the uterine lumen after parturition, and pat hogenic bacteria often persist, causing uterine disease, a key cause of infertility in cattle. The presence of pathogenic bacteria in the uterus causes inflammation, histological lesions of the endometrium, delays ut erine involution, and perturbs embryo survival (Sheldon et al., 2006). Subclinical endometritis can be defined as endometrial inflammation of the uterus, usually determined by cytology, in the absence of purulent material in the vagina. In animals without signs of clinical endometritis, subclinical endometritis is diagnosed by measuring the proportion of neutroph ils present in a sample collected by flushing the uterine lumen. Subclinical endometritis has been defined by the presence of >18% neutrophils in uterine cytology samples collected 20 to 33 d postpartum or > 10% neutrophils at 34 to 47 days postpartum (Kasimanickam et al., 2004). Nutritional strategies to reduce the incidence of uterine infecti on should improve reproductive performance. In the present experiment, primiparous cows fed LSO had lo wer neutrophil counts based upon uterine cytology compared to primiparous cows fed CaVeg (3.1 vs. 6.5 log units) whereas those of multiparous cows did not differ (5.0 vs. 5.2 log units; CaVeg vs. LSO by parity interaction, P = 0.04; Table 4.3). This suggests that primiparous cows fed LS O had a healthier uterine environment compared to primiparous cows fed CaVeg. This was unexp ected since omega-3 fatty acids have been reported to suppress PGFM concentrations (Mat tos et al., 2004; Petit et al., 2004) and reduce neutrophil function during the early postpartum (Thatcher et al., 2006). Another explanation is that the lower number of neutrophils in primip arous cows fed LSO was due to a reduction or

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129 clearance of apoptotic neutr ophils by macrophage engulfment dur ing inflammation (Cox et al., 1995). This might delay endometrial repair ( Kaitu'u-Lino et al., 2007) in the pospartum period. Acute Phase Proteins Acute phase proteins are produced in the liver in response to inflamma tion or stress and are released in the blood to help the immune syst em. Haptoglobin acts in plasm a as a scavenger molecule for free hemoglobin (Lim et al., 2000). Haptoglobin concentrati ons in plasma were increased in cows with fatty liver (Yoshino et al., 1992; Nakagawa et al., 1997; Petersen et al., 2004) or mastitis (Grnlund et al ., 2005; Eckersall et al., 2006; kerstedt et al., 2007). However, little is known about the effects of lipid supplementation on acute phase proteins in dairy cows. Across treatments, plasma concentrations of haptoglobin were greater during the first 5 DIM (DIM, Figure 4.29, P < 0.001) whereas those of acid solubl e protein were greater the first 2 wk postpartum (DIM, Figure 4.32, P = 0.09), possibly reflecting th e stress of parturition. The stress of calving and adjustments associated with lactation appeared to be greater for primiparous compared to multiparous cows as evidenced by greater (P < 0.001) mean plasma concentrations of haptoglobin (0.0247 vs. 0.0187 arb itrary units) and acid soluble protein (3.55 vs. 3.40 g/mL; Table 4.3, Figure 4.31, and 4.34, respectively for ha ptoglobin and acid soluble protein). Primiparous cows fed CaTRANS tended to have gr eater mean concentrations of haptoglobin in plasma compared to those fed HOSFO (0.029 vs 0.023) whereas multiparous cows did not differ (0.017 vs 0.018, HOSFO vs. CaTRANS by parity interaction, P = 0.06; Figure 4.30). In contrast to the present experiment, Bazinet et al. (2004) reported that pigs supplemented with omega-3 fatty acids had reduced haptoglobin concentrations in plasma compared to a control group fed a diet rich in omega-6. Haptoglobi n was not affected by energy or starch concentrations in newlyreceived feedlot calves (Berry et al., 2004). Diets enriched in omeg a-3 (ground flaxseed),

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130 omega-6 (soybeans) or omega-9 (tallow) fatty aci ds did not affect plasma concentrations of haptoglobin in beef stee rs injected i.v. with E. coli lypopolysaccharide (LPS) (Farran et al., 2008). Acid soluble protein (also called alpha 1-acid glycoprotein) is a minor acute phase protein constitutively expressed by the liver and us ually found in blood (Lecchi et al., 2008) and is increased with systemic inflammation (Hochepied et al., 2003). As in the case of haptoglobin, primiparous cows fed TRANS had greater mean plasma concentrations of acid soluble protein compared to primiparous cows fed HOSFO ( 37.9 vs. 33.2 ng/mL) (HOSFO vs. TRANS by parity interaction; P = 0.03) but the opposite was true for cows (28.5 vs. 33.3 ng/mL; Figure 4.33). This effect might be mediated through the nuc lear receptor retinoid X receptor (RXR) as shown by Mouthiers et al. (2004). The authors repo rted that alpha 1-acid glycoprotein gene was activated by retinoic acid through a DR1 responsiv e element that involves RXR. Since fatty acids are known to modulate gene expression th rough RXR nuclear receptors, the effect of the TRANS supplement on acid soluble pr otein could be mediated via RX R. In addition, interleukins are modulators of the alpha 1-acid glycoprotei n (Fournier et al., 2000) More studies on the effects of fat source on cytokines and acute phase proteins warra nt investigation. Fat supplementation affects animal performan ce during different stages of the production cycle. The effects are mediated by changes in hormone, metabolites, gene expression, and markers of immune function. The integrati on of nutrition, physiol ogy, endocrinology, and immune function of the dairy cow will lead to si gnificant advances in field of dairy science. Further investigations on the mechanisms by which different fat sources influence metabolism will be beneficial to dairy nutritionists fo r maximizing the nutritional effects of lipid supplementation on integrated performa nce of periparturient dairy cows.

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131 Conclusions The CaTRANS supplement had a proinflamm atory effect on prim iparous cows as evidenced by increased mean concentrations of plasma haptoglobin and acid soluble protein. The PUFA supplements also appeared to be proi nflammatory, but only in the first few days after calving based upon greater plasma concentrations of haptoglobin. The omega-3 supplement may have had anti-inflammatory effects later in la ctation based upon fewer numbers of neutrophils recovered from a uterine flushing. Mean concentrations of plasma insulin were lower for primiparous cows fed PUFA accompanied by increas ed mean concentrations of plasma NEFA. Indeed, concentration of plasma NEFA were grea ter at 2 wk postpartum for all parities fed the omega-3 fat source. Concentrations of plasma IG F-1 rose at a faster rate after calving when animals were fed PUFA sources. However this fast er rise did not result in an earlier return to ovarian activity or a greater calculated accumula tion of progesterone over time. Accompanying the more rapid rise in IGF-1 concentrations was an increased expres sion of mRNA for IGFBP-3 in liver tissue as well as an increased rise in PC and PEPCK in liver tissue when animals were fed the omega-3 versus the omega-6 fat source. Supplementing fat sources that differ in fatty acid profile can modulate the conc entrations of plasma metabolites, plasma hormones associated with the IGF system, and expression of mR NA of gluconeogenic enzymes and IGF-binding proteins of the hepatic tissue of lactating da iry cows. In several cases, primiparous cows responded differently than multiparous cows to the fat supplements. These changes have the potential to impact producti on, immunity, and repr oduction of lactating dairy animals.

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132 Table 4-1. Concentration of plasma metabolite s of Holstein cows fed diets supplemente d with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegeta ble oil (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Treatments1 Orthogonal contrasts 2, P value Measure HOSFO TRANS CaVeg LSO SE Pa rity Time Treatment*Time A B C D E F P3 M4 P M P M P M Glucose, mg/dl 62.5 56.5 65.1 55.9 65.5 59.1 59.2 58. 3 2.6 0.01 <.001 0.09 0.79 0.70 0.20 0.26 0.50 0.27 Blood urea nitrogen, mg/dl 8.7 9.4 9.4 9.3 8.3 8.7 8.7 8.5 0.7 0.69 <0. 001 0.38 0.18 0.67 0.88 0.85 0.54 0.69 NEFA, meq/L 401 588 472 541 588 421 635 624 86 0.75 <0.001 0.05 0.28 0.89 0.16 0.08 0.48 0.38 1 HOSFO = high oleic sunflower oil (Trisun, Hum ko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairla wn, OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (A rcher Daniels Midla nd, Redwing, MN). 2 Orthogonal contrast of means were the following: A = MUFA (HOSFO + TRANS) vs. Poly (C aVeg + LSO), B = HOSFO vs. TRANS, C = CaV eg vs. LSO, D = contrast A by parity, E = contrast B by parity, and F = contrast C by parity. 3 Primiparous cows. 4 Multiparous cows.

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133 Table 4-2. Concentration of plasma hormone s of Holstein cows fed diets supplemente d with high oleic sunf lower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegeta ble oil (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Treatments1 Orthogonal contrasts 2, P value Measure HOSFO TRANS CaVeg LSO SE Parity A B C D E F P3 M4 P M P M P M Insulin, ng/mL 0.52 0.41 0.50 0.42 0.44 0.50 0.44 0.44 0.04 0.36 0.86 0.88 0.58 0.06 0.82 0.54 IGF-1, ng/mL 144 99 135 134 116 133 146 129 11 0.10 0.63 0.20 0.20 0.10 0.04 0.09 GH, ng/mL 7.9 7.3 8.2 6.7 7.0 7.5 6.1 5.9 1.1 0.54 0.26 0. 87 0.27 0.42 0.67 0.75 IGF-1/GH ratio 24.2 18.5 22.2 24.7 21. 1 25.2 29.6 28.0 4.8 0.95 0.30 0.65 0.26 0.68 0.38 0.56 1 HOSFO = high oleic sunflower oil (Trisun, Hum ko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN). 2 Orthogonal contrast of means were the following: A = MUFA (HOSFO + TRANS) vs. Poly (C aVeg + LSO), B = HOSFO vs. TRANS, C = CaV eg vs. LSO, D = contrast A by parity, E = contrast B by parity, and F = contrast C by parity. 3 Primiparous cows. 4 Multiparous cows.

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134 Table 4-3. Log of total neutrophil count in the uterine flusing and acute phase proteins of Holstein cows fed diets supplemente d with high oleic sunflower oil ( HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium sa lts of vegetable oil (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Treatments1 Orthogonal contrasts 2, P = Measure HOSFO TRANS CaVeg LSO SE Parity Time Treatment*Time A B C D E F P3 M4 P M P M P M Log of Total Neutrophils 7.3 5.6 6.7 5.0 6.5 5.2 3.1 5.0 0.8 0.25 0.30 0.98 0.01 0.20 0.79 0.04 Haptoglobin (Arbitrary units) 0.0232 0.0179 0.0288 0.0172 0.0246 0.0221 0.0224 0.0179 0.0022 <0.001 <0.001 0.03 0.42 0.10 0.13 0.11 0.07 0.50 Acid soluble Protein ( g/mL) 33.2 33.3 37.9 28.5 31.8 29.0 36.5 29.3 1.1 <0.001 0.09 0.74 0.15 0.76 0.16 0.46 0.03 0.27 1 HOSFO = high oleic sunflower oil (Trisun, Hum ko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairla wn, OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (A rcher Daniels Midla nd, Redwing, MN). 2 Orthogonal contrast of means were the following: A = MUFA (HOSFO + TRANS) vs. Poly (C aVeg + LSO), B = HOSFO vs. TRANS, C = CaV eg vs. LSO, D = contrast A by parity, E = contrast B by parity, and F = contrast C by parity. 3 Primiparous cows. 4 Multiparous cows.

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135 Table 4-4. Least squares means for hepati c PC, PEPCK, IGF-2, IGFBP-2, and IGFBP3 mRNA abundance normalized to 18S rRNA of cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO) from 4 wk prepartum to 15 wk postpartum. Treatments1 Orthogonal contrasts 2, P value Measure3 HOSFO TRANS CaVeg LSO A B C D E F DIM of liver biopsy 2 14 28 2 14 28 2 14 28 2 14 28 SE Parity Day Treatment*Day PC mRNA 0.89 0.83 0.78 0.81 0.74 0.75 0.75 0.72 0.71 0.76 0.77 0.80 0.07 0.57 <0.001 0.01 0.33 0.29 0.49 0.94 0.79 0.99 PEPCK mRNA 1.01 1.01 0.97 0.93 0.94 0.94 0.88 0.94 0.93 0.99 0.98 1.06 0.06 0.23 0.06 0.01 0.93 0.43 0.24 0.89 0.67 0.69 IGF-2 mRNA 0.83 0.85 0.73 0.72 0.75 0.69 0.69 0.77 0.66 0.76 0.74 0.75 0.06 <0.01 0.01 0.07 0.57 0.38 0.63 0.38 0.61 0.99 IGFBP-2 mRNA 0.82 0.87 0.81 0.78 0.79 0.77 0.75 0.80 0.78 0.78 0.83 0.85 0.04 0.09 0.01 0.16 0.82 0.39 0.48 0.54 0.82 0.57 IGFBP-3 mRNA 1.16 1.17 1.12 1.07 1.07 1.06 1.01 1.10 1.11 1.20 1.18 1.28 0.11 0.29 0.14 0.07 0.72 0.58 0.35 0.80 0.69 0.80 1 HOSFO = high oleic sunflower oil (Trisun, Hum ko Oil, Memphis, TN); CaTRANS = Ca salts of trans C18:1 (EnerG TR, Virtus Nutrition, Fairlawn OH); CaVeg = Megalac-R (Church & Dwight Co, Princeton, NJ); LSO = linseed oil (Archer Daniels Midland, Redwing, MN). 2 Orthogonal contrast of means were the following: A = MUFA (HOSFO + TRANS) vs. Poly (C aVeg + LSO), B = HOSFO vs. TRANS, C = CaV eg vs. LSO, D = contrast A by parity, E = contrast B by parity, and F = contrast C by parity. 3 All measures are normalized to 18S rRNA.

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136 Figure 4-1. Least squares means for plasma glucose of Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oi l (CaVeg) or linseed oil (LSO). The asterisks indicate that animals fed CaVeg had higher plasma glucose concentration than animals fed LSO at weeks 1 (P = 0.03) and 2 (P = 0.04) postpartum.

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137 Figure 4-2. Least squares means for plasma BUN from lactating primiparous (A) and multiparous (B) Holstein cows fed diets s upplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO ). Treatments did not differ.

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138 Figure 4-3. Concentration of NEFA in plasma of lactating Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or lins eed oil (LSO). The treatment x parity x week interaction was significant ( P = 0.03). The asterisks indicates tha cows fed LSO had greater concen trations of NEFA in plasma at wk 2 ( P = 0.02) and 5 ( P = 0.08).

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139 Figure 4-4. Polynomial regression cu rves (first order) of concentra tions of plasma insulin from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The data was best described by a first order polynomial. The HOSFO vs. CaTRANS x parity x DIM interaction was significant (P < 0.05).

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140 Figure 4-5. Polynomial regression cu rves (first order) of concentra tions of plasma insulin from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with MUFA (HOSFO + CaTRANS) or PUFA (CaVeg + LSO). The data was best described by a first order polynomial. The MUFA vs. PUFA x parity x DIM interaction was significant (P < 0.05).

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141 Figure 4-6. Polynomial regression cu rves (first order) of concentra tions of plasma insulin from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with MUFA (HOSFO + CaTRANS) or PUFA (CaVeg + LSO). The data was best described by a first order polynomial. The HOSFO vs. CaTRANS x parity x DIM interaction was significant (P < 0.05).

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142 Figure 4-7. Polynomial regression cu rves (first order) of concentr ations of plasma IGF-1 from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The data was best described by a first order polynomia l. The MUFA vs. PUFA x parity x DIM interaction was significant (P < 0.05).

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143 Figure 4-8. Polynomial regression cu rves (first order) of concentr ations of plasma IGF-1 from lactating Holstein cows fed diets supple mented with MUFA (HOSFO + CaTRANS) or PUFA (CaVeg + LSO). MUFA vs. PUFA x DIM interaction wa s significant (P < 0.001).

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144 Figure 4-9. Polynomial regression curves (seco nd order) of concentrations of plasma accumulated progesterone from lactati ng primiparous (A) and multiparous (B) Holstein cows fed diets supplemented w ith high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The da ta includes anestrous cows The proportion of anestrous cows was not different among treatments. The data was best described by a second order polynomial. The HOSFO vs. CaTR ANS x parity x DIM interaction was significant (P < 0.05).

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145 Figure 4-10. Days to first ovula tion of lactating primiparous (A ) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The data includes anestrous cows The proportion of anestrous cows was not different among treatments. Effect of treatment was not significant.

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146 Figure 4-11. Polynomial regression curves (first order) of concentrations of plasma growth hormone from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oil (CaVeg) or linse ed oil (LSO). The data was best described by a first order polynomial. The MUFA vs. PUFA x parity x DIM interaction was significant (P < 0.05).

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147 Figure 4-12. Polynomial regression curves (first order) of concentrations of plasma growth hormone from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oil (CaVeg) or linse ed oil (LSO). The data was best described by a first order polynomial. The Caveg vs. LSO x parity x DIM interaction was significant (P < 0.05).

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148 Figure 4-13. Polynomial regression curves (first order) of concentrations of plasma growth hormone from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oil (CaVeg) or linse ed oil (LSO). The data was best described by a first order polynomial. The MUFA vs. PUFA x parity x DIM interaction was significant (P < 0.05).

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149 Figure 4-14. Polynomial regression curves (second order) of ratio of IGF-1/GH from lactating primiparous (A) and multiparous (B) Holste in cows fed diets supplemented with high oleic sunflower oil (HO SFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The data was best described by a second order polynomial. The Caveg vs. LSO x parity x DI M interaction was significant (P < 0.05). PUFA vs. MUFA by pa rity by DIM interaction was significant ( P < 0.05).

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150 Figure 4-15. Polynomial regression curves (second order) of ratio of IGF-1/GH from lactating primiparous (A) and multiparous (B) Holste in cows fed diets supplemented with high oleic sunflower oil (HO SFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The data was best described by a second order polynomial. PUFA vs. MUFA by parity by DIM interaction was significant ( P < 0.05).

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151 Figure 4-16. Polynomial regression curves (second order) of ratio of IGF-1/GH from lactating primiparous (A) and multiparous (B) Holste in cows fed diets supplemented with high oleic sunflower oil (HO SFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The data was best described by a second order polynomial. The Caveg vs. LSO x parity x DI M interaction was significant (P < 0.05).

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152 Figure 4-17. Polynomial regression curves (s econd order) of concentrations of plasma prostaglandin F2 metabolite from lactating prim iparous (A) and multiparous (B) Holstein cows fed diets supplemented w ith high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The data was best described by a second order polynomial. The HOSFO vs. CaTRANS x parity x DIM in teraction was significant (P < 0.05).

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153 Figure 4-18. Effect of supplemental fat sour ce on pyruvate carboxylas e (PC) mRNA expression in liver of Holstein cows (n = 8 per treat ment) biopsied at 2, 14, and 28 DIM. Dietary fat supplements were high oleic sunflo wer oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellular RNA were subj ected to Northern blot analysis (A). Representative Northern blots for PC mR NA expression are shown. The CaVeg vs. LSO x DIM interacti on was significant ( P = 0.04). A B

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154 Figure 4-19. The mRNA expression of pyruvate car boxylase (PC) in liver of Holstein cows (n = 8) biopsied at 2, 14, and 28 DI M. Dietary fat supplements were high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Ten micr ograms of total cellular RNA were subjected to Northern blot analysis (A ). Representative Northern blots for PC mRNA expression are shown. The lin ear effect of DIM was significant ( P = 0.001). A B

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155 Figure 4-20. Effect of supplemental fat s ource on phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in liver of Holstein co ws (n = 8 per treatment) biopsied at 2, 14, and 28 DIM. Dietary fat supplements we re high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellular RNA were subjected to Northern blot analysis (A). Representative Northern blots for PEPCK mRNA expression are shown. The CaVeg vs. LSO x DIM quadratic interaction was significant ( P = 0.01). A B HO

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156 Figure 4-21. The mRNA expression of phosphoe nolpyruvate carboxykinase (PEPCK) in liver of Holstein cows (n = 8) biopsied at 2, 14, and 28 DIM Dietary fat supplements were high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellular RNA were subjected to Northe rn blot analysis (A). Representative Northern blots for PEPCK mRNA expres sion are shown. The DIM effect was significant ( P = 0.03). A B HO

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157 Figure 4-22. The mRNA expression of insulin-like growth factor-2 (IGF-2) in liver of Holstein cows (n = 8) biopsied at 2, 14, and 28 DIM. Dietary fat supplements were high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellular RNA were subjected to Northern blot analysis (A). Representative Northern blots for IGF-2 mRNA expression are shown. The DIM effect was significant ( P = 0.01). A B HO

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158 Figure 4-23. Effect of supplemental fat source on insulin-like growth factor-2 (IGF-2) mRNA expression in liver of Holstein cows (n = 4 per treatment) biopsied at 2, 14, and 28 DIM. Dietary fat supplements were high ol eic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellula r RNA were subjected to Northern blot analysis (A). Representative Northern blot s for IGF-2 mRNA expression are shown. The CaVeg vs. LSO by DIM quadratic interaction was significant ( P = 0.01). A B HO

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159 Figure 4-24. Effect of parity on insulin-like growth factor-2 (IGF-2) mRNA expression in liver of Holstein cows (n = 4 per treatment) biopsied at 2, 14, and 28 DIM. Dietary fat supplements were high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oils (CaVeg), and linseed oil (LSO). The parities differed (P < 0.001).

