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Dietary Strategies to Modulate Performance, Health, and Immune Responses in Holstein Calves

Permanent Link: http://ufdc.ufl.edu/UFE0043757/00001

Material Information

Title: Dietary Strategies to Modulate Performance, Health, and Immune Responses in Holstein Calves
Physical Description: 1 online resource (240 p.)
Language: english
Creator: Perdomo, Milerky C
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

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

Notes

Abstract: A series of in vitro experiments were conducted to examine the effect of exogenous conjugated linoleic acid (CLA) and peroxisome proliferator-activated receptor gamma (PPAR-?) agonist, rosiglitazone, on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF-a) production in cultured bovine blood, and to identify the signaling pathway through which LPS and PPAR-? interact to alter TNF-a biosynthesis in vitro. Additionally three in vivo experiments were conducted in Holstein calves. The first experiment was an observational prospective cohort study conducted with 1,247 calves from 7 dairy farms to determine the effects of passive immunity at 48 h of life on performance, morbidity and mortality risk, survival time and rearing costs in preweaned Holstein calves. The second and third experiment tested the effects of feeding pomegranate extract and Saccharomyces cerevisiae fermentation products (SFP), respectively, on performance, health, and immunocompetence of Holstein calves during the preweaning period. Co-incubation with trans-10, cis-12 CLA isomer or rosiglitazone decreased LPS-induced TNF-a production. Rosiglitazone-induced TNF-attenuation was reversed when blood was treated with both rosiglitazone and GW9662, a selective PPAR-? antagonist. Addition of rosiglitazone to the culture medium tended to reduce nuclear factor kappa Bp65 concentration in nuclear and cytosolic extracts isolated from cultured PBMC. Calves classified as having adequate passive transfer (APT; serum IgG = 1.0 g/dL) had greater total DM intake and feed conversion ratio during the preweaning period. Daily grain intake and body weight were greater in APT calves during the first 30 d of age. Calves with APT had reduced risk to develop morbidity, pneumonia, and multiple diseases, and had less number of disease events per calf. Calves with failure of passive transfer (FPT) had 3.6 times greater hazard of dying in the first 75 d of age. Rearing costs and income per calf were greater for calves with APT than those with FPT. The probability of death within the first 75 d of age decreased as IgG concentration in serum increased. Increase in STP reduced the probability of death, but values above 7.0 g/dL increased the probability of death. Serum IgG as percent of STP was the best predictor of probability of death than STP and IgG concentration. Feeding 15 mg of gallic acid equivalent/kg of body weight or subtherapeutic doses of antibiotics (200 mg oxytetracycline and 200 mg neomycin/d) to preweaned calves did not benefit performance, health, and immune and antioxidant response. Supplementing dairy calves with SFP added to milk up to 4 g/d in the first 2 months of age did not affect performance. Calves fed SFP had an increased proportion of neutrophils with capacity for oxidative burst, but this effect did not influence measures of health based on daily fecal, nasal, ocular, cough and attitude scores, and rectal temperature.
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 Milerky C Perdomo.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Badinga, Lokenga.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043757:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043757/00001

Material Information

Title: Dietary Strategies to Modulate Performance, Health, and Immune Responses in Holstein Calves
Physical Description: 1 online resource (240 p.)
Language: english
Creator: Perdomo, Milerky C
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

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

Notes

Abstract: A series of in vitro experiments were conducted to examine the effect of exogenous conjugated linoleic acid (CLA) and peroxisome proliferator-activated receptor gamma (PPAR-?) agonist, rosiglitazone, on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF-a) production in cultured bovine blood, and to identify the signaling pathway through which LPS and PPAR-? interact to alter TNF-a biosynthesis in vitro. Additionally three in vivo experiments were conducted in Holstein calves. The first experiment was an observational prospective cohort study conducted with 1,247 calves from 7 dairy farms to determine the effects of passive immunity at 48 h of life on performance, morbidity and mortality risk, survival time and rearing costs in preweaned Holstein calves. The second and third experiment tested the effects of feeding pomegranate extract and Saccharomyces cerevisiae fermentation products (SFP), respectively, on performance, health, and immunocompetence of Holstein calves during the preweaning period. Co-incubation with trans-10, cis-12 CLA isomer or rosiglitazone decreased LPS-induced TNF-a production. Rosiglitazone-induced TNF-attenuation was reversed when blood was treated with both rosiglitazone and GW9662, a selective PPAR-? antagonist. Addition of rosiglitazone to the culture medium tended to reduce nuclear factor kappa Bp65 concentration in nuclear and cytosolic extracts isolated from cultured PBMC. Calves classified as having adequate passive transfer (APT; serum IgG = 1.0 g/dL) had greater total DM intake and feed conversion ratio during the preweaning period. Daily grain intake and body weight were greater in APT calves during the first 30 d of age. Calves with APT had reduced risk to develop morbidity, pneumonia, and multiple diseases, and had less number of disease events per calf. Calves with failure of passive transfer (FPT) had 3.6 times greater hazard of dying in the first 75 d of age. Rearing costs and income per calf were greater for calves with APT than those with FPT. The probability of death within the first 75 d of age decreased as IgG concentration in serum increased. Increase in STP reduced the probability of death, but values above 7.0 g/dL increased the probability of death. Serum IgG as percent of STP was the best predictor of probability of death than STP and IgG concentration. Feeding 15 mg of gallic acid equivalent/kg of body weight or subtherapeutic doses of antibiotics (200 mg oxytetracycline and 200 mg neomycin/d) to preweaned calves did not benefit performance, health, and immune and antioxidant response. Supplementing dairy calves with SFP added to milk up to 4 g/d in the first 2 months of age did not affect performance. Calves fed SFP had an increased proportion of neutrophils with capacity for oxidative burst, but this effect did not influence measures of health based on daily fecal, nasal, ocular, cough and attitude scores, and rectal temperature.
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 Milerky C Perdomo.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Badinga, Lokenga.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043757:00001


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1 DIETARY STRATEGIES TO MODULATE PERFORMANCE, HEALTH, AND IMMUNE RESPONSES IN HOLSTEIN CALVES By MILERKY CRISTINA PERDOMO LOZADA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILL MENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Milerky Cristina Perdomo Lozada

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3 To Carlos, for his love, encouragement and support, and to my daughters: Veronica Milena, my strength, and C

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4 ACKNOWLEDGMENTS I would like to thank Dr. Lokenga Badinga for his precious guidance with the laboratory techniques. I am grateful to him for encouraging me as a scientist during these four years. I thank Dr. Jos E.P. Sa ntos for accepting me as his graduate student, for his critical thinking, and for sharing with me his knowledge and scientific inspiration. I am also grateful to my supervisory committee members: Dr. Charles S taples, Dr. Alan Ealy, and Dr. Art Donovan, fo r time they devoted to this dissertation. Special thanks go to Dr. Ealy for his advice and help during my in vitro experiments. I also want to thank to all people from the University of Florida Dairy Unit, especially Eric Diepersloot and Sherry Hay, who al ways were available to give me a friendly helping hand during my experiments. Thanks also go to Dr. Sally Willia m s, Dr. Joel Yelich Dr. Willia m Thatcher, Dr. Klibs Galvo and Dr. Jorge Hernandez for providing the laboratory support to complete my researc h. Many thanks are extended to Mariana Ragazoni, Maur cio Favoreto, Ana L cia Michelle Smith, Lucas Furtado, Pedro Bueno, Luis Fe rnando Becker, Vin cius Rezende, Armando Schlaefli Alana Calaa, Gabriel Gomes and Samara Soa res for being efficient and enthusiastic helpers during my experiments. I want to thank to all people from the Department of Animal Sciences, especially Sergei Sennikov for his wonderful help with laboratory analyses. Thanks also go to Sabrina Robinson, Jo hnnie Salvino Joyce Hayen Cristina Dore and Joan Fisher for their help in many different ways.

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5 Great appreciation goes to my lab mates: Dr. Cristina Caldari Torres, Dan Wang, Eduardo Ribeir o, Fbio Lima, Jae Hyeong Shin Leandro Greco, Natalia Martinez and Rafael Bisinotto for always being available to help me. I am also thankful to Dr. Maria Beatriz Padua for her unconditional friendship and support to my family and me from our first day in Gainesville. Thanks to my special fri ends Miria m Garcia and Tao Sha for always being available to listen to me. Great appreciation goes to my friends Dr. Katty Arriola, Jacob Bubolz, Juan J. Romero and Miguel Zarate I especially want to thank Universidad Centroccidental Lisandro Alvarado for the opportunity to mov e abroad to obtain my doctoral degree. Gratitude is extended to Dr. Gustavo Nouel and Mr. Odriom Escobar for assuming my academic responsibilities during my absence in these four years away from Venezuela. I am grateful to Dr. Monica Prado and Dr. Andres Kowalski for encouraging me to do my doctoral studies when I was in Venezuela. Last, but perhaps most important ly I am appreciative for the sacrifices and unconditional love of my parents, Victor and Josefa, and of my sisters: Mirielly, Mildred, and Milen y. I am especially thankful to my husband Carlos Torrealba, for his love, support and for his encouragement in all my endeavors. I am grateful to my loving daughter Veronica for giving me the opportunity to perform the most important role for me, be a Mom. Thank you God for Y our overflowing generosity to me

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .............. 4 LIST OF TABLES ................................ ................................ ................................ ......................... 9 LIST OF FIGURES ................................ ................................ ................................ ..................... 11 LIST OF ABBREVIATIONS ................................ ................................ ................................ ...... 14 ABSTRACT ................................ ................................ ................................ ................................ 17 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ................. 19 2 REVIEW OF LITERATURE ................................ ................................ ............................... 22 Overview of the Immune System ................................ ................................ ...................... 22 Innate Immune System ................................ ................................ ............................... 22 Adaptive Immune System ................................ ................................ ........................... 25 Immunity of Newborn Cattle ................................ ................................ .............................. 28 Passive Immunity in Newborn Cattle ................................ ................................ ............... 29 Factors Affecting Passive Transfer of Colostral Immunoglobulin ........................ 30 Effect of Adequate Passive Transfer ................................ ................................ ........ 34 Modulation of Immune Response of Calves by Other Colo strum Constituents ................................ ................................ ................................ .............. 35 Feeding Conjugated Linoleic Acid to Dairy Cattle ................................ ......................... 36 Effect of Dietary CLA on Animal Performance ................................ ............................... 38 Effect of CLA on Milk Fat Depression ................................ ................................ ....... 42 Effect of CLA on Reproductive Performance in Dairy Cattle ................................ 43 Effect of Dietary CLA on Health ................................ ................................ ........................ 44 Proposed Mechanisms of Beneficial Properties of CLA ................................ ........ 57 F eeding Pomegranate to Dairy Cattle ................................ ................................ ............. 58 Effect of Dietary Pomegranate on Animal Performance ................................ ............... 60 Effect of Pomegranate on Health ................................ ................................ ..................... 62 Proposed Mechanisms of Beneficial Properties of Pomegranate ........................ 72 Feeding Saccharomyces cerevisiae to Dairy Cattle ................................ ...................... 72 Effect of Dietary Saccharomyces cerevisiae on Animal Performance ....................... 74 Effect of Dietary Saccharomyces cerevisiae on Health ................................ ................ 83 Proposed Mechanisms of Beneficial Properties of Saccharomyces cerevisiae ................................ ................................ ................................ ................... 87 3 TRANS 10, CIS 12 CONJUGATED LINOLEIC ACID AND THE PPA R AGONIST ROSIGLITAZONE ATTENUATE LIPOPOLYSACCHARIDE INDUCED TNF .............................. 88

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7 Introduction ................................ ................................ ................................ .......................... 88 Materials and Methods ................................ ................................ ................................ ....... 90 Media and Reagents ................................ ................................ ................................ ... 90 Animals and Blood Sampling ................................ ................................ ..................... 90 Peripheral Blood Mononuclea r Cell Isolation ................................ .......................... 90 Lymphocyte Proliferation Assay ................................ ................................ ................ 91 Lipopolysaccharide Stimulation of Tumor Necrosis Factor Alpha Production in Whole Bovine Blood ................................ ................................ ............................ 92 Fatty Acid Treatment of Whole Blood ................................ ................................ ....... 92 Peroxisome Proliferator Activated Receptor Gamma Modulation of Tumor Necrosis Factor Alpha Production ................................ ................................ ......... 92 Tumor Necrosis Factor Alpha Assay ................................ ................................ ........ 93 Isolation of Cytosolic and Nuclear Proteins ................................ ............................. 93 Western Blot Analysis of Nuclear Factor Kappa B ................................ ................. 94 Statistical Analyses ................................ ................................ ................................ ...... 95 Results ................................ ................................ ................................ ................................ .. 95 Effect of Lipopolysaccharide on Peripheral Blood Mononuclear Cell Proliferation ................................ ................................ ................................ ............... 95 Tumor Necrosis Factor Alpha Response to Lipopolysaccharide ......................... 96 Nuclear Factor Kappa B Response to Lipopolysaccharide ................................ .. 96 Discussion ................................ ................................ ................................ ............................ 96 4 ASSOCIATION OF PASSIVE TRANSFER WITH PERFORMANCE, INCIDENCE OF DISEASES, AND RISK OF DEATH IN PREWEANED HOLSTEIN CA LVES ................................ ................................ ................................ ........ 106 Introduction ................................ ................................ ................................ ........................ 106 Materials and Methods ................................ ................................ ................................ ..... 107 Animals, Housing and Feeding ................................ ................................ ................ 107 Body Weight and Blood Sam pling and Analyses ................................ ................. 108 Incidence of Health Disorders, Treatments and Costs Associated with Treatments ................................ ................................ ................................ .............. 108 Economic Analysis of Calf Rearing ................................ ................................ ......... 109 Statistical Analyses ................................ ................................ ................................ .... 110 Results ................................ ................................ ................................ ................................ 112 Discussion ................................ ................................ ................................ .......................... 114 Conclusions ................................ ................................ ................................ ........................ 121 5 EFFECT OF FEEDING POMEGRANATE EXTRACT ON GROWTH, HEALTH, AND IMMUNE RESPONSES OF HOLSTEIN CALVES ................................ ............. 130 Introduction ................................ ................................ ................................ ........................ 130 Materials and Methods ................................ ................................ ................................ ..... 133 Calves and Treatments ................................ ................................ ............................. 133 Milk and Grain Sampling Nutrient Analyses, and Quantification of Bacteria in Milk ................................ ................................ ................................ ....................... 134 Body Weight, Height and Scoring ................................ ................................ ........... 135

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8 Evaluation of Incidence of Health Disorders ................................ ......................... 135 Blood Sampling ................................ ................................ ................................ .......... 136 Hematocrit, Serum Total Protein and Total Immunoglobulin G Quantification 137 Assays for Plasma Metabolites ................................ ................................ ............... 137 Antioxidant Enzymes and Quantification of Unstable Lipid Hydroperoxides ... 138 Immunization with Ovalbumin and Assay for Anti OVA IgG ............................... 139 Leukocyte Population Quantification ................................ ................................ ...... 140 Phagocytosis and Oxidative Burst Assays ................................ ............................ 140 Experimental Design and Statistical Analyses ................................ ...................... 141 Results ................................ ................................ ................................ ................................ 143 Discussion ................................ ................................ ................................ .......................... 147 Conclusions ................................ ................................ ................................ ........................ 151 6 IMPACT OF FEEDING DIFFERENT AMOUNTS OF SACCHAROMYCES CEREVISIAE FERMENTATION PRODUCTS ON IMMUNE STATUS, HEALTH AND PERFORMANCE OF HOLSTEIN CALVES ................................ ........................ 173 Introduction ................................ ................................ ................................ ........................ 173 Materials and Methods ................................ ................................ ................................ ..... 175 Calves, Housing and Colostrum Feeding ................................ .............................. 175 Treatments, Feeding and Measurements ................................ .............................. 175 Grain and Milk Sa mpling, and Nutrient Analyses ................................ ................. 176 Scoring, Incidence of Health Disorders, and Costs Associated with Treatments ................................ ................................ ................................ .............. 177 Blood Sampling ................................ ................................ ................................ .......... 179 Hematocrit, Serum Total Protein and Total Immunoglobulin Quantification .... 179 Assays for Plasma Metabolites ................................ ................................ ............... 1 80 Immunization with Ovalbumin and Assay for Anti OVA IgG ............................... 180 Leukocyte Population Quantification ................................ ................................ ...... 182 Phagocytosis and Oxidative Burst Assay ................................ .............................. 182 Fecal Shedding of E. coli and Salmonella spp ................................ .................... 183 Experim ental Design and Statistical Analyses ................................ ...................... 183 Results ................................ ................................ ................................ ................................ 185 Discussion ................................ ................................ ................................ .......................... 187 Conclusions ................................ ................................ ................................ ........................ 191 7 GENERAL DISCUSION AND CONCLUSIONS ................................ ........................... 212 LIST OF REFERENCES ................................ ................................ ................................ ......... 218 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ..... 240

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9 LIST OF TABLES Table page 4 1 Descriptive statistics according to farm (mean SD or proportion) ..................... 122 4 2 Association between passive transfer and performance of Holstein calves in the preweaning period ................................ ................................ ................................ 123 4 3 Association between passive transfer and health of Holstein calves in the preweaning period ................................ ................................ ................................ ........ 124 4 4 Association between passive transfer and rearing cost of Holstein calves in the preweaning period ................................ ................................ ................................ 125 5 1 Nutrient composition and micro bial contamination of pasteurized milk fed to calves1. ................................ ................................ ................................ .......................... 153 5 2 Nutrient composition (mean SD) of the starter grain (DM basis)1 .................... 154 5 3 Effect of feeding pomegranate extract or antibiotics on performance and growth of preweaned calves ................................ ................................ ....................... 155 5 4 Effects of feeding pomegranate extract or antibiotics on h ealth of preweaned calves ................................ ................................ ................................ ............................. 156 5 5 Effect of feeding pomegranate extract or antibiotics on incidence of diseases in preweaned calves ................................ ................................ ................................ .... 157 5 6 Effect of feeding pomegranate extract or antibiotics on concentration of white blood cells of preweaned calves, 103/L SEM ................................ .................... 158 5 7 Effect of feeding pomegranate extract or antibiotics on the proportion of individual leukocytes in blood of preweaned calves, % SEM. ........................... 159 5 8 Effect of feeding pomegranate extract or antibiotics on superoxide dismu tase (SOD), glutathione peroxidase (GPx), and thiobarbituric acid reactive substances (TBARS) of plasma of preweaned calves ................................ ........... 160 6 1 Nutrient composition and microbial contamination of pa steurized milk fed to calves1 ................................ ................................ ................................ ........................... 192 6 2 Nutrient composition (mean SD) of the starter grain and Saccharomyces cerevisiae fermentation products (SFP) (DM basis) ................................ ............... 193 6 3 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on growth parameters of preweaned calves ................................ ................................ 194

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10 6 4 Effect of feed ing Saccharomyces cerevisiae fermentation products (SFP) on health of preweaned calves ................................ ................................ ...................... 195

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11 LI ST OF FIGURES Figure page 3 1 Interferon gamma (IFN peripheral blood mononuclear cell (PB MC) proliferation. ................................ ........ 99 3 2. Lipopolysaccharide (LPS) stimulates tumor necrosis factor alpha (TNF production in cultured bovine blood in a dose and time dependent manner. 100 3 3 Trans 10, cis 12 conjugated linoleic acid ( t 10, c 12 CLA) isomer attenuates lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNF production in a dose dependent manner. ................................ ............................... 101 3 4 Isomer specific effect of trans 10, cis 12 conjugated linoleic acid ( t 10, c 12 CLA) on lipopolysaccharide (LPS) induced tumor necrosis facto r alpha (TNF ................................ ................................ ..... 102 3 5 Rosiglitazone attenuates lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNF ................................ .................. 103 3 6 Western blot analysis of nuclear nuclear factor kappa B p65 (NF kB p65). Peripheral blood monocytes were cultured without (control) or with lipopolysaccharide (LPS) for 24 h. ................................ ................................ .......... 104 3 7 Western blot analysis of cytosolic nuclear factor kappa B p65 (NF kB p65). Peripheral blood monocytes were cultured without (control) or with lipopolysaccharide (LPS) for 24 h. ................................ ................................ ........... 105 4 1 Survival curves for time to death in dairy calves according to passive transfer. ................................ ................................ ................................ .......................... 126 4 2 Pro bability of death within 75 d of age according to serum immunoglobulin (Ig) G concentrations in dairy calves calculated from the logistic function. ....... 127 4 3 Probability of death within 75 d of age according to serum total protein concentrations in dairy calves calculated from the logistic function. ................... 128 4 4 Probability of death within 75 d of age according to serum IgG as percent of total protein in d airy calves. ................................ ................................ ........................ 129 5 1 Effect of feeding pomegranate extract (POM) or antibiotics on grain intake of preweaned calves. ................................ ................................ ................................ ....... 161 5 2 Effect of feeding pomegranate extract (POM) or antibiotics on body weight of preweaned calves. ................................ ................................ ................................ ...... 162

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12 5 3 Effect feeding pomegranate extract (POM) or antibiotics on average daily gain of preweaned calves. ................................ ................................ .......................... 163 5 4 Effect of feeding pomegranate extract (POM) or antibiotics on plasma concentration of glucose and BHBA in preweaned calves. ................................ .. 164 5 5 Effect of feeding pomegranate extract (POM) or antibiotics on plasma concentration of NEFA and urea N in preweaned calves. ................................ ..... 165 5 6 Effect of feeding pomegranate extract (POM) or antibiot ics on hematocrit and serum total protein in preweaned calves. ................................ ................................ 166 5 7 Effect of feeding pomegranate extract (POM) or antibiotics on fecal score and attitude score of preweaned calve s. ................................ ................................ .. 167 5 8 Effect of feeding pomegranate extract (POM) or antibiotics on rectal temperature during first 14 d of age. ................................ ................................ ........ 168 5 9 Survival curves for the effect of feeding pomegranate extract (POM) or antibiotics to dairy calves in the preweaning period. ................................ ............. 169 5 10 Effect of fe eding pomegranate extract (POM) or antibiotics on neutrophil phagocytosis and oxidative burst in preweaned calves. ................................ ...... 170 5 11 Effect of feeding pomegranate extract (POM) or antibiotics on neutrophil phagocytosi s mean fluorescence intensity and oxidative burst mean fluorescence intensity in preweaned calves. ................................ ........................... 171 5 12 Effect of feeding pomegranate extract (POM) or antibiotics on anti ovalbumin IgG serum titers in response to ovalbumin immunization in preweaned calves. ................................ ................................ ................................ ........................... 172 6 1 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on grain dry matter intake of preweaned calves. ................................ .......................... 196 6 2 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on total dry matter intake of preweaned calves. ................................ ........................... 197 6 3 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on plasma glucose concentrations of preweaned calves. ................................ ........... 198 6 4 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on plasma hydroxybutyric acid (BHBA) concentrations of preweaned calves. .... 199 6 5 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on concentrations of nonesterified fatty acids (NEFA) in plasma of preweaned calves. ................................ ................................ ................................ ........................... 200

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13 6 6 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on concentrations of urea N in plasma of preweaned calves. ................................ .... 201 6 7 Effect of feeding Saccharom yces cerevisiae fermentation products (SFP) on rectal temperature during the first 14 d of age. ................................ ....................... 202 6 8 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on hematocrit of preweaned calves. ................................ ................................ .............. 203 6 9 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on serum total protein concentration of preweaned calves. ................................ ...... 204 6 10 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on white blood cell count of preweaned calves. ................................ .......................... 205 6 11 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on concen trations of neutrophil and lymphocyte in blood of preweaned calves. .... 206 6 12 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on concentrations o f basophil eosinophil and monocyte in blood of preweaned calves. ................................ ................................ ................................ ........................... 208 6 13 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP ) on neutrophil phagocytosis and oxidative burst of preweaned calves. ................... 209 6 14 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on neutrophil phagocytosis mean fluorescence intensity neutrophil oxidative burst mean fluorescence intensity in preweaned calves. ................................ ..... 210 6 15 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on anti ovalbumin IgG in preweaned calves. ................................ .............................. 211 7 1 Proposed model for lipopolysaccharide (LPS) and fatty acid signals in cultured blood cells. ................................ ................................ ................................ ..... 217

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14 LIST OF ABBREVIATIO NS ARA Arachidonic acid ADG Average daily gain AP 1 Activator protein 1 AHR Adjusted hazard ratio AOR Adjusted odd ratio APT Adequate passive transfer CFU Colony forming unit CLA Conjugated linoleic acid ClnA Conjugated linolenic acid ConA Concanavalin A COX Cyclooxygenase DMI Dry matter intake DIM Day in milk DHA Docosahexaenoic acid EPA Eicosapentaenoic acid FPT Failure of passive transfer fMLF N formyl methionyl leucyl phenylalanine GAE Gallic acid equivalent GIT Gastrointestinal tract GM CSF G ranulocyte macrophage colony stimulating factor GPx Glutathione peroxidase IC50 Half maximal inhibitory concentration Ig Immunoglobulin

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15 IGF 1 Insulin like growth factor 1 IL 1 Interleukin 1 LOX Lipoxygenase LPS Lipopolysaccharide LTB4 Leukotriene B4 MCP 1 Monocyte chemoattractant protein 1 NF NO Nitric oxide NFAT Nuclear factor of activated T cell MDA Malondialdehyde MFD Milk fat depression MHC Major histocompatibility complex OVA Ovalbumin p38 MAPK p38 mitogen activated pr otein kinase PBMC Peripheral blood mononuclear cells PGE 2 Prostaglandin E 2 PHA Phytohemagglutinin PMA Phorbol 12 myristate 13 acetate PMN Peripheral blood polymorphonuclear cells PMNL Polymorphonuclear leukocytes PPAR Peroxisome proliferator activa ted receptor PUFA Polyunsaturated fatty acid ROC Receiver operator characteristic

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16 ROS Reactive oxygen species SOD Superoxide dismutase SREBP Sterol response element binding protein STP Serum total protein TAG Triacylglycerol TBARS Thiobarbituric acid reactive substances TGF Transcription growth factor TH T helper cell Treg T regulatory cell TLR Toll like receptor TNF Tumor necrosis factor USDA United States Department of Agriculture

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17 Abstract o f Dissertation Presented t o t he Gr aduate School o f t he University o f Florida i n Partial Fulfillment o f t he Requirements f or t he Degree o f Doctor o f Philosophy DIETARY STRATEGIES TO MODULATE PERFORMANCE, HEALTH, AND IMMUNE RESPONSES IN HOLSTEIN CALVES By Milerky Cristina Pe rdomo Lozada December 2011 Chair: Lokenga Badinga Major: Animal Sciences A series of in vitro experiments were conducted to examine the effect of exogenous conjugated linoleic acid (CLA) and peroxisome proliferator activated receptor gamma (PPAR (LPS) stimulated tumor necrosis factor alpha (TNF production in cultured bovine blood, and to identify the signaling pathway through which LPS and PPAR biosynthesis in vitro Additionally t hree in vivo experiments were conducted in Holstein calves. The first experiment was an obs ervational prospective cohort study conducted with 1,247 calves from 7 dairy farms to determine the effects of passive immunity at 48 h of life on performance, morbidity and mortality risk, survival time and rearing costs in pre weaned Holstein calves. The second and third experiment tested the effects of feeding pomegranate extract and Sac charomyces cerevisiae fermentation products (SFP), respectively, on performance, health, and immunocompetence of Holstein calves during the preweaning period. Co incubatio n with trans 10, cis 12 CLA isomer or rosiglitazone decreased LPS induced TNF Rosiglitazone induced TNF attenuation was reversed when blood was treated with both rosiglitazone and GW9662, a selective PPAR

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18 Addition of rosiglitazo ne to the culture medium tended to reduce nucle ar factor kappa Bp65 concentration in nuclear and cytosolic extracts isolated from cultured PBMC. g/dL) had greater total DM intake and feed conversion ratio during the preweaning period. Daily grain intake and body weight w ere greater in APT calves d ur ing the first 30 d of age Calves with APT had reduced risk to develop morbidity, pneumonia, and multiple diseases, and had less nu mber of disease events per calf. Calves with failure of passive transfer (FPT) had 3.6 times greater hazard of dying in the first 75 d of age. Rearing costs and income per calf were greater for calves with APT than those with FP T T he probability of death with in the first 75 d of age decreased as IgG concentration in serum increased. Increase in STP reduced the probability of death, but values above 7. 0 g/dL increased the probability of death S erum IgG as percent of STP was the best predictor of probability o f death than STP and IgG concentration F eeding 15 mg of gallic acid equivalent /kg of body weight or subtherapeutic doses of antibiotics ( 200 mg oxytetracycline and 200 mg neomycin/d) to preweaned calves did not benefit performance health and immune and antioxidant response Supplementing dairy calves with SFP added to milk up to 4 g/d in the first 2 months of age did not affect performance. Calves fed SFP had an increased proportion of neutrophils with capacity for oxidative burst, but this effect did n ot influence measures of health based on daily fecal, nasal, ocular, cough and attitude scores, and rectal temperature.

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19 CHAPTER 1 INTRODUCTION the ruminant placenta does not p ermit the transfer of maternal immunoglobulins (Ig) to the fetus. Disease resistance in the neonate is therefore dependent upon the passive immunity derived from colostrum. Passive acquisition of maternal Ig is facilitated by the ability of the neonatal en terocytes to non selectively absorb Ig and other macromolecules during the first 24 to 36 h after birth (Weaver et al. 2000). Time of ingestion, quality and quantity of colostrum ingested, and method of colostral administration can influence the passive t ransfer of colostral Ig (Weaver et al., 2000) Adequate transfer of colostral Ig in the calf is represented by a serum IgG concentration of 1.0 g/dL or greater (Tyler et al., 199 6 ). This correlates with a serum total protein (STP) concentration greater th an or equal to 5.2 g/dL (Tyler et al. 1998). In 2007, the National Animal Health Monitoring System (NAHMS) reported that in the USA, almost one of five heifer calves (19.2%) had failure of passive transfer of immunity based on serum IgG testing (IgG < 1. 0 g/dL). In the west region, 21.2% of calves had FPT, whereas in the east region, the corresponding proportion was 18.8% (NAHMS, 2007). Due to the fact that the neonate is dependent upon the passive immunity derived from colostrum, the most critical ti me in the life of the dairy replacement is during the first few days after birth when morbidity and mortality are greatest. A United States Department of Agriculture (USDA) study indicated that pre weaning mortality of calves during preweaning period was 7. 8%, whereas mortality after weaning was only 1.8%. Diarrhea or other digestive problems ac counted for the majority of pre weaned heifer

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20 deaths (56.5%). Respiratory disease was the single largest cause of weaned heifer deaths (46.5%) (NAHMS, 2007). In the Un ited States, subtherapeutic levels of antimicrobials are added to milk or milk replacer for calves as an attempt to reduce the incidence of disease and to improve feed efficiency and g rowth. However, this method has been blam ed for promoting antibiotic res istance to gut bacteria of calves (Langford et al., 2003) and for eliminating beneficial intestinal flora and does not stimulate or support the immune system Strategies to reduce antimicrobial use but maintain calf health and welfare are needed. Conjugat ed linoleic acid is a collective term describing a mixture of positional and geometric dienoic isomers of LA. Interest in CLA research stems from the well documented anticarcinogenic, antiatherogenic, antidiabetic, and antiobesity properties of CLA in rod ent models (Badinga and Greene, 2006). In addition, a rapidly expanding literature in this field suggests that CLA modifies immune function (Hayek et al., 2006 ; Bassaganya Riera et al., 2001a, 2001 b ). Pomegranate is well known for its antimicrobial, antiox idant, antiinflammato ry, and anticancer properties. Yeast cells and compounds produced during fermentation activity might be responsible for the positive effects on performance and health when yeast products are incorporated into the diet of animals (Magal hes et a l ., 2008). However, few studies have evaluated the effect of pomegranate, yeast or CLA on performance on immune system of calves. For this reason, the goal of this research project was to examine strategies to modulate performance, health and immu ne responses in Holstein calves. This dissertation begins with an overview of immune system, followed by review of immune system of newborn cattle, passive transfer of immunity in calves, as well as

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21 information on the effects of supplemental conjugated li noleic acid and pomegranate on productive performance and health on cattle (Chapter 2). Experiments described in Chapters 3 were designed to examine the effect of exogenous CLA and a PPAR agonist on LPS stimulated TNF d, and to identify the signaling pathway through which LPS and PPAR biosynthesis in vitro Chapter 4 presents an analytical observational cohort study to evaluate the effects of serum IgG concentration at 48 h of life on productiv e and economic performance, morbidity and mortality in pre weaned Holstein calves. Chapter 5 summarizes an in vivo study which determined the effects of feeding liquid pomegranate extract on productive performance and health response during the first 63 d o f age. Chapter 6 presents other in vivo study which evaluated the effect of feeding different amounts of SFP on immune status, health and performance of Holstein preweaned calves. The final chapter is a general conclusion and discussion of the major findin gs of this research project (Chapter 7).

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22 CHAPTER 2 REVIEW OF LITERATURE Overview of the Immune System All higher organisms are constantly exposed to microorganisms, but seldom become ill. The immune system protects the host from microbial pathogens, a nd clears origin can be attributed to Edward Jenner who, in 1796, reported the first experimental vaccine (Murphy et al., 2008). The e pithelial layer of the skin and t he mucosal surfaces of the respiratory, urogenital and gastrointestinal tracts (GIT) and eyes constitute the first barriers against pathogen invasion. Their defense mechanisms include the prevention of pathogen adherence and the secretion of antimicrobial enzymes and peptides. When these barriers are overcome, the immune system has two major arms which recognize and eventually eliminate the pathogen, the innate immune system and the adaptive immune system. Innate Immune System The innate immune response is activated within minutes to hours after tissue damage or infection and kills the microbes. This early innate system depends on non specific receptors recognizing common features of pathogen. They do not lead to immunological memory. This innate immune sys tem consists of soluble plasma proteins and inflammatory cells. The main innate immune response is inflammation, which increases the blood flow to the site of trauma, bringing cells to attack and destroy pathogens.

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23 The cellular components of innate immuni ty originate in the bone marrow. These include macrophages and dendritic cells, which are the first line of defense, the cells). Microbial invasion of host tissues is first detected by resident macrophages, which are mature forms of circulating monocytes residing in tissues, and immature dendritic cells. These are antigen presenting cells which are soon reinforced by the recruitment of large numbers of neutrophils to t he site of infection. The first important effect of the interaction between pathogens and macrophages or neutrophils is phagocytosis. This is an active process in which the pathogen is first surrounded by the phagocyte membrane and then internalized in an endocytic vacuole or phagosome. The phagosome becomes acidified, killing most pathogens. Macrophages and neutrophils have granules, and a lysosome that contains enzymes, proteins and peptides that can attack the microbe. During phagocytosis, macrophages an d neutrophils produce a variety of other toxic substances such as antimicrobial peptides, nitric oxide, superoxide anion and hydrogen peroxide that help kill the microorganism. Nitric oxide is produced by nitric oxide synthase. S uperoxide is generated by a NADPH oxidase enzyme in a process known as respiratory burst. This process is accompanied by an increase in oxygen consumption; superoxide anion is converted by the enzyme superoxide dismutase to H 2 O 2. Then H 2 O 2 produces toxic compounds such as the hydr oxyl radical, hypochlorite, and hypobromite (Murphy et al., 2008). Neutrophils are short lived cells whereas macrophages are long lived and continue to generate new lysosomes.

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24 The cells of the innate immune system have different receptors to recognize pat hogens. Some are displayed o n the surface of immune cells, whereas others are produced as secreted molecules. Mannose receptors are found on macrophages; whereas CD14 is found on monocytes and macrophages, which bind to lipopolysaccharide (LPS) present on the surface of Gram negative bacteria and allow it to be recognized by Toll like receptors (TLR). Secreted molecules promote the phagocytosis of pathogens by opsonization or by the activation of the complement system. When microbial presence is detected t hough membrane bound receptors, the transcription factor nuclear factor k appa B (NF directs the expression of molecules associated with the inflammatory response (i.e cytokines, chemokines, prostanoids, leukotrienes, p roteases and acute phase proteins). Also phosphorylation of the members of the mitogen activating protein kinase family modulates cytokines expression. The second important effect of the interaction between pathogens and macrophages or dendritic cells is the production of cytokines that set up the inflammation in the tissue. Inflammation is characterized by pain, redness, heat and swelling at the site of an infection. The objectives of inflammation are: 1) to augment the killing of microorganism s 2) to pr event the spread of infection, and 3) to repair tissues. Cytokines cause the dilation of local blood vessels, leading to the movement of neutrophils and monocytes from the blood vessel into the infected tissue, guided by chemokines produced by activated m acrophages and by adhesion molecules.

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25 The complement system is a group of plasma proteins activated by pathogens or by pathogen bound antibody. There are three pathways: the classical, the lectin, and the alternative pathway. These pathways lead to a casc ade of reactions that occur on the surface of pathogens and generate active components with various effectors functions such as mediator of local inflammation, phagocyte recruitment, opsonization of pathogens, removal of immune complex and, lysis of certai n pathogens and cells through damage to their membrane. Adaptive Immune System When the innate immune system is overcome by the pathogen or infection, the adaptive immune system is activated. Adaptive immune system recognizes a particular pathogen and prov ides enhanced protection against reinfection. There are two major types of lymphocytes: B and T lymphocytes. The B lymphocytes mature in the bone marrow and are the source of circulating antibodies, and have cell surface Ig molecules that act as receptor s for antigen and, once activated, these Ig are secreted as soluble antibody against extracellular pathogen s The T lymphocytes mature in the thymus and recognize peptides from pathogens presented by major histocompatibility complex molecules (MHC) on infect ed cells or antigen presenting cells (i.e., dendritic cells, macrophages, and B cells). There are two types of effectors T cells. CD4+ T cells, called helper T cell, which activate macrophages and B cells, and CD8+ T cells, the cytotoxic T cells, which kil l infected target cells. Lymphocytes are derived from hematopoietic stem cells in the bone marrow. In the bone marrow, the precursor B cells rearrange their Ig genes, an action that is independent of the antigen. Surviving mature B cells carrying an antig en receptor (IgM and IgD) enter secondary lymphoid tissues through the bloodstream, whereas antigens

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26 move from the infection site to secondary lymphoid tissues via lymphatic system. The B cells are activated when they encounter with their specific foreign antigen. Activated B cells proliferate and differentiate into antibody secreting plasma cells and long lived memory cells (Murphy et al., 2008). The T cell precursors migrate from the bone marrow to the thymus, where T cell receptor genes are rearranged. Surviving T cells leave the thymus though bloodstream to peripheral lymphoid organs, where they also may encounter their specific foreign antigen and become activated. Activated T cells proliferate and differentiate into effector T cells (cytotoxic cells and helper cells) or memory T cells (Murphy et al., 2008). Effector cells return via the lymphatics to the bloodstream to arrive at the infection site. Mature B and T cells that have not yet encountered their specific antigens are known as nave T cells (Murphy et al., 2008). Cytotoxic T cells kill target cells that display a peptide fragment of cytosolic pathogen (e.g. virus infected cell). Helper T cells are divided in to T helper 1 (T H 1), T helper 2 (T H 2), T helper 17 (T H 17), and T regulatory cells ( T reg ). The T H 1, T H 2 and T H 17 cells express the CD4+ co receptor and promote inflammatory (T H 1), humoral or allergic (T H 2) or acute (T H 17) responses to pathogens. These different responses depend on the type of cells that they activate. T H 1 cells produce cy tokines that activate macrophages and B cells and the activated B cells produce opsonizing antibodies (IgG). T helper 2 cells produce cytokines that stimulate B cells to produce antibodies involved in anti parasitic and allergic type response (IgE). The Th 17 cells induce local epithelial and stromal cells to produce chemokines to recruit neutrophils to sites of infection early in the adaptive immune response (Murphy et al.,

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27 2008). The T reg cells suppress the other T cell activities and help prevent the deve lopment of autoimmunity during immune response. As mentioned above, CD4+ T cells can differentiate into several cell subsets. The fate is regulated by the signal provided by the antigen presenting cells, principally dendritic cells. CD4+ T cells differenti ate into T H 1, when dendritic cells produce IFN and IL 12. Interleukin 4 induces T H 2 cell development. Interleukin 6 interacts with transcription growth factor beta (TGF H 17 cells. When pathogens are absent, the abundance of TGF 6, IFN 12 favor the development of T reg cells (Murphy et al., 2008). These different types of effector T cells produce molecules with different effects, such as cytokines and proteases. CD8+ effector cells release proteases (i.e. perforin, gran zymes) and IFN to block viral replication and eliminate the virus from infected cells without killing them. Cytokines such as IFN 2 are produced by T H 1 cells, and IL 4 and IL 5 by T H 2 cells. The T H 17 cells produce IL 17 and IL 6, whereas T reg ce lls produce TGF 10. The humoral immune response to infection involves the production of antibody by plasma cells derived from B lymphocytes. Antibodies are proteins that bind to antigens, and there are 5 classes of antibodies: IgG, IgM, IgA, IgD, and IgE. Immunoglobulin G has the greatest concentration in serum. These antibodies are specific and will only bind to the antigens that caused their production. Antibodies protect the host from infection in three ways: neutralization, opsonization and com plement activation. They can inhibit the toxic effect of pathogens by binding to them; they can also coat them (promoting their phagocytosis), and trigger the activation of the complement system which enhances

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28 opsonization and lysis of pathogens. A primary response to antibodies occurs during the first exposure when only a small amount of antibodies is produced. A secondary response occurs with repeated exposure to the pathogen and is associated with greater concentrations of antibody in blood. Immunity of Newborn Cattle The newborn calf starts its life with a competent, but still naive immune system, and specific responses develop over time. Susceptibility of newborns to pathogens is not attributable to any inherent incapacity to raise an immune response, b ut is caused by the fact that their immune system is unprimed. Kampen et al. (2006) showed that both lymphocyte subpopulations and neutrophilic granulocyte functions reached stable levels during the first 6 months of life. This does not mean that a young calf cannot respond to antigens, but the response is weaker, slower, and easier to overcome (Cortese, 2009). Differences between calves and adult animals with regard to the different subpopulations of lymphocytes and their relative importance have been stu died. Calves and heifers have higher absolute numbers of lymphocytes in peripheral blood than adult animals, and young calves generally have a higher proportion of T cells expressing the cells (Kampen et al., 2006) Percentages of CD4+, CD8+ and l ymphocytes stabilize during the first 10 12 weeks of life. The decrease in the relative percentage of T cells is attributable to an increase in the absolute numbers of CD4+ and B cells, rather than a change in absolute Tcell numbers with age. The pro portion of B lymphocytes increases rapidly during the first 6 to 8 weeks of life, followed by an increase in gamma globulin concentration after the age of 10 to 12 weeks (Kampen et al., 2006).