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160 Figure 4-25. The mRNA expression of insulinlike growth factor bind ing protein-2 (IGFBP-2) in liver of Holstein cows (n = 8) biopsied at 2, 14, and 28 DIM. Dietary fat supplements were high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellular RNA were subj ected to Northern blot analysis (A). Representative Northern blots for IGFBP2 mRNA expression are shown. The DIM effect was significant ( P = 0.03). A B HO

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161 Figure 4-26. Effect of supplemental fat source on insulin-like growth fa ctor binding protein-2 (IGFBP-2) mRNA expression in liver of Holstein cows (n = 8 per treatment) biopsied at 2, 14, and 28 DIM. Dietary fat supple ments were high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellular RNA were subjected to Northern blot analysis (A). Representative Northern blots for IGFBP-2 mRNA expression are shown. The MUFA vs. PUFA x DIM linea r interaction was significant ( P = 0.02). A B HO

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162 Figure 4-27. Effect of parity on insulin-like growth factor binding protein-2 (IGFBP-2) mRNA expression in liver of Holstein cows (n = 8 per treatment) biopsied at 2, 14, and 28 DIM. Dietary fat supplements were high ol eic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Primiparous cows tended ( P = 0.09) to have higher IGFBP-2 mRNA expression than multiparous cows.

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163 Figure 4-28. Effect of supplemental fat source on insulin-like growth fa ctor binding protein-3 (IGFBP-3) mRNA expression in liver of Holstein cows (n = 4 per treatment) biopsied at 2, 14, and 28 DIM. Dietary fat supple ments were high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oils (CaVeg), and linseed oil (LSO). Ten micrograms of total cellular RNA were subjected to Northern blot analysis (A). Representative Northern blots for IGFBP-3 mRNA expression are shown. The m onounsaturated fatty acids (HOSFO + CaTRANS; MUFA) vs. polyunsaturated fatty acids (CaVeg + LSO; PUFA) x DIM linear interaction was significant ( P = 0.01). A B HO

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164 Figure 4-29. Concentrations of plasma haptogl obin from lactating Holstein cows fed diets supplemented with high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetabl e oil (CaVeg) or linse ed oil (LSO). The data was best described by a second order polynomial. Effect of treatment was not significant.

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165 Figure 4-30. Concentrations of plasma ha ptoglobin from lactating primiparous (A) and multiparous (B) Holstein cows fed diets s upplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). The HOSFO vs. CaTR ANS x parity interaction tended ( P = 0.06) to be significant.

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166 Figure 4-31. Mean concentra tion of plasma haptoglobin from lactating primiparous and multiparous Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). Parities differed (P < 0.001). Multiparous

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167 Figure 4-32. Concentration of plasma acid soluble protein from l actating Holstein cows fed diets supplemented with high oleic sunflowe r oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegeta ble oil (CaVeg) or linseed oil (LSO). HOSFO vs. CaTRANS by parity interaction was significant ( P = 0.03).

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168 Figure 4-33. Mean concentration of plasma acid soluble protein from lactating primiparous (A) and multiparous (B) Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). HOSFO vs. CaTRANS by parity interaction was significant (P = 0.03).

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169 Figure 4-34. Mean concentration of plasma acid soluble protein from lactating primiparous and multiparous Holstein cows fed diets supplemented with high oleic sunflower oil (HOSFO), calcium salts of trans fatty acids (CaTRANS), calcium salts of vegetable oil (CaVeg) or linseed oil (LSO). Parities differed ( P < 0.001). Primiparous Multiparous

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170 CHAPTER 5 EFFECT OF OMEGA-3 AND OMEGA-6 SUP PLEMENTATION ON ACUTE PHASE PROTEINS IN PLASMA OF PERIPARTURIENT DAIRY COWS Abstract The objective of the study was to evaluate if dietary supplem ental PUFA, enriched in omega-6 or omega-3 fatty acids, can regulate an d improve the immunosuppressive state that is typical of periparturient Holsteins primiparous (n=16) and multiparous (n=29) cows. Treatments were: 1) Control (CO, no fat supplement), 2) Ca salts of fatty acids made from safflower oil (Omega-6, 63% C18:2, PreQuil-21 ), and 3) Ca salts of fatty acids made from palm oil and fish oil (Omega-3, 11% C20:5 plus C22:6, StrataG). Supplem ental fats (Virtus Nutrition, Corcoran, CA) were fed at 1.5% of dietar y DM during pre and pos tpartum periods. Blood samples were taken daily from calving through 10 DIM for determination of PGFM and thrice weekly (Monday-Wednesday-Friday) thereafter through 50 DIM for determination of acute phase proteins (haptoglobin, ceruloplasmin, -acid soluble protein, and fibrinogen). Multiparous cows fed Omega-6 had greater concentrations of plasma fibrinogen (259 vs 206 mg/dl) compared to Omega-3-fed cows, but values were not different for primiparous cows (226 vs. 254 mg/dl; Omega-6 vs. Omega-3 by parity interaction, P < 0.05). Primiparous cows fed Omega-3 had reduced concentrations of plasma cerulop lasmin compared to Omega-6-fed primiparous cows (10.5 vs. 11.9 mg/dl) but values were not different for cows (11.4 vs. 11.1 mg/dl; Omega-6 vs. Omega-3 by parity interaction; P < 0.05). Multiparous cows fed fat supplements had greater (59.8 g/mL) concentrations of acid soluble protein in plasma during the first 3 wk postpartum compared to CO cows (42.9 g/mL), but this was reversed for primiparous cows (51.7 vs. 45.0 mg/dl; CO vs. fats by parity by DIM interaction; P = 0.06). In conclusion, animals fed Omega-3 fatty acids had attenuated acute immune respons e compared to Omega-6 fed animals but this effect varied with parity.

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171 Key Words: omega-3, omega-6, acute phase prot eins, periparturient dairy cow Introduction During the transition period the im mune status of dairy cows is partially suppressed (Goff and Horst, 1997). Nutritional m anagement to improve immune status around parturition is desired in order to reduce incidence of diseases during the postpa rtum period. Dietary fatty acids can alter innate and acquired immunity through several mechanisms (Calder, 2007). Fatty acid supplementation influenced the production of cyto kines in humans (Han et al., 2002), antibodies in bovids (Lessard et al., 2003), and prostaglandins which are i nvolved in uterine involution in bovids (Mattos et al., 2004). Cytokines produced by macrophages and neut rophils, especially tumor necrosis factor alpha (TNF), stimulates the liver to produce acute phase proteins as an acute response to inflammation or other source s of stress (Calder, 2007). Some of the acute phase proteins are haptoglobin, ceruloplasmin, fibrinogen, and acid solubl e protein. Haptoglobin binds hemoglobin so that the ir on in hemoglobin is not availabl e to pathogenic bacteria for replication. Haptoglobin is invol ved in lipid metabolism (Nakag awa et al., 1997). Haptoglobin locus in human chromosome 16 is close to th e loci of lipid-related enzymes such as lecithin:cholesterol acy ltransferase (Reeders and Hildeb rand, 1989). Haptoglobin can modulate the synthesis of prostaglandin in vitro (Jue et al., 1983, Frohlander et al., 1991) which is influenced by omega-3 fatty acid supplement ation (Mattos et al., 2004). Ceruloplasmin is a protein that binds copper and helps prevent oxidative damage to endothelial cells during inflammation (Uriu-Adams and K een, 2005). Ceruloplasmin concentrations in plasma increased with increased bacterial contamin ation of the uterus during the first 2 wk postpartum compared to cows with low bacterial infection (Sheldon et al., 2003). Acid soluble protein is an antiinflammatory agent that helps control inappropriate or extended activation of the immune system (Jafari et al., 2006). It reduced concentration of prostaglandin E2 in plasma of rats (Matsumoto et

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172 al., 2007). Fibrinogen is a sticky, fibrous protein used to ma ke fibrin for blood clotting and tissue repair (Gentry, 2004). In addition, prostagl andins induce blood clotting (Marx, 1977). Polyunsaturated fatty acids ( PUFA ) of the n-3 and n-6 family can be important modulators of immune reactions (Calder et al., 2002). Dietary fats rich in n3 or n-6 PUFA modulated the inflammatory responses of guin ea pigs (Pomposelli et al., 1989) and of humans experiencing chronic disease (Stenson et al., 1992). In mice fed an enriched n3 PUFA diet, inflammatory reactions were reduced, and different types of antibody response to antigenic stimulations were developed compared with mice fed an n-6 enriched diet (Albers et al., 2002) However, little is known about the effects of omega-3 and omeg a-6 fatty acids on acute phase proteins concentrations in plasma of newly-calved dairy cows. Objective of this experiment was to determ ine if source of dietar y supplemental PUFA enriched in omega-6 or omega-3 fatty acids w ould influence the acute phase response by dairy cows around parturition such that the immunosuppressive state that is typical of periparturient cows may be improved. We hypothesized that th e acute phase proteins associated with prostaglandin would be reduced in an imals fed omega-3-enriched diets. Material and Methods Animals, Treatments, and Sampling Experim ent was conducted at the University of Florida dairy research unit (Hague, FL) during the months of October 2006 through April 2007. All experimental animals were managed according to the guidelines approved by the Universi ty of Florida Animal Research Committee. Periparturient Holstein primiparous (n = 16) and multiparous (n = 29) cows were assigned to treatment at approximately 34 7 d prior to their actual calving date. Dietary treatments were the following: 1) control diet ( CO ; no fat supplement), 2) Ca salts of fatty acids made from safflower oil (Omega-6, 63% linol eic acid; EnerG HL, Virtus Nutr ition, Corcoran, CA), and 3)

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173 Ca salts of fatty acids made from palm oil a nd fish oil (Omega-3, 11% eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA], Stra taG 0.5, Virtus Nutrition, Corcoran, CA). Supplemental fats were fed at 1.5% of dietary DM during the pr epartum and postpartum periods. Parity (primiparous or multiparous), BW and milk production of the previous year for multiparous cows were used to assign cows to the 3 treatment groups. Prepartum cows were housed in pens with a sod base and feeding ar ea equipped with fans, sprinklers, and shaded Calan gates (American Calan In c., Northwood, NH). Postpartum cows were housed in a sandbedded, free-stall barn equipped with fans, sprinkle rs, and Calan gates. Cows were milked twice daily at 1030 and 2230 h. Prepartum cows we re fed TMR twice daily at 1000 and 1400 h whereas postpartum cows were fed twice dail y at 0900 and 1300 h to allow 5 to 10% feed refusals. One sample of corn silage was collected weekly and immediately dried for 1 h using a Koster (Koster Crop Tester, Inc., Strongsville, OH) to calculate the concentration of DM in order to maintain the formulated forage to concentrate ratio in the ration. Prepartum diets consisted of ~32% corn silage, 12 to 19% bermud agrass hay, and 49 to 56% concentrate in order to feed isocaloric diets whereas postpartum diet s consisted of 38% corn silage, 12% alfalfa hay, and 50% concentrate (DM basis). Representative samples of corn silage, bermudagrass hay, alfalfa hay, and concentrate mixes were collected on a weekly basis. Weekly samples were composited on a monthly basis and ground through a 1-mm Wiley mill screen (A. H. Thomas, Philadelphia, PA). Blood (10 mL) was collected at 0700 h daily from coccygeal vessels into sodium heparinized tubes (Vacutainer, Becton Di ckinson, Franklin Lakes, NJ) from day of parturition until 10 DIM and on a Monday, Wednesda y, and Friday schedule thereafter until 47 3 DIM. Samples were put immediately on ice until centrifuged at 2619 x g at 5C for 30 min (RC-3B refrigerated centrifuge, H 600A rotor, So rvall Instruments, Wilmington, DE). Plasma

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174 was separated and frozen at -200C for subsequent analysis of pr ostaglandin F metabolite on days 1 to 10 pospartum and for haptoglobin, cerulopla smin, fibrinogen, and acid soluble protein on the thrice weekly samples. Sample Analysis Feed sam ples (corn silage, bermudagrass hay, alfalfa hay, and concentrate mixes) were analyzed for mineral and fat composition (Dai ry One, Ithaca, NY), NDF (Mertens, 2002), ADF (Van Soest et al., 1991), and CP using a macro elemental analyzer vario MAX CN (Elementar Analysensysteme GmbH, Hanau, Germany). Prepartum diets contained 1.61, 1.62, and 1.63 Mcal of NEL; 14.9, 14.9, and 14.9% CP and 4.6, 5.5, and 5.4% EE. Postpartum diets contained 1.77, 1.83, and 1.83 Mcal of NEL; 18.1, 18.4, and 18.7% CP; 4.5, 5.8, and 5.8% EE for control, Omega-6 and Omega-3 diets, respectively. Plasma Hp concentrations were determined in duplicate samples by measuring haptoglobin/hemoglobin complexing by the estimati on of differences in peroxidase activity (Makimura and Suzuki, 1982). Results are expres sed as arbitrary units resulting from the absorption reading at 450 nm. Plasma ceruloplasmin oxidase activity was measured in duplicate samples using colorimetric procedur es described by Demetriou et al. (1974). The intra-assay CV of duplicate samples was c ontrolled to values <10%. Ceruloplasmin concentrations were expressed as mg/dL as de scribed by King (1965). Inte r-assay variation of both acute phase protein assays were controlled by CV limits <10%, as a result of a control sample analyzed in duplicate within each individu al assay run. When the inter-assay CV of any specific run exceeded 10%, all samples contained in the individual run were re-analyzed. Plasma acid soluble protein was extracted from plasma with 0.6 M perchloric acid and analyzed with the bicinchoninic acid kit (Sigma-Aldri ch, Saint Louis, MO). Plasma fibrinogen concentrations were

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175 determined using a fibrinogen determination kit (Sigma Diagnostics, St. Louis, MO). The intraassay CV was <5%. Statistical Analysis Repeated measures d ata (PGFM) were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) according to the following model: Yijkl = + Ti + Pj + TPij +Ck (i j) +Dl + TDil + PDjl + TPDijl + Eijkl where Yijkl is the observation, is the overall mean, Ti is the fixed effect of treatment (i = 1, 2, and 3), Pj is the fixed effect of parity (j = 1 and 2), TPij is the interaction of treatment and parity, Ck (i j) is random effect of cow within treatment and parity (k = 1, 2, n), Dl is the fixed effect of DIM (l = 0, 1, 2, ), TDil is the interaction of treatment and DIM, PDjl is the interaction of parity and DIM, TPDijl is the three way interaction of treatment, parity and DIM, and Eijkl is the residual error. Orthogonal contrasts used to detect treatment differences were the following: 1) Control vs. Omega-6 + Omega-3 and 2) Omega-6 vs. Omega-3. Acute phase protein data (plasma concentr ations of Hp, ceruloplasmin, acid soluble protein, and fibrinogen) did not have a normal distribu tion and were analyzed by PROC GLIMMIX of SAS. Results and Discussion The postpartum pattern of plasma concentrations of the acute phase pr oteins indicate that parturition is a proinflammatory event. Immediat ely after calving, plasma concentrations of all acute phase proteins rose but thereafter their pattern thereafter differed among the acute phase proteins (Figure 5.1). After peaking at 3 DIM, haptoglobin decreased gradually until plateauing at approximately 11 DIM. Acid soluble prot ein concentrations peaked at about 7 DIM and continued to decline until 21 DIM at which time concentrations leveled off. Likewise plasma

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176 concentrations of fibrinogen increased rapidly during the first week but, unlike acid soluble protein, concentrations continued to increase slow ly for the next 6 wk. Ceruloplasmin appears to be an exception to the other acute phase proteins in that the pattern of the plasma concentrations appeared relativel y constant during the 7 wk pos tpartum after the first rise. Therefore ceruloplasmin may not be a sensitive marker to the stress of early lactation. Fat supplementation did not affect the pattern over time (treatment by DIM interaction) or mean concentrations of plasma haptoglobin (Figure 5.3). A diet enriched in omega-3 fatty acids, mainly C18:3, reduced plasma concentrations of haptoglobin in pigs (Bazinet et al., 2004). In ruminants, however, the biohydrogenation of the omega-3 fatty acids in the rumen might not allow the escape of sufficient amounts of PUFA to tissues that will influence prostaglandin secretion or haptoglobin concentrat ion in plasma as seen in the present experiment. Indeed, there was no treatment effect on plasma concentrations of PGFM in the present study (Figure 5.2). In vitro studies using bovine endometrial cells incubated with different fatty acids showed that EPA and DHA s uppressed synthesis of PGF2 (Mattos et al., 2003). Using an in vivo model, Mattos et al. (2004) fed diets containing either fish oil or olive oil from 21 d before the expected calving date until parturition (2% of dietary DM) and from parturition until 21 d postpartum (1.8% of dietary DM). Authors re ported that cows fed fish oil had reduced concentration of plasma PGFM in the first 2.5 DIM compared with cows fed olive oil. Midlactation multiparous Holstein cows were fe d Megalac (2.8% of dietary DM), formaldehydetreated whole linseed (6.7%), a 50:50 (oil basis) mixture of formaldehyde-treated whole linseed and fish oil (4.6% of dietary DM) or were infused with linseed oil into the duodenum (500 g/d; Petit et al. 2002). Cows were injected i.v. with oxytocin to stimulate th e uterus to release PGF2 Cows receiving fish oil tended to have a greater concentration of plasma PGFM than those fed

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177 linseeds alone or infused with linseed oil. Moussavi et al. (2007b) reported no effect of fish meal (1.25, 2.5, or 5% of dietary DM) or Ca salts of a mxture of palm o il and fish oil (2.3% of dietary DM) on plasma concentration of PGFM of mu ltiparous cows given oxytocin i.v. on d 15 of a synchronized estrous cycle. Wams ley et al. (2005) observed th at fish meal supplementation had no effect on the secretion of PGF2 by nonlactating primiparous cows having normal concentrations of plasma proge sterone and only decreased plasma PGF2 in those having reduced progesterone concentrations. Mean ceruloplasmin concentrations in plas ma of primiparous cows fed supplemental omega-3 fatty acids were reduced compared to primiparous cows fed supplemental omega-6 fatty acids (Figure 5.4A, P < 0.05) but did not differ in multiparous cows were (Omega-6 vs. Omega-3 by parity interaction, Figure 5.4B, P < 0.05). This could indicate a better immune status of primiparous cows fed Omega-3 or indi cate a reduced ability of animals to resist infection. Animals fed the omega-3 fat source were diagnosed with a more severe infection of metritis at 5 and 10 DIM compared to those fed the omega-6 fat source (Table 6.10) suggesting that the latter explanation was true. Plasma concentrations of acid soluble protein rose right after partur ition and were greater in primiparous cows on 3, 5, and 7 DIM (parity by DIM interaction; Figure 5.5, P < 0.05) compared to cows which might indicate that parturition was a more severe stressor in primiparous cows than in cows. Mean plasma concentrations of acid soluble protein tended (P = 0.06) to be reduced in primiparous cows fed supplemental fat compared to primiparous cows fed the control diet (45.0 vs. 51.7g/mL) but the opposite occurred in multiparous cows (54.2 vs. 42.5 g/mL; parity by control vs. fat interaction). Acid soluble protein has a dual immuno modulatory effect in which it

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178 can activate monocytes and i nduce cytokine secretion (Su and Yeh, 1996) or can cause immunosuppression (Bennet and Schmid, 1980). Thus primiparous cows fed the control diet could possibly have experienced a greater challenge on the immune sy stem such that acid soluble protein was increased to regulat e normal immune status. On the other hand, fat could have had an immosuppresive effect such that acid so luble protein increased to normalize the immune system. If PUFA sources were stimulating th e immune system of multiparous cows, then the circulating concentrations of acid soluble prot ein were elevated in order to modulate this stimulatory effect. Dietary PU FA may be having the opposite e ffect in lactating primiparous cows. That is, the PUFA were partially suppre ssing the immune system of primiparous cows and so the circulating concentratio ns of acid soluble protein were lower. This appears to match the ceruloplasmin response for the primiparous cows fed the Omega-3 fat source. A lower plasma concentration of ceruloplasmin may sugg est an immunosuppressive effect of the Omega3 fat source. We hypothesized that animals fed Omega-6 woul d have greater fibrinogen concentrations in plasma via stimulation of prostaglandins by the Omega-6 fatty acid supplementation. Despite a lack of effect of Omega-6 on PGFM, multipar ous cows fed Omega-6 had greater (259 vs. 206 mg/dL) concentrations of fibrinogen in plasma compared to multiparous cows fed Omega-3 but no treatment effect was detected in primip arous cows (226 vs. 254 mg/dL, figure 5.7). One mechanism by which Omega-6 supplementation could increase fibrinogen concentrations in cows may be due to up regulation of IL-6 (Meerara ni et al., 2003) which is the major inducer of fibrinogen in hepatocytes (Albrecht et al., 2007). Conclusions In summ ary, the feeding of fat to primiparous cows (the parity that exhibited greater stress due to parturition and lactation, Fi gure 5-5), resulted in lower circul ating concentrations of acid

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179 soluble protein (Figure5-6A). This suppressive response agreed with the suppressive effects of omega-3 fat on concentrations of plasma cerulopl asmin of primiparous cows (Figure 5-4A). In the case of multiparous cows, feeding fat was immunostimulatory (Figure 5-6B). Likewise, omega-6 fats were immunostimulatory in multiparous cows based upon fibrinogen concentrations in plasma (Figure 5-7B). Although the effects of PUFA were not identical across parities, this study supplies eviden ce to demonstrate that suppleme nting with omega-6 fatty acids can stimulate the production of acid soluble protein and fibr inogen whereas supplementation with omega-3 fatty acids can suppress the produc tion of ceruloplasmin and acid soluble protein. Whether these effects have a beneficial effect on the health of the early postpartum cow needs further investigation.

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180 Figure 5-1. Average plasma concentrations of ac ute phase proteins of Holstein cows after calving. The average is across all treatments.

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181 Figure 5-2. Effect of supplementa l fat source on concentrations of prostaglandin F metabolite (PGFM) in plasma of primiparous (A) a nd multiparous (B)Holstein cows fed control (open circle), omega-6 (open square), or omega-3 (closed triangle). There was no treatment effect on PGFM secretion. Prim iparous cows fed omega-3 fatty acids had greater concentrations of PGFM in plasma at 6 DIM compared to omega-6 fed primiparous cows (panel A; Tr eatment by parity interaction: P < 0.05). However, there was no difference among treatments in multiparous cows (panel B).

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182 Figure 5-3. Mean plasma concentrations of haptoglobin from lactating Hols tein cows fed control (white bar), omega-6 (black bar), or omega-3 (gray bar) from calving through 48 DIM. There was no treatment effect on mean plasma concentrations of haptoglobin.

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183 Figure 5-4. Plasma concentrations of ceruloplasmin from lactating Holstein primiparous (A) and multiparous (B) cows fed no supplemental fat (Control, white bar), Omega-6 (black bar), or Omega-3 (gray bar) diets. Prim iparous cows supplemented with Omega-3 fatty acids had reduced concentrations of ceruloplasmin in plasma compared to Omega-6 fed primiparous cows (panel A) whereas that of multiparous cows were not affected by diet (panel B). (treat ment by parity interaction, P < 0.05).

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184 Figure 5-5. Concentration of acid soluble protei n in plasma of primiparous and multiparous cows. Primiparous cows (closed circle) ha d greater concentrati on of acid soluble protein on 3, 5 and 7 DIM compared to multiparous cows (open diamond) (Parity effect: P < 0.05).

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185 Figure 5-6. Plasma concentrations of acid solubl e protein from lactati ng Holstein primiparous (A) and multiparous (B) cows fed control ( open circle), omega-6 (open square), or omega-3 (closed triangle). Primiparous cows fed control diet tended to have greater concentrations of acid soluble protein in plasma compared to cows fed supplemental fat (panel A; Control vs. fat by parity interaction: P = 0.06). However, cows fed control diet had lower concentration of aci d soluble protein in plasma compared to cows fed supplemental fat source (panel B).

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186 Figure 5-7. Plasma concentrations of fibrinogen from lactating Holstein primiparous (A) and multiparous (B) cows fed control (white bar), omega-6 (black bar), or omega-3 (gray bar). Multiparous cows fed omega-3 fatty acids had reduced concentrations of ceruloplasmin in plasma compared to omega-6 fed multiparous cows (panel B; Treatment by parity interaction: P < 0.05). However, there was no difference among treatments in primiparous cows (panel B).

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187 CHAPTER 6 EFFECT OF OMEGA-3 AND OMEGA-6 SU P LLEMENTATION ON IMMUNITY AND PERFORMANCE OF PERIPARTURIENT DAIRY COWS Abstract Objective was to evaluate two sources of supplemental lipid enriched in om ega-6 or omega-3 fatty acids for influence on production, metabolism, milk composition, and immunity of periparturient Holsteins primiparous (n=16) a nd multiparous cows (n=29). Treatments were the following: 1) Control diet (no fat supplement), 2) Ca salts of fatty acids made from safflower oil (Omega-6, 63.6% linoleic acid; EnerG HL), and 3) Ca salts of fatty acids made from palm oil and fish oil (Omega-3, 5.4% C20:5 and 5.3% C 22:6, StrataG 0.5). Supplemental fats (Virtus Nutrition, Corcoran, CA) were fed at 1.5% of dietary DM. Blood was taken daily for first 10 DIM for PGFM analysis and thrice weekly ther eafter until 49 DIM for measures of plasma metabolites. Phagocytotic and oxida tive burst activities of neutr ophils were measured using flow cytometry in whole blood samples taken at -18, 0, 7, and 40 DIM. Milk yield was recorded twice daily and weekly samples were taken for milk composition. Milk samples from wk 5, 6, and 7 were pooled for fatty acid analys is. Orthogonal contrasts were Cont rol vs. (Omega-6 + Omega-3) and Omega-6 vs. Omega-3. Animals fed Omega-3 tended to consume less DM (% of BW) and produce less milk fat compared to animals fed Omega-6. Mean values for DMI prepartum (13.4, 13.7, and 13.5 kg/d; SE = 0.6), milk yield (32.8, 34.4, and 31.3 kg/d; SE = 1.5), milk protein concentration (3.0, 2.9, and 2.9%; SE = 0.1), BW (603, 593 and 593 kg; SE = 18), BCS (3.12, 3.26, and 3.15; SE = 0.10), plasma glucose ( 68.8, 69.5, and 69.5 mg/dL; SE = 1.8), plasma NEFA (459, 391, and 433 Eq/L; SE = 38), and plasma BHBA (5.7, 5.7, and 5.9 mg/dL; SE = 0.5) for treatments Control, Omega-6, and Om ega-3, respectively were not different among treatment groups. Concentration of milk fat from cows fed Omega-6 (3.52%) or Omega-3 (3.21%) was lower than that from control cows (3.76%) and that of Omega-3 was lower than

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188 Omega-6. These decreases were likely due to gr eater milk fat concentrations of CLA isomers and C18:1 trans -10. Omega-3 had immunosuppressive e ffects; namely, lowering blood concentrations of W BC (8,796 vs. 11,492 WBC/L; P = 0.06) and neutrophils (2463 vs. 3495 per L; P < 0.01), decreasing the intensity of neutrophil action against E. coli, and decreasing the production of cytokines by isolat ed lymphocytes in vitro. The omega-6 enriched fat had immunostimulatory effects, namely preventing the decrease in concentration of blood neutrophils at 7 DIM that occurred in the other treatments an d stimulating the humoral response (IgG) postpartum to ovalbumin injections. It also appeared to have im munosuppressive effects on production of cytokines by concanavalin-A-stimu lated lymphocytes, simila r to that of cows fed the omega-3 fat source. Introduction Because of their h igh energy density, fats are incorporated into dairy cow diets to improve production, growth, and reproduction. However, due to the essentiality of specific fatty acids and their role as precursors of hormones or on ge ne expression, it is possible that reproduction (Santos et al., 2008) and immune function (Calder, 2007) may be in fluenced more by the type of fat fed than by feeding fat per se. Fat sources differ in the composition of their fatty acids which in tu rn can differentially affect production and composition of milk (Juchem et al., 2008), incorporation of specific fatty acids in several tissues of da iry (Bilby et al., 2006c) and beef cows (Burns et al., 2003), circulating concentrations of hormones such as progesterone (Staples et al., 1998) and prostaglandins (Mattos et al., 2004 ), and consequently can affect cells of the immune system (Calder, 2007). Bharatan et al. (2008) reported incr eases in C20:5 and a tendency ( P = 0.07) for increases in C22:6 in milk fat of cows fed fish oil at 0.5% of dietary DM. Shingfield et al. (2006) reported

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189 that cows fed fish oil at 1.5% of dietary DM ha d greater concentrations of C20:5 and C22:6 in milk fat compared to cows fed a control diet. Mid lactating dairy cows fed extruded soybeans at 2% of dietary DM had greater concentration of C18: 2 in milk fat compared to cows fed fish oil at the same proportion of the diet (AbuGhazaleh et al., 2002). Bilby et al. (2006c) reported that cows fed fish oil had greater incorporati on of C20:5 (0.10 vs. <0.01%) and C22:6 (1.42 vs. 0.92%) into endometrium of dairy cows compared to those fed whole cottonseed. The increase in the omega-3 fatty acids incorporated into the en dometrium is concomitant with a decrease in the C20:4 proportion in the endometrium of cows fed fish oil. Since C20:4 is the precursor of PGF2 fish oil supplementation reduced PGF2 secretion in early postpartum dairy cows compared to olive oil supplementation (Mattos et al., 2004). Childs et al. (2008) reported that the overall mean concen tration of progesterone in plasma of beef primiparous cows fed diets of 6.67% fish oil was greate r during the 16 d of the estrus cycle than that of cows fed fish oil at 1.67% of dietary DM. This may be due to an increase in the concentrations of cholesterol in plasma which has been consistently shown in fat supplemented cows (Staples et al., 1998). Many effects mediated by PUFA on immune cells appear to be exerted in an eicosanoidindependent manner. There is now eviden ce that the omega-3 fatty acids have immunosuppressive effects (Shaikh and Edidi n, 2008) whereas the omega-6 fatty acids are immunostimulators of immune cells (Kang et al., 2007). The objective of this experiment was to evalua te the effects of two sources of supplemental lipid enriched in omega-6 or omega-3 fatty acids on production, metabolism, milk composition, and immunity of periparturient Holstein cows.

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190 Material and Methods Animals, Treatments, and Sampling Experim ent was conducted at the University of Floridas dairy research unit (Hague, FL) during the months of October 2006 through April 2007. All experimental animals were managed according to the guidelines approved by the Universi ty of Floridas Animal Reseach Committee. Periparturient Holstein primiparous (n = 16) and multiparous (n = 29) cows were assigned to treatment at approximately 34 7 d prior to their actual calving date. Dietary treatments were the following: 1) Control diet ( CO ; no fat supplement), 2) Ca salts of fatty acids made from safflower oil (Omega-6, 63% linol eic acid; EnerG HL, NutriScien ce, Fairlawn, OH), and 3) Ca salts of fatty acids made from palm oil and fish oil (Omega-3, 5.4% eicosapentaenoic acid [EPA] and 5.3% docosahexaenoic acid [DHA], Stra taG 0.5, Virtus Nutrition, Corcoran, CA). Supplemental fats were fed at 1.5% of dietary DM during the pr e(Table 6.1) and postpartum period (Table 6.2). Mean values for calculated ca lving date, parity (primiparous or multiparous), BW and milk production of the previous year for multiparous cows were similar among treatment groups. Prepartum animals were housed in sod-based pens equipped with fans, sprinklers, and shaded Calan gates (American Calan Inc., Northwood, NH). Postpartum animals were housed in a sand-bedded, free-stall barn equipped with fans, sprinklers, and Calan gates. Prepartum cows were fed TMR twice daily at 1000 and 1400 h and postpartum cows were fed twice daily at 0900 and 1300 h to allow 5 to 10% feed refusals. Intake of DM was measured daily. Corn silage was collected weekly and i mmediately dried for 1 h using a Koster (Koster Crop Tester, Inc., Strongsville, OH) to calculate the concentration of DM in order to maintain the formulated forage to concentrate ratio in the ra tion. Animals were milked twice daily at 1030 and 2230 h. Cows were weighed and body condition scor ed (Edmonson et al., 1989) at -8, -5, -3, and 0 weeks relative to calving. After calving cows were weighed and body condition scored weekly

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191 after the 1030 h milking and before eating. Rect al temperature was measures at 4, 7, and 12 DIM. Energy balance was calculated using the following equation: Energy balance = net energy of intake (net energy of maintenance + net energy of lactation) Net energy of intake was calculated by multip lying weekly DMI by the calculated energy value of the diet. Energy requirement for body maintenance was calculated using the following equation (NRC, 2001): Net energy of maintenance = 0.08 x BW0.75 Milk energy was estimated by the following equation: Net energy of lactation = [(0.0920 x %fat ) + (0.0547 x %protein ) + 0.192] x milk weight Feed efficiency was cal culated as follows: Feed efficiency = kg of 3.5% FCM / kg of DMI. Sample Collection and Analysis Representative sam ples of corn silage, bermudagrass hay, alfalfa hay, and concentrate mixes were collected on a weekly basis. Weekly samples of concentrates were composited on a monthly basis and ground through a 1-mm Wiley mill screen (A. H. Thomas, Philadelphia, PA) whereas forage samples were ground first and then composited. Composited feed samples were analyzed for mineral and fat (acid hydrolys is) composition (Dairy One, Ithaca, NY), NDF (Mertens, 2002), ADF (AOAC, 1995), and CP using a macro elemental analyzer vario MAX CN (Elementar Analysensysteme GmbH, Hanau, Germany). Caruncles were collected from all cows in the study within 12 h after calving by manual extraction via the vagina. Briefl y, the perineal area was washed with chlorhexidine diacetate (Novalsan, Fort Dodge, WI), iodine, and dried with paper towel. A shoulder-length sleeve was

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192 used with sterile lubrication to access the carunc le intravaginally and manually remove it. After collection, caruncles were washed w ith saline, dried with sterile filt er paper, snap frozen in liquid nitrogen, and stored at -80oC until fatty acid analysis. Milk samples were collected weekly from 2 consecutive milkings using bronopol-B-14 as a preservative. Milk was measured for fat, true protein, and SCC by Southeast Milk lab (Belleview, FL) using a Bently 2000 NIR analyzer. Daily concentra tions of fat and protein were calculated after adjusting for milk production duri ng those 2 milkings. M ilk without preservative was collected at 2 consecutive milkings at 5, 6, and 7 wk postpartum, and frozen for fatty acid analysis. To determine the fatty acid profile of milk fat, milk samples were composited (final volume of 45 mL) from wk 5, 6, and 7 postp artum according to milk production. Fat was isolated from milk by centrif ugation of thawed milk at 17,800 x g for 30 min at 8C. Fatty acids from about 325 mg of isolated fat were extrac ted using a 3:2 (vol/vol) hexane/isopropanol solvent mixture (18 mL / g of fat). The extracted fatty acids were converted to methyl esters (Chouinard et al., 1999). Approxima tely 200 mg of the methyl esters were transferred into an acid-washed 15-mL glass tube to which 2 mL of hexane and 40 l of methyl acetate (Fisher Scientific, Hampton, NH, USA) were added. The tube was vortexed unti l fat was dissolved. Forty l of sodium methylate solution (Fishe r Scientific, Hampton, NH, USA) was added, the tube was vortexed, and allowed to react for 10 min at room temperature. Sixty microliters of oxalic acid solution (Fisher Scientific, Hampton, NH, USA) was added to terminate the reaction and the tubes were centrifuged at 2,000 x g for 5 min at 5oC. The top hexane layer containing the fatty acids in the methyl ester form was transferred to 2 mL crimp-top vials (Fisher Scientific, Hampton, NH, USA) for fatty acid analysis.

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193 The fatty acid extraction and methylation proced ure (Kramer et al., 1997) were the same for dietary fat supplements and caruncle. Fat s upplements (approximately 200 mg) and caruncle tissue (approximately 200 mg of fr esh weight) were freeze-dried for 24 h. One mg of internal standard (C19:0) was added in order to calculate total fatty acid concentration. Lipid was extracted by adding 2 mL of sodium methoxi de (Acros, New Jersey, USA), vortexing, and incubating in a 50oC water bath for 10 min. After cooli ng for 5 min, 3 mL of 5% methanolic HCl (Fisher Scientific, Hampton, NH, USA) were added and the tubes vortexed. The tubes were incubated in an 80oC water bath for 10 min, removed from wa ter bath, and allowed to cool for 7 min. One mL of hexane and 7.5 mL of 6% K2CO3 were added. The tubes were vortexed and centrifuged at 194 x g for 5 min. The upper layer was transfer red into 10 mL glass tubes. The solvent was completely evaporated under N gas. He xane (100 l) was added in order to redisolve methylated fatty acids and the solution was transferred to a crimp-top vial. Fatty acid methyl esters were determined using a Varian CP-3800 gas chromatograph (Varian Inc., Palo Alto, CA) equipped with an auto-sampler (Varian CP-8400), flame ionization detector, and a Varian capillary column (CP-S il 88, 100 m x 0.25 mm x 0.2 um ). The carrier gas was He, the split ratio was 10:1, and the injector a nd detector temperatures were maintained at 230C and 250C, respectively. One l of sample was injected via the au to-sampler into the column. The oven temperature was initially se t at 120C for 1 min, increased by 5C/min up to 190C, held at 190C for 30 min, increased 2C/min up to 220C, and held at 220C for 40 min. The peak was identified and calculated based on the retention time and peak area of known standards. The desaturase index for cis -9 C16:1 and cis -9 C18:1 was defined as follows: [product of 9 desaturase] / [product of 9 desaturase + substrate of 9 desaturase]. For example, the cis -9 C16:1

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194 desaturase index would be calculated as [ cis -9 C16:1] / [ cis -9 C16:1 + C16:0] (Kelsey et al., 2003). Blood (10 mL) was collected at 0700 h daily from parturition until 10 DIM and on Monday, Wednesday, and Friday thereafter until 47 3 DIM from coccygeal vessels into sodium heparinized tubes (Vacutainer, Be cton Dickinson, Franklin La kes, NJ). Samples were put immediately on ice until centrifuged at 2619 x g at 5C for 30 min (RC-3B refrigerated centrifuge, H 600A rotor, Sorvall Instruments, Wilmington, DE). Plasma was separated and frozen at -200C for subsequent metabolite, and hormone analysis. Plasma concentrations of NEFA (NEFA-C kit; Wako Fine Chemical Industries USA, Inc., Dallas, TX; as modified by Johnson, 1993) and -hydroxy butyric acid (BHBA) (Pointe Scientific Inc., Lincoln Park, MI) were determined once weekly for 7 wk. A T echnicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to determine weekly concentrations of plasma BUN (a modification of Coulombe and Favreau, 1963 and Marsh et al., 1965) and plasma glucose (a modification of Gochman and Schmitz, 1972). Concentrations of progesterone were determin ed on every plasma sample collected using Coat-A-Count Kit (DPC Diagnostic Products Inc., Los Angeles, CA) solid phase 125I RIA. The sensitivity of the assay was 0.1 ng/mL and the intraand interassay coefficien ts of variation were 5.1 and 7.3%, respectively. A pol yethylene glycol RIA procedur e described by Meyer et al. (1995) was used to analyze for the concentr ation of 15-keto-13,14-dihydro-prostaglandin F2 metabolite ( PGFM ) in each plasma sample collected duri ng the first 10 DIM. Sensitivity of the assay was 31.2 pg/mL and the intraand interass ay coefficients of variation were 9.3 and 5.9%, respectively.

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195 Immune Status Neutrophil function Blood (6 mL) collected in vacutainer (Vacuta iner, Becton Dickinson, Franklin Lakes, NJ) tubes containing acid citrate dextro se were collected from coccyg eal vessels at 18, 0, 7, and 40 DIM. Neutrophil numbers and function were accessed within 3 h af ter blood collection. Neutrophil concentration in whole blood was esti mated using a hemacytometer. In order to measure phagocytosis and oxidative burst of neutrophils, whole bl ood (100 l) was pipeted into each of 3 tubes. Then, 10 l of 50 M dihydr orhodamine 123 (DHR) (Sigma-Aldrich, Saint Louis, MO) was added to all tubes. Tube s were vortexed and in cubated in oven at 37oC for 10 min with constant rotation using the Clay Adam s nutator (BD, San Jose, CA). Ten l of 20 g/mL solution of phorbol 12-myristate, 13-acetat e (PMA) (Sigma-Aldrich) was added to tube number 2 only. An Escherichia coli bacterial suspension (106 cells/mL) labeled with propidium iodide (Sigma-Aldrich) was added to tube number 3 to establish a bacteria to neutrophil ratio of 40:1. Tubes were vortexed and incubated in oven at 37oC for 30 min with constant rotation using the Clay Adams nutator (BD, San Jose, CA ). Then all tubes were removed and placed immediately on ice to stop phagoc ytosis and oxidative burst activit y. Tubes were processed in a Q-Prep Epics immunology workstation on the 35 sec cycle. Cold distilled water (500 l) and 0.4% tryphan blue (10 l) were added to each tube Then tubes were vortexed, kept on ice, and 10,000 cells were read at the F acsort flow cytometer (BD bios ciences, San Jose, CA). The amount of bacteria that each neutrophil phagocytized was measured by median fluorescence intensity (MFI) using the flow cytometer. Bovine peripheral blood mononuclear cel l (PB MC) isolation and stimulation Six tubes of blood (10 mL each) were collected from each cow from the coccygeal vessels using heparinized tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) on 10, 20, and 30

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196 DIM. The samples were transported to the laborat ory at ambient temperature and isolation of the PBMC was initiated within 3 h after blood collection. Tubes were centrifuged (Damon/IEC Division, IEC HN-SII Centrifuge) at 1006 x g for 30 min at room temperature. The buffy coat was removed from 2 vacutainer tubes of the same cow a nd transferred with sterile transfer pipettes to one 13 mL tube (Sarstedt Inc., Newton, NC) c ontaining 2 mL of medium 199 (M-199) (SigmaAldrich, Saint Louis, MO). The buffy coat s and M-199 were mixed by pipetting up and down several times. This cell suspension was transf erred slowly on top of 2 mL of Fico/Lite LymphoH (Atlanta Biologicals, Lawrencevill e, GA) to prevent mixing. The cell suspension/Fico/Lite LymphoH solution was centrifuged at 252 x g for 30 min at room temperature. Mononuclear cells were collected from the Fico/Lite interface and transferred to pre-labeled 13 mL culture tubes containing 2 mL of Red Blood Cell Lysing Buffer (SigmaAldrich, Saint Louis, MO). Exactly 20 sec after transferring, the solution was neutralized with 8 mL 1X DPBS (Sigma-Aldrich, Saint Louis, MO). The so lution was centrifuged at 252 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 (m ononuclear cells) was resusp ended in 2 mL of M199 by pipetting up and down 10 times with a ster ile transfer pipette. The solution was centrifuged at 112 x g for 3 min at room temperature. The supernatant was removed and the pellet was resuspended in modified M-199 (M -199 supplemented with 5% horse serum, 500 U/mL penicillin, 0.2 mg/mL streptomycin, 2 mM glutamine, 10-5 M -mercaptoethanol (all reagents from Sigma-Aldrich, Saint Louis, MO )). The PBMCs were counted using the Trypan blue dye (Sigma-Aldrich, Saint Louis, MO) excl usion method. The cell suspension was adjusted to 2 x 106 cells/mL. Cell suspension was plated in duplicate with modified M-199 media and stimulated or not stimulated with 10 g/mL of concanavalin A (ConA) (Sigma-Aldrich, Saint