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29 Phagocytosis by polymorphonuclear leukocytes is the first and major defense mechanism against bacterial and fungal pathogens. After colostrum ingestion, the polymorphonuclear leukocyte activity increases rapidly. Phagocytosis, respiratory burst and bactericidal activity of calf neutrophils is intact and functional fr om the first week of life. Decreased phagocytic capacity against Escherichia coli has been demonstrated in neutrophils from neonatal calves before colostrum ingestion compared to 3 9 week old calves, whereas monocytes of neonates exhibited an enhanced ph agocytic activity. The oxidative burst activity of neutrophils, as well as of monocytes was higher in newborn calves than in old calves (Menge et al., 1998). Kampen et al. (2006) found high and stable percentages of phagocytosis during the first 29 week s o f life, whereas the oxidative burst is high during the first 2 weeks, decreases until 12 weeks, and then increases again. Passive Immunity in Newborn Cattle Calves at birth are agammaglobulinemic because the syndesmochorial placenta of the cow forms a syn cytium between the maternal endometrium and the fetal trophectoderm. This syncytium separates the maternal and fetal blood supplies, and prevents the transmission of Ig. Consequently, calves need to ingest and absorb colostral Ig to acquire passive immunit y. Colostrum is the first secretion from the mammary gland for the first 24 h after parturition. Its composition differs from milk composition. Bovine colostral lactose concentration is inversely proportional to other colostral constituents. Percentages of protein, fat and lactose in colostrum and milk have been reported to be 13.3, 6.4 and 2.5 vs. 3.9, 3.2, and 4.9% respectively (Rauprich et al. 2000). In addition to nutrients, colostrum contains antibodies, antibacterial proteins, lactoferrin, cytokine s, hormones,

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30 growth factor and immune cells. Because of these properties, colostrum has short and long term impacts on calves. Colostrum is necessary to assure good passive transfer in newborn calves, reducing morbidity and mortality in the pre weaning and weaning periods ( Beam et al., 2009; Tyler et al., 1998; Robinson et al., 1988) It also has a positive impact on health and production of replacement heifers ( DeNise et al., 1989 ). The most important components of colostrum are Ig. Although colostrum con tains several types of Ig (IgG, IgA, IgM), IgG represents approximately 85% of the Ig in colostrum. In the cows, transport of Ig from serum to the mammary gland starts weeks before parturition and reaches a peak 1 3 d before parturition. Concentration of I g in colostrum is facilitated by receptors on the mammary alveolar epithelial cells. Glandular epithelial cells cease expressing this receptor at the beginning of lactation. Transfer of Ig from the dam to the neonate is called passive transfer. Failure of passive transfer (FPT) is not a disease, but is a condition that predisposes the neonate to the development of diseases. Due to the fact that in 2007 20% of dairy calves are estimated to suffer from FPT (Beam et al., 2009), this condition is one factor tha t must be considered when raising calves. Factors Affecting Passive Transfer of Colostral Immunoglobulin The timing of colostrum ingestion, Ig concentration of colostrum, method and volume of colostrum offered, metabolic disturbances and environment condit ions have all been reported as the most important effectors of Ig passive transfer. During the first 12 h of life, the enterocytes of the small intestine nonselectively absorb a variety of macromolecules by pinocytosis. Immunoglobulins are transported acro ss the cell and into the lymphatics by exocytosis. The Ig then access the circulatory system through thoracic duct (Weaver et al., 2000).

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31 The termination of macromolecule absorption could be explained by a decrease in pinocytotic ability of a more mature population of gut epithelial cells. Immunoglobulin transfer across the epithelium is optimal within the first 4 h of life and begins to decline rapidly after 12 h. Calves fed with colostrum the first 4 h of life have significantly higher serum IgG concent ration than those fed later when similar concentrations and volumes of colostrum are fed. In a study conducted by Matte et al. (1982), percentages of ingested IgG that appeared in plasma were 65.8, 46.9, 11.5, 6.7 and 6% at the age of 6, 12, 24, 36 and 48 h after birth, respectively, whereas total fecal IgG increased linearly with age. The concentration of Ig in colostrum is one of the most important factors for successful transfer of maternal Ig. There is variation of colostral Ig concentration of the fir st milking between cows. In regard to Ig concentration, colostrum can be of good or poor quality. When the colostrum has 50 mg/mL or more of Ig, it is considered to be of good quality, and is direct used to feed newborn calves, or frozen thawed before use The most important factors associated with the Ig concentration in Holstein cows are time after calving, and parity. There are contrasting results relating to Ig concentration and time after calving. Some studies have shown that Ig concentration is highe st in the first colostrum immediately after calving and drops rapidly thereafter, whereas other studies have show n that the Ig concentration in colostrum drops slightly in the first 24 h after calving and drops sharply thereafter. The time of colostrum col lection could explain this discrepancy and previous nursing by the calf may also be important modulator of colostral Ig concentration

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32 There are contrasting results in relation to colostral Ig concentration based on parity. Shearer et al. (1992) reported t hat cows in their second, third or fourth or greater parity were 2.4, 2.7 and 2.7 times more likely to have good quality colostrum compared to cows in their first parity. There was no significant difference in the quality of colostrum among parities after the first one. Tyler et al. (1999 a ) found that cows in their third or greater lactation had higher colostral IgG concentrations than cows in their first or second lactation. They found no significant difference in colostral IgG concentration among cows in the first or second lactation. The method of feeding colostrum can affect passive transfer of colostral IgG and of failure of passive transfer has been reported in calves allowed to obtain colostrum via nursing. This FP T may be explained by the fact that the calf may ingest an inadequate quantity of colostrum and/or the time of sucking is delayed. Beam et al. (2009) determined that the odds ratio of FPT when the calves are allowed to nurse was 2.4. The most common method s of colostrum feeding are esophageal tube feeder and nipple bottle. The use of esophageal tube feeder is convenient and quick, but the esophageal groove reflex is not triggered, resulting in colostrum deposition in the forestomachs, and delaying the arriv al of colostrum to the small intestine for its absorption. When a nipple bottle or bucket is used, the esophageal groove channels colostrum into the omasum and abomasum ( Lateur Rowet and Breukink, 1983 ) Comparison of nipple bottle and esophageal tube adm inistration of equal volume and IgG mass of colostrum demonstrated that calves allowed to suck a nipple bottle have slightly higher average serum IgG concentration when a small (1.5 L) volume of

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33 colostrum is fed (Godden et al., 2009). These authors found that feeding a large volume (3L) of colostrum, resulted in APT regardless of the feeding method used, and suggested that the selection of methods of feeding must depend on the volume of colostrum. The apparent efficiency of IgG absorption in newborn calv es is less than 40% ( Arthington et al., 2000 ). This efficiency depends of the volume offered and feeding method. God den et al. (2009) reported efficiencies of 51 and 40% when calves were fed with small volume of colostrum (1.5 L) using nipple bottle or es ophageal tube, respectively, and 41 and 39% when 3 L were offered. The volume of colostrum used really depends on the amount of IgG in the colostrum, and the body weight of the calf. To reach at least 10 mg/mL of IgG, a newborn calf weighing 40 kg needs t o be fed with 2.1 L of colostrum with at least 50 mg/mL of IgG. When a fixed amount of colostrum is fed, the Ig concentration is the only determinant of total Ig delivered, and higher serum IgG concentrations are reached by feeding higher IgG colostrum. R espiratory acidosis can develop during prolonged parturition. Calves with prolonged calving time or dystocia are more likely to have respiratory acidosis than calves resulting from a normal parturition ( Szenci 1983) However, Drewry et al. (1999) failed t o find a correlation between respiratory acidosis and IgG absorption in Holstein calves. The increased rate of FPT seen in animals born following abnormal calving may be related to the incapacity of calf to get up and drink colostrum though bottle, rather than as a result of abnormalities in absorptive capacity ( Weaver et al., 2000 ).

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34 H igh air temperatures during late pregnancy and the early postpartum period markedly affected the composition of colostrum from primiparous dairy cows (Nardone et al 1997). In addition environmental stress may in some way interfere with the natural suckling of colostrum by neonatal calves. Under Florida environmental conditions, Donovan et al. (1986) found better Ig absorption during February and March and poorest during Jul y and August. It may be speculated that calves born during periods of heat stress are less vigorous and therefore less lik ely to nurse from their dams or via artificial feeding, resulting in inferior rates of passive transfer even with higher concentration s of Ig in the colostrum. Effect of Adequate Passive Transfer Passive immunization through the absorption of colostral Ig is an essential process for the well being of the bovine neonate. Passively acquired Ig assists the calf through its early stages of growth and development. Although the half life of colostrum derived Ig is short, approximately 21 d, it directly influences the course of disease in the calf for the first 4 to 6 months (Robinson et al., 1988) Some studies have been performed in Holstein calves using STP or IgG as a measure of passive transfer to evaluate th e short and long term effect of APT (Beam et al., 2009; Tyler et al., 1998; DeNise et al., 1989; Robinson et al., 1988). Failure of hat extends into the juvenile period. Tyler et al. (1998) evaluated the relationship between STP in the first week of life and survival to 16 weeks of age in 3,479 Holstein replacement heifers over a period of 10 years. They found that 34% of calves had S TP concentration less than 5.0 g/dL and 60.5% had STP less than 5.5 g/dL. Calves with inadequate passive transfer (STP less than 5.0 g/dL) experienced increased mortality until at least 10 weeks of age.

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35 The APT of IgG not only reduces mortality but also in creases the average daily gain (ADG) during the first 6 month of life (Robinson et al. 1988). Serum IgG concentrations greater than 1.2 g/dL at 24 to 48 h of age in heifer calves are associated with increased daily weight gain up to 180 d of life and grea ter weaning weights compared to calves with low serum IgG concentration. Appropriate growth and development during the first 6 months is critical to subsequent growth, development, reproduction and production of the dairy replacement heifer. A study condu cted at the University of Arizona demonstrated that heifers with FPT had significantly lower mature equivalent milk production during the 1 st lactation and had a greater tendency to be culled in the 1 st lactation when compared with those with APT. Failure of passive transfer had no effect on age at first calving ( DeNise et al., 1989 ). Modulation of Immune Response of Calves by Other Colostrum Constituents There is evidence that antibodies from colostrum remain ing in the lumen after feeding also provides loc al immunity against enteric viral infections and diarrhea cause d by bacterial enterotoxins. Berge et al. (2009) conducted a study to evaluate the effect of supplementing colostrum to the milk replacer during the first 14 d. Although no differences in morta lity or incidence of respiratory disease were detected, colostrum supplementation was effective in reducing diarrheal disease and antimicrobial treatments. Antibody transfer to newborn calves through colostrum is not the only way to transfer maternal imm unity. Other constituents of colostrum such as cytokines, leukocytes, and proteins such as lactoferrin have the ability to modulate the immune response in calves. B ovine colostrum contains viable leukocytes which are immunologically active. Their effects on the immune system can be mediated by soluble

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36 factors (i.e. cytokines) produced by maternal leukocytes or exerted directly when these maternal leukocytes cross the intestinal barrier and interact with the neonatal immune system in neonat al circulation or Tenorio et al., 2002). Evaluating the effect of maternal cells transferred with colostrum on cellular responses to pathogen antigens in neonatal calves, Donovan et al. (2007) found that live maternal leukocytes can transfer to neonate the ability to respond against antigens to which the dam has developed immune response. This ability occurs during the first 24 h after colostrum ingestion, and appears to have no further direct effect by 7 d after ingestion. This posi tive effect is absent when calves receive frozen colostrum. Studies have not evaluated the viability of colostral leukocytes after pasteurization. Colostrum contains high concentration o f cytokines (IL 6, TNF and INF which may be produced and secreted in the mammary gland and may influence neonatal immunity (Hagiwara et al., 2000). The proinflammatory cytokine, IL contributes to the defense against pathogens by generating fever, activating lymphocytes, and promoting mobilization of leukocyt es into the sites of infection. In vitro studies indicated that cytokines in bovine milk and bovine colostrum have the ability to activate the oxidative burst of polymorphonuclear leukocytes (Sugisawa et al., 2003). Feeding Conjugated Linoleic Acid to Dair y Cattle Conjugated linoleic acid (CLA) designates a series of geometric and positional isomers of linoleic acid (C18:2), of which cis 9, trans 11 CLA isomer and trans 10, cis 12 CLA isomer are predominant. Contrary to the methylene interrupted double bond s of octadecadienoic acids, the double bonds in CLAs are separated by a single carbon carbon bond, and these double bonds can occur at multiple locations along the carbon cis or the tran s

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37 configuration. Conjugated linoleic acids are found predominantly in ruminant derived products and can be produced industrially by partial hydrogenation of linoleic acid. Conjugated linoleic acids originate from incomplete biohydrogenation of polyunsatura ted fatty acids (PUFA) in the rumen (Chouinard et al., 1999 ) or from the desaturation of trans vaccenic acid in the adipose tissue or mammary gland (Griinari et al., 2000). When ruminants consume feeds, dietary lipids are hydrolyzed to free fatty acids by microbial lipases, and then the unsaturated free fatty acids are hydrogenated by the microorganisms in the rumen. The cis 9, trans 11 CLA isomer is the first intermediate from isomerization of linoleic acid produced by Butyrivibrio fibrisolvens and other bacterial species. Some of the cis 9, trans 11 CLA isomer is quickly reduced to trans 11 C18:1 vaccenic acid or C18:0 stearic acid, which are available for absorption by the small intestine. Rumen metaboli sm linolenic acid ( cis 9, trans 12, cis 15 octadecatrienoic acid) is characterized by reduction of the cis double bonds to trans 11 vaccenic acid. Cis 9, trans 11 CLA isomer and trans 11 vaccenic acid can escape the rumen to be incorporated into fat in m ilk and meat (Chouinard et al., 1999). 9 9 desaturase introduces a cis double bond between carbons 9 and 10 of fatty acids. This pathway involves the desaturat ion of trans 11 vaccenic acid. Although some of the of cis 9, trans 11 CLA isomer originated in the rumen, the major portion of cis 9, trans 11 CLA isomer in milk and meat comes from this endogenous synthesis (Griinari et al., 2000).

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38 Conjugated linoleic acids are found in plant oils (e.g. coconut and safflower oil), and animal tissues. The main dietary source of CLA is animal products, of which ruminants are the greatest contributors. Seventy six percent of the CLA in meat from ruminants is the cis 9, tra ns 11 CLA isomer (Chin et al., 1992). Among ruminant animals, the lamb has the highest concentration of CLA with 5.6 mg CLA/g fat, followed by beef which ranges from 2.9 to 4.3 mg CLA/g fat, whereas the veal has the lowest with 2.7 mg CLA/g fat (Chin et al ., 1992). Dairy products contain considerable levels of CLA. Concentrations of CLA in natural cheese are elevated and comparable to those in milk (5.5 mg CLA/g fat), ranging from 2.9 to 7.1 mg CLA/g fat. The Cis 9, trans 11 CLA isomer represents 80 90% of the total CLA in milk fat and more than 83% in natural cheeses (Chin et al., 1992). Foods derived from non ruminants are far lower in CLA content except for turkey, which contains 2.5 mg CLA/g fat (Chin et al., 1992). The content in seafood ranges from 0 .3 to 0.6 mg CLA/g fat. Plant oils contain from 0.1 to 0.7 mg CLA/g fat which is far less CLA than animal products. The two major CLA isomers in plant oils are the cis 9, trans 11 CLA isomer and trans 10, cis 12 CLA isomer with 43 and 40%, respectively (Ch in et al., 1992). Effect of Dietary CLA on Animal Performance Conjugated linoleic acids have positive effects on BW of piglets at weaning when sows are supplemented with 0.5% of CLA from 7 d before parturition until 7 d postpartum (Corino et al., 2009). It has been reported that CLA supplementation al. (2000) fed 36 barrows (initially 37.6 2.8 kg) with 0.5% of CLA 60 (Tonalin CLA 60). Pigs fed with CLA had growth perform ance similar to that of the control pigs. Pigs fed

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39 CLA 60 had greater saturation of fatty acids in the adipose tissue at the 10th rib than pigs fed the control diet. These results were confirmed by Wiegand et al. (2002) who supplemented growing finishing barrows with 0.75% of CLA 60. Additionally, these authors found that CLA supplementation increased loin muscle area and improved the marbling and firmness. The results of sensory panel showed that characteristics of loin chops were not changed by dietary CLA. Weber et al. (2006) supplemented young female pig s with 1 % CLA (CLA 60) or 1% soybean oil. The predicted carcass lean percentage was increased in pigs fed CLA. Abdomen of gilts fed CLA was subjectively and objectively firmer. Dietary CLA increased th e concentration of saturated fatty acids and decreased the concentration of unsaturated fatty acids of the abdominal fat, both layers of backfat, and loin muscle. Evaluating the effect of CLA supplementation in beef cattle, Gillis et al. (2004) supplement ed heifers with 2% rumen protected conjugated linoleic acid during 32 or 60 d, and found that ADG, dry matter intake (DMI), feed efficiency, and carcass characteristic did not differ with dietary treatment or supplementation length. Studies have evaluated the effect of supplemental CLA on transition Holstein cows (Odens et al., 2007; Selberg et al., 2004 ; Perfield et al., 2002) or on lactating Holstein cows ( v on Soosten et al., 2011; de Veth et al., 2005). The CLA supplementations were performed by rumina l infusion (de Veth et al., 2005), abomasal infusion (Shingfield et al., 2009; Perfield et al., 2002), or using rumen protected (RP) CLA (Odens et al., 2007; Casta eda Gutierrez et al., 2005; Selberg et al., 2004; Perfield et al., 2002). Although these stu dies evaluated different doses of CLA (from 6 g/d to 600 g/d in prepartum and

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40 postpartum period), DIM, milk yield, and the yield and content of true protein and lactose in milk were unaffected by CLA supplementation. All aforementioned studies reported mi lk fat reduction. Selberg et al. (2004) reported that RP CLA mix (150 g/d 28 d prior to expecting calving date and 225 g/d in the postpartum period) d ecreased milk fat percentage by 17%. Casta eda Gutierrez et al. (2005) found that adding RP CLA (32 and 63 g/d) to the diet decreased milk fat percentage by 10.2% and 19.4%, respectively. Odens et al. (2007) reported that rumen inert CLA treatments (600 g/d) during the transition period decreased overall milk fat content by 26.0%; CLA induced milk fat depressi on (MFD) becomes significant by d 8 postpartum. v on Soosten et al. (2011) found that supplementation of 100 g/d of CLA mix to transition cows decreased milk fat content in period 1 (1 to 42 DIM) by 14.1% and in period 2 (from 42 to 105 DIM) by 25.4%. Conj ugated linoleic acid isomers have similar effect on MFD. d e Veth et al. (2005) evaluated supplementation of trans 10, cis 12 CLA isomer in the form of calcium salts of CLA or formaldehyde protected CLA in mid to late lactation Holstein cows. They reported that calcium salt and formaldehyde protected CLA decreased milk fat percentage by 38.4% and 54.3%, respectively. Abomasal infusion of 6 g/d of trans 10, cis 12 CLA isomer over a 5 d period reduced milk fat secretion by 41.5% ( Shingfield et al., 2009). O ther CLA isomers have been postulated to contribute to diet induced MFD. Abomasal infusion (5 g/d) of a 9, 11 CLA mix ( trans 9, cis 11 CLA isomer; cis 9, trans 11 CLA isomer; and tran 9 trans 11 CLA isomer), a trans 9, trans 11 CLA isomer supplement, or trans 10, cis 12 CLA isomer supplement were evaluated by Perfield et

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41 al. (2007). Milk fat yield was reduced by 15% for the CLA mix and by 27% for the trans 10, cis 12 CLA isomer, but trans 9, trans 11 CLA isomer had no effect on milk fat yield. On the basis that cis 9, trans 11 CLA isomer did not alter milk fat concentration, they suggested that trans 9, cis 11 is the CLA isomer in the 9, 11 CLA mix responsible for the reduction in milk fat synthesis, although to a lesser magnitude than that observed f or trans 10, cis 12 CLA isomer. Other studies have shown CLA supplementation to dairy cows reduces short to medium chain fatty acid (C4 to C14) concentrations and increases linoleic and linolenic acid concentrations in milk fat (de Veth et al., 2005; Selb erg et al ., 2004). However, Perfield et al. (2002) reported that CLA supplement reduced the secretion of fatty acids of all chain lengths. Dietary supplementation of CLA has also been reported to increase trans 10, cis 12 CLA concentration in milk fat (Ode ns et al., 2007; Casta eda Gutierrez et al., 2005; de Veth et al., 2005; Selberg et al., 2004; Perfield et al 2002). On the other hand, Bernal Santos et al. (2003) and Selberg et al. (2004) reported that concentration of cis 9, trans 11 CLA isomer in milk fat was similar in control and CLA treatments, whereas Odens et al. (2007) Casta eda Gutierrez et al. (2005), and de Veth et al. (2005) and reported an increase of cis 9, trans 11 CLA isomer following CLA supplementation. Perfield et al. (2007) found th at abomasal infusion of trans 10, cis 12 CLA isomer reduced short to medium chain fatty acid concentrations whereas linoleic and linolenic acid concentrations were increased in milk fat. This isomer did not affect the cis 9, trans 11 CLA isomer concentra tion in milk fat, whereas trans 10, cis 12 CLA isomer was increased.

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42 Effect of CLA on Milk Fat Depression Milk fat depression occurs in dairy cows when they are fed highly fermentable rations or dietary supplements of plant or fish oils. Milk fat depressio n can result in a reduction in milk fat yield of up to 50%, and the decrease involves fatty acids of all chain lengths (Bauman et al., 2008). The biohydrogenation theory has been proposed to explain the diet induced MFD. This theory stipulates that inhibit ion of mammary lipid synthesis is caused by specific fatty acids that are intermediates in the biohydrogenation of dietary PUFA and are produced only under certain conditions of rumen fermentation. Trans 10, cis 12 CLA isomer is the first CLA isomers asso ciated with reduction in milk fat yield (Bauman et al., 2008). In addition, trans 9, cis 11 and cis 10, trans 12 CLA isomers were identified as inhibitors of milk fat synthesis (Perfield et al., 2007). Whereas cis 9, trans 11 CLA isomer did not inhibit mil k fat synthesis. At similar doses, cis 10, trans 12 CLA isomer was similarly or slightly more effective in reducing milk fat yield than trans 9, cis 11 CLA isomer. Expression of lipogenic enzymes is coordinately stimulated by a class of transcription facto rs known as master regulators of lipid synthesis, and one of these is the sterol response element binding protein (SREBP) family. Therefore, transcription of mammary genes associated with milk fat synthesis is coordinately down regulated during CLA and di et induced MFD (Bauman et al., 2008). Acetyl CoA carboxylase, fatty acid synthase, lipoprotein lipase, fatty acyl 9 desaturase are SREBP1 regulated lipogenic enzymes that exhibited a reduction in expression during treatment with trans 10, ci s 12 CLA or diet induced MFD (Bauman et al., 2008).