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197 Louis, MO) on a 6-well plate (C orning Inc., Corning, NY) for production of the cytokines: interferon-gamma (IFN) and tumor necrosis alpha (TNF). Ovalbumin challenge All cows were injected s.c. with 1 mg of ovalbumin (Sigma-Ald rich, Saint Louis, MO) diluted in Quil A adjuvant (0.5 mg of Quil A/ mL of PBS) (Accurate chemical & Scientific Corp. Westbury, NY) at -8 wk relative to calving and at parturition. At -5 wk relative to calving ovalbumin was mixed in the JVac (Merial, Athens GA) vaccine so that the total amount of ovalbumin injected was 1 mg. Blood samples for se rum analysis of IgG were collected at -8, -5, -3, 0, 1, 2, 3, 4, and 7 wk relative to calving. Sa mples were taken in vacutainer (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) tubes c ontaining no anticoagulant before the ovalbumin injection. Serum concentration of anti-oval bumin IgG was measured by an Enzyme Linked ImmunoSorbent Assay (ELISA) as described by Mallard et al. (1997). Briefly, flat bottom 96well polystyrene plates (Immulon 2, Dynex Tech., Chantilly, VA) were co ated with a solution of OVA dissolved in carbonate-bicarbonate coa ting buffer (1.4 mg OVA/ mL of carbonatebicarbonate buffer). Plates were incubated at 4C for 48 h, then washed with PBS and 0.05% Tween-20 solution (pH = 7.4). Plat es were blocked with a PBS-3% Tween-20 and bovine serum albumin (Sigma Chemical, St. Louis, MO) solution and incubated at room temperature for 1 h. Plates were washed and diluted sera samples and control sera (l/50 and l/200) were added in duplicate using a quadrant system (Wright, 1987). Po sitive and negative c ontrol sera to antiovalbumin IgG were obtained from a pool of sera of known high (21 d after the third injection of ovalbumin) and low (pre-ovalbumin injection) concentrations, respectively. All samples from the same cows were analyzed in the same plate a nd plates contained balanced number of animals from each diet group. Plates were incubated at room temperature for 2 h and washed with

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198 previously described buffer solution. Subsequent ly, alkaline phosphatase conjugate rabbit antibovine IgG whole molecule (Sigma Chemical, St Louis, MO) was dissolved in wash buffer, added to the plates and incubated for 2 h at r oom temperature. After incubation, plates were washed 4 times and substrate solution [P-Nitrophenyl Phosphate Disodium (Sigma Chemical, St. Louis, MO)] was added and the plate was incubate d at room temperature for 30 min. Plates were read on an automatic ELISA plate reader (MRX Revelation; Dynex Technologies Inc., Chantilly, VA) and the optical density was recorded at 405 nm and the reference at 650 nm. Prior to the initiation of experimental serum sample analyses 3 plates containing pos itive controls for antiOVA IgG were analyzed at 1/50 and 1/200 dilutions in order to calculate an initial mean and SD. Further positive controls obtained from plat es containing experimental samples were subsequently added to calculate the total mean and standard deviation. Plates with positive control mean above or below 1.5 SD of total accumulated positive controls were repeated. Inter assay CV for positive control samples was 9%. Pl ates were also repeated when the CV of positive control samples were above 20% within a plate. A correction factor was calculated for each plate by dividing the total mean from the accumulated positive control results to the total mean of the positive controls from each plate. Experimental sample results were obtained from the product of the sum of the av erage of each duplicated sample dilution by the correction factor of each plate. Cytokines (IFN, and TNF) were analyzed by Enzyme-L inked ImmunoSorbent Assays (ELISA). The IFN(Mabtech, Cincinnati, OH), and TNF(Endogen, Rockford, IL) cytokines were analyzed as recommended by the manufacturer. Vaginoscopy Cows were evaluated for cervical discharg e on days 5 and 10 postpartum using the metricheck (Metricheck, Simcro, New Zealand). The vulva was first cleaned using a povidone-

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199 iodine scrub (0.75% titratable iodine and 1% povidone soluti on, Agripharm, Memphis, TN, USA) and dried off with a clean paper towel. The metricheck was inserted in the vagina all the way close to the cervix. The floor of the vagina was scraped, the discharge collected in a 50-ml conical tube (Fisher Diagnostics, Middletow n, VA), and assigned a score of 0 (clear or translucent mucus), 1 (mucus containing flecks of white or off-white pus), 2 (discharge containing 50% white or off-white mucupurulent ma terial), or 3 (discharge containing 50% purulent material usually white or yeallow but occasionally sanguineous) according to Sheldon et al. (2006). Uterine cytology At 37 3 DIM, a single assessm ent of ut erine cytology was conducted. Cows were flushed using a 53.3 cm silicon Fo ley catheter (i.e., 18 Fr and 5 cc) The vulva was cleaned with chlorhexidine ciacetate (NolvasanFort Dodge, Overland Park, KS) a nd dried with a paper towel. The catheter was introduced through the cervix into the previously pregnant uterine horn. The air balloon was placed approximately 1 cm past th e bifurcation of the uterine horn and inflated with air to a volume consistent with the size of the uterine horn. Sterile saline (20 mL of 0.9%) was infused into the uterine horn and aspirated back using a syringe with a Foley connector. The aspirated solution was placed into a sterile 50-mL conical tube and vortexed. A 50-l aliquot of flushed solution was placed into a bullet tube and mixed with 50 l of a trypan blue solution (0.4%) for 1 min. A 10 l sample of the solution was placed in each side of a hemacytometer in order to count total white blood cells (WBC) and to determine cel l viability using a microscope set to a magnification of 40x. Ce lls not stained were considered live and cells stained with trypan blue were considered dead. Concen tration of WBC was determined by counting 5 squares (i.e., each square = 0.2 0.2 mm2) from each side of the hemocytometer in the large middle square using a magnification of 40x. Con centration of WBC was calculated as follows:

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200 WBC (number / mL) = total WBC count in 10 s quares/2 50,000 dilution factor 2. Proportion of cell viability was calculated as follows: cell viability ( %) = number of WBC not stained (viable) / number of stained a nd not stained WBC 100. After dete rmination of total and viable WBC, 20 l of flushed solution was pipetted onto a gla ss slide and smeared (2 slides per uterine flush). Smear was air-dried and stained using the Diff-Quick (F isher Diagnostics, Middletown, VA) stain. Slides were examined for WBC and neutrophil nu mbers at magnification of 40x. Number of total WBC as well as number of neutr ophils were counted and percent of neutrophils calculated as follows: % neutroph ils = total number of neutrophils / total number of WBC 100. The percentage of neutrophils was used to estimate the number of ne utrophils per mL of flush solution using the hemacytometer results as follows: number of neutrophils / mL of flush = total WBC count / mL (hemacytometer) % neutrophils. Statistical Analysis Measurem ents of daily DMI during the pre and postpartum periods, milk production, and milk composition were reduced to weekly means before statistical analyses were performed. Repeated measures data (DMI, milk production, milk fat, milk protein, BW, BCS, rectal temperature, IFN, TNF, concentration of plasma NEFA, BHBA, BUN, glucose, progesterone, and serum IgG) were analyzed using MIXED procedure of SAS (SAS Institute Inc., Cary, NC) according to the following model: Yijkl = + Fi + Pj + FPij +Ck (i j) +Wl + FWil + PWjl + FPWijl + Eijkl where Yijkl is the observation, is the overall mean, Fi is the fixed effect of dietary fat source (i = 1, 2, and 3), Pj is the fixed effect of parity (j = 1 and 2), FPij is the interaction of fat source and parity, Ck (i j) is random effect of cow within fat source and parity (k = 1, 2, n), Wl is the fixed effect of week (l = 0, 1, 2, ), FWil is the interaction of fat source and week, PWjl

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201 is the interaction of parity and week, FPWijl is the three way interaction of fat source, parity and week, and Eijkl is the residual error. Data were tested to determine the structure of best fit, namely AR (1), ARH (1), CS, or CSH, as indicated by a lower Schw artz Baesian information criteri on value (Littell et al., 1996). Orthogonal contrasts used to det ect treatment differences were the following: 1) Control vs. (Omega-6 + Omega-3) and 2) Omega-6 vs. Omega-3. Vaginoscopy data were analyzed using proc logistic of SAS. Data that did not have a normal distribution (uterine cyto logy data, and blood neutrophils and WBC) were analyzed by proc Glimmix of SAS using Poisson distribution. Progesterone data (cyclic, DIM at first ovulation, number of cycles, peak progesterone in the first cycle, first cycle length, mean progesterone concentration in the first cycle, and total progesterone concentration of first cycle) were analyzed using PROC GLM of SAS. Milk and caruncle fatty acids were analy zed using PROC GLM of SAS. The model contained treatment, parity and treatment by parity interaction. The orthogonal contrasts mentioned above were also used to test for treatm ent effects. Differences were considered to be significant at P < 0.05 for all analyses. Results and Discussion The om ega-6 fat source was an excellent so urce of C18:2, containing 63.6% C18:2; other fatty acids present in relative high concentra tions were C18:1 at 16.7% and C16:0 at 12.9% (Table 6.3). The omega-3 fat source contained 1 2.5% omega-3 fatty acids (sum of C18:3, C20:5, C22:5, and C22:6; Table 6.3). Othe r fatty acids present in signifi cant concentrations were C16:0 (38.7%) and C18:1 (30.8%).

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202 DMI, Milk Production, and Milk Compositio n As expected, primiparous cows had lower DMI prepartum (11.3 vs. 15.8 kg/d) and postpartum (15.3 vs. 20.4 kg/d) compared to multiparous cows (parity effect; P < 0.001) (Figure 6.1). Dry matter intake did not differ among tr eatments during the prepartum or postpartum period (Table 6.4). However, postpartum intake of diets containing the omega-3 supplement tended to be lower ( P = 0.09) compared to cows fed diet s containing the omega-6 supplement when expressed as a percentage of BW (2.93 vs. 3.22%). Allred et al. (2006) reported a nonsignificantly lower DM intake by lactating dairy cows fed the same fat source as used in the current study at 2.7% of dietary DM compar ed to cows fed no fat supplement (28.6 vs. 26.9 kg/d). Andersen et al. (2008) reported no effect of fat supplementation up to 1.5% of dietary DM on prepartum DMI. In addition, Allred et al. (2006) reported that cows fed Ca salts of a mixture of palm and fish oils at 2.7% of dietary DM had similar intakes as cows not fed this fat source. Decreased DMI was reported when dairy cows we re fed an unprotected mix of fish oil and sunflower oil (1:2) at 4.5% of dietary DM (Shingfield et al., 2006). Infusing unprotected fish oil into the rumen of lactating dairy cows also de pressed DM intake compared to ruminal infusion of a Ca salt mix of fish oil a nd palm oil at equal deliveries of fish oil of 145 g/d (CastanedaGutierrez et al., 2007). Fat supplementation did not influence milk yi eld over the 7-wk postpartum period (Table 6.4; Figure 6.2). Although mean concentration of milk protein (3.0, 3.0, and 2.9%), yield of milk protein (0.96, 0.99, and 0.92 kg of protein/d), 3. 5% FCM (33.8, 35.3, and 29.7 kg/d) (Figure 6.3), 3.5% FPCM (33.02, 34.4, and 29.3 kg/d) and ECM ( 33.4, 34.8, and 29.6 kg/d, respectively for control, Omega-6, and Omega-3 treatments) we re unchanged by fat supplementation, cows fed Omega-6 tended to have greater m ilk fat concentration (3.5 vs. 3.2%; P = 0.06) and milk fat yield (1.19, and 1.00 kg of fat/d; P = 0.10) compared to cows fed Omega-3. In addition, cows fed

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203 Omega-6 fat had greater concentr ation of milk fat at wk 1, 2, and 3 postpartum compared to Omega-3-fed cows (Omega-6 vs. Omega-3 by week interaction; P < 0.05) (Figure 6.4). Similarly, Bharathan et al. (2008) reported that cows fed fish oil at 0.5% of dietary DM had lower (3.3 vs. 3.6%) milk fat concentration and yield of ECM (32.1 vs. 34.5 kg/d) compared to cows fed a control diet without fish oil. In contrast, Bu et al. (2007) reported that cows supplemented with soybean oil or flaxseed oil at 4% of dietary DM had similar ECM yield compared to cows fed a control diet without fat which was likely due to the lack of effect of oil supplementation on milk fat concentration althou gh it was numerically lower for cows fed oils (3.3%) compared to control (3.5%). The difference between the results of Bharathan et al. (2008) and Bu et al. (2007) may have been due to the gr eater feed intake (25 vs. 16 kg/d) by cows used in the Bharathan et al. (2008) study. Greater feed intake may have resulted in a more acidic ruminal environment, which leads to greater formation of the CLA and trans C18:1 isomers that are associated with milk fat depression. Cows fed fish oil at an increasing rate of 0.33, 0.67, and 1.00% of dietary DM with soybeans to provide the balance of 2% added fat in the diet had lower milk fat compared to cows fed a control diet without fat supplement (Whitlock et al., 2006). The milk fat depression is likely due to greater co ncentration of trans -10, cis -12 CLA (0.065 vs. 0.036%) in milk fat of cows fed Omega-3 compared to that of Omega-6 fed cows (Table 6.5). These isomers had potent effects on lowering milk fa t concentration when infused postruminally (Baumgard, et al., 2000). Mean energy balance was lower for multiparous cows compared to primiparous cows ( P < 0.001; Figure 6.5). Multiparous cows were in NEB during all 7 wk postpartum whereas primiparous cows returned to positive EB at least once between wk 4 and 7 postpartum although parity by time interaction was not significant. Mean EB across 7 wk postpartum was not different

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204 among treatments (Figure 6.5). Primiparous cows fed Omega-3 had a numerically higher EB through wk 4 compared to primiparous cows fed Omega-6. Feed efficiency declined from wk 1 (2.2 kg of 3.5% FCM/ kg of DMI) until 6 wk postpartum (1.6 kg of 3.5% FCM/ kg of DMI) (Figure 6.6) but did not differ among the treatments (Table 6.4). In early lactation feed efficiency reflects BW losses, high milk fat percentages, and low DMI. The lowest feed efficien cy occurs in late lact ation where cows divert more of their DMI to BW gain, support of repr oduction and growth in young animals than for milk production. The decrease in feed efficiency duri ng late lactation is more from a decrease in milk production without a proportion decrease in feed intake. Mean feed efficiency across 7 wk postpartum was not different among treatments. Similarly, Bharathan et al. (2008) reported no effect of feeding fish oil (0.5% of dietary DM) on feed effien cy of lactating Holstein cows. Primiparous cows had lower feed efficiency (1.73 vs. 2.00 kg of 3.5% FCM/ kg of DMI) than multiparous cows which was likely due to the usage of nutrients from the diet for growth as well as for milk by primiparous cows. As expected, multiparous cows were heavie r (673 vs. 520 kg) than primiparous cows (parity effect, P < 0.001). Primiparous cows lost BW (Fi gure 6.8) in the first 2 wk postpartum whereas multiparous cows lost BW through 3 wk postpartum. Mean body condition decreased through the first 3 wk postpartum and then pl ateaued until 7 wk postpartum. Loss of body condition averaged 0.4 units. Fatty Acid Profile Milk Milk f at contains fatty acids derived from de novo synthesis by the mammary gland (C4:0 to C14:0 plus a portion of C16:0) and from mammary uptake of preformed fatty acids (a portion of C16:0 and all longer chain fatty acids). Fat suppl ementation decreased (P < 0.05) the

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205 concentration of most of the fatty acids synthesized de novo [C6:0 (1.67 vs. 2.02%), C8:0 (1.19 vs. 1.49%), C10:0 (2.57 vs. 3.40%), C12:0 ( 2.89 vs. 3.82%), C14:0 (10.81 vs. 12.12%), and C16:0 (28.58 vs. 31.10%)], tended ( P = 0.06) to decrease C15:0 (0.97 vs. 1.07%) but did not affect C4:0 (1.58 vs. 1.64%; Table 6.5). Fatty acids synthesized de novo (C4 to C14) were reduced by 15% and the long chain fatty acid s (C16 to C22) increased by 14% in cows supplemented with fat compared to control cows (Table 6.5). Exogenous fatty acids compete for esterification with newly synthesized short-chain fatty acids in mammary cells, and could lead to feedback inhibition of lipogenic enzymes. Thus, greater uptake and secretion of dietary and rumen-derived fatty acids may account for the majority of the reduction in de novo synthesis in cows fed unsaturated oils (Palmquist et al., 1993 ) likely due to a decrease in abundance of the nuclear sterol regulatory element-binding prot ein 1 (Peterson et al., 2004) and consequent reduction in the mammary acetyl-CoA carboxylase a nd fatty acid synthase activity as well as a reduction in acetyl-CoA carboxylase mRNA abunda nce (Piperova et al., 2000). Consequently, milk fat of cows fed supplemental fat had a 22 and 21% increase in MUFA and PUFA, respectively. Moate et al. (2008) reported that there is a positive quadratic relati onship between intake of fish oil fatty acids and production of total trans octadecenoic acids with the maximum production of the trans isomers occurring with an intake of approximately 350 g/d of fish oil fatty acids. This supports our finding s of the increase of C18:1 isomers trans 6 to 8 (0.044 vs. 0.032%), trans -10 (7.92 vs. 6.18%), and a tendency ( P = 0.10) for increasing trans -9 (0.80 vs. 0.70%) in cows fed Omega-3 compared to cows fed Omega-6. Recent evidence from principal component and multivariate analys is of milk long chain fatty acid composition during milk fat depression indicated that trans 6-8 C18:1 might be more important than trans -10 C18:1 in

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206 inducing milk fat depression (Kadegowda et al., 2008). Thus, the lower mik fat concentration of cows fed Omega-3 was also likely due to an increase in trans 6-8 C18:1 in milk fat compared to cows fed Omega-6. However, increases in concen tration of octadecaenoi c acids alone does not appear to be sufficient to depress milk fat as evidenced by greater concentration of these fatty acids in milk fat of cows fed the omega-6 suppl ement compared to that of control cows yet without changes in milk fat concentration. The trans -10, cis -12 CLA isomer may be a more potent agent than the octadecaenoic acids as concen tration of this CLA isomer was only different for the group of cows experiencing milk fat depression, namely those fed omega-3 fats. Production of C20:5 and C22:6 in milk fat are strongly and positively related to the intake of fish oil fatty acids (Moate et al., 2008). The concentration of C20:5 and C22:6 increased 4 and 94 fold, respectively, in milk fat of cows fed Omega-3 compared to cows fed Omega-6 (Table 6.5). Bharatan et al. (2008) report ed a slight increase in concentration of C20:5 and a tendency (P = 0.07) for increasing C22:6 in milk fat of cows fed fish oil at 0.5% of dietary DM. It is important to note that in the present ex periment fish oil was fed in the form of Ca salts which might explain the greater incorporation of C20:5 and C22:6 into milk fat. Similarly, Shingfield et al. (2006) reported that cows fed fish oil at 1.5% of dietary DM had greater concentration of C20:5 and C22:6 in milk fat comp ared to cows fed a cont rol diet. In contrast, Bilby et al. (2006c) reported increa sed concentrations of C22:6 in milk fat of cows fed a Ca salt of palm and fish oils compared to cows fed whole cottonseed but no treatment effect on concentration of C20:5 in milk fat was detected. Concentration of C18:2 in milk fat was greater (3.83 vs. 3.50%) in animals fed fat compared to controls but did not differ betw een animals fed the fat sources (3.94 vs. 3.72%, respectively for Omega-6 and Omega-3). This effect was due to multiparous cows. The

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207 concentration of C18:2 was increased in milk fat of multiparous cows (4.30 vs. 3.41%) but not primiparous cows (3.35 vs. 3.57%) fed control vs fat supplements (parity by control vs. fat interaction, P < 0.001). This may be explained by the re sults of AbuGhazaleh and Jenkins (2004) who reported that the addition of either EPA or DHA to mixed ruminal cultures resulted in reduced biohydrogenation of C18:2. When lact ating dairy ewes were supplemented with sunflower oil at a greater proportion (6% of diet ary DM), concentration of C18:2 in milk fat increased (3.76 vs. 2.87%) compared to ewes fed a control diet without fat (Hervs et al., 2008). Mid lactation dairy cows fed extruded soybeans at 2% of dietary DM had a greater concentration of C18:2 in milk fat compared to cows fed fish oil at the same proportion of the diet (AbuGhazaleh et al., 2002). The lack of a larg er response of Omega-6 supplementation on the concentration of C18:2 in milk fat was likely due to the biohydr ogenation of C18:2 in the rumen. Lundy et al. (2004) reported that ruminal biohyd rogenation of linoleic ac id averaged 95% for unprotected soybean oil and 92% for the Ca salts of soybean oil leading to an additional 14 g/d of C18:2 delivered to the omasum. Harvatine and Allen (2006a) supplemented Ca salts of unsaturated fatty acids and us ed a kinetic approach to es timate the extent of ruminal biohydrogenation in lactating cows They reported that protecti on of the 18-carbon PUFA from biohydrogenation was minimal in a commercial source of protected fat (Ca salts of fatty acids). Likewise in sheep, Fotouhi and Jenkins (1992) observed the extent of ruminal biohydrogenation of linoleic acid was 93% for free linoleic acid and 95% for Ca salts of linoleic acid. Despite the extensive biohydrogenation of C18:2 in the rumen, su fficient quantities left the rumen to increase the C18:2 concentration in milk fat. Concentration of cis -9, trans -11 CLA (0.42, 0.86, and 1.13%, respectively for control, Omega-6, and Omega-3) was increased in milk fat of cows supplemented with fat and the

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208 increase was greater in cows fed Omeg a-3 compared to cows fed Omega-6 ( P < 0.001). In contrast, AbuGhazaleh et al. ( 2002) reported no effect of fish oil supplementation on proportion of cis -9, trans -11 CLA in milk fat but concentration of trans -10, cis -12 CLA was increased compared to cows fed extruded soybeans. Bharathan et al. (2008) reported a 47% increase in total CLA concentration in milk fat of cows fed fish oil (0.5% of dietary DM) compared to cows not fed fish oil. Juchem et al. (2008) fed Ca salts of fish and palm oils or tallow at 0.95 and 0.90% of dietary DM, respectively, for the firs t 25 DIM and at 1.90 and 1.80% thereafter until 145 DIM. Cows supplemented with Ca salts of fi sh and palm oil had gr eater concentration of cis 9, trans -11 CLA in milk fat (0.76 vs. 0.53%) compared to cows fed tallow. Ruminal infusion of fish oil at 1.2% of dietary DM (276 g of menha den oil) increased 5 fold the concentration of cis 9, trans -11 CLA in milk fat compared to those fed a control diet without fat (Loor et al., 2005b). As a result of these shifts in omega-6 and omega-3 fatty acids, cows fed Omega-3 had a lower (7.18 vs. 10.90) n6/n3 ratio in milk fat co mpared to Omega-6-fed cows as reported by others (Bilby et al., 200 6c; Petit et al., 2007). The desaturase index is a measurement of the 9-desaturase activity. The 9 desaturase acitivity regulates the conve rsion of C16:0 into C16:1 cis -9 and C18:0 into cis -9 C18:1 (Bauman et al., 1999). Kelsey et al. ( 2003) defined the desaturase inde x using the following equation: (product of 9-desaturase)/ (product of 9-desaturase + substrate of 9-desaturase). The desaturase indices for C18:1 (di 181) and C16:1 (di 161) were greater ( P = 0.01) for cows fed Omega-3 compared to cows fed Omega-6 (0.66 vs. 0.61 and 0.023 vs. 0.019 for di 181 and di 161 for Omega-3 and Omega-6, respectively). It is interesting to note that the increase in the di 181 was mainly due to the efficiency of the desaturase enzyme to produce cis C18:1 from smaller concentrations of substrate C18:0 for cows fed Omega-3 compared to those fed Omega-6

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209 (10.49 vs. 13.10%; P < 0.001) although no difference existed for cis C18:1 product between cows fed Omega-3 or Omega-6 fat supplements (20.67 vs. 20.46%). In contrast, di 161 was greater for cows fed Omega-3 compared to thos e fed Omega-6 due to an increase in the product formed cis C16:1 (0.70 vs. 0.54%; P < 0.001) although no difference existed in the substrate (29.13 vs. 28.03%). Chouinard et al. (1999) report ed that di 161 and di 181 were increased linearly in milk fat of cows fed increasing doses of CLA suppl ements (0, 50, 100, or 150 g/d of CLA supplement). Perfield et al. (2006) reported that cows infused with CLA trans -10, trans -12 (5 g/d) into the abomasum had lower di 161 (0.032) and di 181 (0.590) compared to those infused with ethanol (0.39 and 0.66 for di 161 and di 181, respectively) or with trans -10, cis -12 CLA (0.37 and 0.63 for di 161 and di 181, respectively). The fatty acid profile differed somewhat be tween parities. Compared to multiparous cows, the milk fat of primiparous cows contained a greater ( P < 0.01) proportion of C15:0 (1.07 vs. 0.93%), C17:0 (0.79 vs. 0.74%), C20:0 (0.20 vs. 0.16%), trans -10, cis -12 CLA (0.049 vs. 0.038%) and tended ( P < 0.10) to have a greater proportion of C18:0 (12.31 vs. 11.45%) and C20:5 (0.049 vs. 0.039%). In addition, primiparous co ws had a lower (P < 0.05) concentration of cis -9 C18:1 (19.83 vs. 21.85%), of C18:2 (3.43 vs. 4.00%), of PUFA (4.94 vs. 5.60), of the n6/n3 ratio (8.40 vs. 9.53), and of di 181 (0.62 vs. 0.65) in milk fat compared to multiparous cows. Similarly, the proportion of cis -9, trans -11 CLA and PUFA were increased ( P < 0.05) in milk fat of multiparous cows (1.09 vs. 0.36% and 6.18 vs. 4.43%, respectively for proportion of cis -9, trans -11 CLA and PUFA) but not primiparous cows (0.49 vs. 0.90% and 4.80 vs. 5.02%, respectively for proportion of cis -9, trans -11 CLA and PUFA) fed control vs. fat supplements (parity by control vs. fat interaction, P < 0.05).

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210 Caruncle The predominant fatty acid in caruncular tissue (Table 6.6) w as C18:0 (25.63%), followed by cis -9 C18:1 (19.38%), C16:0 (18.79%), C18:2 (13.61%), and C20:4 (10.58%) which are similar to the major fatty acids found in th e endometrium of dairy (Bilby et al., 2006c) and beef cows (Burns et al., 2003). Cows fed supplemental fat had a lower ( P < 0.05) concentration of C15:0 (1.81 vs. 2.04%) and C16:0 (18.31 vs. 19.76%) but a greater concentration of cis -9 C16:1 (0.53 vs. 0.47%) in caruncular tissue compared to cows fed the c ontrol diet. Due to the greater concentration of the product ( cis C16:1) and lower concentr ation of the substrate (C16:0), cows fed supplemental fat had greater di 161 ( P < 0.001) compared to control cows. Primiparous cows fed the control diet had greater concentration of C16:0 (19.82 vs. 17.19%) in caruncular tissue compared to those fed fat but C16:0 did not differ among multiparous cows (19.69 vs. 19.42%; parity by control vs. fat interaction, P < 0.01). In addition multiparous cows fed Omega-3 tended to have greater concentration of C16:0 (20.29 vs. 18.57%) in caruncular tissue compared to multiparous cows fed Omega-6 but C16:0 did not differ among primiparous cows (17.25 vs. 17.13%; parity by Omega-6 vs. Omega-3 interaction, P = 0.07). Multiparous cows fed Omega-3 tended to have a lower concentration of C18:0 (23.19 vs. 26.37%) compared to multiparous cows fed Omega-6 but C18:0 did not differ among primiparous cows (25.98 vs. 25.76%; parity by Omega-6 vs. Omega-3 interaction, P = 0.10). All C18:1 trans isomers detected in caruncles ( trans -9 C18:1 (0.14, 0.16, and 0.24%), trans -10 C18:1 (0.61, 1.34, and 2.02%), and trans -11 C18:1 (0.11, 0.22, and 0.34%, for control, Omega-6 and Omega-3, respectively)) were greater ( P < 0.01) in cows fed fat compared to control cows and the increase was greater ( P < 0.001) in cows fed Omega-3 compared to cows fed Omega-6. In addition, concentration of cis -9, trans -11 CLA in caruncles of cows followed

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211 this same pattern. This increase in cis -9, trans -11 CLA was likely due to the greater production of this isomer in the rumen via microbial biohy drogenation and preferential incorporation of this fatty acid into the caruncle. Similarly, the increase of cis -9, trans -11 CLA in milk fat of cows fed Omega-3 compared to cows fed Omega-6 was due likely to greater production of cis -9, trans -11 CLA in the rumen as well as the conversion of C18:1 trans -11 into cis -9, trans -11 CLA by 9 desaturase in the mammary gl and (Griinari et al., 2000). Incorporation of C18:2 into the caruncle was greater ( P < 0.001) for cows fed Omega-6 (14.87%) or Omega-3 (14.29%) compared to cont rol cows (11.66%) but did not differ between cows fed the fat sources. This was possibly due to partial inhibition of biohydrogenation of C18:2 in the rumen by the presence of C20:5 and C22:6 (AbuGhazaleh and Jenkins, 2004) or greater elongation and desaturati on of C18:2 into C20:4 for cows fed Omega-6 compared to Omega-3-fed cows (10.81 vs. 9.40%; P = 0.02). Concentration of C20: 4 in caruncular tissue was greater for cows not fed fat compared to those fed supplemental fat (11.54, 10.81, and 9.40%, respectively for control, Omega-6, and Omega-3). Caruncles from cows fed Omega-3 had a greater ( P = 0.001) proportion of the omega-3 fatty acids, C18:3 (0.47 vs. 0.37%), C20:5 (1.22 vs. 0.88%), C22:5 (2.49 vs. 1.87%), and C22:6 (1.06 vs. 0.41%) compared to cows fed Omega-6 (Table 6.6). As a result the n6/n3 ratio was lower (P < 0.001) in caruncles from cows fed the Om ega-3 compared to those fed the Omega-6 fat source (4.6 vs. 7.4). Similarly, Bilby et al. (2006c) reported that cows fed a Ca salt mix of palm oil and fish oil had greater incorporat ion of C20:5 (0.10 vs. <0.01%) and C22:6 (1.42 vs. 0.92%) in endometrium of lactating dairy cows. Fat supplementation increased ( P = 0.005) the proportion of PU FA (29.24 vs. 26.86%) in caruncle compared to control cows mainly due to the incorporat ion of PUFA from the dietary

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212 sources. Therefore, the PUFA/MUFA ratio tended to be greater ( P = 0.06) in caruncles from animals fed the fat supplements. The fatty acid profile of caruncles differed somewhat between parities. Compared to multiparous cows, primiparous cows had lowe r concentrations of C16:0 (18.07 vs. 19.52%), trans -9 C18:1 (0.16 vs. 0.20%) and trans -11 C18:1 (0.21 vs. 0.24%), and tended ( P = 0.08) to have a lower concentration of C18:2 (13.13 vs. 14.09%). In contrast, primiparous cows had greater ( P < 0.05) concentrations of trans -10 C18:1 (1.41 vs. 1.23%), C22:5 (2.20 vs. 1.98%), and C22:6 (0.67 vs. 0.58%). Hormones and Metabolites Glucose Concentrations of glucose in plasm a decrease d dramatically from calving to the first week postpartum, then plateaued until 4 wk and slight ly increased until 7 wk postpartum (Figure 6.10). However, treatments did not affect mean plasma concentration of gluc ose (Table 6.7). As expected, multiparous cows had lower ( P < 0.001) concentrations of glucose (66 vs. 73 mg/dl) in plasma compared to primiparous cows (Figure 6.11). Selberg et al. (2004) reported that supplementation of Ca salts of CLA or trans -octadecenoic acid isomers did not affect plasma concentrations of glucose compared to cows fe d no supplemental fat. Similarly, Moallem et al. (2007) reported that supplementation with saturated (Energy Booster 100, Milk Specialties, Dundae, IL) or unsaturated (Megalac-R, Church and Dwight, Princeton, NJ) fat did not affect concentration of plasma gluc ose prepartum (65.4 vs. 66.1 mg/dL) or postpartum (59.7 vs. 59.7 mg/dL). Moussavi et al. (2007) repo rted that cows fed fish meal at 5% of dietary DM or Ca salts of a mix of palm oil and fish oil at 2.3% of dietary DM from 5 to 50 DIM had greater concentration of glucose in plasma (57.6 and 57.3 mg/dL, respectively for fish and Ca salts of fish oil) than cows fed fish meal at 2.5% of dietary DM (51.1 mg/dL) or a control group not fed

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213 fat (53.4 mg/dL) but did not differ from cows fed fish meal at 1.25% of dietary DM (55.3 mg/dL). The authors attributed the increase in plasma glucose to a greater production of propionate in the rumen of cows fed fish oil as reported by others (Wachira et al., 2000 and Fievez et al., 2003). Since propionate is the single most important s ubstrate for gluconeogenesis ( Drackley et al., 2001), fish oil apparently shifts rum inal fermentation by decreasing methanogenesis that conserves energy and yields more propionate (Fievez et al., 2003). These effects seemed to be determined by the amount of the unique PUFA (i.e., EPA and DHA) present in fish oil products rather than simply by the total amount of PUFA fed. Blood urea nitrogen Cows fed supplem ental fat tended ( P = 0.06) to have lower mean concentration (12.9 vs. 13.8 mg/dL) of plasma BUN compared to control co ws (Table 6.7). In contrast, other researchers reported no effect of fat supplementation on plas ma concentrations of BUN (Moussavi et al., 2007; Petit et al., 2007). Feeding fat often has a negative effect on protozoal numbers in the rumen (Onetti et al., 2001). If that happened in the current study, then engulfment of bacteria would be reduced and possibly result in a re duction in ammonia produced in the rumen. The concentration of plasma BUN changed overtime ( P < 0.001; Figure 6.12). Concentrations were high initially, possibly due to the catabolism of tissue protei n for gluconeogenesis, then decreased by wk 1 followed by a gradual increa se over the remaining weeks due to increasing intake of dietary prot ein as DMI increased. Beta hydroxy butyric acid Concentration of plasma BHBA ave raged 5.8 mg/dL across 7 wk postpartum and did not differ among treatments (Table 6.7). Concentrations of BH BA generally decreased with increasing week postpartum (Figure 6.13) reflecting an improving energy balance. As expected, multiparous cows had a greater mean ( P = 0.01) plasma concentration (6.5 vs. 5.0 mg/dL) of

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214 BHBA compared to primiparous cows (Figure 6.14) due to a more negative energy balance. In addition, multiparous cows experienced an initial rise followed by a gradual decrease in plasma concentration of BHBA whereas th at of primiparous cows remained farily constant (parity by week interaction, P = 0.04; Figure 6.14). Nonesterified fatty acid Concentration of plasma NEFA dec lined from calving (807 Eq/L) through 7 wk postpartum (197 Eq/L) (Figure 6.15) and did not differ among treatments (Table 6.7). Moallem et al. (2007) reported no e ffect of saturated or unsaturated fa t supplementation to lactating dairy cows on concentrations of NEFA in plasma (588 vs. 600 Eq/L). Fish meal or Ca salts of palm and fish oil supplemented to multiparous Holste in cows from 5 to 50 DIM did not affect concentration of NEFA in plasma (Moussavi et al., 2007). In contrast, Peti t et al. (2007) reported that multiparous cows fed saturated fat at 1.7 and 3.5% of dietary DM pre and postpartum respectively had greater concentration of NEFA in plasma compared to multiparous cows fed whole flaxseed at 3.3 and 11.0% of dietar y DM during the pre and postpartum period, respectively, but diets had no effect on concentrat ion of NEFA in plasma of primiparous cows (parity by treatment interaction). As expected, multiparous cows mobilized more fat (P < 0.001) than primiparous cows (503 vs. 353 Eq/L) across the 7 wk postpartum (Figure 6.16) as reported by others (Vandehaar et al., 1999; Petit et al., 2007). Progesterone A greater proportion of prim ip arous cows had an estrous cycle during the first 42 DIM when a fat supplement was fed compared to those not fed fat (50 vs. 90%) whereas fat supplements had the opposite effect on multipar ous cows (75 vs. 100%, Table 6.8; parity by control vs. fat interaction; P = 0.02). As a result, the number of cycles followed the same pattern ( P = 0.03). Primiparous cows fed supplemental fa t had more cycles (1.25 vs. 0.7 cycles) during

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215 the 7 wk postpartum compared to those fed a c ontrol diet (Table 6.8) whereas multiparous cows did not differ (1.0 vs. 1.3 cycles; pa rity by control vs. fat interaction, P < 0.03). If an animal did not cycle, she was assigned 42 DIM as the day of first cycle. Animals fed the omega-3 fat source cycled about 6 d earlier than those fed the omega-6 fat source (17.8 vs. 24 DIM, P = 0.05). The pattern of accumulated concentrations of plasma progesteron e supports this earlier return to estrus by omega-3-fed animals (Figure 6 .17). This earlier return may have been due to a better energy status for this group of cows during the first 4 wk pos tpartum (Figure 6.5). Primiparous cows fed the omega-3 fat experienced the most rapid rise in plasma progesterone followed by those fed the omega-6 fat followed by th e control cows. Howeve r the rise in plasma progesterone for multiparous cows was more si milar among treatments although those fed the omega-6 fat appeared to lag behind those fed th e other two treatments (parity by treatment by DIM interaction, P < 0.01; Figure 6.17). Childs et al. (2008) reported that the overall mean concentration of progesterone in plasma of beef primiparous cows fed a diet of 4.15% rumenprotected fish oil was greater dur ing the 16 d of the estrous cycle than that of primiparous cows fed the fish oil at 1.04% of dietary DM. Authors attributed this increase to increased concentration of plasma choleste rol and a larger CL on day 7 of the cycle. An increase in the concentration of cholesterol in plasma has been shown consistently in fat-supplemented cows (Ryan et al., 1992; Hawkins et al ., 1995; Staples et al., 1998). Cholesterol is a precursor for the synthesis of progesterone by ovarian cells (Grummer and Carrol, 1991). Thus, the hypercholesterolemia may increase CL steroidoge nesis which in turn increases progesterone concentrations in plasma. Fat feeding not only increased plasma progesterone concentration but also reduced progesterone clea rance (Hawkins et al., 1995). St aples and Thatcher (2005) summarized the effects of fat supplementation on the size of the dominant follicle and reported

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216 an average increase of 3.2 mm (23%) in the dom inant follicle of cows fed supplemental fat compared to control cows not fed fat. A larger dominant follicle will form a larger CL which in turn synthesizes more progesterone (Vascon celos et al., 1999; Sart ori et al., 2002). Other researchers (Bilby et al., 2006b, Moussavi et al., 2007) reporte d no effect of fish oil supplementation on plasma concentration of proges terone of dairy cows. It is important to emphasize that the differences in the response to fat supplementation are likely due to the proportion of fat in the diet, duration of fat feed ing, time of initiation of fat feeding, stage of lactation, or days of the estrous cycle, all of which could influen ce the uptake of the fatty acids by the tissues as well as their turnover. The remaining 3 measures of progesterone and length of first cycle in Table 6.8 included only those cows that ovulated. Length of the first cycle (16.1, 15.1, and 16.6 d), peak concentration of progesterone in the first cycle (5.9. 5.9, and 6.7 ng/mL), mean concentration of progesterone in the first cycle (3.9, 3.1, and 3.3 ng/mL), and total concentration of progesterone in the first cycle postpartum (23.6, 18.5, and 21.9 ng/mL) did not differ among treatments (Table 6.8). Therefore the CL activity from the first ovulation of cycling cows was not affected by diet. Ultrasonography of ovaries was not done in the cu rrent study and first cycle responses may not be good indicators of later cycle dynamics. Neutrophil Concentration and Function in Whole Blood Peripheral blood neutrophil function of peripartur ient dairy cows is im paired relative to non-parturient cows (Kehrli et al., 1989; Cai et al., 1994). Blood neutrophil function begins to decline prior to parturition, reaches a nadir shor tly after parturition, and returns to prepartum activity by about 4 wk postpartum (Kehrli et al., 1989). Neutrophil phagocyt osis is the initital and most important defense mechanism in the control of bacterial a nd fungal infections.

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217 Parity did not influence any of the immune re sponses measured in this study, nor was the test of parity by treatment interaction significant. Therefore only results of treatment and day of sampling will be reported. The c oncentration of blood neutrophils changed overtime with the greatest concentrations occurr ing at calving (effect of day, P < 0.01; Figure 6.18). The mean concentration of blood neutrophils across days was lower ( P < 0.01) for animals fed the omega-3 fat source compared to those fed the omega-6 fat source (2463 vs. 3462 neutrophils/L of blood) with the greatest difference occurring at 7 DIM (1808 vs. 3620 neutrophils/L of blood, P < 0.01; Figure 6.18). The lack of change in neutrophil concentration between 0 and 7 DIM (3834 vs. 3620 neutrophils/L of blood) for the omega-6 fat-fed group may reflect an immunostimulatory effect. On the other hand, as expected, neutrophil conc entrations decreased from 0 to 7 DIM for both control cows (4604 vs 2762 neutrophils/L of bl ood) and those fed the omega-3 fat source (3046 vs. 1808 neutrophils/L of blood; P < 0.01). The proportion of neutrophils in wh ole blood that phagocytized labeled E. coli (Figure 6.19) was greater ( P = 0.01) at calving (82, 91, 82, and 83% for -18, 0, 7, and 40 DIM) but did not differ among the treatments (85, 86, and 83% respectively for control, Omega-6, and Omega-3). Calder et al. (1990) reported that macrophages enriched with C20:5 and C22:6 had lower phagocytic activity than what would be expected due to the degr ee of unsaturation of the fatty acid. Similar to our results, Ballou and DePeters (2008) fed 51 Jersey bu ll calves (5 1 d of age) milk replacers supplemented with 2% fatty acids having a 3:1 mix of corn and canola oils, a 1:1 mix of fish oil and the 3:1 mi x of corn and canola oils, or fish oil only. Authors reported that fish oil supplementation had no effect on the ability of neutrophils from the calves to phagocytose E. coli. According to Calder (2007), studi es that investigate the number or proportion of phagocytes involved in engulfing the target material are not likely to detect an

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218 effect of PUFA because it is unlikely that su ch manipulation will completely stop phagocytes from engaging in the process of phagocytosis. However, PUFA might affect the phagocytic activity, i.e. the amount of target material engul fed by those cells that are active (Calder, 2007). Indeed, the amount of bacteria that each neutrophil phagocytized as measured by the median fluorescence intensity tended ( P = 0.07) to be lower for Omega-3 compared to Omega-6-fed cows (263 vs. 335) (Figure 6.20) which indicates a lower phagocytic efficiency for neutrophils from cows fed Omega-3. In addition, phagocytic efficiency was greater ( P < 0.01) at calving compared to prepartum or postpartum phagocyt ic efficiencies (266, 355, 282, and 271 for -18, 0, 7, and 40 DIM) (Figure 6.20). The production of reactive oxygen species by the action of NADPH oxidase of neutrophils is a critical mechanism to kill phagocytosed ba cteria, a process called oxi dative burst.The ability of the neutrophils to undergo oxidative burst (F igure 6.21) was greater at calving (day effect; P < 0.01) but did not differ among treatments (85, 88, and 87%, respectively for control, Omega-6, and Omega-3). Supplementing rabbits with a high dose of fish oil decreased neutrophil oxidative burst by approximately 30%; however, a lower dose of fish oil had no influence on superoxide generation ( DAmbola et al., 1991) which corroborates the results of this experim ent in which cows were fed the Ca salt of fatty acids at only 1.5% of dietary DM. In contrast, elderly men supplemented with either a low (1.35 g/d), modera te (2.7 g/d), or high ( 4.05 g/d) dose of EPA had suppressed oxidative burst by neutrophils but no effect was detected in young men (Rees et al., 2006). Bartelt et al. (2008) reported that hea lthy males, aged 18 to 40 years supplemented daily with capsules containing fish oil (166 mg of C20:5 and 119 mg of C22:6) for 8 wk had an immune-stimulating effect on neutrophil oxidative burst compared to subjects supplemented with olive oil. Discrepancies in the effects of the omega-3 fatty aci ds on immune function are