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43 Effect of CLA on Reproductive Performance in Dairy Cattle A multi study analysis evaluated the efficacy of CLA for improving reproduction in early lactation dairy cows (de Veth et al., 2009) The data wer e obtained from 5 studies (Mann et al., 2007; Castaeda Gutierrez et al., 2007, 2005; de Veth et al., 2005; Bernal Santos et al., 2003). This analysis found that supplementation with trans 10, cis 12 CLA isomer was associated with an increase in the probab ility of cows becoming pregnant and a decrease in the time to conception with increasing dose. There was a quadratic effect of CLA dose on pregnancy, increasing up to an optimum dose of 10.1 g/d trans 10, cis 12 CLA isomer, after which the beneficial effec ts of CLA decreased. In relation to time to first ovulation, this study found that increasing the rate of trans 10, cis 12 CLA supplementation led to an 8 d decrease in time to first ovulation at dose of 8.0 g/d. This dose was slightly lower (by approximat ely 2 g/d) than the predicted value to maximize pregnancy with CLA (de Veth et al., 2009). Conjugated linoleic acid supplementation in lactating cows allow to spare energy, decreasing the extent of negative energy balance after parturition or repartitionin g of nutrients toward milk protein and milk lactose synthesis. Changes in metabolic and endocrine signaling may be occurring with CLA induced MFD in the early lactation dairy cow. Insulin like growth factor 1 (IGF 1) is postulated to be responsible for CLA effect on reproductive performance in cow, because IGF 1 concentration were increased when supplemental trans 10, cis 12 CLA isomer was provided to early lactating dairy cows (Castaeda Gutirrez et al., 2007). These authors proposed that the mechanism t hrough which CLA affects reproduction may involve improved ovarian follicular steroidogenesis by increased circulating concentrations of IGF 1. Casta eda Guti rrez et al. (2007) reported that plasma concentrations of IGF 1 were highly correlated to

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44 plasma estradiol concentrations. It has been suggested that estradiol produced by the growing follicle triggers the events leading to the LH surge and first ovulation. Effect of Dietary CLA on Health Conjugated linoleic acid decreases body fat mass and increases lean body mass in some animal models (Wargent et al., 2005; Ostrowska et al., 1999; Park et al., 1997). Park et al. (1997) fed mice with 0.5% CLA (50% cis 9, trans11 CLA isomer and 50% trans 10, cis 12 CLA isomer) and reported a decrease in body fat and an increase in lean mass. The same effect was reported in pigs (Ostrowska et al., 1999). Evaluating these isomers individually in mice, Wargent et al. (2005) found the trans 10, cis 12 CLA isomer to be responsible for the loss of fat mass, and not the cis 9, trans 11 CLA isomer. H uman studies have not reflected the findings obtained in rodent and pig models. Zambell et al. (2000) examined the effects of 3 g/day intake of CLA for 64 d in humans. They reported no differences in fat mass. In another study, supp lementation of 1.7, 3.4, 5.1, and 6.8 g CLA/day for 12 weeks to overweight and obese people decreased fat mass without significantly affecting body weight (Blankson et al., 2000). Bhattacharya et al. (2006) evaluated the effects of CLA on events associated with body weight. They reported that some short and long term studies in healthy and obese, sedentary and exercised humans showed the beneficial effects of CLA in reducing fat mass without affecting body weight. According to the authors, the effects seen in animal studies, especially in mice, have not been reflected in human studies. This may be partly because CLA doses used in human studies are much lower than those used in animal studies. Moreover, most animal studies have been in young growing mice or rats, whereas studies in humans were mostly in mature volunteers.

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45 Animal studies have shown that CLA has many positive effects on cardiovascular risk factors reducing plasma cholesterol and triacylglycerol (TAG) concentration and improving insulin sensiti vity (Roche et al., 2001). A rabbit study indicated that supplementing 0.5 g/ d of CLA reduced atherosclerosis (Lee et al., 199 4 ). Rabbits were fed with 14% fat and 0.1% cholesterol as atherogenic diet. These authors demonstrated that CLA reduced plasma TA G and LDL cholesterol concentrations, and cholesterol deposition in the aortas. d e Deckere et al. (1999) investigated the relative efficacy of diets enriched with a CLA blend, cis 9, trans 11 CLA isomer or trans 10, cis 12 CLA isomer on lipoprotein metabol ism. The CLA mix and trans 10, cis 12 CLA isomer reduced LDL and HDL cholesterol concentrations and increased VLDL TAG concentrations. In addition, Gavino et al. (2000) reported that a diet providing cis 9, trans 11 CLA isomer only had no significant effe ct on plasma lipoprotein metabolism, whereas plasma cholesterol and TAG concentrations were significantly reduced in hamsters fed a diet enriched with a CLA mix (trans 10, cis 12 CLA isomer and cis 9, trans 11 CLA isomer). Valeille et al. (2005) reported t hat hamsters fed with 20% butter fat for 12 weeks showed lower aortic lipid deposition when the diet was supplemented with 1% cis 9, trans 11 CLA isomer. This study indicated that the individual isomers of CLA may be more effective against atherosclerosis when diet is high in saturated fats. In h u man studies no changes in plasma lipid or lipoprotein concentrations were found after intake of CLA from 2.1 to 4.2 g/d (Petridou et al., 2003; Benito et al., 2001; Smedman and Vessby, 2001). Noone et al. (200 2 ) c onducted a double blind placebo controlled CLA supplementation study in human subjects to investigate the effect of

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46 CLA on lipoprotein metabolism. They supplemented 3 g/d for 8 weeks of two isomeric mix of CLA (50:50 or 80:20) of the cis 9, trans 11 CLA is omer and trans 10, cis 12 CLA isomer in normolipemic subjects. The 50:50 mix reduced plasma TAG concentrations. In addition, Mougios et al. (2001) showed that using a lower concentration of CLA (0.7 g/day for 4 weeks) decreased serum TAG and HDL cholestero l. The beneficial effects of CLA on cardiovascular risk factors such as plasma lipids and lipoproteins are inconsistent and do not necessarily correlate with beneficial effects on incidence of atherosclerosis (Bhattacharya et al., 2006). This indicates th at more studies are necessary with different ratios of CLA isomers to clearly establish their antihyperlipidemic and antiatherosclerotic activities in humans. Several studies describe the anticarcinogenic effects of CLA in various experimental models (Cesa no et al., 1998; Visonneau et al., 1997). Using dairy products or commercial sources of CLA, Ip et al. (1999) fed rats from weaning until 50 d of age. Reduction in tumor incidence and number occurred regardless of whether the CLA sources. Song et al. (200 4) reported that CLA inhibits cellular proliferation and modulates protein kinase C isoforms in human prostate cancer cells. In 2006, the same group reported induction of apoptosis and inhibition of NF prostate cancer cells by the cis 9, trans 11 CLA isomer but not the trans 10, cis 12 CLA isomer. More recently, Lau and Archer (2010) found that trans 10, cis 12 CLA isomer (100 M), but not cis 9, trans 11 CLA isomer, inhibited fatty acid synthase expression and enzyme activity in human breast, colon, and prostate cancer cells. This inhibition may contribute to growth inhibition of cancer cells but only at relatively high concentrations.

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47 In humans, no clear association has been established between dietary CLA and decreased risk of breast cancer (Larsson et al., 2009; Bhattacharya et al., 2006). In regard to colon cancer, one human study suggested that intake of high fa t dairy foods and CLA may reduce the risk of colorectal cancer (Lars s on et al., 2005). So far, there have been no published reports on the effect of CLA on the risk of prostate cancer in human. Eicosanoids, including prostaglandins and leukotrienes, are au tocrine and paracrine signaling molecules that are generated primarily through oxidative metabolism of arachidonic acid (ARA) (Harizi et al., 2008) They play an important role in regulating many aspects of immune function, including cytokine production, an tibody formation, antigen presentation and differentiation, proliferation and migration of immune cells ( MacRedmond and Dorscheid, 2011) Production of eicosanoid mediators (i.e. prostaglandins, thromboxanes, leukotrienes) is determined primarily by the av ailability of substrate fatty acids in the membrane phospholipids and by the amount of n 6 PUFA relative to other 20 carbon PUFAs (mostly n 3 PUFA) (Calder, 2006). Increased consumption of the long chain n 3 PUFAs, eicosapentaenoic acid (EPA) and docosahex aenoic acid (DHA), typically found in fish oils results in increased proportions of these PUFAs in membrane phospholipids, partly at the expense of ARA (Healy et al., 2000) Similar to n 3 PUFAs, CLA can compete with dietary n 6 PUFAs (primarily linoleic a cid) at the level of elongases and desaturases for the biosynthesis and membrane incorporation of ARA ( MacRedmond and Dorscheid, 2011) Conjugated linoleic acid is itself incorporated into the membrane phospholipids where it competes with ARA as a

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48 substrat e for phos p holipase A2 resulting in reduced free ARA release ( Urquhart et al., 200 2 ). Conjugated linoleic acid may additionally reduce eicosanoid production by transcriptional regulation of cyclooxygenase 2 (COX 2) (Ringseis et al., 2006). Conjugated lino leic acid has been reported as more potent than EPA or DHA in reducing inflammatory eicosanoids release from a variety of human cell types including vascular smooth muscle cells and immune cells ( Urquhart et al., 200 2 ). On the other hand, peroxisome proli ferator activated receptors (PPARs) are a family of nuclear receptors which directly and indirectly regulate transcription of a wide variety of genes important in energy regulation, homeostasis and immune responses (MacRedmond and Dorscheid, 2011). The act ivation of PPAR an important negative regulator of inflammatory responses through a number of mechanisms including transcriptional regulation of cytokines, chemokines and cell survival factors and repression of pro inflammatory tra nscription factors including NF B and activator protein 1 (AP 1) (Daynes and Jones, 2002). The expression of PPAR inflammatory cells including eosinophils (Denning and Stoll, 2006; Belvisi and M itchel, 2009). Peroxisome proliferator activated receptor gamma is bound and activated to a variable degree by a number of endogenous lipophilic compounds, largely free fatty acids, oxidized lipids and eicosanoids (Kersten et al., 2000). Synthetic agonists of PPAR treatment of diabetes mellitus due to effects on insulin sensitivity. They are also known to reduce of inflammation by a variety of mechanisms including regulation of

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49 proinflammatory, anti inflammatory and modulation of NF B activity (MacRedmond and Dorscheid, 2011). Conjugated linoleic acid reduces the proportion of n 6 PUFA in immune cells, and thus affects the production of inflammatory mediators. In rats, Sugan o et al. (1998) studied the effect of CLA on the concentration of chemical mediators in peritoneal exudate cells, spleen and lun g, and the concentration of Ig i n mesenteric lymph node, splenic lymphocytes and in serum. Rats were fed diets containing 0, 0.5 or 1.0% CLA for 3 wk. They found a trend toward a reducti on in the release of LTB4 from exudates cells. Supplementation of CLA reduced splenic LTB4, lung LTC4 and serum prostaglandin E 2 (PGE 2 ) levels. Evaluating the effect of CLA on human saphenous vein endothelial cells, Urquhart et al. (2002) found that CLA mixture ( 50:50, cis 9, trans 11 CLA isomer: trans 10, cis 12 CLA isomer) and the individual isomers ( cis 9, trans 11 CLA or trans 10, cis 12 CLA) inhibited the resting production of PGF by 50, 43 a nd 40%, respectively. A dose dependent inhibition of stimulated prostaglandins was observed with the CLA mixture ( half maximal inhibitory concentration, IC 50 : cis 9, trans 11 CLA isomer and trans 10, cis 12 (50 M) CLA isomers individually inh ibited the overall production of stimulated prostaglandin. When tested at a high concentration (100 M), cis 9, trans 11 CLA isomer was found to inhibit the eicosanoid production, whereas trans 10, cis 12 CLA isomer caused stimulation. The results of this study suggest that both isomers inhibit eicosanoid production, and although trans 10, cis 12 CLA isomer exhibits pro inflammatory activity at high concentrations, the CLA mixture maintains its beneficial anti inflammatory action.

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50 In regard to cytokine pr oduction, Lee et al. (2009) examined how different CLA isomers regulate gene expression in mouse macrophage raw cells. The cells were treated with five different CLA isomers ( trans 9, trans 11 CLA, cis 9, trans 11 CLA, cis 9, cis 11 CLA, trans 10, cis 12 CLA, and cis 11, trans 13 CLA). Trans 9, trans 11 CLA; cis 9, trans 11 CLA, trans 10, cis 12 CLA, and cis 11, trans 13 CLA isomers decreased production of proinflammatory cytokines such as IL 6. In addition, trans 9, trans 11 CLA enhanced the anti inflammatory cytokine IL 1Ra. The CLA isomers did not decrease the production of TNF ained by the use of INF vs. LPS as macrophage activators. As mentioned earlier, the anti inflammatory properties of CLA are mediated, at least in part, by the nuclear hormone receptor PPAR Yu et al. (2002) found that 200 M CLA isomers (including cis 9, trans 11 CLA isomer and trans 10, cis 12 CLA isomer) activated PPAR decreased IFN induced mRNA expression of COX 2 and TNF the production of PGE 2 TNF 6. In re gard to other functions of innate immune cells such as phagocytosis, Kang et al. (2004) found that direct treatment with trans 10, cis 12 CLA isomer had no effect on the phagocytic capacity of porcine peripheral blood polymorphonuclear cells (PMN). However the phagocytic capacity of these cells was markedly enhanced in the culture supernatant from porcine peripheral blood mononuclear cells (PBMC) treated with this isomer In 2007, the same authors conducted a study to determine whether trans 10, cis 12 CLA promote s the production phagocytosis promoting factors in porcine PBMC. They hypothesized that factors such as TNF

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51 porcine PMN. Different concentrations of trans 10, cis 12 CLA (from 2.5 to 20 M) increased TNF A expression and its production by PBMC. The phagocytic capacity of PMN was enhanced in either culture supernatant fraction from PBMC treated with trans 10, cis 12 CLA or recombinant porcine (rp) TNF rpTNF polyclonal antibody inhibited the enhance ment of PMN p hagocytic capacity. This isomer also up regulated PPAR PPAR trans 10, cis 12 CLA stimulating effects on TNF by PBMC, but also decreased the enhancement of PMN phagocytic capacity by the trans 10, cis 12 CLA stimulated PBMC culture supernatant fraction. These results indicate that trans 10, cis 12 CLA isomer has an immunostimulating effect on porcine PMN phagocyt ic capacity, which is mediated by TNF dependent pathway. The effect CLA supplementation on immune function during the early stages of life has also been evaluated. Because CLA is one the fatty acid s found in breast milk, Ramirez Sa ntana et al. (2009) evaluated the influence o f CLA on immune function during the early stage of life. Rat were fed with 1% of 80:20 mixture of cis 9, trans 11 and trans 10, cis 12 CLA isomers during the last week of gestation (2 wk. ) and suckling period (3 wk. ). Pups received dietary CLA from dams through the placenta and breast milk (group A) or by oral administration (group B). Pups from group C only received CLA during suckling by oral administration. Group D constituted the reference group. Milk from da ms fed the CLA diet had a high content of CLA and higher IgA and IgG concentrations than rats fed the control diet. The plasma of pups from groups A, B and C showed 6, 12 and 9 fold higher contents of the cis 9, trans 11 CLA isomer than those

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52 of the group D pups. Rats from group A exhibited higher serum IgG concentrations than rats from the rest of the groups, whereas rats from groups A and B showed approximately 2 fold higher splenocyte IgM production than rats from groups C and D. Conjugated linoleic acid supplementation did not influence significantly the splenocyte proliferative response or cytokine secretion. Dietary CLA can prevent food allergy reaction. This reaction is initiated by the production of allergen specific IgE. Immunoglobulin A, in contra st, serves as an antiallergic factor by interfering with the intestinal absorption of allergen, and IgG also works as an antiallergic factor through the competition with binding of allergen to the receptor on the surface of the target cells such as mast ce lls and basophiles. In the study conducted by Sugano et al. (1998), splenic concentration of IgA, IgG, and IgM increased while those of IgE decreased significantly in rat s fed with 1.0% CLA diet. The levels of IgA, IgG, and IgM in mesenteric lymph node lym phocytes increased as CLA content of the diet increased, whereas IgE was reduced in those fed the higher CLA concentration. Yamasaki et al. (2000) conducted a similar study using different doses of CLA (0, 0.05, 0.1, 0.25, 0.5%) due to CLA level in an Amer ican type diet correspond to about 0.03% of experimental diet. They found that CLA increased IgG, IgM production of spleen lymphocytes in a dose dependent manner, and these levels reached a plateau at 0.03% of CLA incorporation in the diet. However, dietar y CLA did not affect serum Ig concentrations. As discussed earlier, Sugano et al (1998) and Yamasaki et al. (2000) reported that dietary CLA increased I g A, IgG, and IgM in cultured rat lymph node cells, whereas IgE was reduced. Yang and Cook (200 3 ) propo sed that CLA may promote Th1

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53 cytokine and inhibit Th2 cytokine production. Cytokines produced by Th2 (i.e. IL 4 or IL 13) are required for Ig class switch from IgG to IgE. They injected LPS to CLA fed (0.5%) mice to validate their hypothesis. Plasma TNF level after 2 h of LPS injection was lower in CLA fed group. Concentration of IL 4 was decreased in CLA fed mice when splenocytes were stimulated with concanavalin A (ConA) for 44 hr. However, IL 2 and the IL 2:IL 4 ratio were elevated, confirming their hy pothesis that CLA affects Th2 cytokine production without affecting the Th1 cytokine synthesis. Because the aforementioned rodent studies used a mixture of cis 9, trans 11 and trans 10, cis 12 CLA isomers, Yamasaki et al. (2003) conducted an experiment to determine which isomers alters IL 4, IgE, IL 2, IgM and IgG production. Mice were fed experimental diets containing 0% CLA, 1% cis 9 trans 11 CLA isomer, 1% trans 10 cis 12 CLA isomer or a 1:1 mixture (0.5% + 0.5%) of these two CLA isomers for 3 wk. Spl een lymphocytes from the mice fed trans 10 cis 12 CLA isomer produced more IgA and IgM but not IgG when stimulated with ConA compared with controls. On the contrary, cis 9 trans 11 CLA isomer did not affect the production of any of the Ig subclasses. Th e proportion of B cells in the spleen lymphocyte population was significantly lower in the cis 9 trans 11 CLA group, and higher in the trans 10 cis 12 CLA group than in the controls. Compared with the control group, the percentage of CD4+ T cells was low er in the trans 10 cis 12 CLA group. There are contrasting results relative to the effect of CLA on human immunity. Ten healthy woman were supplemented with ~3.9 g/d CLA mix ( trans 10, cis 12 CLA isomer, 22.6 %; cis 11, trans 13 CLA isomer, 23.6%; cis 9, trans 11 CLA isomer, 17.6%; trans 8, cis 10 CLA isomer, 16.6%; and other isomers 19.6%) during 93 d

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54 (Kelley et al., 2001; 2000). No effect of CLA was detected on lymphocyte proliferation, serum antibody titers, delayed type hypersensitivity, ex vivo LPS s timulated secretion of PGE 2 LTB4, IL phytohemagglutinin (PHA) induced IL 2 production. However, Albers et al. (2003) repo rted that supplementation of 71 healthy males with ~1.6 g/d 50:50 blend of the cis 9, trans 11: trans 10, cis 12 CLA iso mers for 12 weeks augmented seroprotective antibody levels in response to hepatitis B, suggesting that CLA may enhance B cell function in humans. The apparent discrepancy between these two studies could be explained by a different isomer composition of the CLA test product and by the limited number of subjects in the former study. Supplementing CLA to young healthy volunteers, Song e t al. (2005) reported a decrease in TNF inflammatory IL 10 production. Nugent et al. (2005) supplemented 55 healthy volunteers with ~ 1.9 g/d CLA blend of different levels (50:50 vs. 80:20) of the cis 9, trans 11 and tr ans 10, cis 12 CLA isomers. The 80:20 CLA isomer blend enhanced phytohemagglutinin(PHA) induced lymphocyte proliferation and increased PHA induced IL 2 and TNF However, similar results were observed with linoleic acid supplementation. Conjug ated linoleic acid supplementation had no significant effect on IL 4 production by PBMC, or plasma PGE 2 or LTB4. The immunomodulatory properties of CLA in pigs may be correlated with health status and the environmental condition of the animal. Wiegand et al. (2011) reported that feeding CLA up to 1.0% to growing finishing pigs did not significantly alter either innate or cell mediated immune system. They found no differences on white blood cell or lymphocyte distribution (CD4+, CD8+, CD2+) across CLA treat ments. Evaluating the

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55 effect of CLA supplementation (up to 2%) in nursery pigs in a dirty or clean environment on immune respons e, Bassaganya Riera et al. (2001a ) found that CLA induced a linear increase in percentage of CD8 + lymphocytes in pig from the di rty room. The same group evaluated the effect of CLA using a pig model after infection with type 2 porcine circovirus (PCV2). The infection with PCV2 induced a depletion of B cells, which was more accentuated in pigs fed the control diet, in which IL 2 mRN A expression was downregulated. Histopathologic improvements found in lungs of pigs fed CLA were correlated with greater numbers of CD8+T cells ( Bassaganya Riera et al., 200 2 ). One the most important aspects used to evaluate the effect of CLA on immunity i s cytokine production. The production of proinflammatory cytokines is a normal response to infection but inappropriate amounts or overproduction is dangerous. To investigate the anti inflammatory role and mechanisms of action of CLA in weaned pigs, Changh ua et al. (2005) created a model of acute inflammation. This model consisted of challenging pigs with LPS after 14 d of CLA mix (2%) supplementation. Dietary CLA alleviated growth depression and decreased mRNA expression of proinflammatory cytokines (such as IL expression of IL 10 and PPAR the spleen and thymus Changhua et al. (2005) performed in vitro experiments to elucidate which isomers of CLA are responsible for the reduction in proinf lammatory cytokine production and the mechanism of action of CLA. Peripheral blood mononuclear cells were isolated from weaned pigs and cultured in media containing cis 9, trans 11 and trans 10, cis 12 CLA isomers. Each CLA isomer suppressed the productio n and expression of proinflammatory cytokines and enhanced PPAR