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219 likely due to the methodology used for measuring th e immune status such as isolation of the neutrophils from whole blood prio r to lipid incubation vs. anal ysis in whole blood, amount and duration of lipid supplementation, age related effects, etc. White Blood Cells in Whole Blood As in the case of neutroph il concentration, concentration of WBC in whole blood tended ( P = 0.07) to be lower for Om ega-3 compared to Omega-6-fed cows (8,796 vs. 11,290 WBC/ l) (Figure 6.22). This effect was likely due to chan ges in composition of plasma fatty acids which in turn may influence the producti on of mediators such as leukot rienes and prostaglandins that regulate lymphocyte function (R occa and FitzGerald, 2002). Rectal Temperature Increased rectal tem perature is an indication of infection or inflammation. Mean rectal temperature on any one day was not greater than 38.90 C indicating that cows were mostly healthy. Rectal temperature taken on 4, 7, and 12 DIM did not differ among treatments (Figure 6.23). Uterine Flushing Endom etritis in dairy cows occurs during the postpartum period and is associated primarily with contamination of the reproductive tract involving Arcanobacter pyogenes together with Gram-negative anaerobes (Dhaliwal et al., 2001). The presence of pathogens triggers an innate immune response including neutrophil migration, cytokine release, macrophage, and lymphocyte increases in order to overcome the local infe ction. Concentration of alive WBC (21.8, 4.2, and 1.4 x 1,000 cells/mL, respectively for control, Omega-6, and Omega-3) or dead WBC (1.4, 2.7, and 1.0 x 1,000 cells/mL, respectively for control, Omega-6, and Omega-3) or cell viability (73, 50, and 60%, respectively for control, Omega-6, and Omega-3) in the uterine flushing at 37 3 DIM did not differ among treatments (Table 6.9). In the present e xperiment, the concentration of

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220 neutrophils in the uterine flushing (9.6, 1.9, a nd 0.3 x 1,000 cells/mL, respectively for control, Omega-6, and Omega-3), neutrophil as a proporti on of total cells (13.3, 12.3, and 12.4) or as a proportion of WBC (17.3, 15.6, and 18.8%), a nd proportion of other WBC (58.6, 60.3, and 53.2%) did not differ among treatment s (Table 6.9). The lack of treatment effect was likely due to the high SE associated with the variables. Ho wever, the concentration of neutrophils in the uterine flushing of cows fed fat was numerically lower ( P = 0.21) than for cows fed no fat. Others have reported an immunosuppre ssive effect of dietary fish o il (Calder, 1997; Pizato et al., 2006; Calder, 2007). Thatcher et al (2006) reported that peripart urient dairy cows fed a fat source enriched in C18:2 (28% C18:2) at 2% of dietary DM had a slower decline in concentration of PGFM in plasma and fewer health problems in the first 10 d postpartum compared to cows not fed fat prepartum. The aut hors reported that the uterus and cells of the immune system had greater potential to secrete prostaglandins because of the increase in the supply of linoleic acid to tissues which likely enhanced postpartum uterine health and immunocompetence of the cow. Proliferation of endometrial epithelial cells in vivo occurs in response to estrogens and is inhibited by progesterone (Clark e and Sutherland, 1990). Although the proportion of epithelial cells (22, 28, and 33%) in the uterine flushing did not differ among treatments, primiparous cows had a greater ( P = 0.01) proportion (34.5 vs. 20.6%) of epithe lial cells compared to multiparous cows. This was likely due to a lower ( P < 0.10) concentration of progesterone in plasma compared to multiparous cows (Table 6.9). Cytokines Produced By Lymphocytes One characteristic of inf lammatory responses is the great induction of diverse cytokines (Grinble, 1998). Cytokines are soluble proteins that are releas ed from immune cells (mainly monocytes and macrophages) in response to infec tion, injury, or foreign substances. Liberation

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221 of cytokines is indispensable for the initiation of the immune response and for the regulation of the multidirectional communication between the diffe rent cells involved (Seematter et al., 2004). The main pro-inflammatory cytokines are interle ukin-1 (IL-1b), interleuki n-6 (IL-6), interleukin2 (IL-2), interleukin-8 (IL-8), TNF, and IFN(Grinble, 1998), while the anti-inflammatory ones are IL-1ra, IL-4, IL-10, and IL-13 (Zhang and An, 2007). Lymphocytes were isolated from whole blood at 10, 20, and 30 DIM and stimulated with concanavalin A or not stimulated (control) for cytokine production. The difference between cytokine production of the stimulated and contro l was used for statistical analysis. TNF-alpha secretion by lymphocytes increased over time across treatments (day effect; P < 0.01). Lymphocytes isolated from cows fed supplemen tal fat and stimulated with concanavalin A secreted less ( P < 0.05) TNF(80 vs. 165 pg/mL, Figure 6.24) and IFN(2531 vs. 4313 pg/mL, Figure 6.25) compared to cows not suppl emented with fat. In addition, lymphocytes isolated from primiparous cows secreted less IFNcompared to those from multiparous cows (2230 vs. 3976 pg/mL) (parity effect; P < 0.05). Dietary supplementation with EPA and DHA for 1 to 6 mo in humans diminished (Endres et al., 1989; Me ydani et al. 1991; Caughey et al., 1996) or did not affect (Cooper et al., 1993, Kew et al., 2004) ex vivo production of TNFby peripheral blood mononuclear cells. Si erra et al. (2008) reported th at lymphocytes from mice fed diets enriched in EPA and DHA produced less TNFcompared to mice fed a diet containing 53.8% C18:2. Lessard et al. (2004) fed flaxseed (5.9% of dietary DM), Ca salts of palm oil (2.7% of dietary DM) or micronized soybeans (9.4% of dietary DM) to Holstein cows from 6 wk prepartum until 6 wk postpartum. They reported that stimulated lymphocytes isolated from primiparous cows secreted more TNFcompared to those from multiparous cows but supplemental fat source had no effect on TNFsecretion. Discrepancies among experiments

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222 could be due to difference among species, amount and source of n-3 PU FA added to diets, and physiological state of the animal. Lymphocytes isolated from physically stressed swimmers receiving 2.5 g of fish oil per day (0.9 g of EPA and 0.5 g of DHA) for 6 wk produced less TNFand IFNcompared to those receiving a placebo (mineral oil) (Andrade et al., 2008). Differences in blood composition of n-3 and n-6 PUFA may influence production of mediators such as leukotrienes and prostaglandi ns which are known to help regulate cytokine production and consequently the resp onse of immune cells to stimu li. Fatty acids are precursors of prostaglandins. The n-3 and n-6 fatty acids lea d, respectively, to the synt hesis of series 3 and series 2 prostaglandins (Yaqoob and Calder, 199 5). Moreover, many effects mediated by PUFA on immune cells appear to be exerted in an eicosanoid-independent manner. The n-3 and n-6 PUFA may affect immune cell f unctions by regulating the expre ssion of key genes encoding for molecules involved in the signal transduction pathway such as nuclear transcription factorB and peroxisome proliferatoractivat ed receptors (Calder et al., 2002). Humoral Response The acquired immune response involves lym phocyt es and is highly specific to a certain antigen. Following activation, several days are needed to beco me effective but the response persists after removal of the source of the in itiating antigen. This persistence gives rise to immunological memory which is the basis for a st ronger and more effective immune response to re-exposure to the same antigen. The humoral response deals with ex tracellular pathogens through B lymphocytes which are characterized by their abil ity to produce immunoglobulins specific for an individual antigen (Calder, 2007). As expected, all treatment gr oups had the same IgG concentration (0.18 OD) at -8 wk relative to calving, just before the first ovalbumin injection. Cows were injected (s.c.) with ovalbumin at -8 and -3 wk relative to calving and at parturition. Mean concentration of IgG

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223 across weeks was greater for animals consuming the omega-6 fat source compared to those fed the omega-3 fat source (0.65 vs. 0.49 OD; P < 0.01). This increase was evident at 7 of the 8 times points after initial inject ion (Figure 6.27). After calving, th e ovalbumin response was 49% greater in cows fed Omega-6 compared to cows fed Omega-3. Cows fed Omega-3 had lower humoral response as measured by antibody secreti on against ovalbumin challenge compared to cows fed Omega-6. According to SLICE analysis, treatments differed at wk 1 ( P = 0.05), 2 ( P = 0.07), and 4 ( P < 0.01) postpartum with cows fed th e omega-6 fat source having greater concentrations than cows fed the other 2 treatment s. Lessard et al. (2003) fed primiparous (n = 8) and multiparous (n = 22) Holstein cows whole fl axseed (10.4% of dietary DM), Megalac (3.8% of dietary DM), or micronized soybean (17.7% of dietary DM) from calving to 105 DIM. At insemination (between 60 and 72 DIM), cows were injected with ovalbumin and serum samples were taken at 0, 10, 20, and 40 d post AI for anal ysis of immunoglobulin response to ovalbumin. Diet did not affect this humoral response. In another study to evaluate the effect of fat feeding during the prepartum period on humoral response, Lessard et al. (2004) injected cows with ovalbumin at 6 and 3 wk before parturition. They reported that multiparous cows fed micronized soybeans at 9.4% of dietary DM from 6 wk prior to calving until parturition had greater IgG concentration in colostrum compared to cows fed Megalac (2.7% of dietary DM) or flaxseed (5.9% of dietary DM) but there was no effect of diet on antibody secreti on against ovalbumin in serum. Vaginoscopy In dairy cows, uterine function is often co m promised by bacterial contamination of the uterine lumen after parturition. Pathogenic bacteria frequently persist, cau sing uterine disease, a key cause of infertility (Sheldon and Dobson, 2004) Bacteria can be cultured from samples collected from the uterine lumen of most dairy cattle in the firs t 2 wk after parturition in many

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224 situations. Although many cows eliminate these bacter ia during the first 5 wk after parturition, in 10 to 17% of animals, persistence of bacteria l infection causes uterin e disease detectable by physical examination (Le Blanc et al., 2002). The pr esence of pathogenic bacteria in the uterus causes inflammation, histological lesions of the endometrium, delays uterine involution, and perturbs embryo survival (Sheldon et al., 2006). Thus, uterine dis ease is associated with lower conception rates, increased interv als from calving to first servic e or conception, and more cattle culled for failure to conceive. During parturition, eicosanoids are produced in substantial quantities and play an important role in the regulation and control of parturition, and expulsi on of the placenta and uterine contents through opening of the cervix an d contractions of the uterus (Santos et al., 2008). Prostaglandin F2 is an important eicosanoid involved in the regulation of CL lifespan and likely influences retention of fe tal membranes and consequently uterine health. Arachidonic acid (C20:4, omega-6) is the precursor of the potent pr ostaglandin PGF2 The more C20:4 in the endometrial tissue available for ei cosanoid synthesis, the more PGF2 is likely to be secreted, which in turn may influence uterine health. Fat supplementation did not a ffect the incidence of metritis as measured by the vaginoscopy scores (83.9, 84.4, and 83.3%, respectively for control, Omega-6, and Omega-3; Table 6.10). However, the severity of metritis was lower (63.0 vs. 92%) for cows fed Omega-6 compared to cows fed Omega-3. In the present experiment, cows fed Omega-6 had a greater concentration of C20:4 in caruncular tissue compared to cows fed Omega-3 (Table 6.6). Even though the concentrations of PGF2 metabolite (PGFM) in plasma did not differ among treatments, cows fed Omeg a-6 had better uterine health and immunecompetence compared to cows fed Omega-3. Similarly, Cullens et al. (2004) reported that cows supplemented with Ca salts of PUFA rich in omega-6 fatty acids starting at 4 wk prepartum had

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225 reduced incidence of postpartum diseases includi ng retained placenta, metritis and mastitis (8.3 vs. 42.9%) compared with cows not fed fat prepar tum. Juchem (2007) reported that cows fed diets of 2% Ca salts of long chai n fatty acids of either palm o il or a blend of C18:2 prepartum had reduced odds of puerperal metritis (8.8 vs 15.1%) although the in cidence of retained placenta did not differ between tr eatments and averaged 6.6%. Conclusions Supplem enting periparturient dairy cows with diets enriched in omega-6 or omega-3 fatty acids affected postpartum performance as well as endocrine and immunological variables. Cows fed the omega-3 fat source tended to consume le ss DM (% of BW) and produce less milk fat due to a depression in milk fat concentration when compared to cows fed the omega-6 fat source. This reduction in milk fat likely was due to increased production of trans fatty acids in the rumen as reflected by increased concentration of trans fatty acids in milk fat. Milk and caruncular fatty acids reflected the increase d feeding of omega-6 and omega-3 fat sources. Based upon concentrations of plasma progesterone, animals fed the omega-3 fat source cycled about 6 d earlier than those fed the omega-6 fat sour ce. The Omega-3 enriched dietary fat had immunosuppressive effects, namely lowering th e concentration of bl ood neutrophils and WBC postpartum, decreasing the intens ity of neutrophil action against E. coli in blood, and decreasing the production of cytokines by isolated and in vitro stimulated lymphocytes. The omega-6 enriched fat had immunostimulator y effects, namely preventing the decrease in concentration of blood neutrophils at 7 DIM that otherwise occurred in the other treatments and stimulating the humoral response (IgG) postpartum to ovalbumin injections. It also appeared to have immunosuppressive effects on production of cytoki nes by stimulated lymphocytes, similar to that of cows fed the omega-3 fat source.

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226 Table 6-1. Ingredient and chemical compositi on of TMR fed to Holstein cows during the prepartum period. Treatments Control Omega-6 Omega-3 Ingredient (% of dietary DM) Corn silage 32.0 31.2 31.2 Bermudagrass hay 11.5 18.6 18.6 Ground corn 11.4 10.0 10.0 Citrus pulp 12.7 6.5 6.5 Soybean meal 13.5 13.8 13.8 Soy Plus1 2.4 1.7 1.7 Cottonseed hulls 10.1 10.4 10.4 Mineral and vitamin premix2 6.4 6.3 6.3 EnerG HL3 1.5 StrataG 0.54 1.5 Component NEL, Mcal/kg of DM 1.61 1.62 1.63 CP, % of DM 14.9 14.9 14.9 NDF, % of DM 37.0 38.1 37.5 ADF, % of DM 24.0 24.7 23.5 Ether extract, % DM 4.63 5.50 5.41 Ca, % of DM 2.2 2.31 2.31 P, % of DM 0.34 0.34 0.36 Mg, % of DM 0.33 0.33 0.35 K, % of DM 1.67 1.72 1.74 Na, % of DM 0.17 0.17 0.19 S, % of DM 0.53 0.52 0.52 Cl, % of DM 0.87 0.89 0.92 Fe, mg/kg of DM 222 245 264 Zn, mg/kg of DM 46 49 45 Cu, mg/kg of DM 21 24 24 Mn, mg/kg of DM 40 46 47 1 West Central Soy, Ralston, IA. 2 Mineral and vitamin premix contained 22.8% CP, 22.9% Ca, 0. 20% P, 0.2% K, 2.8% Mg, 0.7% Na, 2.4% S, 8% Cl, 147 mg/kg of Mn, 27 mg/kg of Fe, 112 mg/kg of Cu, 95 mg/kg of Zn, 7 mg/kg of Se, 8 mg/kg of I, 11 mg/kg of Co, 268,130 IU of vitamin A/kg, 40,000 IU of vitamin D/kg, and 1129 IU of vitamin E/kg (DM basis). 3 Calcium salts of fatty acids made from saff lower oil (Virtus Nutrition, Corcoran, CA). 4 Calcium salts of fatty acids made from palm oil and fish oil (Virtus Nutrition, Corcoran, CA).

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227 Table 6-2. Ingredient and chemical compositi on of TMR fed to Holstein cows during the postpartum period. Treatments Control Omega-6 Omega-3 Ingredient (% of dietary DM) Corn silage 38.0 38.0 38.0 Alfalfa hay 12.0 12.0 12.0 Ground corn 22.4 20.6 20.6 Citrus pulp 5.0 5.0 5.0 Soybean meal 9.0 9.1 9.1 Soyplus1 9.0 9.2 9.2 Mineral and vitamin mix2 4.6 4.6 4.6 EnerG HL3 1.5 StrataG 0.54 1.5 Component NEL, Mcal/kg of DM 1.77 1.83 1.83 CP, % of DM 18.1 18.4 18.7 NDF, % of DM 26.7 26.8 26.6 ADF, % of DM 17.3 17.6 17.4 Ether extract, % DM 4.52 5.80 5.75 Ca, % of DM 1.23 1.29 1.32 P, % of DM 0.48 0.39 0.39 Mg, % of DM 0.30 0.34 0.36 K, % of DM 1.72 1.88 1.85 Na, % of DM 0.46 0.49 0.52 S, % of DM 0.24 0.22 0.23 Cl, % of DM 0.42 0.35 0.41 Fe, mg/kg of DM 242 199 213 Zn, mg/kg of DM 122 116 129 Cu, mg/kg of DM 39 38 45 Mn, mg/kg of DM 90 96 129 1 West Central Soy, Ralston, IA. 2Mineral and vitamin mix contai ned 26.4% CP, 10.2% Ca, 0.90% P, 3.1% Mg, 1.5 % S, 5.1% K, 8.6 % Na, 1500 mg/kg of Zn, 512 mg/kg of Cu, 339 mg/kg of Fe, 2231 mg/kg of Mn, 31 mg/kg of Co, 26 mg/kg of I, 7.9 mg/kg of Se, 147,756 IU of vitamin A/kg, and 787 IU of vitamin E/kg (DM basis). 3 Calcium salts of fatty acids made from saff lower oil (Virtus Nutrition, Corcoran, CA). 4 Calcium salts of fatty acids made from palm oil and fish oil (Virtus Nutrition, Corcoran, CA).

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228 Table 6-3. Fatty acid profile of the fat supplements (% of identified fatty acids). Fat supplements1 Omega-6 Omega-3 C14:0 1.01 4.53 C16:0 12.92 38.67 cis -9 C16:1 0.13 0.03 C17:0 0.11 0.32 C18:0 4.16 4.63 cis -9 C18:1 16.70 30.82 C18:2 n-6 63.62 8.01 C18:3 0.20 0.95 C20:4 ND2 0.40 C20:5 0.61 5.40 C22:5 ND 0.90 C22:6 0.36 5.26 1 Omega-6 = Calcium salts of fatty acids made from saffl ower oil (EnerG HL, Virtus Nutrition, Corcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 ND= non detected.

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229Table 6-4. Dry matter intake, milk yiel d, milk composition, feed efficiency, postp artum body weight, and postpartum body condi tion score of Holstein cows fed control diet (CO) and diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Treatme nt by parity by time interaction was not significant ( P < 0.05) for any dependent variable. Treatments1 Contrasts 2, P values Measure Control Omega-6 Omega-3 SE Parity Week Treatment*Week A B C D P3 M4 P M P M Dry matter intake, kg/d Prepartum 10.90 15.90 11.24 16.18 11.85 15.21 0.90 <0.001 <0.001 0.77 0.78 0.84 0.60 0.38 Postpartum 15.15 20.86 16.34 21.00 14.49 19.29 1.07 <0.001 <0.001 0.69 0.80 0.13 0.59 0.95 Dry matter intake, % BW Postpartum 2.92 3.05 3.14 3.29 2.92 2.94 0.15 0.43 <0.001 0.72 0.50 0.09 0.88 0.71 Milk yield, kg/d 26.87 38.73 27.18 41.68 24.16 38.41 2.12 <0.001 <0.001 0.87 0.97 0.17 0.48 0.96 3.5% FCM5, kg/d 27.06 40.62 26.20 41.90 22.57 37.32 2.57 <0.0001 <0.0001 0.29 0.41 0.13 0.71 0.86 3.5% FPCM6, kg/d 26.65 39.39 25.80 40.82 22.39 36.80 2.37 <0.0001 <0.0001 0.29 0.45 0.13 0.63 0.90 ECM7, kg/d 26.94 39.81 26.08 41.26 22.63 37.20 2.4 <0.0001 <0.0001 0.29 0.45 0.13 0.63 0.90 Milk fat, % 3.55 3.83 3.34 3.70 3.01 3.39 0.15 <0.01 <0.001 0.02 0.02 0.06 0.76 0.93 Milk protein, % 3.09 2.95 2.94 2.96 2.90 2.98 0.08 0.85 <0.001 0.90 0.33 0.93 0.19 0.77 Milk fat yield, kg/d 0.95 1.47 0.89 1.49 0.73 1.28 0.11 <0.0001 <0.0001 0.20 0.22 0.10 0.78 0.81 Milk protein yield, kg/d 0.80 1.12 0.79 1.19 0.70 1.13 0.07 <0.0001 <0.0001 0.50 0.81 0.30 0.41 0.88 Milk SCC score 1.98 0.16 0.26 0.48 2.25 0.32 0.03 0.11 <0.001 0.76 0.98 0.37 0.27 0.20 Feed efficiency8 1.92 1.96 1.70 2.05 1.58 1.99 0.12 <0.01 <0.001 0.80 0.21 0.46 0.06 0.79 BW, kg 525 681 522 664 512 673 25 <0.001 <0.001 0.46 0.65 1.00 0.92 0.72 BCS 2.94 3.31 3.34 3.17 3.00 3.29 0.14 0.15 <0.001 0.19 0.51 0.47 0.21 0.14 1 Control diet = no supplemental fat; Omega-6 = Calcium salts of fatty acids made from safflower oil (EnerG HL, Virtus Nutrition Corcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 Orthogonal contrast of means were the following: A = Control vs. fat (Omega-6 + Omega-3), B = Omega-6 vs. Omega-3, C = contras t A by parity interaction, and D = contrast B by parity interaction. 3 Primiparous cows. 4 Multiparous cows. 5 3.5% FCM = (0.4324*milk yield) + (16.216*milk fat yield). 6 3.5% fat and protein corrected milk = (12.82 kg of fat) + (7.13 kg of protein) + (0.323 kg of milk).