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56 cultured PBMCs. At the molecular level, the inhibitory actions of CLA on these cytokines were attributable mainly to trans 10, cis 12 CLA isomer and the ant i inflammatory properties of CLA were mediated, at least in part, through a PPAR dependent mechanism. Evidence suggests that adult immune defenses develop during the first phase of life and are influenced by the intake of PUFA (Field et al., 2000). Bonte mpo et al. (2004) proposed CLA as an immunologic activator in young pigs that have not yet developed a more specific immune response repertoire. They studied the effects of CLA on immunologic variables in late gestating sows (6 d before parturition), lacta ting sows and piglets. Dietary supplementation of 0.5% CLA to sows increased the IgG concentration of colostrum. CLA supplementation to sows increased serum IgG in piglets. Serum IgG was also increased by CLA supplementation to p ostweaning piglets indepen dent of whether or not the sow was supplemented with CLA. These results were later confirmed by Corino et al. (2009), wh o also found an increase in IgA and IgM in CLA supplemented pigs. The effect of CLA on the immune system in ruminant s has not been well investigated. In contrast to the results reported in pigs and rodents, humoral immunity of small ruminant s is not affected by CLA supplementation. Castro et al. (2006) evaluated the effect of dietary CLA (20 g/kg of DM) on IgG concentration in blood serum of goats from the third month of gestation until 96 h postpartum and in postpartum colostrum. Serum IgG concentration w as higher in CLA supplemented goats than in control animals, but colostrum IgG concentration was not different between dietary groups. T err et al. (2011) found no differences in antibody response to ovalbumin

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57 when weaned lambs (15 2.9 kg of BW, 41 6.9 d old) were supplemented with 2.5 g/kg or 10 g/kg of rumen protected CLA mixture (50:50, cis 9, trans 11 and trans 10, cis 12 CLA isome rs). Proposed Mechanisms of Beneficial Properties of CLA Some of the mechanisms suggested to be involved in milk fat reduction with CLA intake are increased energy expenditure, increased fat oxidation, decreased adipocyte size, decreased energy intake and inhibition of enzymes involved in fatty acid metabolism and lipogenesis (Bhattacharya et al., 2006). Some of the proposed mechanisms involve in antiatherosclerotic and lipid lowering effects of CLA include their role on PPARs, sterol regulatory element bin ding proteins and stearoyl CoA desaturase. Peroxisome proliferator activated receptors regulate the expression of genes that control lipid and glucose homeostasis, and therefore modulate the metabolic disorders predisposing to atherosclerosis. Peroxisome p roliferator activated receptors exert additional anti inflammatory and lipid modulating effects in the arterial wall (Bhattacharya et al., 2006). Little information is available on the mechanisms by which CLA prevents or inhibits cancer. It has been prop osed that CLA has the potential to act at many points of cancer development, including tumor o genesis, promotion, mitogenesis, mutagenesis, carcinogen activation and detoxification, and signal transduction (McGuire and McGuire, 2000). The activation of PPAR of inflammatory responses through a number of mechanisms including transcriptional regulation of cytokines, chemokines and cell survival factors and repression of pro inflammatory transcription fact ors including NF B and AP 1. Conjugated linoleic acid

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58 may affect the heterodimer binding of PPAR element, compete with other endogenous PPAR phosphorylation (MacRedmond and Dorscheid 2011). Feeding Pomegranate to Dair y Cattle Punica granatum commonly known as the pomegranate, is a member of the Punicaceae (Pomegranate family). Pomegranate is a tree that grows up to 5 m in height. The fruit has a leathery pericarp and its interior i s compartmentalized by walls. The locules are packed with arils, each of which contains one seed and juicy pulp. Conventional pomegranate juices normally constitute 45 to 65% of the whole fruit. The arils consist of approximately 80% juice pulp and 20% see ds; traditionally the entire aril is crushed to make the juice so the expressed juice contains liquid from the seeds and the surrounding aril juice. In the USA, some juices (i.e. POM Wonderful juice) do not contain any seed constituents because the seed s a re not crushed. The arils contain acids, sugars, vitamins, polysaccharides, phenols compounds and minerals (Melgarejo et al., 2000) Phenol compounds are also detected in the pericarp, leaf and flower. In seeds, triglycerides constituted the oil, with a hi gh content of punicic acid. However, several factors may contribute to the composition including cultivars, environmental conditions, ripening, storage and postharvest treatments, which may affect fruit quality and health beneficial compounds (Schwartz et al., 2009). Phenolic compounds contain one or more phenol units. They are reducing agents, and together with other dietary reducing agents, such as vitamin C, vitamin E, and 2008). Phenolic compounds are divided in to polyphenols and phenols. Phenols and polyphenols are characterized by the presence of one or more phenol units in their

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59 structure, respectively. P olyphenols includes flavonoids, condensed tannins and hydrolysable tannins (Gil et al., 2000) Hydrolysable tannins are the predominant polyphenols found in pomegranate juice and account for 92% of its antioxidant activity (Gil et al., 2000) These tanni ns are mainly represented by ellagitannins, which are concentrated in the peel, membranes, and arils of the fruit. Punicalagins are the major ellagitannins in the whole fruit, which is unique to pomegranates ( Jurenka, 2008) Punicalagins can be hydrolyze d to ellagic acid and other smaller polyphenols in vivo (Landete, 2011) The flavonoids are mainly represented by anthocyanins (i.e. delphinidin, cyanidin, and pelargonidin), which are present in the peel or juice. Anthocyanins constitute 10% of total poly phenols and have potent antioxidant properties. Other flavonoids of possible interest include flavonols (kaempferol, quercetin) and flavones (luteolin), also found in pomegranate peels and in commercially available juices ( Jurenka, 2008) Extracts of all p arts of the pomegranate appear to have therapeutic properties due to their composition. Pomegranate juice is composed of anthocyanins, glucose, ascorbic acid, ellagic acid, gallic acid, caffeic acid, catechin, epigallocatechin gallate quercetin, rutin, minerals (particularly Fe ), and amino acids. Pomegranate seed oil contains 80% of punicic acid, and the other constituents include ellagic acid, other fatty acids, and sterols. Pomegranate pericarp (peel, rind) are represented by phenolic punicalagins, g allic acid and other fatty acids, catechin, epigallocatechin gallate quercetin, rutin, and other flavonols, flavones, flavonones, anthocyanidins. Pomegranate leaves contain tannins (punicalin and punicafolin) and flavones glycosides, which include luteoli n and apigenin. Pomegranate flower has gallic acid,

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60 ursolic acid, triterpenoids, including maslinic and asiatic acid, and other unidentified constituents. Pomegranate roots and bark are made by ellagitannins and punicalin ( Jurenka, 2008) Studies have inv estigated the bioavailability, absorption, and metabolism of pomegranate components. Consumption of 180 mL pomegranate yielded 31.9 ng/mL plasma ellagic acid at one hour, with rapid plasma clearance by 4 h post ingestion (Seeram et al., 2004). Two pomegra nate juice metabolites were detected in plasma (i.e. urolithin A and B) when subjects consumed one liter of pomegranate juice (containing 4.37 g/L punicalagins and 0.49 g/L anthocyanins). Maximum excretion rates occurred 3 4 d after juice ingestion. Signif icant variability of urinary metabolite concentration was observed among subjects and may be attributable to differences in colonic microflora, where the ellagitannins are believed to be metabolized (Cerd et al., 2004). These authors indicated that the pe rsistence of urolithin A and B in the urine may polyphenols found in the juice. In another study, subjects were given 800 mg of pomegranate extract daily containing 330.4 mg pu nicalagins and 21.6 mg ellagic acid The maximum concentration of plasma ellagic acid was 33.8 12.7 ng/mL at 1 h post ingestion (Mertens Talcott et al., 2006). Effect of Dietary Pomegranate on Animal Performance There are few published data evaluating th e effect of pomegranate on animal performance. The effect of pomegranate seed pulp feeding on performance was evaluated in goats (Modar esi et al., 2011). Twenty seven multiparous cross bred goats (averaging 71 12.5 DIM, 1.09 0.13 kg of milk/day, and 2 8 2.5 kg of BW), were fed

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61 with a diet containing 0, 6 and 12% of pomegranate seed pulp (DM basis) for 45 d. Results showed that the use of pomegranate seed pulp did not affect DMI and ADG of goats. Milk production tended to decrease with increasing level of pomegranate seed pulp in the diet. Milk fat concentration of goats fed diets containing 6 and 12% pomegranate seed pulp, increased by 8 and 15%, respectively but milk fat yield, milk protein concentration and yield, and milk solids no fat concentration and yield were not affected by diets. Milk lactose concentration in goat s fed supplemental pomegranate seed pulp was increased compared with control group. Feeding pomegranate seed pulp had no effects on blood glucose, cholesterol, urea N, triglyceride an d lipoproteins. Fresh pomegranate peels containing up to 3% (w/w) of arils were offered ad libitum to Holstein Friesian bull calves (average body weigh 380 kg) for 8 weeks (Shabtay et al., 2008). The fresh peel contained 140 mg total polyphenols/g materia l on a DM basis, of which hydrolysable and condensed tannins constituted 3.35 and 34.4 mg/g, respectively (on a DM basis). Results revealed a linear increase of peel consumption over time, which demonstrated pomegranate peel palatability to beef calves. In spite of the higher feed intake of the peel consuming calves, ADG only tocopherol concentrations in peel consuming calves were greater than those in control calves. Oliveira et al. (2010) evaluat ed the effect of feeding a dried pomegranate juice extract on performance of calves during the first 70 d of age. Holstein calves were supplemented with 5 or 10 g pomegranate juice extract /d. Juice extract contained 16.9% gallic acid equivalent (GAE) resul ting in intakes of 0, 850 and 1,700 mg of GAE/d. Feeding pomegranate juice extract had no effect on intake or BW gain in the

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62 first 30 d of age, but after 30 d of age, both grain DMI and BW gain decreased with increasing addition of pomegranate juice extrac t. Feeding juice extract did not influence DM, OM, or starch digestibility, but reduced crude protein and fat digestion. Plasma concentrations of glucose and 3 hydroxybutyrate were similar among treatments throughout the first 70 d of age. Measures of calf health such as fecal and attitude scores, risk of fever, and rectal temperature were not altered by dietary treatments. Effect of Pomegranate on Health Pomegranate is well known for its antimicrobial, antiinflammatory, antioxidant, and anticancer properti es. Punica granatum has antimicrobial activity which is attributed to its high tannins concentration (Jayaprakasha et al., 2006) AL Zoreky (2009) studied the in vitro antimicrobial effect of pomegranate peels extract and found that the peels extract was a potent inhibitor of Listeria monocytogenes S taphylococcus aureus Escherichia coli and Yersinia enterocolitica Similarly, Choi et al. (20 09 ) investigated the in vitro and in vivo antimicrobi al activity of pomegranate peel extract on 16 strains of Salmon ella The in vivo ant ibacterial activity of the peel extract was examined in a S. typhimurium infection mouse model. Mice were initially infected with S. typhimurium (day 1) and 5 mg peel extract was then administered daily. The extract had significant eff ects on all tested S almonella strains. Mice mortality and the numbers of viable S. typhimurium recovered from feces were reduced in the peel extract treated group. The antimicrobial activity of pomegranate is likely due to its chemical constituents. Reddy et al. (2007) reported that ellagic acid, gallagic acid, punicalins, and punicalagins isolated from pomegranate juice had antimicrobial activity when tested against E. coli Pseudomonas aeruginosa Candida albicans Cryptococcus neoformans S aureus Aspe rgillus fumigatus and Mycobacterium intracellulare

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63 The consumption o f pomegranate products may lead to a considerable accumulation of ellagitannins in the large intestine where they interact with complex gut microflora (Bialonska et al., 2009 a ). These a uthors found that byproducts of pomegranate inhibited the growth of pathogenic clostridia and S aureus in vitro whereas the probiotic lactobacilli and bifidobacteria were not affected by ellagitannins. According to the authors, the growth inhibition of p athogenic bacteria could be attributed to the lower pH of media due to the presence of punicalagins. In other studies the antimicrobial effect detected in vivo contrast with those detected in vitr o in vitro antiplasmo dial activity of methanolic extracts of a tannin enriched fraction and of metabolites of the immature fruit of Punica granatum. They concluded that metabolic extracts of pomegranate inhibited parasite growth in vitro Nevertheless, an in vivo study showed that the extracts were not effective in the murine model. Additionally, Van Parys et al (20 10 ) found a strong action of hydrolysable tannin s against Salmonella spp in vitro but not in vivo. It is suggested that lack of effect of pomegranate extracts in v ivo might be attributed to the low bioavailability as well as the kinetic of conversion of ellagic acid to the metabolites Pomegranate has also been evaluated for its antiinflammatory benefits. Inflammatory disorders ar e due to excessive production of pro inflammatory mediators such as TNF macrophage colony stimulating factor (GM CSF), IL 1, IL 6, IL 8, leukotriene B4, and reactive oxygen species (ROS). Reactive oxygen species is produced by neutrophils, w hich play a key role in host defense against invading microorganisms. Bachoual et al. (2011) conducted an experiment to examine the effect

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64 of aqueous pomegranate peel extract on ROS production by human neutrophils, and its effect on LPS induced lung inflam mation in mice. Human neutrophils were incubated with pomegranate peel extract (0, 25, 100, 200 ng/ml) and stimulated with N formyl methionyl leucyl phenylalanine (fMLF) or phorbol 12 myristate 13 acetate (PMA) to measure ROS, and superoxide production. Th e mice received intraperitoneal injections of 200 mg/kg peel extract for two consecutive days. On the second day, 5 g LPS/mouse was provided by intratracheal instillation, and 24 h later, the mice were euthanized. The aqueous peel extract inhibited ROS i n both resting and stimulated neutrophils. However, this extract had no effect on superoxide or H2O2 concentration. The aqueous extract attenuated LPS induced lung inflammation in mice. Shukla et al. (2008a) evaluated the anti inflammatory properties of po megranate juice extract. They used collagen induced arthritis (CIA) in mice, an animal model of rheumatoid arthritis. The mice were fed juice extract (13.6 mg/kg or 34 mg/kg) by intubation for 10 d before immunization with chicken type II collagen. Consump tion of pomegranate juice extract delayed the onset and reduced the incidence of CIA in mice. Severity of arthritis was also lower in juice extract fed animals. Histopathology of the arthritic joints from juice extract fed mice demonstrated reduced joint i nfiltration by the inflammatory cells, and the destruction of bone and cartilage were alleviated. Concentrations of the inflammatory cytokine IL 6 were decreased in the joints of juice extract fed mice with CIA. Additionally, pomegranate juice extract (20 g/mL) inhibited LPS induced production of nitric oxide, the MAPK signal transduction pathways and the activation of NF B in mouse macrophages.

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65 Using ex vivo experiments, Shukla et al. (2008b) determined the effect of rabbit plasma obtained after ingestion of a polyphenols rich extract of pomegranate fruit on COX enzyme activity and the IL induced production of NO and P GE2 in chondrocytes in vitro Rabbits were given 10 mL of extract of pomegranate (34 mg/Kg) by intubation. Plasma from pomegranate extract fed rabbits inhibited IL induced PGE2 and NO production in chondrocytes. The same plasma samples also inhibited b oth COX 1 and COX 2 enzyme activity ex vivo, but the effect was more pronounced on the COX 2 activity. The authors suggested that pomegranate extract derived bioavailable compounds may exert an anti inflammatory effect by inhibiting the inflammatory cytoki ne induced production of PGE2 and NO in vivo. The pomegranate properties could be mediated by in vivo produced metabolites in addition to the original phenolic compounds present in the fruit. Larrosa et al. (2010) conducted an experiment to evaluate wheth er the beneficial effects of pomegranate were due to the ellagitannins or to their microbiota derived urolithins. Male Fisher rats were fed daily 250 mg of pomegranate extract/kg or 15 mg urolithins/kg/day for 25 d. To promote inflammatory bowel disease de xtran sodium sulfate (5%) (DSS) was administered for 5 d prior to euthanasia Both pomegranate extract and urolithins decreased inflammation markers (i.e. iNOS, COX 2, PGE2, and prostaglandin E synthase in colonic mucosa) and modulated favorably the gut mi crobiota. The urolithin group showed down regulation of the inflammatory response pathway. Only urolithins preserved colonic architecture. The normal formation of urolithins in rats fed with pomegranate extract was prevented during inflammation. This findin g indicates that the pomegranate extract effects were not due to the accumulation of urolithins in the colon,

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66 whereas the effect observed in DSS urolithin group was clearly attributed to this metabolite. The authors suggested that urolithins may be the mos t active antiinflammatory compound derived from pomegranate ingestion in healthy subjects, whereas in colon inflammation, the effects could be due to the non metabolized ellagitannin related fraction. However, independent of the main compound responsible f or the action, these results indicate that pomegranate components could provide protection against colon inflammation before and during the disease process. Oxidation is a metabolic process that leads to energy production necessary for essential cell activ ities. However, metabolism of oxygen in living cells also leads to the unavoidable production of oxygen derived free radicals, commonly known as ROS, which are involved in the onset of many diseases. These free radicals attack the unsaturated fatty acids o f membranes, which results in lipid peroxidation and the destruction of proteins and DNA, which causes a series of deteriorative changes in the biological systems leading to cell inactivation (Chidambara Murthy et al., 200 2 ). Commercial pomegranate juices showed an antioxidant activity three times higher than those of red wine and green tea (Gil et al., 2000). The activity was higher in commercial juices extracted from whole pomegranates than in experimental juices obtained from the arils only. Analyses of the juices revealed that commercial juices c ontained more punicalagin (1500 to 1900 mg/L) than those in experimental juice obtained from arils (Gil et al., 2000). In rats, Chidambara Murthy et al. (2002) found that extract of pomegranate peel (50 mg/ kg BW for 14 d) caused preservation of catalase, peroxidase, and superoxide dismutase to values comparable with control values, whereas lipid peroxidation was

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67 reduced by 54% when compared to controls Mertens Talcott ( 2006) investigated the antioxidant effect s of a standardized extract from pomegranate in healthy human volunteers after consumption of 800 mg of extract. The antioxidant capacity was increased (32% after 0.5 h) whereas the generation of ROS was not affected by dietary treatments. The inflammatory biomarker IL 6 was not affected after 4 h after the consumption of the extract. Pomegranate consumption has not been reported to possess deleterious health effects in humans. Heber et al. (2007) evaluated the safety and antioxidant activity of a pomegran ate ellagitannin enriched polyphenol dietary supplement in overweight individuals with increased waist size. In their first study, the subjects consumed either one or two pomegranate extract capsules per day providing 710 mg (435 mg of GAE) or 1420 mg (870 mg of GAE) of extracts, respectively, and placebo (0 mg of GAE). Study 2 was designed for antioxidant activity assessment in 22 overweight subjects by administration of two capsules per day providing 1000 mg (610 mg of gallic acid equivalents) of extract versus baseline measurements. There was evidence of antioxidant activity through a significant reduction in thiobarbituric acid reactive substances ( TBARS ). There were no serious adverse events in any subject studied at either site. These studies demonstra te the safety of a pomegranate ellagitannin enriched polyphenol dietary supplement in humans during a 4 week study and provide evidence of antioxidant activity in humans. Another report in humans indicated that pomegranate juice had antioxidant capacity su perior to that of apple juice. Guo et al. (2008) found that 250 mL of pomegranate juice administered to healthy elderly subjects daily for four weeks

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68 increased plasma antioxidant capacity from 1.33 m M to 1.46 m M while subjects consuming apple juice exper ienced no significant increase in antioxidant capacity. Plasma vitamin E, ascorbic acid, and glutathione values did not differ significantly between groups, leading researchers to conclude that pomegranate phenolics may be responsible for the observed resu lts. The systemic antioxidant effect of pomegranates is due to the polyphenols and their metabolites (i.e. urolithins). In a cell based assay, Bialonska et al. (2009 b ) showed that urolithins also have antioxidant properties. They found that the most potent antioxidants are urolithins C and D with IC50 (concentration of compound that cau ses a In vitro studies have shown that pomegranate can inhibit the progression of br east, prostate, colon, lung and skin cancer. In regard to breast cancer, Mehta and Lansky (2004) evaluated the chemopreventive efficacy of purified pomegranate juice and whole pomegranate seed oil in a mouse mammary organ culture. Pomegranate juice resulte d in 42% reduction in the number of lesions compared with controls Grossmann et al. (2010) showed that punicic acid from pomegranate also is capable of inhibiting breast cancer proliferation in vitro The effects of pomegranate on prostate cancer have bee n investigated in a phase II clinical study in humans. Pantuck et al. (200 6 ) evaluated in vitro antiproliferative activities of punicalagin, ellagic acid, pomegranate juice, and a pomegranate tannin extract on colon and prostate cancer. They recruited pati ents with rising prostate specific antigen after surgery or radiotherapy and gave them 8 ounces of pomegranate

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69 juice daily until disease progression. Mean prostate specific antigen doubling time increased with treatment from a mean of 15 months at baseline to 54 months posttreatment. A major disadvantage of this study was the absence of a proper placebo control; however, significant prolongation of PSA doubling time suggested a potential for pomegranate mediated prevention of human prostate cancer. Kohno et al. (2004) evaluated the effect of pomegranate on colonic cancer in rats by subcutaneous injections of 20 mg azoxymethane/kg body weight for 2 wk. At 1 wk before the azoxymethane treatment, mice were placed on a diet containing 0.01%, 0.1%, or 1% pomegr anate seed oil for 32 wk. Administration of pomegranate seed oil inhibited the incidence and multiplicity of colonic adenocarcinomas. The inhibition of tumor was associated with increased expression of PPAR tumor mucosa. The pro oxidant/antiox idant balance is an important determinant of immune cell function, not only for maintaining of integrity and functionality of membrane lipids, cellular proteins, and nucleic acids of the immune cell, but also for the control of signal transduction and gene expression (Neyestani 2008). The immune cells are particularly sensitive to oxidative stress because of the rather high percentage of PUFA in their cell membranes. On the other hand, these cells are frequently exposed to this stress because of the free r adical production as a part of their normal function. To overcome this problem, immune cells usually use higher amounts of antioxidants than do other cells. Polyphenols as dietary antioxidants may affect various aspects of the immune system by shifting pr o oxidant/antioxidant balance towards antioxidant. There are contradictory results related to immunomodulatory properties of polyphenol from pomegranate. Some studies showed that polyphenols from

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70 pomegranate improved the immune response, whereas others sho wed immunosuppression. Harikrishnan et al. (2010) evaluated the effect of Punica granatum solvent extracts on immune system and disease resistance in Paralichthys olivaceus exposed to lymphocystic disease virus. Infected fish were intraperitoneally admini stered 0, 5, 50, and 100 mg/kg body weight of leaf extracts of pomegranate. In groups treated with 50 and 100 mg of leaf extracts/kg BW, phagocytic activity, respiratory burst activity, alternative complement activity, and lysozyme activity increased after 8 weeks when compared to control treatment. Administration of pomegranate extracts for 8 weeks reduced the rate of mortality. They concluded that intraperitoneal administration of the leaf extracts of P. granatum at 50 or 100 mg/kg doses enhanced the inna te immune responses and disease resistance after 8 weeks. Pomegranate juice extract may also improve the adaptive immune response, Holstein calves were supplemented with 0, 5, or 10 g/d of pomegranate juice extract containing 16.9% GAE. Neutrophil phagocyt ic and killing activities did not differ among treatments. F eeding pomegranate extract increased synthesis of INF 4 by PBMC and improved total IgG responses to ovalbumin vaccination (Oliveira et al., 2010). Pomegranate extract inhibited the produc tion of proinflammatory cytokines of IL 6 and IL 8 in vitro Basophilic cell lines were stimulated with PMA plus calcium i o nopho re A23187 (PMACI). Pomegranate extract decreased PMACI stimulated expression of IL 6 and IL 8 via modulation of the JNK and E RK MAPKs and NF dependent pathways (Rashe ed et al., 2009).