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2307 Energy corrected milk = (0.327 milk kg) + (12.95 kg of fat) + (7.20 kg of protein). Tyrrel and Reid, 1965. 8 Feed efficiency = kg of 3.5% FCM / kg of DMI.

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231 Table 6-5. Effect of supplemental fat source on concentration of identified fatty acids of milk fat. Fatty acid Treatments1 Control Omega-6 Omega-3 S.E. Contrasts2, P values ------% of identified fatty acids----A B C D C4:0 1.64 1.64 1.52 0.08 0.58 0.33 0.56 0.41 C6:0 2.02 1.81 1.59 0.10 0.01 0.11 0.74 0.67 C8:0 1.49 1.26 1.12 0.07 <0.001 0.15 0.68 0.60 C10:0 3.40 2.70 2.44 0.17 <0.001 0.29 0.34 0.70 C12:0 3.82 3.00 2.78 0.18 <0.001 0.39 0.19 0.76 C14:0 12.12 10.88 10.74 0.36 <0.001 0.79 0.03 0.31 C15:0 1.07 0.96 0.98 0.04 0.06 0.83 0.01 0.49 C16:0 31.10 28.03 29.13 0.49 <0.001 0.13 0.02 0.42 cis -9 C16:1 0.43 0.54 0.70 0.03 <0.001 <0.001 0.19 0.93 C17:0 0.77 0.70 0.83 0.02 0.89 <0.001 0.88 0.99 C18:0 12.07 13.10 10.49 0.41 0.59 <0.001 0.76 0.38 trans 6 to 8 C18:1 0.019 0.032 0.044 0.003 <0.001 0.01 0.43 0.99 trans -9 C18:1 0.32 0.70 0.80 0.04 <0.001 0.10 0.46 0.40 trans -10 C18:1 2.34 6.18 7.92 0.53 <0.001 0.03 0.92 0.62 trans -11 C18:1 1.23 2.35 2.29 0.10 <0.001 0.69 0.67 0.80 cis -9 C18:1 21.38 20.46 20.67 1.03 0.51 0.89 0.11 0.35 C18:2 n-6 3.50 3.94 3.72 0.11 0.01 0.17 <0.001 0.94 CLA cis-9, trans-11 0.42 0.86 1.13 0.06 <0.001 0.01 0.04 0.93 trans-10,cis-12 0.029 0.036 0.065 0.004 <0.001 <0.001 0.34 0.09 C18:3 n-3 0.41 0.39 0.43 0.02 0.95 0.09 0.04 0.87 C20:0 0.16 0.18 0.20 0.01 0.01 0.19 0.62 0.19 C20:4 n-6 0.19 0.16 0.12 0.01 <0.001 <0.001 0.03 0.57 C20:5 n-3 0.023 0.022 0.088 0.005 <0.001 <0.001 0.68 0.94 C22:5 n-3 0.044 0.050 0.103 0.007 <0.001 <0.001 0.63 0.22 C22:6 n-3 0.001 0.001 0.094 0.006 <0.001 <0.001 0.66 0.78 MUFA3 25.73 30.27 32.42 1.06 <0.001 0.16 0.11 0.50 PUFA4 4.62 5.45 5.75 0.14 <0.001 0.16 <0.001 0.95 PUFA/MUFA 0.18 0.18 0.18 0.01 0.96 0.84 0.21 0.63 n6/n3 ratio5 8.81 10.90 7.18 0.30 0.54 <0.001 0.16 0.45 Desaturase index (di)6 di 181 0.64 0.61 0.66 0.01 0.73 0.01 0.20 0.83 di 161 0.014 0.019 0.023 0.001 <0.001 0.012 0.11 0.87 1 Control diet = no supplemental fat; Omega-6 = Calcium salts of fatty acids made from safflower oil (EnerG HL, Virtus Nutrition, Corcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 Orthogonal contrast of means were the following: A = Control vs. fat (Omega-6 + Omega-3), B = Omega-6 vs. Omega-3, C = contrast A by parity, and D = contrast B by parity. 3 MUFA = cis -9 C16:1 + C18:1 trans family + cis -9 C18:1. 4 PUFA = C18:2 + C18:3 + cis -9, trans -11 CLA + trans -10, cis -12 CLA + C20:3 + C20:4 + C20:5 + C22:6. 5 n-6/n-3 ratio = (C18:2 + cis -9, trans -11 CLA + trans -10, cis -12 CLA + C20:4) / (C18:3 + C20:3 + C20:5 + C22:6). 6 Desaturase indexes are ratios of the 9-desaturase product divided by the sum of the 9-desaturase product and substrate. For example, the desaturase index for cis -9 C16:1 would be ( cis -9 C16:1)/( cis -9 C16:1 + C16:0).

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232Table 6-6. Effect of supplemental fat source on co ncentration of identified fatty acids of caruncle. Fatty acid Treatments1 Control Omega-6 Omega-3 S.E. Contrasts2, P values ------% of identified fatty acids----A B C D C14:0 0.96 1.55 0.94 0.28 0.41 0.14 0.41 0.05 C15:0 2.04 1.79 1.84 0.08 0.04 0.65 0.48 0.12 C16:0 19.76 17.91 18.71 0.35 0.01 0.12 0.01 0.07 cis -9 C16:1 0.47 0.50 0.57 0.02 0.03 0.04 0.55 0.93 C17:0 1.64 1.56 1.71 0.04 0.92 0.02 0.80 0.64 C18:0 26.24 26.07 24.59 0.70 0.29 0.15 0.20 0.10 trans -9 C18:1 0.14 0.16 0.24 0.01 0.01 <0.001 0.05 0.27 trans10 C18:1 0.61 1.34 2.02 0.08 <0.001 <0.001 0.86 0.66 trans11 C18:1 0.11 0.22 0.34 0.01 <0.001 <0.001 0.07 0.28 cis -9 C18:1 20.39 18.69 19.05 0.50 0.02 0.62 0.95 0.20 C18:2 n-6 11.66 14.87 14.29 0.46 <0.001 0.39 0.97 0.64 CLA cis -9, trans -11 0.09 0.13 0.17 0.01 <0.001 0.01 0.80 0.72 trans -10, c is -12 0.024 0.022 0.030 0.004 0.75 0.18 0.03 0.68 C18:3 n-3 0.39 0.37 0.47 0.01 0.15 <0.001 0.37 0.88 C20:0 0.78 0.84 0.85 0.03 0.07 0.70 0.09 0.68 C20:4 n-6 11.54 10.81 9.40 0.41 0.01 0.02 0.70 0.45 C20:5 n-3 0.86 0.88 1.22 0.07 0.03 0.001 0.29 0.15 C22:5 n-3 1.91 1.87 2.49 0.06 <0.001 <0.001 0.65 0.42 C22:6 n-3 0.39 0.41 1.06 0.04 <0.001 <0.001 0.39 0.03 MUFA3 21.71 20.92 22.21 0.52 0.82 0.09 0.97 0.19 PUFA4 26.86 29.36 29.13 0.66 0.01 0.81 0.82 0.37 PUFA/MUFA 1.24 1.42 1.33 0.06 0.06 0.23 0.85 0.18 n6/n3 ratio5 6.63 7.41 4.62 0.25 0.05 <0.001 1.00 0.37 Desaturase index (di)6 di 181 0.44 0.42 0.44 0.01 0.59 0.25 0.50 0.11 di 161 0.023 0.027 0.030 0.001 <0.001 0.14 0.07 0.37 1 Control diet = no supplemental fat; Omega-6 = Calcium salts of fatty acids made from safflower oil (EnerG HL, Virtus Nutrition Corcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 Orthogonal contrast of means were the following: A = Control vs. fat (Omega-6 + Omega-3), B = Omega-6 vs. Omega-3, C = contras t A by parity, and D = contrast B by parity.

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2333 MUFA = cis -9 C16:1 + C18:1 trans family + cis -9 C18:1. 4 PUFA = C18:2 + C18:3 + cis -9, trans -11 CLA + trans -10, cis-12 CLA + C20:3 + C20:4 + C20:5 + C22:6. 5 n-6/n-3 ratio = (C18:2 + cis -9, trans -11 CLA + trans -10, cis -12 CLA + C20:4) / (C18:3 + C20:3 + C20:5 + C22:6). 6 Desaturase indexes are ratios of the 9-desaturase product divided by the sum of the 9-desaturase product and substrate. For example, the desaturase index for cis -9 C16:1 would be ( cis -9 C16:1)/( cis -9 C16:1 + C16:0).

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234Table 6-7. Concentration of plasma metabolites from calving to 49 DIM of Holstein cows fed control diet (CO) and diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prep artum to 7 wk postpartum. Treatment by parity by time in teraction was not significant ( P < 0.05) for any dependent variable. Treatments1 Contrasts 2, P value Measure Control Omega-6 Omega-3 SE Parity Time Treatment*Time interaction A B C D P3 M4 P M P M Glucose, mg/dl 72 66 74 65 73 66 3 0.001 <0.001 1.00 0.73 0.99 0.70 0.81 Blood urea nitrogen, mg/dl 13.3 14.2 12.8 13.2 12.6 12.9 0.5 0.24 <0.001 0.05 0.06 0.63 0.59 0.85 BHBA, mg/dL 5.3 6.1 4.4 7.1 5.4 6.4 0.7 0.01 0.11 0.94 0.89 0.83 0.40 0.24 NEFA, meq/L 412 506 320 462 326 539 54 0.001 <0.001 0.78 0.32 0.45 0.38 0.52 1 Control diet = no supplemental fat; Omega-6 = Calcium salts of fatty acids made from safflower oil (EnerG HL, Virtus Nutrition Corcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 Orthogonal contrasts of means were the following: A = Control vs. fat (Omega-6 + Omega-3), B = Omega-6 vs. Omega-3, C = contra st A by parity, and D = contrast B by parity. 3 Primiparous cows. 4 Multiparous cows

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235Table 6-8. Cyclicity, interval to first ovulat ion, number of cycles, and progesterone concentrations during the first cycle of Holstein cows fed control diet (CO) a nd diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Treatments1 Contrasts2, P values Measure Control Omega-6 Omega-3 SE Parity A B C D P3 M4 P M P M Cyclicity (# of animals cycling/ total # of animals) 0.5 (3/6) 1.0 (9/9) 1.0 (5/5) 0.7 (8/11) 0.8 (4/5) 0.8 (7/9) 0.1 0.58 0.56 0.63 0.02 0.42 Number of cycles 0.7 1.3 1.3 1.0 1.2 1.0 0.2 0.77 0.53 0.83 0.03 0.83 DIM at first ovulation5 25.0 23.1 25.6 22.4 16.5 19.1 3.0 0.75 0.27 0.05 0.78 0.33 Peak progesterone in the first cycle, ng/ml 5.3 6.1 5.4 6.4 6.7 6.7 1.2 0.56 0.62 0.53 0.88 0.67 First cycle length, d 13.3 19.0 14.2 16.1 14.3 19.0 2.7 0.07 0.92 0.58 0.62 0.60 Mean progesterone concentration in the first cycle, ng/ml 3.7 3.8 2.7 3.5 2.5 4.1 0.5 0.09 0.30 0.78 0.31 0.53 Total progesterone concentration of first cycle, ng/ml 21.2 24.0 16.2 20.9 15.4 28.5 5.6 0.16 0.64 0.56 0.55 0.47 1 Control diet = no supplemental fat; Omega-6 = Calcium salts of fatty acids made from safflower oil (EnerG HL, Virtus Nutrition Corcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 Orthogonal contrasts of means were the following: A = Control vs. fat (Omega-6 + Omega-3), B = Omega-6 vs. Omega-3, C = contra st A by parity, and D = contrast B by parity. 3 Primiparous cows. 4 Multiparous cows. 5 DIM at first ovulation was determined as th e average of the 2 sampling d in which conc entrations of progesterone were consecuti vely greater than 1 ng/ml.

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236Table 6-9. Uterine cytology (37 3) of Holstein cows fed control diet and diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Treatments1 Contrasts2, P value Measure Control Omega-6 Omega-3 SE Parity A B C D P3 M4 P M P M Alive white blood cells (x1,000/mL) 11.6 41.1 1.4 12.2 2.8 0.7 20 0.73 0.33 0.99 0.42 0.90 Dead white blood cells (x1,000/mL) 0.3 6.1 6.0 1.2 1.2 0.7 3.5 0.97 0.82 0.60 0.41 0.65 Cell viability (%) 80.2 67.0 52.8 48.3 62.8 57. 6 12.6 0.92 0.11 0.34 0.80 0.30 Neutrophil concentration (x1,000/mL) 4.0 23.2 0.5 7.8 0.8 0.1 11.6 0.84 0.21 0.93 0.33 0.62 Other white blood cells (%) 57.6 59.7 56.2 64.4 48.6 57.8 7.6 0.31 0.78 0.36 0.62 0.95 Epithelial cells (%) 33.6 10.4 33.6 21.6 36.2 29.7 6.4 0.01 0.15 0.41 0.22 0.68 Neutrophil (% of total cells) 8.4 21.2 10.6 14.3 17.0 9.0 5.3 0.64 0.95 0.91 0.14 0.16 Neutrophil (% of white blood cells) 13.2 22.8 13.5 18.1 27.7 12.8 6.6 0.88 0.96 0.96 0.18 0.19 1 Control diet = no supplemental fat; Omega-6 = Calcium salts of fatty acids made from safflower oil (EnerG HL, Virtus Nutrition Corcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 Orthogonal contrasts of means were the following: A = Control vs. fat (Omega-6 + Omega-3), B = Omega-6 vs. Omega-3, C = contra st A by parity, and D = contrast B by parity. 3 Primiparous cows. 4 Multiparous cows.

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237Table 6-10. Incidence and severity of metritis as measured by vaginoscopy scores at 5 and 10 DIM of Holstein cows fed control d iet (CO) and diets supplemented with omega-6 (Omega-6) or omeg a-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Treatments1AOR (95% CI)2 Control Omega-6 Omega-3 Control vs. Fat Omega-6 vs. Omega-3 -------------------------% (no./no.)------------------------Incidence3 83.9 (26/31) 84.4 (27/32) 83.3 (25/30) 0.94 (0.06, 17.74) 0.78 (0.30, 5.03) Severity 4,5 76.9 (20/26) 63.0 b (17/27) 92.0a (23/25) 0.64 (0.05, 7.67) 0.15 (0.03, 0.78) 1 Control = no supplemental fat; Omega-6 = Calcium salts of fatty acids made from safflower oil (EnerG HL, Virtus Nutrition, Co rcoran, CA), and Omega-3 = Calcium salts of fatty acids made from palm oil and fish oil (StrataG 0.5, Virtus Nutrition, Corcoran, CA). 2 AOR = adjusted odds ratio; CI = confidence interval. 3 Vaginoscopy scores 2 and 3 were considered infected compared to scores 0 and 1 (not infected). 4 Vaginoscopy score 3 was considered severe compared to score 2. 5 The incidence of puerperal metritis (metritis associated with rectal temperature 39.5oC within the first 12 DIM) was 7% (1/15), 0% (0/16), and 0% (0/14) for Control, Omega-6, and Om ega-3, respectively. a,b Superscripts in the same row differ (P < 0.05).

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238 Figure 6-1. Least squares means for dry matter intake of primiparous (A) and multiparous (B) Holsteins cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Parities differed (P < 0.001).

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239 Figure 6-2. Least squares means for milk yield of primiparous (A) and multiparous (B) Holsteins cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Parities differed (P < 0.001).

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240 Figure 6-3. Least squares means for 3.5% fat corrected milk (FCM) by Holsteins cows fed control diet (no fat supplement) or diet s supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Cows fed Omega-6 tended (P = 0.13) to have greater produc tion of 3.5% FCM compared to cows fed Omega-3.

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241 Figure 6-4. Least squares means fo r concentration of fat in milk of Holsteins cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. The asterisks indicate that cows fed Omega-6 had greater (P < 0.05) concentration of fat in milk at wk 1, 2, and 3 postpartum compared to cows fed Omega-3 (Omega-6 vs. Omega-3 by week interaction; P < 0.05).

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242 Figure 6-5. Least squares means for energy balance of primiparous (A) and multiparous (B) Holsteins cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Parities differed ( P < 0.001).

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243 Figure 6-6. Least squares means for feed efficien cy of Holsteins cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Effect of treatment was not significant.

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244 Figure 6-7. Least squares means for body weight of Holsteins cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Effect of treatment was not significant.

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245 Figure 6-8. Least squares means for body weight of Holsteins primiparous (open triangle) and multiparous (closed square) cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or om ega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Parities differed (P < 0.01).

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246 Figure 6-9. Least squares means for body condition sc ore of Holsteins cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega3) fatty acids from 4 wk prepartum to 7 wk postpartum. Effect of treatment was not significant.

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247 Figure 6-10. Least squares means for concentra tion of plasma glucose of Holstein cows fed control diet (no fat supplement) or diet s supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk pr epartum to 7 wk postp artum. Effect of treatment was not significant.

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248 Figure 6-11. Least squares means for plasma gluc ose of Holstein cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Parities differed (P < 0.001).

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249 Figure 6-12. Least squares means for plasma BU N of Holstein cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Effect of treatment was not significant.

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250 Figure 6-13. Least squares means for plasma BHBA of Holstein cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Effect of treatment was not significant.

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251 Figure 6-14. Least squares means for plasma BH BA of Holstein primiparous (open diamond) or multiparous (closed square) cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or om ega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Parities differed ( P < 0.05). Parity by week differed ( P = 0.04).

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252 Figure 6-15. Least squares means for plasma NEFA of Holstein cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Effect of treatment was not significant.

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253 Figure 6-16. Least squares means for plasma NEFA of Holstein primiparous (open triangle) or multiparous (closed square) cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or om ega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Parities differed ( P < 0.001).

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254 Figure 6-17. Least squares means for acucumu lated progesterone in plasma of Holstein primiparous (A) or multiparous (B) cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or om ega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Primiparous cows fed Omega-3 started had greater concentration of accumulated progesterone earlier than primparous cows fed Omega6 (treatment by parity by DIM interaction; P < 0.01). Anestrus cows were included in the data.

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255 Figure 6-18. Least squares means for concentration of neutrophils in blood of Holstein cows at 18, 0, 7, and 40 DIM fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Cows fed omega-3 had lower concentration of neutrophils in blood compared to cows fed omega-6 (P < 0.01) Concentration of neutrophil decreased between 0 and 7 DIM for control cows a nd those fed Omega-3 but were unchanged for cows fed Omega-6 ( P < 0.05).

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256 Figure 6-19. Least squares means for percentage of neutrophils that carri ed out phagocytosis of E. coli in vitro in blood of Holstein cows fe d control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or om ega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Treatments di d not differ (P > 0.05). Effect of day was significant (P < 0.01).

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257 Figure 6-20. Least squares means for median fl uorescence intensity of ne utrophils in blood of Holstein cows fed control diet (no fat s upplement) or diets supplemented with omega6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Median fluores cence intensity was measured at -18, 0, 7 and 40 days relative to calving. Neutrophils from cows fed omega-3 tended ( P = 0.07) to phagocytize less bacteria compared to cows fed the omega-6 fat source. Effect of day was significant ( P < 0.01).

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258 Figure 6-21. Least squares means for oxidative bu rst of neutrophils in bl ood of Holstein cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Oxidative burst was measured at -18, 0, 7 a nd 40 days relative to calving. Effect of treatment was not significant. Effect of day was significant ( P < 0.01).

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259 Figure 6-22. Least squares means for concentra tion of white blood cells in blood of Holstein cows fed control diet (no fat supplemen t) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. White blood cells were measur ed at -18, 0, 7 and 40 days relative to calving. Cows fed omega-3 tended ( P = 0.07) to have lower con centrations of white blood cells compared to cows fed omega6. Effect of day was not significant (P > 0.05).

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260 Figure 6-23. Least squares means for rectal temperature of Holstein primiparous (A) and multiparous (B) cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum. Treatments did not differ ( P > 0.05).

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261 Figure 6-24. Least squares means for producti on of tumor necrosis factor alpha (TNF) produced by lymphocytes isolated at 10, 20, and 30 DIM from blood of Holstein cows fed control diet (no fat supplemen t) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum and stimulated in vitro with concanavalin A or not stimulated. TNF-alpha concentration is expressed as the difference in the concentration of TNF-alpha of the stimulated cells and not stimulated cells Lymphocytes from cows fed supplemental fats had reduced secretion of TNFcompared to cows fed control diet (P < 0.05). Effect of day was significant ( P < 0.01).

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262 Figure 6-25. Least squares means fo r production of interferon gamma (IFN) produced by lymphocytes isolated at 10, 20, and 30 DIM from blood of Holstein cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum and stimulated in vitro with concanavalin A. Lymphocytes fr om cows fed supplemental fats had reduced secretion of IFNcompared to cows fed control (P < 0.05). Treatment by DIM interaction was significant (P < 0.05).

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263 Figure 6-26. Least squares means fo r production of interferon gamma (IFN) produced by lymphocytes isolated at 10, 20, and 30 DIM from blood of Holstein cows fed control diet (no fat supplement) or diets supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum and stimulated in vitro with concanavalin A. Parities differed ( P < 0.05).

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264 Figure 6-27. Least squares means for production of immunoglobulin G by Holstein cows fed control diet (no fat supplement) or diet s supplemented with omega-6 (Omega-6) or omega-3 (Omega-3) fatty acids from 4 wk prepartum to 7 wk postpartum and challenged with injections of ovalbumin at -8, -3, and 0 wk relative to calving. Cows fed Omega-6 had greater response to oval bumin challenge compared to cows fed Omega-3 (P < 0.05). The asterisks indicat e that omega-6 fed cows had greater response to ovalbumin challenge at wk 1 ( P = 0.05), 2 ( P = 0.07), and 4 ( P < 0.01) postpartum compared to those fed the other 2 treatments according to SLICE analysis.