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71 Gracious et al. (2001) studied the immunomodulatory activity of Punica granatum in rabbit. The fruit rind powder of Punica granatum was offered at dose of 100 mg/kg BW for 10 d by intragastric intubation. Al l rabbits were injected with 0.1 mL of purified protein on day 1. The pomegranate group showed a delayed hypersensitivity reaction and an increased antibody titer response to thymus dependent antigen. Punicalagin is the major component responsible for th e antioxidant activity of pomegranate juice. In an in vitro study, Lee et al. (2008) identified punicalagin as a potent immune suppressant based on its inhibitory action on the activation of the nuclear factor of activated T cells (NFAT). The transcription factor NFAT plays an essential role in the expression of the autocrine growth factor IL 2, which promotes T cell proliferation by interacting with the IL 2 receptor (IL 2R). Lee et al. (2008) found that punicalagin (5 40 M) diminished the mRNA expressi on and protein production of IL 2 and cell proliferation of murine splenic CD4+ T cells. In another study, Lee et al. (2008) evaluated the effects of Punicalagin on PMA induced ear edema. They injected PMA to ears of mice (n = 10/treatment group) for 10 d to induce ear edema, and punicalagin (5 mg/kg or 10 mg/kg) was daily administered intraperitoneally from day 5 to day 10. Mice were sacrificed on day 10. Edema weight and thickness were reduced in the punicalagin treated groups. Different components of Pu nica granatum have been evaluated for their immunomodulatory proprieties. Pomegranate seed oil contains 80% of punicic acid ( cis 9, trans 11, cis 13; C18:3), a conjugated linolenic acid (ClnA) isomer. Fatty acids with conjugated double bonds like CLA and C lnA have attracted attention because of their

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72 suggested health benefits. Although punicic acid is not a polyphenolic compound, it may have some immunomodulatory properties. Yamasaki et al. (2006) studied the dietary effect of punicic acid from pomegranate seed oil on humoral response. Mice were fed experimental diets containing 0, 0.12, or 1.2% pomegranate seed oil for 3 wk. Splenocytes isolated from seed oil fed mice produced larger amounts of IgG and IgM but not IgA. Dietary pomegranate seed oil did not affect the percentages of B, CD4+ or CD8+ T cells in splenocytes. Concentration of IL 4, INF and TNF groups. Proposed Mechanisms of Beneficial Properties of Pomegranate The proposed mechani sms involved in antiinflammatory effects of pomegranate or their metabolites include the reduction of oxidative stress, inhibition of the p38 mitogen activated protein kinase (p38 MAPK) pathway, and inhibition of the activation of NF Activation of p38 MAPK and NF expression of TNF 1 (MCP1), the inducible nitric oxide synthase (NOS), COX and LOX, agents that are critical mediators of inflammation (Shukla e t al., 2008a). It has been proposed that the antioxidant effects of pomegranate and/or their metabolites may involve up regulation of the expression of genes encoding the enzymes such as superoxide dismutase, catalase, or glutathione peroxidase. Feeding S accharomyces cerevis i ae to Dairy Cattle Yeasts are unicellular fungi that are referred to as saprophytic organism due to their inability to manufacture organic nutrients by photosynthesis. Some yeasts do not affect their host s (i.e. Saccharomyces cerevis i a e ), while others may serve as potential

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73 pathogens (i.e. Candica albicans ). Saccharomyces cerevis i ae is often added to animal feeds as a source of proteins, vitamins ( NRC, 19 9 8) or as a probiotic. Vitamins present in yeasts include biotin, folic acid, niaci n, pantothenic acid, thiamin and vitamins B 2 and B 6 ( NRC, 1998 ). The cell wall of S. cerevisiae is made of mannan, glucan, and chitin. Mannan, a soluble branched polymer of mannose, occurs at the outer surface of the cell wall and represe nts about 30 % of the dry cell wall weight ( Kollar et al., 1997 ). Mannan is a 1,6 linked ba ckbone. Glucan and chitin are the main cell wall stru ctural polysaccharides of yeast. Glucan is an insoluble 1,6 glucosidic lin kages and accounts for 50 to 60 % of the dry cell wall weight. Glucan is found in the inner cell wall and is 1,6 glucan appears to be the central molecu 1,3 glucan, mann oprotein and part of the chitin 1,4 linked N acety l glucosamine, comprises 1 to 2 % of the cell wall ( Kollar et al., 1997 ). Yeast products are used in animal nutrition. The most common products are: yeast culture, active dry yeast, mineral enriched yeast, and Saccharomyces cerevis i ae fermentation product. Yeast culture is defined as a dried product composed of yeast and the media on which it was grown, dried in such a manner as to preserve the fermenting activity of the yeast. In order to improve rumen fermentation, cultures rely on dead yeast cells, the media on which the yeast was grown and the metabolites made by the yeast during a fermentatio n process.

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74 Active dry yeast is defined as viable yeast which has been dried in such a manner as to preserve a large portion of its fermenting power. It must contain no added cereal or filler and must contain at least 15 billion live yeast cells per gram. A ctive dry yeast is chosen from a specific strain and separated from its culture medium at the end of production. A natural turnover of live yeast in the rumen occurs over time due to environmental conditions and predatory organisms, such as Protozoa Yeast population is maintained through a combination of reproduction and daily dietary supplementation over time. A mineral enriched yeast is grown on a mineral rich substrate. Yeast incorporates the minerals into its own cell structure. Some companies use yea st to supply trace elements (i.e. selenium, copper, manganese and zinc) The mineral is much more bioavailable to the animal, with less risk of toxicity and environmental pollution. Saccharomyces cerevisiae fermentation products are produced during ferment ation of an unmodified strain of Saccharomyces and include s the products of fermentation, yeast cells, yeast cell wall fragments, and the media utilized during fermentation (Shen et al., 2011). Effect of Dietary Saccharomyces cerevis i ae on Animal Performan ce Studies have evaluated S. cerevis i ae as an alternative to antimicrobial growth promoter in piglet and calf diets. Van der Peet Schwering et al. (2007) fed yeast culture (0.125%) or yeast culture (0.125 %) plus cell wall products (0.2 %) to weanling pigs. Average daily gain body weight at 35 d and feed efficiency were improved in yeast supplemented piglets. Blood cell composition, villous length, crypt depth, and microbial composition i n the gut were unaffected by dietary treatment.

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75 Shen et al. (2009) det ermined the effect of dietary supplementation of yeast culture on the performance, nutrient digestibility, intestinal morphology, intestinal microflora, an d immune response in weanli ng pigs. Pigs (7.5 0.2 kg of BW) were allotted to 6 treatments: control, antimicrobial growth promoter (chlortetracycline, 80 mg/kg) and 2.5, 5 .0 10 and 20 g/kg of yeast culture. Average daily gain of pigs fed with 5 g/kg of yeast culture was greater than that of pigs in the control and other yeast culture groups. There was n o difference between the yeast culture and antimicrobial growth promoter groups. Pigs supplemented with 5 or 10 g/kg of yeast culture, and antimicrobial growth promoter had greater daily feed intakes than the control group. Feed efficiency was not affected by dietary treatment. In a following experiment, three treatments were evaluated: control, antimicrobial growth promoter, and 5 g/kg yeast culture. Although yeast yielded a better result than of the control treatment, no differences in performance, digest ibility, or gut morphology were observed among pigs fed yeast and antimicrobial growth promoter. Using a low concentration of yeast culture, Kornegay et al. (1995) reported that yeast supplementation (0.75%) did not improve ADG, daily feed intake, feed eff iciency, apparent digestibility of DM or N in weanling pigs. Addition of S. cerevisiae fermentation products to gestation and lactation diets of the sow may improve litter BW gain during lactation. Shen et al. (2011) evaluated the effect of supplementing S cerevisiae fermentation products in sow diets on performance of sows and nursing piglets. Sows were supplemented with S. cerevisiae fermentation products from 5 d before breedi ng until lactation (12 and 15 g /d through gestation and lactation, respectivel y) No difference was observed in reproductive

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76 performance between treatments. Sows in the fermentation products group tended to have increased total litter weaning weight and litter BW gain. Concentration of plasma urea nitrogen was numerically greater in control than fermentation products treated sows on d 110 of gestation. Apparent total tract nutrient digestibility of ash, CP, DM, and ether extract were not affected by adding fermentation product. Protein and fat contents in colostrum and milk did not d iffer between dietary treatments. Colostrum from sows in the fermentation products group contained a greater amount of ash than that control sows. Immunoglobulin G concentration in colostrum, milk, and plasma of piglets did not differ among control and fer mentation products treated dams. The beneficial effect of S. cerevisiae fermentation products on gastrointestinal environment has also been reported. Kiarie et al. (2011) evaluated the effect of S. cerevisiae fermentation products on growth performance and gastrointestinal microbial ecology in weanling pigs orally challenged with Escherichia coli K88+. Six treatments were evaluated: control, carbadox (55 mg carbadox/kg of feed), and S. cerevisiae fermentation products offered at 0.2 % of the diet. Pigs were orally challenged with E. coli 8 d after initiation of the supplementation. Pigs receiving fermentation products had greater daily feed intakes than control pigs on d 3, and d 7 post challenge. Pigs receiving carbadox ate more (at d 7 post challenge) than pigs receiving fermentation products Pigs receiving fermentation products showed a smaller number of ileal mucosa adherent E. coli than control pigs. The fermentation products showed increased richness and diversity of bacteria and small prevalence of ent erobacteria in the ileal digesta reduced E. coli numbers adhering to the mucosa

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77 Saccharomyces cerevisiae is extensively used as a fe ed additive in ruminant rations, principally for dairy cattle to increase milk production (Nocek and Kautz, 2006). Yeast studies in pre ruminant calves are limited (Pinos Rodr i guez et al., 2008). Cole et al. (1992) supplemented Holstein calves with 0, 0. 75, 1.125, or 1.5 % of yeast culture added to the starter. Yeast culture supplementation did not affect DMI, ADG or feed eff iciency. The same results were reported when Jersey calves were supplemented with 0.2 % of yeast culture added to the starter (Quigley et al.,1992), when Jersey and Holstein calves were supplemented with 4 g/d of live yeast added to the milk ( Hill et al., 2 009). Lesmeister et al. (2004) offered yeast culture with starter at 0, 1, or 2% of DM. Inclusion of yeast culture at 2% increased starter and total DMI, ADG, and hip width change. Daily change in hip height was also greater for calves receiving 2% than fo r calves fed with 1% of yeast culture Magalhes et al. (2008) evaluated the effect of addition 2% of yeast culture (on DM basis) to the grain. G rain intake, nutrient intake, BW change, feed efficiency, glucose and BHBA concentrations were similar between treatments. Pinos Rodri guez et al. (2008) also reported no effect of yeast culture on BW, ADG and feed efficiency when calves were fed 1 g/d of S. cerevisiae or S. boulardii Yeast was administered orally with a syringe before milk feeding to assure that yeast was deposited in the rumen. Galv o et al. (2005) evaluated the effect of feeding live yeast products to calves with FPT (IgG concentration less than 0.73 g/dL) on productive performance. Four treatments were evaluated: 1) Control, 2) 0.5 g of S. cer evisiae added to the grain for 84 d, 3) 0.5 g of S. boulardii and 0.5 g of S. cerevisiae added to the milk for 42 d + 0.5 g of S. cerevisiae added to the grain for 84 d. Calves receiving treatment 2 showed higher

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78 grain DM intake weight gain prior to weani ng, feed efficiency and plasma glucose concentrations than control calves. Few studies have evaluated the effect of supplemental yeast culture on rumen development. Leismeister et al. (2004) examined the effect of up to 2 % yeast culture added to the start er on rumen development of dairy calves. They reported that rumen wall thickness, papillae length and papillae width did not differ among treatments in calves euthanized at 5 or 6 wk of age. P apillae length and papillae width were greater in calves fed 2% yeast than in calves receiving the control starter at weaning These data indicate that the addition of yeast culture in a dairy calf starter at 2% may improve rumen development in dairy calves. In contrast, Hill et al. (2009) reported lack of effect of li ve yeast product ( 4 g/d added on milk) on rumen papillae development. The effect of yeast on rumen development may vary, depending on compartment of gastrointestinal tract where yeast is deposited or the type of yeast used. Pinos Rodr i guez et al. (2008) ev aluated the rumen fermentation pattern in calves after feeding live S. cerevis i ae or S. boulardii (1g/d). Yeast was administered orally with a syringe Ruminal ammonia N, propionate and butyrate increased with S. cerevis i ae but not with S. boulardii Resu lts indicated that S. cerevis i ae may modify ruminal fermentation, but does not impact performance of calves. Agarwal et al. (2002) evaluated the rumen enzyme profile of calves fed live S cerevis i ae. Calves (averaging 23 kg of BW) were fed 10 6 CFU /m L Acti vities of amylase, protease, urease and pH of the rumen liquor were unaffected by S. cerevis i ae supplementation.

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79 In adult ruminants, the use of yeast culture as a dietary supplement has bee n suggested as a useful tool to stabilize ruminal fermentation (Williams et al., 1991). One of the consequences of feeding high concentrate diets is the occurrence o f subclinical ruminal acidosis Low pH in the rumen over long periods inhibits DMI and fibe r digestion. In addition, the VFA profile in rumen fluid is altered with low acetate to propionate ratio, and sometimes with increase in lactic acid concentration (Williams et al., 1991) Callaway and Martin (1997) indicated that yeast culture provides sol uble growth factors (i.e., organic acids, B vitamins, and amino acids), which stimulate growth of ruminal bacteria that utilize lactate and digest cellulose (i.e. Selenomonas ruminantium, Megasphaera elsdenii, Fibrobacter succinogenes, and Ruminococcus fla vefaciens, respectively) Desnoyers et al. (2009) reviewed the effect of live S. cerevis i ae supplementation on ruminal parameters and milk production of ruminants. They found that live yeast supplementation increased rumen pH and rumen VFA concentration, and tended to decrease rumen lactic acid concentration, but had no influence on acetate to propionate ratio. Rumen pH and VFA concentration increased linearly with live yeast concentration. These authors reported that the positive effect of yeast supplemen tation on rumen pH tended to be increased by DMI. The proportion of concentrate in the diet increased this effect, whereas the proportion of NDF decreased it. The positive effect of live yeast supplementation on rumen VFA concentration was increased by DMI proportion of concentrate, NDF, and CP in the ration. The negative effect of live yeast supplementation on rumen lactic acid concentration tended to be attenuated by the

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80 proportion of concentrate in the ration and DMI. There was no influence of dietary N DF or CP on the effect of yeast on rumen lactic acid concentration. Desnoyers et al. (2009) also found that live S. cerevis i ae supplementation increased the organic matter digestibility in a dose dependent manner. The positive effect of live yeast on organ ic matter digestibility was decreased by the proportion of concentrate in the diet and increased by the proportion of dietary NDF. No influence of dietary CP percentage or DMI was detected. Different strains of S. cerevisiae fed as active dried yeast may vary in their ability to modify the rumen fermentative pattern. Chung et al. (2011) reported differing effects of 2 active dried yeast ( S. cerevisiae ) strains on ruminal acidosis and methane production in nonlactating dairy cows Cows were assigned to con trol, yeast strain 1, or yeast strain 2 (a novel strain selected for enhanced in vitro fiber degradation). Both strains were provided at 1 10 10 CFU /day. Dry matter intake, BW, and apparent total digestibility of nutrients were not affected by yeast trea tment. Strain 2 decreased the average daily minimum, mean, and maximum ruminal pH, and prolonged the time the ruminal pH remained below 5.8 compared with the control or strain 1. The molar percentage of acetate was lower and propionate was greater in the r uminal fluid of cows receiving strain 2 compared with cows receiving no yeast or strain 1. Enteric CH 4 production did not differ between either yeast strain compared with the control and tended to be reduced by 10% when strain 2 was compared with strain 1. Yeast culture could also influence ruminal fermentation and microbial p opulation Using an in vitro study, Fortina et al. (2011) evaluated the effect of inactiv at ed yeast on rumen fermentation. They added yeast culture (1 g) to the rumen fluid and incuba ted for

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81 different times. Yeast culture increased crude protein and NDF digestibility after 48 of incubation, but did not influence DM digestibility. Yoon and Stern (1996) examined the effect of yeast culture supplementation on ruminal fermentation. They fe d Holstein cows with 57 g/d of yeast. Ruminal pH, ammonia N concentration, and total VFA concentrations were not affected by treatment. Yeast culture increased ruminal organic matter and CP digestion and decreased organic matter and N flow to the duodenum. Fiber digestion was not affected by yeast supplementation. Yeast culture increased proteolytic and cellulolytic bacterial counts. Hristov et al. (2010) fed Holstein cows with 556 g S. cerevisiae fermentation products daily The fermentation products had no effects on ruminal fermentation, nutrient digestibility, or N losses. Supplementation with fermentation products tended to reduce ruminal ammonia concentration and to increase microbial protein synthesis in the rumen. Manure from cows fed the fermentati on products supplemented ration had decreased ammonia and methane emitting potential. Desnoyers et al. (2009) reported that live S. cerevisiae supplementation increased DMI and milk yield and tended to increase milk fat content. Live yeast supplementation did not influence milk protein content. Dry matter intake and milk yield tended to increase linearly with yeast dose, but milk fat and protein contents were not affected by yeast dose. The positive effects of live yeast supplementation on DMI was increased by the proportion of concentrate in the ration but was not altered by the proportion of NDF or CP in the diet. The effect of live yeast supplementation on milk production increased with DMI, proportion of concentrate, NDF, and CP in the ration. The yeast effect on milk

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82 fat content was not influenced by DMI, dietary NDF, or CP percentage, but it tended to be increased by dietary concentrate proportion. Piva et al. (1993) supplemented mild lactating Holstein Friesian cows with yeast culture (10 g/d) for 4 wk Milk production, FCM, and milk fat were increased by dietary treatment. Blood plasma components were not affected by yeast supplementation Robinson and Garrett (1999) fed multiparous and primiparous Holstein cow with yeast culture (57 g/d) for approxima tely 23 d prepartum and 56 d postpartum. Cows supplemented with yeast had numerically higher DMI, milk production and milk components than control cows. Body weight and body condition score were not affected by treatment. Ramsing et al. (2009) fed multipa rous and primiparous Holstein cows with yeast culture at 0, 57 or 227 g/d from approximately 21 d prepartum to 21 d postpartum. Cows fed 57 g/d had a greater prepartum DMI than those fed 227 g/d. Postpartum DMI and pre and postpartum BW were similar for a ll groups. Milk production was greater in yeast supplemented than control cows. Energy corrected milk, 3.5% FCM and milk fat yield tended to be greater for yeast supplemented cows than control cows. Milk protein production, milk protein percentage, milk fa t percentage, and milk somatic cell scores were not influenced by dietary treatment. There were no effects of yeast supplementation on plasma BHBA, glucose, or nonesterified fatty acid concentrations pre or postpartum. Dann et al. (2000) supplemented Jers ey cows with yeast culture (60 g/d) for approximately 21 d prepartum and 140 d postpartum. Supplementation of yeast increased DMI. Treatment by day interaction indicated that cows supplemented with yeast lost BW less rapidly postpartum, reached peak milk p roduction more quickly than did nonsupplemented cows. However, total milk produced

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83 during the first 140 d of lactation did not differ between treatments. Concentrations of fat, protein, lactose, total solids, and urea N in milk, as well as somatic cell cou nt, were not affected by YC. In addition, Hristov et al. (2010) reported that daily supplementation with 556 g S. cerevisiae fermentation products had no effects on milk production or composition. Effect of Dietary Saccharomyces cerevis i ae on Health Mortal ity of young calves and piglets is an important problem on farms, and diarrhea is the most common disease. During the pre weaning period, the young animal is susceptible to many infectious pathogens causing the primary damage to the intestine. Antibiotic a re used to decrease the incidence of diarrhea and mortality. The overuse of antibiotics in animal husbandry may influence the antibiotic resistance of potential human pathogens (Fey et al., 2000) by exerting the selective pressures which render antibiotics ineffective in controlling bacterial diseases (Amabile Cuevas et al., 1995). Saccharomyces cerevis ia e has been evaluated as an alternative to antibiotics for the prophylaxis of diarrhea in piglets and calves. Yeast culture is a rich source of nutritiona l metabolites, mannan glucans, and other yeast fermentation metabolites. These compounds may prevent the interaction between pathogenic bacteria and intestinal cells, as well as strengthen the immune system (Shen et al., 2009: Gao et al., 2008). Price e t al. (2010) evaluated the use of S. cerevi sia e fermentation products on growth performance and microbiota of weaned pigs during and after an oral challenge with Salmonella. Weaned pigs (21 d of age) were assigned in a 2 2 factorial arrangement consistin g of diet (control or 0.2% fermentation products) and inoculation

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84 (sterile broth or Salmonella ). On d 14, pigs were orally inoculated with 10 9 CFU of Salmonella or sterile broth. Growth performance and alterations in the gastrointestinal microbial ecology were measured during pre inoculation (0 to 14 d), sick (14 to 21 d), and post inoculation (21 to 35 d) period. Diets had no effect on BW, ADG, or rectal temperature during any period. Inclusion of S cerevis ia e fermentation products tended to increase Sal monella shedding in feces during the sick period. This effect may indicate a rapid elimination of the pathogen from the gastrointestinal tract, which may reduce infection rates and enhance clearance of the pathogens. Consumption of fermentation products al tered the composition of the gastrointestinal microbial community, resulting in increased populations of Bacteroidetes and Lactobacillus after Salmonella infection. During the post inoculation period, an interaction between diet and inoculation on ADG indi cated that pigs infected with Salmonella grew better when eating fermentation products than the control diet. The addition of fermentation products to the diets of weanling pigs resulted in greater compensatory BW gains after infection with Salmonella than in pigs fed conventional nursery diets. This increase in BW gain was likely associated with an increase in beneficial bacteria within the gastrointestinal tract. In calves, addition of yeast culture ( 2%) to the grain reduced the number of days with watery feces, decreased the incidence of fever and diarrhea, minimized the frequency of health treatments, and the risk of health disorders and of mortality ( Magalhes et al., 200 8). Galvo et al. (2005) also reported a reduction in diarrhea day s when calves wer e fed live yeast. In contrast, Lesmeister et al. (2004) reported no effect of yeast culture on the number of days scoured in calves Similarly, Pinos Rodriguez et

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85 al. (2008) reported no effect of treatment on the incidence of diarrhea and pneumonia in calv es supplemented with 1 g/d of S. cerevis i ae and S. boulardii. Supplementation of sick calves with yeast culture can have beneficial effects. Cole et al. (1992) offered 0 or 0.75% yeast to calves challenged intranasally with bovine rhinotracheitis virus. Ca lves fed yeast culture tended to maintain heavier weights and DMI, and fewer sick days than those fed control diet during infection. Yeast or yeast cell walls may be able to modulate the immune system ( Van der Peet Schwering et al ., 2007). In humans and mi glucans have been shown to stimulate immune responses glucans stimulate ROS production by neutrophils and monocytes (Rubin Bejerano et al., 2007). In piglets, Davis et al. (2004) reported an improvement in performance and a modulation of the immu ne system after supplementing the diet with phosphorylated mannans derived from S. cerevisiae Blood neutrophils tended to be reduced whereas lymphocytes were increased in mannan fed group compared with control group. The authors concluded that the observe d tendency of neutrophils to decrease in the blood of mannan fed piglets may be due to reduced inflammatory challenge imposed on the piglets. In contrast, Van der Peet Schwering et al (2007) failed to detect an effect of dietary treatment (0.125 % of yea st culture) on the concentration of white blood cells and the percentage of lymphocytes or neutrophils in piglets. In calves, Magalhes et al. ( 200 8) reported that supplementation with 2% of yeast culture to grain tended to increase the number of phagocyti zed bacteria and killing of phagocytized bacteria but did not influence humoral immune response.

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86 Shen et al. (2009) reported that plasma IFN pigs supplemented with yeast culture (2.5, 5, 10 and 20 g/kg of YC) and antibio tic growth promoter (80 mg/kg) compared with control pigs on d 7, and CD4+ was decreased in pig s supplemented with yeast and antibiotic on d 14. Oral administration of S. boulardii may reduce the mortality associated with immune responses to Escherichia coli endotoxin in pigs. Collier et al. (2011) evaluated the effects of active dry yeast S. boulardii on the i mmune response and subsequent mortality to Escherichia coli LPS administration in newly weaned piglets. Piglets were assigned to 0 or 200 g of S. boulardii /ton of feed On d 16, all piglets were challenged yeast treated piglets, cumulative ADG increased and LPS induced piglet mortality was reduced compared with control piglets. Total white blood cells, lymphocytes, and neutrophils were increa sed in yeast treated animals before LPS challenge compared with control piglets. There were no effects of treatment on neutrophils phagocytosis and oxidative burst. Production of IL administration tended to be reduced in treated piglets co mpared with contro ls. Serum IL 6 was numerically reduced in the yeast group when compared with control group; the concentration of IL 6 did not differ between groups at 2.5 h. Concentration of TNF production in yeast treated animals was greater than con trol piglets only at 1.5 h after LPS challenged. The concentration of IFN treated animals than in control piglets. 0 ) reported immunomodulating effects of yeast on the non specific and specific cellular and humoral immunity in lambs. Lambs (30 3 d of age) were supplemented with 0.3 % dried brewer's yeast. High gamma globulin

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87 concentration, and elevated lysozyme and ceruloplasmin activity were detected in yeast fed lambs compared with control lambs. No di fferences in serum total protein were found between the control and experimental groups. High levels of potential killing activity of phagocytes, and elevated proliferative response of blood lymphocytes were detected after stimulation with LPS and ConA in the yeast supplemented group compared with control group. Proposed Mechanisms of Beneficial Properties of Saccharomyces cerevisiae The reduction in incidence of diarrhea may be mediated by structural components of the yeast, such as mannan oligosaccharides glucans, and other yeast fermentation metabolites whereas the effect of yeast o n rumen development may be explained by increased butyric acid production, decrease lactic acid production, and increased rumen pH (Kumar et al., 1997; Quigley et a l., 1992; Williams et al. 1991). The mechanisms by which yeast improve animal performance are not completely understood. Specific strains are metabolically active in the rumen and can have three main modes of action: 1) Competition with other microorganisms for foo d, thus reducing lactic acid production, 2) Provision of nutrients and soluble growth factors (amino acids, organic acids such as malic acid, B vitamins) which stimulate growth of other microorganisms that will help digestion and fulfill the metabolizable protein needs of the animal, and 3) Scavenging oxygen to help the growth of beneficial anaerobic bacteria, which remove lactic acid from the rumen and help improve fiber digestibility ( Callaway and Martin, 1997 )

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88 CHAPTER 3 TRANS 10, CIS 12 CONJUGATED LINO LEIC ACID AND THE PPAR ROSIGLITAZONE ATTENUATE LIPOPOLYSACCHARIDE INDUCED TNF PRODUCTION BY BOVINE IMMUNE CELLS Introduction The outermost membrane of gram negative bacteria is composed of LPS which includes an inner lipid portion (referred to as lipid A) and an outer polysaccharide (Fenwick, 1995). When LPS is released from gram negative bacteria, it binds to plasma membrane proteins (Parrillo, 1993). Th is complex then interacts with host cells via specific receptors that subsequently induce the production and release of inflammatory mediators (Aderem and Ulevitch, 2000). These include IL 1 and TNF ultimately induce the systemic production of other inflammatory mediators (e.g., prostaglandins, leukotrienes, platelet activating factor etc). Whether and the extent to which supplemental LPS induces TNF s to be elucidated. Conjugated linoleic acid is a collective term describing a mixture of positional and geometric dienoic isomers of LA. I nterest in CLA research stems from the well documented anticarcinogenic, antiatherogenic, antidiabetic, and antiobesity properties of CLA in rodent models (Badinga and Greene, 2006). In addition, rapidly expanding literature in this field suggests that CLA modifies immune function (Hayek et al., 2006 ; Bassaganya Riera et al., 2001 a,b ) and may reduce immune mediated catabolism in animal models (Cook et al., 1993; Miller et al., 1994). Specific mechanisms by which CLA modulates immune function are complex and may involve regulation of prostaglandin, cytokine and PPAR pathways. In this regard, supplemental CLA reduced production of eicosanoid products such as prostaglandin E 2 (PGE 2; Li and Watkins,

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89 1998), which are involved in early inflammation events. There also is evidence that dietary CLA suppresses the synthesis of proinflammatory cytokines at both the protein and mRNA levels (Changhua et al., 2005) and that this effect may be mediated, in part, via activation of the PPAR b ). The PPAR activated transcription factors that classically has been characterized for its role in adip ocyte differentiation and fat metabolism (Sanchez Hidalgo et al., 2007). However, evidence is rapidly accumulating that PPAR cytokine expression by antagonizing the activities of c jun NH 2 terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK) in vivo (Desreumaux et al., 2001) and by interfering with the transcription factor activation such as nuclear factor signal transducers and transcription activators (STAT), activating protein 1 (AP 1) and the nuclear factor of activated T cells (NFAT), all of which regu late cytokine gene expression ( Dubuquoy et al., 2002; Delerive et al., 2001). Because CLA and PPAR ligands downregulate inflammatory gene expression in various experimental models (C hanghua et al., 2005; Jiang et al., 1998), we hypothesized that these molecules may attenuate LPS induced cytokine production by bovine blood cells. The primary objective of this study was to examine the effect of exogenous CLA and PPAR agonist, rosigli tazone, on LPS stimulated TNF secondary objective was to identify the signaling pathway through which LPS and PPAR in vitro

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90 Materials and Methods Media and Reagents The mediu mercaptoethanol, fatty acid free bovine serum albumin (BSA) and LPS from Escherichia coli (serotype 0111:B4) were from Sigma Aldrich (St. L ouis, MO). Fico/LiteTM LymphoH was from Atlanta Biologicals (Lowrenceville, GA). RPMI 1640 was from Invitrogen (Carlsbad, CA). Linoleic acid, c9,t11 CLA (purity 96%), t10, c12 CLA (purity 98%), rosiglitazone, and GW9662 were from Cayman Chemical Co. ( Ann Arbor, MI). The ELISA kit for bovine TNF analysis (No. DY2279) was purchased from R&D Systems, Inc. (Minneapolis, MN). Monoclonal anti rabbit NF actin and HRP conjugated IgG were obtained from Cell Signaling Technology (Danvers, MA). The enhanced chemiluminescence (ECL) kit was from Perkin Elmer, Inc. (Walthan, MA) Animals and Blood Sampling Single blood samples (~50 mL per animal) were collected by puncture of the jugular veins from clinically healthy Holstein heifers (mean age = 5.5 m onths, n = 3 different animals/treatment, and transported to the laboratory at environment temperature The heifers were fed the same diet. The TMR (DM basis) consisted of bermuda silage (23.0%), corn silage bag (40.8%), brewers grain (11.2%), soyhulls (23 .0%), and heifer mineral (2%) The daily amount offered was 10 kg of DM/heifer. Peripheral Blood Mononuclear Cell Isolation Heparinized blood samples were brought to the laboratory within 1 h of blood collection and centrifuged at 600 x g for 30 min at ro om temperature. The buffy coat was carefully removed and transferred to pre labeled tubes containing 2 mL of Medium

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91 199. After thorough mixing, the cell suspension was layered on top of 2 mL of Fico/Lite LymphoH and centrifuged at 600 x g for 30 min at r oom temperature. Mononuclear cells were collected from the Fico/Lite interface and transferred to fresh tubes containing 2 mL of red blood cell lysing buffer. The mixture then was neutralized with 8 Buffered Saline and centrifuged at 600 x g for 15 min at room temperature. The supernatant was discarded and the pellet resuspended in 2 mL of Medium 199. The solution was centrifuged for 3 min at room temperature and the pellet resuspended in Medium 199, containing 5% (v/v) horse ser um, 500 U/mL penicillin, 0.2 mg/mL streptomycin, 2 mM glutamine, and 10 5 mercaptoethanol. Lymphocyte Proliferation Assay Proliferative response of PBMC to LPS was examined by incubating freshly isolated mononuclear cells (10 6 cells/mL) with LPS ( 0 or 1 0 g/mL), IFN 0 or 5 ng/mL), or a combination of these two molecules in 96 well flat bottom tissue culture plates for 72 h at 37 o C in a 5% CO 2 environment. Lipopolysaccharide concentration was chosen based on previous observations that 10 g or 1 g/mL of LPS maximally stimulate d TNF al., 2010; Hulbert et al., 2011). During the last 24 h of culture, cell s were pulsed with 0.2 Ci/well [ 3 H] thymidine (6.7 Ci/mmol; MP Biomedical, Santa Ana, CA). Incorporation of [ 3 H] thymidine into DNA was assessed by liquid scintillation counting in a Beckman LS6000 counter (Pegassus Scientific Inc., Rockville, MD). Greater incorporation of [ 3 H] thymidine reflected a greeater proliferation of PBMC. Ea ch treatment was run in triplicates and results are expressed as fold stimulation over untreated cells.

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92 Lipopolysaccharide Stimulation of Tumor Necrosis Factor Alpha Production in Whole Bovine Blood To evaluate the effect of exogenous LPS on TNF cells, from clinically healthy Holstein heifers (n = 3 different animals) Diluted blood (1: 5 in RPMI Medium) was incubated with increasing doses of LPS (0, 0.1, 1.0 and 10.0 g/ mL) for 24 h or with 1 g/mLof LPS for increasing times (0, 4, 12 and 24 h). After LPS challenge, culture plates were centrifuged at 600 x g for 3 min and the supernatant stored at 80oC for subsequent TNF Fatty Acid Treatment of Whole Bl ood To examine CLA modulation of LPS induced TNF of diluted whole blood (1: 5 in RPMI Medium) were treated with increasing doses (0, 10, 50, 100, and 200 M) of the two CLA isomers for 24 h and then challenged with 1g/mL of LPS for 24 h. Fatty acids were complexed with BSA (1:2 ratio) for 2 h prior to addition to the cultures. The time point for FA treatment (24 h) was based on previous studies in our laboratory which ind icated that CLA and other PUFA elicited maximal p hysiological effects at 24 h of treatment of bovine endometrial (BEND) cells (Rodriguez Sallaberry et al., 2006). To test the isomer specificity of CLA effect, diluted blood samples were incubated with 100 M each of BSA complexed LA, c9,t11 CLA or t10,c 12 CLA for 24 h. Cultures then were challenged with LPS (1 g/mL) for an additional 24 h. After LPS treatment, culture plates were centrifuged at 600 x g for 3 min and the supernatant stored at 80oC for subsequent TNF Peroxisome Proliferator Activated Receptor Gamma Modulation of Tumor Necrosis Factor Alpha Production To evaluate the involvement of PPAR the PPAR

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93 addition of LPS To determine whether the effect of rosiglitazone on TNF depended on PPAR added to a set of wells 30 min before rosiglitazone addition to the cultures. A set of wells received no treatment and served as control in the experiment. All culture wells received the same amounts of treatment vehicles. After 24 h treatment with LPS, culture plates were centrifuged at 600 x g for 3 min and the supernatant stored at 80oC for subseque nt TNF Tumor Necrosis Factor Alpha Assay Concentration of TNF run in duplicates and the entire experiment repeated with two to four heifer calves. The least detectable concentration was 125 pg/mL and intra and inter assay coefficients of variation were less than 5%. The assay used in this study is bovine TNF specific and has been reported to show no cros s reactivity with either human or mouse recombinant TNF For each experiment all three heifers and treatments were included in a single assay to adjust for differences among sampl es due to between assay variations. Isolation of Cytosolic and Nuclear Proteins Cytosolic and nuclear proteins from PBMC were isolated using a commercial kit (NE PER, Pierce Biotechnology, Rockville, IL). Briefly, control (medium alone) and treated (LPS, LPS + rosiglitazone, and LPS + rosiglitazone + GW9662) PBMC were collected and washed with ice cold phosphate buffered saline at 500 x g for 3 min. The supernatant was discarded and the pellet resuspended in 100 L of ice cold Cytosolic Extration Reagent I (CER I, Pierce Biotechnology). The cell suspension was

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94 vigorously mixed and then incubated on ice for 10 min. After incubation, 5.5 L of CER II were added to all tubes and the mixture incubated on ice for an additional 1 min. Cell extracts then we re centrifuged at 15,000 x g for 5 min. The supernatant, containing cytosolic proteins, was immediately transferred to pre chilled tubes and stored at 80oC until Western blot analysis. The pellet from cytosolic protein extraction was resuspended in 50 L of Nuclear Extraction Reagent (NER). The mixture was incubated on ice for a total of 40 min, with vigorous mixing every 10 min. Nuclear extracts were centrifuged at 15,000 x g for 10 min and the supernatant immediately frozen at 80oC until Western im munoblotting. Western Blot Analysis of Nuclear Factor Kappa B Protein concentrations in nuclear and cytosolic extracts were determined using the Pierce BCA protein assay kit (No. 23225, Pierce Biotechnology, Rockford, IL). s evaluated by Western immunoblotting as previously described (Rodriguez Sallaberry et al., 2006). Briefly, 20 g of nuclear or cytosolic proteins were resolved on a 10% SDS PAGE and transferred electrophoretically to Immobilon P membranes (Millipore Corp ., Bedford MA). The membranes were blocked with 5% (w/v) non fat dry milk in Tris buffered saline containing 0.1% Tween 20 (TBST) and incubated overnight at 4oC with the primary monoclonal antibody against rabbit NF kBp65 at the dilution of 1:1,000. The actin (diluted to 1:1,000) was used as loading control. After incubation, the membranes were washed for 20 min with TBST and further incubated with HRP conjugated secondary antibody at the final dilution of 1:3,000 for 1 h. Primary antibodies (anti rab bit NF actin) were diluted in 3% (w/v) BSA in TBST, whereas the secondary antibody (anti rabbit HRP conjugated IgG) was diluted in 1% (w/v) nonfat dry milk in TBST. Target proteins were detected by

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95 enhanced chemiluminescence and the relative ab undance of each protein was quantified using ImageJ software (Version 1.43) developed by NIH (Rasband, 1997 2009). Statistical Analyses Proliferation, TNF and NF procedure in the SAS software package (SAS Inst itute Inc., Cary, NC). The statistical models included the effects of treatment, heifer and treatment x heifer interaction. When appropriate, treatment was replaced by incubation time or FA dose in the statistical models. The heifer effect was considere d random and, therefore, the variance of treatment x heifer, time x heifer or dose x heifer was used to test the main effect of treatment, time or dose, respectively. The relationships between dose and time of LPS treatment and TNF ultures were further examined using polynomial contrasts. Differences were considered to be significant at P < 0.05, whereas tendencies were discussed at P > 0.05 but < 0.15. Data are presented as least squares means SEM. Results Effect of Lipopolysacc haride on Peripheral Blood Mononuclear Cell Proliferation Addition of LPS (10 M) to the culture medium increased (P < 0.01) PBMC proliferation up to 2.5 fold (Figure 3 1). Co incubation with IFN stimulated ( P < 0.01) the proliferative response of PBMC to LPS (Figure 3 1). There were no detectable differences between basal and IFN induced PBMC proliferation (Figure 3 1).

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96 Tumor Necrosis Factor Alpha Response to Lipopolysaccharide Blood TNF P < 0.01) linearly as the amount of LPS in the cul ture medium increased from 0 to 10 g/mL (Figure 3 2A). There was a curvilinear relationship between the time of LPS treatment and TNF TNF P < 0.01) between 0 and 12 h of LPS stimulation and then leveled off by 24 h after LPS addition to the cultures (Figure 3 2B). Addition 100 M of LA and c 9, t 11 CLA isomer to the culture medium had no detectable effects on LPS induced TNF (Fig Figures 3 3A and 3 4). Conversely, the t 10, c 12 CLA isomer (100 M) decreased ( P < 0.01) LPS stimu lated TNF (Figures 3 3B and 3 4). Inclusion of rosiglitazone, a PPAR agonist, in the culture medium attenuated ( P < 0.01) LPS in duced TNF 5). The PPAR induced TNF attenuation was reversed ( P < 0.05) when blood was treated with both rosiglitazone and GW9662, a selective PPAR 5). Nuclear F actor Kappa B Response to L ipopolysaccharide Addition of rosiglitazone to the culture medium tended ( P = 0.12) to decrease NF extracts isolated from cultured PBMC (Figures 3 6 and 3 7). There were no significant differences between basal and LPS induc ed NF concentration in either nuclear or cytosolic extracts. Discussion Lipopolysaccharide, a surface component of gram negative bacteria released upon host infection, is one of the strongest stimuli that induce TNF immune cells (Morr ison and Ryan, 1998; Raetz et al., 1991). Consistent with in vivo studies (Bruzzone et al., 2003; Yu et al., 2002 a ; Goldberg et al., 2008; Farran et al.,

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97 2008; Kahl et al., 2009), LPS stimulated PBMC proliferation and induced TNF production by whole bov ine blood cells in a dose and time dependent manner. The molecular mechanisms by which LPS affects cytokine biosynthesis are complex and likely involves activation of intracellular signaling molecules such as myeloid differentiating factor (MyD) 88, IL 1 R associated kinase (IRAK), and TNFR associated factor (TRAF) 6. This process ultimately results in the activation of two different pathways that involve the JNK and p38 MAPK family and the Rel family transcription factor NF zio et al., 1998) Pretreatment of whole bovine blood with t 10, c 12 CLA isomer attenuated TNF response to LPS by 55%. The effect was isomer specific, because the c9, t 11 CLA isomer or LA had negligible effects on LPS induced TNF ine blood. Results suggest that the net effect of CLA on immune function of domestic mammals may vary depending on isomeric composition of CLA test products. Exact molecular mechanisms by which CLA alters the release of proinflammatory cytokines are not fully understood. Available dietary intervention and in vitro studies would suggest that exogenous CLA may inhibit TNF 2 supplemen tal CLA may induce intracellular release of intermediate molecules which subsequently alter immune responses in mammalian species. In support of this hypothesis, Cho and co workers (Cho et al., 2008) showed that, in dogs, immunoenhancing effects of t 10, c 12 CLA isomer on the phagocytic capacity and oxidative burst activity of neutrophils were mediated by TNF t 10, c 12 CLA treated PBMC. Furthermore, in the current study, the effects of t 10, c 12 CLA on

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98 LPS induced TNF regulate TNF s that do not involve this fatty acid and were not examined in the present study. Peroxisome proliferator activated receptors are a family of nuclear receptors that regulate the expression of genes involved in energy homeostasis and immune function. In th e present study, incubation of whole bovine blood with rosiglitazone, a selective PPAR induced TNF induced TNF both rosiglita zone and GW9662, a selective PPAR mechanisms through which PPAR complex and likely involve several cytosolic and nuclear signaling proteins (Kota et al., 2005). Available experimen tal models indicate that PPAR proinflammatory cytokine production by antagonizing the activities of various signaling molecules such as JNK and p38 MAPK in vivo (Desreumaux et al., 2001) or by interfering with transcription factor activatio n such as NF 1 and NFAT (Dubuquoy et al., 2002; Delerive et al., 2001). The observation that rosiglitazone tended to decrease cytosolic and nuclear NF suggest that the PPAR induc ed TNF by interfering with activation and nuclear translocation of NF

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99 Figure 3 1. Interferon gamma (IFN induce peripheral blood mononuclear cell (PBMC) proliferation. Results are expressed as fold stimulation over untreated cells. Freshly isolated PBMC were stimulated with interferon gamma (IFN combination of these two molecules for 72 h. During the last 24 h of culture, cells were pulsed with [ 3 H ] thymidine and incorporation of label into DNA was assessed by liquid scintillation counting. Different letters above histograms indicate statistical differences at P < 0.05.