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265 CHAPTER 7 GENERAL DISCUSSION AND CONCLUSION Lipid by definition is any of various substances that are soluble in nonpolar organic solvents (as chloroform and ether), that are usually insoluble in water, that with proteins and carbohydrates constitute the principal structural components of liv ing cells. Lipids include fats, waxes, phosphatides, cerebrosides, and related a nd derived compounds. Just as amino acids are the individual units making up the class of nutrien ts called proteins, so fatty acids (FA) are the major individual units of measure of what is broa dly called lipids. Just as each amino acid has a distinct structure and function in protein build ing, so each FA has a distinct structure and possibly function in metabolism (Staples et al., 2002). Thus, from a nutritional standpoint, the term lipid is a general term which lacks specific ity for the modern nutritionist who is interested in the fatty acid composition of lipids. In 1929, George O. Burr and his wife were the first to describe the essentiality of FA in rats (Burr and Burr, 1929; 1930). The authors reported that growing rats fed diets devoid in fat ceased growing and experienced health problems and irregular ovulation, which were reversed after feeding fat sources rich in the PUFA, C18: 2 and C18:3 (Burr and Burr, 1930). At that time, the concept of essentia l FA (EFA) was established. Later it was understood that C18:2 and C18:3 could not be synthesized by mammalian cells due to the lack of desaturase enzymes active beyond the 9th carbon in the acyl chain. Thus, due to the e ssentiality of these 2 FA and the role of specific FA on several processes, supplemental li pid sources enriched with specific FA have been the gold key in nutrition to improve incorpor ation of specific FA in milk, and tissues, and influence several physiological pr ocesses such as reproduction a nd immune function. However, accomplishing this in ruminant animals is ma de more challenging due to the population of

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266 ruminal microorganisms that biohydrogenate unsatur ated FA such that the uptake of specific unsaturated FA is reduced considerably compared to its content in the diet. Don Palmquist, working at Th e Ohio State University, was the pioneer in developing the Ca salt form of lipids. This product was a dry pr oduct which allowed fats to be fed more easily. More importantly, lipids in the Ca salt form provided some protection of the unsaturated FA from microbial biohydrogenation. Others have developed protective mechanisms such as creating amides of FA (Tom Jenkins at Clems on University) or harnassing the Maillard reaction by steaming cracked oil seeds with a xylose solution to prevent microbial biohydrogenation (Abel-Caines et al., 1998). Regardless of th e method used to protect the FA, biohydrogenation of unsaturated FA still exceeds 60%, and often reaches 90% (Loor et al., 2005; Harvatine and Allen, 2007a). However, supplementation with thes e more inert fat sources has increased the incorporation of specific FA into milk, plasma, liver, and caruncles of dairy cows. How these changes may influence physiological processes su ch as reproductive and immune functions are of current interest in animal biology. The work of Cullens et al. (2004) at the Univ ersity of Florida docum ented that initiating fat supplementation prior to calvi ng was advantageous. The feeding of a Ca salt of a fat source enriched in C18:2 (2% of dietary DM) was initiated either 4 wk prepartum, at parturition, at 28 DIM or not at all. Authors repo rted that cows supplemented with this fat source (Megalac-R) starting prepartum had a lower incidence of di seases including retained fetal membranes, metritis, and mastitis compared with cows not fed fat prepartum. In addition, cows fed fat prepartum produced more milk in the subsequent lactation compared to cows which were started on fat postpartum.

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267 Initial results from a study by Ju chem et al. (2008) indicated that a Ca salt mixture of trans C18:1 isomers and C18:2 improved embryo quality and pregnancy rate of lactating dairy cows. As a result, we designed a study to evalua te the effect of feedi ng lipid sources, starting prepartum, enriched in either trans C18:1 isomers (CaTRANS), C18:2 (Ca salt of palm and soybean oils), or C18:3 (linseed oil, LSO). A fourth diet served as a positive control, in that it contained a lipid source enriched in cis C18:1 (a genetically engin eered sunflower plant whose seeds would produce an oil very high in C18:1) Therefore, 2 monounsatur ated FA (MUFA) and 2 PUFA sources were evaluated. Fats were fe d at 1.35% of dietary DM prepartum and 1.5 to 1.75% of dietary DM postpartum until 105 DIM. We wanted to know if feeding these particular FA at relatively low feeding rates to Holstein cows would affect DM I, milk production and composition, and change the FA profile of plasma, liver, and milk (Chapter 3), influence plasma concentrations of hormones, metabolites, acute phase proteins, and hepatic gene expression (Chapter 4), and influence oocyte quality and folli cular development (Bilby et al., 2006a). In a second study, different dietary fat sources were fed to Holstein cows from approximately 4 wk prior to calving through 7 wk postpartum. Gr eater amounts of C18:2 were supplemented by using a premarket version of a Ca salt of safflow er oil (63% C18:2). In addition, a different source of omega-3 FA was fed; namely, a marketed Ca salt mixture of palm and fish oils (11% C20:5 and C22:6). Fats were fe d at 1.5% of dietary DM and we re compared against a negative control group not fed supplemental fat. The influence of these fat supplements on cow performance, FA profiles of m ilk and caruncles, plasma hormones and metabolites, and immune status were of measured. In both studies, primiparous and multiparous animals were used and parity by fat supplement interactions occu rred for several dependent variables.

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268 Cows fed the omega-3 fat source, LSO, consumed more DM (% of BW) towards the end of the study compared to cows fed CaTRANS. In addition they tended to produce more 3.5% fat-corrected milk due to an improvement in con centration of milk fat compared to cows fed the C18:2 fat source. However, when the omega-3 fat source was of marine origin (Chapter 6) rather than of vegetable origin (Chapt er 3), DMI (% of BW) and concentr ation of milk fat tended to be lower compared to cows fed the omega-6 fat sour ce. As reported by other researchers, the FA, C20:5 and C22:6, can be potent nutrients that re duce milk fat synthesis. Body weight, BCS, nor energy balance were affected by fat source in either study. Feeding increasing amounts of a FA often result ed in an increased concentration of that FA in milk/tissues of animals. In Chapter 3, feeding supplemental C 18:1 increased the C18:1 content of plasma fat but not of liver fat. Oleic acid in milk fat of multiparous but not primiparous cows was increased by feeding supp lemental oleic acid. Cows fed CaTRANS fats had greater concentrations of trans C18:1 isomers in plasma (1.5%), liver fat (1.4%), and milk fat (5.8%) compared to cows fed cis C18:1. Feeding supplemental C18:2 increased concentrations of C18:2 in milk fat (Chapter 3 and 6) and in caruncles (Chapter 6) but not in liver fat or plasma fat (Chapter 3). Arachidon ic acid (C20:4) is synthesized from C18:2 in tissues. However feeding additional C18:2 did not increase concentrations of C20:4 in fat of milk, plasma, liver, or caruncles in either study. Enrichment of the diet with C18:3 using LSO resulted in greater incorporati on of C18:3 and C20:5 in plasma, liver, and milk fat but C22:6 was detected in increased concentrati on only in liver, thus demonstra ting that tissues can synthesize C20:5 from C18:3. When the unique FA of fish oil were fed in study 2, concentrations of C20:5 increased in milk fat by 400% and in caruncles by 50% whereas that of C22:6 increased in milk fat from 0.001 to 0.094% and in caruncles by 27 0%. Although the proportional increases are

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269 impressive, concentrations of C20:5 and C22:6 never got above 1.2% of the total FA. Lastly, concentrations of CLA were in creased routinely by supplementati on with PUFA, indicating that these sources of PUFA were acting as modifiers of nor mal biohydrogenation pathways. Concentrations of cis -9, trans -11 CLA in milk fat and caruncles were increased by both supplemental C18:2 and fish oil when compared against cows not fed supplemental fat (Chapter 6). However when all cows were fed a fat supplement (study 1), concentrations of cis -9, trans -11 CLA in fat of milk or plasma were unc hanged. Only hepatic concentrations of cis -9, trans -11 CLA were increased when cows were fed C18:2 or LSO. In summary, the FA profile of milk, caruncles, and liver of dairy cows can be modifi ed to reflect the FA profile of dietary FA supplements fed at relatively low amounts but most changes are not substantial. Most plasma metabolites measured during the first 7 wk postpartum were unchanged by feeding fat. Mean plasma concentrations of glucose, BUN, and BHBA were not different among treatments in either study. Plasma concentra tions of NEFA were unchanged in Chapter 6, but were elevated at 2 and 5 wk postpartum of animals fed LSO in Chapter 4; however they were not high enough to be indicative of metabolic stress. Indeed, these variables indicate that cows fed these fat sources at these rates were not in a subk etotic or energy-deprived state. These increases in plasma NEFA concentrations of cows fed LSO were accompanied by the upregulation of the mRNA of key enzymes for synthesis of gl ucose (pyruvate carboxylase and phosphoenolpyruvate carboxykinase) at the liver during this same time period. These responses of lipid mobilization and gluconeogenesis suggest a coordinated effort by the cows metabolism to meet metabolic energy needs in the ear ly postpartum period. Plasma concentrations of the insulin family of hormones were measured only in Chapter 4 and they were positively affected by the feedi ng of PUFA sources. Concentrations of plasma

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270 IGF-1 increased at a faster rate postpartum for animals fed PUFA vs. MUFA sources; this was accompanied by a faster rate of increase in plasma insulin of multiparous cows fed PUFA sources. Almost all IGF secreted from the liver circulates as a bound complex and the majority of it (> 90%) is associated with IGFBP-3 (C lemmons, 1997). Cows fed PUFA experienced a rise in the expression of hepa tic IGFBP-3 mRNA overtime compar ed to cows fed MUFA which remained unchanged. This would be expected based upon IGF-1 results. IGFBP-3 is produced mainly in the liver and is the major transporter of IGF-1 in the peripheral circulation (Burger et al., 2005). The increase in IGFBP-3 mRNA expr ession in the liver pos sibly prevented IGF-1 degradation and potentially incr eased availability to other tissues by providing a reservoir of IGF-1. This faster rise in plasma IGF-1 did not result in an earlie r return to ovarian activity or a greater calculated accumulation of progesterone ove r time for these cows. However in Chapter 6, animals fed fish oil cycled about 6 d earlier than those fed the omega-6 fat source. If PUFA supplements can improve IGF-1 status of cows ear ly in lactation, improved fertility may ensue. The effect of fat supplements on the health of the cow was examined mainly in Chapter 6. Indicators of a responsive immune system th at were examined included acute phase proteins, neutrophil numbers and activity, immunoglobulins, and cytokine production. Prostaglandins are mediators of inflammation as they are produ ced by macrophages and are thought to be an attractant for neutrophils to the site of infection. At the time of parturition PGF2 is produced by the uterus and its concentration is measured as its metabolite (PGFM) in plasma. The secretion of PGF2 is associated with involution of the uter us and to help reduce the risk of uterine infection during this vulnerable time. Those cows exhibiting great er plasma concentrations of PGFM around parturition may be better able to combat pathogenic microorganisms due to a greater production of PGF2 The precursor of PGF2 is arachidonic acid (C20:4) which is made

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271 from C18:2. Concentrations of plasma PGFM were greater at parturition and decreased at a faster rate postpartum in primiparous cows fed CaTRANS vs. those fed oleic acid in Chapter 4 which agrees with the work of Rodriguez-Sallaberry et al. (2007) at Florida. However fat source did not affect plasma PGFM in study 2. Therefore feeding additional omega-6 as a precursor of PGFM or feeding omega-3 as a suppressor of PGFM did not occur in these studies. Nevertheless, uterine health was affected. Feeding LSO in study 1 resulted in fewer neutrophil numbers in uterine flushing collected at 40 DIM from primiparous cows. In study 2, neutrophil concentrations were numerically but not significantly lo wer in uterine flushings from animals fed fat (16.4, 5.2, and 0.3 x 1000 neutroophils/mL for control, omega-6, and omega-3 treatments, respectively). A greater proportion of the animals fed fish oil in study 2 were diagnosed with a more severe case of metritis at 5 and 10 DIM compared to the other treatments. It appears that consumption of omega-3 supplements were suppressive of the immune system. Measures of immune status in the blood of cows in Chapter 6 also support this effect of omega-3 on immunity of dairy cows. Cows fed fi sh oil had lower blood c oncentrations of WBC and neutrophils and had circulating neutrophils that consumed fewer E. coli per neutrophil when measured on -18, 0, 7, and 40 DIM. In addition, th ese same cows had circulating lymphocytes that produced fewer cytokines when isolated a nd stimulated in vitro on 10, 20, and 30 DIM. On the other hand, the omega-6 enriched fat fed in Chapter 6 had immunostimulatory effects on lactating dairy cows. These cows did not experience the decrease in concentration of blood neutrophils at 7 DIM that occurred in the other treatments. In addition, these cows had a greater humoral response of IgG concentrations in serum postpartum to ovalbumin injections. Unexpectedly, the lymphocytes from these cows collected on 10, 20, and 30 DIM produced fewer cytokines when stimulated in vitro, similar to that of cows fed the omega-3 fat source.

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272 Cytokines are soluble proteins that are released from i mmune cells (mainly monocytes and macrophages) in response to in fection, injury, or foreign subs tances. Liberation of cytokines is indispensable for the initiation of the immune response and for the regulation of the multidirectional communication between the diff erent cells involved. Cytokines stimulate the liver to produce acute phase proteins as an acu te response to inflammati on or other sources of stress which circulate in the blood. Plasma concen trations of acute phase proteins measured in both studies included haptoglobin and acid soluble protein whereas fibrinogen and ceruloplasmin were added in Chapter 5. Based upon greater pl asma concentrations for these acute phase proteins, primiparous cows were under greater stre ss from parturition and lactation compared to multiparous cows. Concentrations of plasma hapt oglobin were not influenced by fat supplements in either study although primiparous cows fed CaTRANS tended to have greater values than those fed oleic acid in Chapter 4. This may be linked to the grea ter concentrations of plasma PGFM early postpartum in this gr oup of cows. In agreement was the response of concentrations of plasma acid soluble protein, with greater va lues for primiparous cows fed CaTRANS in study 1. In study 2, evidence of immunosuppressive effect s of fish oil was detected in that plasma ceruloplasmin was lower in primiparous cows whereas the omega-6 fat source appeared stimulatory based upon elevated concentrations of plasma fibrinogen in multiparous cows. Lipid supplementation of a specific FA influen ces the FA profile of several tissues which in turn differentially influences metabolism a nd physiology of the dairy cow. The challenge of the ruminant nutritionist is to understand and manage the extent of biohydrogenation in the rumen of unsatured FA so that more of a speci fic FA can be deposited into blood and tissues. These changes may improve the physiological point of interest such as reproduction or immunity

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273 which will be mediated by hormones, metabolites and gene expression among other things. The physiological state of the animal such as parity will affect the impact of FA.

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275 Allen, M. S. 2000. Effects of diet on short-term regul ation of feed intake by lactating dairy cattle. J. Dairy Sci. 83:1598-1624. Allred, S. L., T. R. Dhiman, C. P. Brennand, R. C. Khanal, D. J. McMahon, and N. D. Luchini. 2006. J. Dairy Sci. 89:234-248. Milk and cheese from cows fed calcium salts of palm and fish oil alone or in combination with s oybean products. J. Dairy Sci. 89:234-248. Andersen, J. B., C. Ridder, and T. Larsen. 2008. Priming the cow for mobilization in the periparturient period: effects of supplementing the dry cow with satura ted fat or linseed. J. Dairy Sci. 91:1029-1043. Andrade, P. M. M., B. G. Ribeiro, M. T. Bozza, L. F. B. C. Rosa, and M. T. do Carmo. 2008. Effects of fish oil supplementation on the im mune and inflammatory responses in elite swimmers. Prostaglandins, Leukotriens and Essential Fatty Acids 77:139-145. Anhadi, C. E., N. Beswick, L. Delbecchi, J. J. Kennely, and P. Lacasse. 2002. Addition of fish oil to diets for dairy cows. II. Effects on milk fat and gene expression of mammary lipogenic enzymes. J. Dairy Res. 69:521-531. 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. Ballou, M. A. and E. J. DePeters. 2008. Supplemen ting milk replacers with omega-3 fatty acids from fish oil on immunocompetence and health of jersey calves. J. Dairy Sci. 91:3488-3500. Bartelt, S., M. Timm, C. T. Damsgaard, E. W. Hansen, H. S. Hansen, and L. Lauritzen. 2008. The effect of dietary fish oil-supplement ation to healthy young men on oxidative burst measured by whole blood chemiluminescence. Br. J. Nutr. 99:1230-1238. Bauman, D. E., L. H. Baumgard, B. A. Co rl, and J. M. Griinari. 1999. Biosynthesis of conjugated linoleic acid in ruminants. Pages 1 in Proc. Am. Soc. Anim. Sci., Indianapolis, IN. Bauman, D. E. and J. M. Griinari. 2003. Nutritio nal regulation of milk fat synthesis. Annu. Rev. Nutr. 23:203-27. Baumgard, L. H., B. A. Corl, D. A. Dwyer, A. Saebo, and D. E. Bauman. 2000. Identification of the conjugated linoleic acid isomer that inhi bits milk fat synthesis. Am. J. Physiol. 278(Suppl.):R179R184. Baumgard, L. H., E. Matitashvili, B. A. Corl, D. A. Dwyer, and D. E. Bauman. 2002. trans -10, cis -12 conjugated linoleic acid de creases lipogenic rates and expr ession of genes involved in milk lipid synthesis. J. Dairy Sci. 85:2155. Bazinet, R. P., H. Douglas, E. G. McMilla n, B. N. Wilkie, and S. C. Cunnane. 2004. Dietary 18:3 omega-3 influences immune function and th e tissue fatty acid response to antigens and adjuvants. Immunol. Lett. 95:85-90.

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276 Beam, S. and W. Butler. 1997. Energy balance and ova rian follicle development prior to the first ovulation postpartum in dairy cows receiving th ree levels of dietary fat. Biol. Reprod. 56:133-142. Bec-Villalobos, D., I. Garca-Tornad, G. Shroed er, E. E. Salado, G. Gagliostro, C. Delavaud, Y. Chiliard, I. M. Lacau-Mengido. 2007. Effect of fat supplementation on leptin, insulin-like growth factor I, growth hormone, and insu lin in cattle. Can. J. Vet. Res. 71:218-225. Bell, A. W. 1981. Lipid metabolism in liver a nd selected tissues a nd in the whole body of ruminant animals. In: W. W. Christie (Ed.) Lipid Metabolism in Ruminant Animals. p 363. Pergamon Press, Oxford, U. K. Benchaar, C., H. V. Petit, R. Berthiaume, D. R. Ouellet, J. Chiquette, and P. Y. Chouinard. 2007. Effects of essential oils on digestion, ru minal fermentation, rumen microbial populations, milk production, and milk composition in dairy co ws fed alfalfa silage or corn silage. J. Dairy Sci. 90:886-897. Bennet, M., and K. Schmid. 1980. Imm unosuppression by human plasma 1-acid glycoprotein:importance of the carbohydrate mo iety. Proc. Natl. Acad. Sci. USA. 77:6106113. Berry, B. A., A. W. Confer, C. R. Krehbiel, D. R. Gill, R. A. Smith, and M. Montelongo. 2004. Effects of dietary energy and starch concentrations for newl y received feedlot calves: II. Acute phase protein response. J. Anim. Sci. 82:845-850. Bharathan, M., D. J. Schingoethe, A. R. Hippen, K. F. Kalscheur, M. L. Gibson, and K. Kargest. 2008. Conjugated linoleic acid increases in milk fr om cows fed condensed distillers soluble and fish oil. J. Dairy Sci. 91:2796-2807. Bhattacharya A, Banu J, Rahman M, Causey J, Fernandes G. 2006. Bi ological effects of conjugated linoleic acids in health and disease. J Nutr Biochem. 17:789 Bilby, T. R., J. Block, B. C. do Amaral, O. Sa Filho, F. T. Silvestre, P. J. Hansen, C. R. Staples, and W. W. Thatcher. 2006a. Eff ects of dietary unsaturated fatty acids on oocyte quality and follicular development in lactating dairy cows in summer. J. Dairy Sci. 89:3891-3903. Bilby, T. R., A. Sozzi, M. M Lopez, F. T. Silvestre, A. D. Ealy, C. R. Staples, and W. W. Thatcher. 2006b. Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows:I. Ovarian, conceptus, and growth hormone-insulin like growth factor system response. J. Dairy Sci. 89:3360-3374.. Bilby, T. R., T. Jenkins, C. R. Staples, and W. W. Thatcher. 2006c. Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lact ating dairy cows:III. Fatty acid distribution. J. Dairy Sci. 89:3386-3399. Bichell, D. P., K. Kikuchi, and P. Rotwein. 1992. Growth hormone rapidly activates insulin-like growth factor I gene transcri ption in vivo. Mol Endocrinol 6:1899.

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297 BIOGRAPHICAL SKETCH Bruno Cesar do Am aral was born in So Carl os, So Paulo, Brazil, on March 29, 1977. He attended elementary school and high school in So Carlos, So Paulo. Bruno went on to Universidade de Lavras, Lavras, Minas Gerais, Brazil in 1996 where he earned his B.S. (2001) and M.S. (2003) degrees. In Fall 2003 Bruno moved to Gainesville with his wife Michelle to begin his Ph.D. program in dairy nutrition. Afte r graduation, he will fini sh the post-doctoral position he accepted in summer 2007 with Dr. Geoff Dahl at the University of Florida and in May 2009 he will move to Ann Arbor, Michigan where he will work as a dairy nutritionist for Van Beek Nutrition.