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100 Figure 3 2. Lipopolysaccharide (LPS) stimulates tumor necrosis factor alpha (TNF production in cultured bovine blood in a dose (A) and time (B) dependent manner. Heparinized blood from clinically healthy Holstein heifers was incubated with increasing doses (A) of LPS or with 1 g of LPS for increasing times (B). Differ ent letters above histograms indicate statistical differences at P < 0.05.

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101 Figure 3 3. Trans 10, cis 12 conjugated linoleic acid ( t 10, c 12 CLA) isomer attenuates lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNF pr oduction in a dose dependent manner. Diluted blood samples were incubated with increasing doses of c 9, t 11 CLA (A) or t 10, c 12 (B) conjugated linoleic acid isomers for 24 h. Cultures then were challenged with LPS for 24 h. Results are expressed as percentag es of LPS stimulation in the absence of fatty acids. Different letters above histograms indicate statistical differences at P < 0.01.

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102 Figure 3 4. Isomer specific effect of trans 10, cis 12 conjugated linoleic acid ( t 10, c 12 CLA) on lipopolysaccharide (L PS) induced tumor necrosis factor alpha (TNF without (control) or with 100 M each of linoleic acid (LA), c 9, t 11 or t 10, c 12 CLA for 24 h. Cultures then were challenged with LPS fo r 24 h. Results are expressed as percentages of LPS stimulation in the absence of fatty acids. Different letters above histograms indicate statistical differences at P < 0.01.

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103 Figure 3 5. Rosiglitazone attenuates lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNF were cultured without (control) or with LPS for 24 h. One set of cultures was treated with a selective peroxisome proliferator activated receptor gamma (PPAR osiglitazone) for 1 h before LPS addition and another set received both PPAR 1.5 h, respectively, prior to LPS addition. Results are expressed as fold stimulation over control cultures. Different letters above histograms indicate statistical differences at P < 0.05.

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104 Figure 3 6. Western blot analysis of nuclear nuclear factor kappa B p65 (NF kB p65). Peripheral blood monocytes were cultured without (control) or with lipopolysaccharide (LPS ) for 24 h. One set of cultures was treated with a selective peroxisome proliferator activated receptor gamma (PPAR (rosiglitazone) for 1 h before LPS addition and another set received both PPAR respectively, prior to LPS addition. Rossiglitazone tended ( P = 0.12) to reduce the nuclear NF kB tr anslocation compared to lipolysaccharide treatment.

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105 B Figure 3 7. Western blot analysis of cytosolic nuclear factor kappa B p65 (NF kB p65). Peripheral blood monocytes were cultured without (contr ol) or with lipopolysaccharide (LPS) for 24 h. One set of cultures was treated with a selective peroxisome proliferator activated receptor gamma (PPAR (rosiglitazone) for 1 h before LPS addition and another set received both PPAR respectively, prior to LPS addition.

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106 CHAPTER 4 ASSOCIATION OF PASSIVE TRANSFER WITH PERFORMANCE, INCI DENCE OF DISEASES, AND RISK OF DEATH IN PREWEANED HOLSTEIN CALVES Introduction The syndesmochorial placenta of cow prevents transfer of Ig to the fetus; as a consequence, calves are agammaglobulinemic at birth. Colostrum management is often indicated as o ne of the most important fact ors in determini ng calf health and survival (Go dden et al., 2009). The ingestion and absorption of colostral Ig is essential for establishing passive immunity. Failure of passive transfer predisposes the neonate to development of disease (Ber ge et al., 2009; Wittum and Per ino, 1995). Measurement of calf serum IgG concentration is used for determining the level of passive transfer of immunity. In 2007 the National Animal Health Monitoring System (NAHMS) reported that in the US A almost one out of five heifer calves (19.2%) had FPT of immunity (IgG < 1 g/dL) In the western states of the USA, 21.2% of calves had FPT, whereas in the eastern states, the corresponding proportion was 18.8% (NAHMS, 2007). Some studies have indicated that FPT is related with risk of mortality and performance of weaned calves and replacement heifers. Tyler et al. (1998) reported that calves with FPT experienced increased mortality until at least 10 weeks of age. Robinson et al. (1988) found that heifers with FPT had greater mortality during the postweaning period compared to those with APT. Wittum and Per ino (1995) found that beef calves with FPT had inc reased risk of neonatal and preweaning mortality, and pre weaning mo r b id ity than those with APT. Adequ ate passive transfer of IgG not only reduces the mortality but also increases productive performance. Serum IgG concentrations greater than 1.2 g/dL at 24 to 48 h

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107 of age in heifer calves were associated with increased ADG up to 180 d of life and greater we aning weights compared with calves with low er serum IgG concentration (Robinson et al. 1988). DeNise et al. (1989 ) found that heifers with FPT produced less milk during the first lactation and tended to have increased risk of culling before completing the first lactation when compared with those with APT. Failure passive transfer was not associated with age at first calving. Calves with low STP or Ig concentrations generally are accepted to be at great risk for morbidity and mortality. Nevertheless, some s tudies have failed to identify any significant increase in risk of mortality or morbidity associate d with low serum IgG (Tyler, 1998; Rea et al., 1996). In the current study, calves from multiple farms under diverse management practices were evaluated for the association of passive transfer with measures of growth performance, feed efficiency, health and costs associated with pre weaning rearing. We hypothesized that FPT compromises growth, feed efficiency, health, survival, and income from calves at weanin g. Materials and Methods Animals, Housing and Feeding An observational prospective cohort study was conducted with 1,247 Holstein calves, 317 males and 930 females, from 7 dairy farms In all farms, calves were housed in individual hutches and fed 1.9 L o f frozen thawed colostrum in the first 4 h of life and an additional 3.6 L in the following 20 h. In 3 sites, pasteurized milk was fed at 6 L split into three feedings daily of 2 L each from 1 to 21 d of age, and then 4 L split into two feedings from 22 to 60 d of age, whereas in the other 4 sites calves were fed 4 L of pasteurized milk split into 2 daily feedings for the first 56 d of life. Calves remained in the hutches until 64 to 75 d of age, according to farm, when the study ended.

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108 Calves had ad libitu m access to water and a starter grain throughout the study. The DM content of calf grain was monitored weekly to determine grain DM intake. Grain intake and refusals were measured daily, and amounts offered were adjusted to allow for 10% orts (minimum of 1 00 g) during the study. Milk was sampled 3 to 4 times weekly and analyzed for nutrient content to determine the amount of milk DM consumed by each calf. Body Weight and Blood Sampling and Analyses Calves were weighed o n 2 consecutive days at 1, 30 and 64 d of age and the average BW for each of the 3 time points was used for statistical analyses. Blood was sampled by puncture of the jugular vein using evacuated tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) without any additive for serum separatio n. Blood was kept at ambient temperature until clotting and then placed on ice until transported to the laboratory. Tubes were centrifuged at 3000 rpm at 4C for 20 min. Sera were separated and the total protein determined using a clinical refractometer (R eichert Jung, Buffalo, NY). Sera was then frozen and later analy zed for total IgG concentration using a bovine IgG radial immunodiffusion kit from Veterinary Medical Research and Development Inc. (Pullman, WA). T he assay was performed according to manufact percent of total protein in serum represented by IgG was also calculated. Incidence of Health Disorders, Treatments and Costs Associated with Treatments Calves were observed daily for diagnosis of health disorders. Rectal temperatur e was monitored daily using an electronic thermometer (M750, GLA Agricultural Electronics, St. Louis Obispo, CA) for the first 14 d of age. Fever was considered when rectal te mperature was greater than 39.4 o C. Fecal scores were evaluated daily and calves w ith runny or watery feces (Magalhes et al., 2008; Larson et al., 1977) for at

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109 least 2 consecutive days were considered as having diarrhea. Pneumonia was characterized by presence of respiratory distress, increased respiratory frequency and nasal discharge with or without fever. Treatment of diseases was according to each farm standard operating procedures based on veterinary recommendation. Treatments were performed by farm staff, and records of each medication used in each calf were collected daily by the research team for later analyses of cost of health treatments. Economic Analysis of Calf Rearing An economic analysis of cost of rearing calves was performed Measures included cost of grain consumed by each calf, cost of milk consumed by each calf, trea tment costs for health problems, labor costs associated with feeding calves and the value of a calf that survived at the end of the study. The input values in the calculation were as follows: $0.37/kg of grain DM; $0.15/L of pasteurized milk. Labor cost wa s computed at actual local cost of $0.45/calf/d based on an hourly wage of $10 and the measurements in the 7 farms in which the average employee was able to handle the daily acti vities of 23 calves/h for feeding milk and grain, replacing water, treating si ck calves, and bedding hutches. The value of a newborn male calf was assumed at $100 and a female at $500. Therefore, a dead male represented an opportunity cost of at least $100, whereas a female at least $500. The value of a live calf at 75 d of age was computed at $300/male and $750/female. Rearing cost was calculated from the summation of costs with feeding, health treatments, and labor. The value of a calf was computed for males and females based on the respective weaning value (male = $300; female = $ 750) or the respective cost of a dead calf (male = $100; female = $500). Income per calf was calculated based on the value of a calf minus the rearing cost.

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110 Statistical Analyses The study was an observational prospective cohort study. Receiver operator cha racteristic (ROC) curve was calculated using the ROC curve analysis of MedCalc software (MedCalc ver. 10.0.1.0 software, Mariakerke, Belgium) Serum IgG cut point was selected based on results by others in the literature (Berge et al., 2005; Tyler et al., 1996). Calves were classified as having FPT when serum IgG < 1.0 g/dL or APT when if Th e ROC curve was used to determine the sensitivity and specificity of the selected cutoff using the preweaning mortality data Categorical respons es were analyzed using the GLIMMIX procedure of SAS (SAS/STAT ver. 9.2, SAS Inst. Inc., Cary, NC) fitting either binary distribution with a logit link or Poisson distribution with a log link. Multivariate models were built and included the fixed effects of passive transfer (APT vs. FPT), calf gender (male vs. female), season of birth (spring, summer, fall, and winter), and the random effect of farm by passive transfer interaction Additional explanatory variables were used when appropriate. The interaction between passive transfer and calf gender was included in the models when P < 0.05. PHREG procedure of SAS. The adjusted hazard ratio (AHR) estimates the relative rate of mortality according to the explanatory variables used in the model. The time variable used in the model was the interval in days between birth and death or when calves remaining alive were censored at the end of the pre weaning period. Proportionality was assessed by evaluating the Kaplan Meier curves. The mean ( SEM) survival time were calculated using the LIFETEST procedure of SAS. Survival plots were generated with

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111 MedCalc. Explanatory variables in the model included passive transfer, calf gender, season of birth, and farm. Continuous data were analyzed by the GLIMMIX procedure of SAS fitting a normal distribution function. Statistical models included the fixed effects of passive transfer, calf gender, season of birth, and the random effect of farm. Additional expl anatory variables were used when appropriate. The interaction between passive transfer and calf gender was included in the models when P < 0.05. The association of serum IgG concentrations on the probability of death was modeled using logistic regression c urves with the intercept and the coefficient estimates from the analysis applied to the logistic formula: P = 1/[1+(e(a+b1X1 + a+b2X2 + Additional logistic regression analyses were performed to determine if STP, IgG, or the percent of total protein a s IgG influenced risk of death in pre weaning dairy calves. Initially, the statistical multivariate models included the effects of either STP or serum IgG or the percent of total protein as IgG. The models were built using GLIMMIX and also included the fixe d effects of gende r and season. Farm was a random effect in the model. The model was fitted with binary distribution with a logit link function. After that, a final multivariate logistic regression model was built and included the fixed effects of STP, ser um IgG, percent of total protein as IgG, gender and season, and the random effect of farm. Least square means, proportions and adjusted odds ratios (AOR), AHR, and medians are reported. Treatment differences with P and 0. 05 < P

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112 Results Results from ROC analysis indicated that when cut point of serum IgG < 1.0 g/dL is used to predicted pre weaning mortality the sensitivity and specificity is 26.9% an d 93.7%, respectively (area under the cur ve of 0.62; 95% CI = 0.59 0.64; P < 0.001). Serum concentration of IgG 1.0 g/dL is the same value suggested by others (Tyler et al., 1996; Berge et al., 2005) to categorize calves as not having received APT. The high specificity indicates that 93.7% of t however, the low sensitivity indicates that only 26.9% of the calves that died had serum IgG < 1.0 g/dL. The descriptive statistics according to farm is presented in the Table 4 1. As anticipated, the conc entrations of total protein and IgG in sera of calves were variable according to farm of origin, and they averaged 6.0 and 2.6 g/dL, respectively. The proportion of evaluated calves classified as having FPT ranged from 0 to 32.7%. Overall, 7.6% of the calv es had serum IgG < 1.0 g/dL. Morbidity was high and affected 75.1% of the calves, and 6.3% died before d 75 of age. Body weight on day 1 was not different between FPT and APT calves (Table 4 2). On day 30, APT calves weighed 1.3 kg more ( P < 0.01), but th is advantag e was no longer present at 64 d of age. The ADG in the first 30 d of age was greater ( P < 0.01) for APT than FPT calves, but after 30 d of age, calves gained at a similar rate of BW gain, averaging 726 g/d. The daily grain DM intake was greater ( P < 0.01) for APT than FPT calves in the first 30 d of age, but similar afterwards. Despite the similar daily grain DM intake from 31 to 64 day of age APT calves consumed more ( P < 0.01) total kg of grai n and milk DM in the entire pre weaning period (Tabl e 4 2). The DM conversion ratio was improved for calves classified to have APT than those with FPT.

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113 As anticipated, calves classified as having APT had greater ( P < 0.01) STP and IgG than those classified as FPT (Table 4 3). Four hundred twenty three cal ves had IgG greater to 3 .0 g/dL. Morbidity in all calves was high, particularly because of diarrhea, but APT calves tended ( P = 0.06) to have less morbidity than FPT calves. For the individual diseases, incidences of fever and diarrhea did not differ betwe en APT and FPT, but pneumonia tended ( P = 0.08) to affect more FPT than APT calves. As a result, calves suffering from FPT had increased ( P < 0.01) incidence of multiple diseases and a greater ( P = 0.01) mean number of disease cases per calf. Calves class ified as ATP were less likely to have multiple diseases than FPT calves. Mortality increased ( P < 0.01) 4 fold in calves with FPT (Table 4 3). Similarly, calves with FPT had 3.6 fold greater ( P < 0.01) hazard of dying in the first 75 d of life than calves with APT (Figure 4 1). When the statistical model included serum concentration of IgG in place of FPT, the probability of death in the first 75 d of age decreased as the serum IgG concentration increased (Figure 4 2). Although serum IgG and total protein were closely related (r = 0.79; P < 0.001) and, as STP increased so did serum IgG (IgG = 4.535 + 1.190 STP; r 2 = 0.53, P < 0.001), probability of death responded differently for STP (Figure 4 3). As STP increased, the probability of death declined until approximately 5.5 g/dL. At that concentration of STP, probability of death reached its nadir which was maintained until STP of approximately 7. 0 g/dL; however, probability of death increased again when STP increased beyond 7. 0 g/dL. Interestingly, when ser um IgG, STP, and the percent of STP as IgG were all included in the statistical model to predict risk of death, only the percent of IgG as STP influenced ( P < 0.001) risk of death, but not STP

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114 ( P = 0.21) or serum IgG ( P = 0.13). In fact, Figure 4 4 depicts the decline in probability of death in the pre weaning period as the percent of STP as IgG increases. The cost of feeding calves differed between APT and FPT (Table 4 4). The cost of feeding milk was greater ( P < 0.01) for FPT than APT; however, because g rain intake was greater, the cost of feeding grain/d was greater ( P < 0.01) for APT compared with FPT. Daily treatment costs and the overall cost/d of rearing a pre weaning calf did not differ between groups. When costs were calculated per calf during the e ntire pre weaning period, then raising APT calves was more expensive. Calves considered as having APT consumed more kg of milk and grain DM (Table 4 2), which resulted in greater costs associated with feeding and labor per calf (Table 4 4). These difference s resulted in a 13.6% increase in the rearing cost per calf for APT. Nevertheless, the improved weight gain in the first 30 d of age and survival during the pre weaning period resulted in more ( P < 0.01) kg of calf weaned in APT than FPT. Because of improve d survival, APT calves had a greater ( P < 0.01) value for males and females, and they also generated greater ( P < 0.0 1) income at the end of the pre weaning period. Discussion The transfer of Ig from the dam to the neonate is critical for protection from infectious diseases. Colostrum feeding provides more than passive immunity as it contains twice to three times the fat content of milk, four to five times the protein, and many growth factors important for intestinal development in the young calf ( Blum and Hammon, 2000). In the USA, approximately 19.2% of the dairy calves have FPT based on serum IgG concentrations (IgG < 1.0 g/dL; NAHMS, 2007). Increased neonatal mortality caused by FPT is a well accepted consequence of inadequate colostrum intake or IgG ab sorption (Berge et al., 2009; Weaver et al., 2000). The results of this

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115 study showed that calves with FPT experienced increased incidence of multiple disease s, and mortality during the pre weaning period, and tended ( P = 0.06) to be increased morbidity whic h corroborates with results from Wittum and Perino (1995) in beef and Robinson et al. (1988) in dairy calves. Calves classified as having APT had improved measures of growth performance in the first 30 d of age. Calves with APT had improved daily grain in take, which increased ADG and body weight at 30 d of age. Bovine colostrum has more total solids, fat, casein, minerals and vitamins than milk (Go d den, 2008). Although all calves received the same amount of colostrum, it is possible that composition varied and absorption of IgG was not similar among calves, which explains the variability in serum IgG concentrations of calves at 48 h of age. Colostrum also contains o ther important components that might improve calf performance such as growth factors (i.e. tr ansforming growth factor beta 2 ,TGF insulin like growth factor I, IGF 1) and white blood cells (Go d den, 2008). Colostral IGF 1 may be a key regulator in the development of gastrointestinal tract of bovine neon ates, including stimulation of mucosal growth, brush border enzymes, intestinal DNA synthesis, increased villous size, and glucose uptake (Bhler et al., 1998). In addition to stimulating tissue development, IGF 1 also participates in ensuring an adequate immune response by the calf, because it fosters proliferation and development of lymphocytes (Oda et al., 1989). Hammon and Blum (1997) reported that serum IGF 1 concentrations were greater in calves fed colostrum compared with those only fed milk replacer In the current study, calves were fed a specific amount of colostrum in the first then it is likely because the

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116 composition of colostrum may have varied Calves with increased serum IgG might have also received more nutrients, growth factors and hormones allowing an improved grain intake and growth rate during the first weeks of life. Calves with FPT suffered from greater multiple diseases and had more disease cases than calves with APT. Decreased appetite during infections is mediated in part via cytokines produced by the immune system (Johnson, 19 9 8) The most common diseases that affect calves in the first weeks of life involve the GIT (NAHMS, 2007). Diarrhea and other enteric diseases impair nu trient absorption by affecting the villous epithelial cell of the intestine s ( Parreo et al., 2004) which is expected to reduce weight gain and compromise feed efficiency. Inflammation generated during the development of disease results in the release of cytokines, which can also activate homeo r rhe t ic responses to partition nutrients away from tissue deposition and toward immune response (Colditz, 2004). As a consequence, the possible difference in amount of nutrients, growth factors, and hormones ingested from colostrum, and the incidence of disease s between groups might explain the reduce d measures of growth performance during 30 d of age. In fact, calves with FPT had reduced DM convers ion ratio during the entire pre weaning period confirming th at feed eff iciency is reduced as reported by Robinson et al. (19 8 8). Robinson et al. (19 8 8) studied 1000 dairy heifers in a single farm and observed that as serum IgG concentration increased, ADG in the first 6 months of age also improved. Similarly, Berge et al. (2 009) reported that calves with FPT (IgG < 1 .0 g/dL) showed less ADG during the 60 d than APT calves. In contrast and confirming the results found in the current study, Virtala et al. ( 1996 ) observed that FPT reduced ADG

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117 in the first month of life of calve s but during the second month, FPT was not associated with ADG. In the current study, calves had similar body weight at weaning regardless of being classified as APT or FPT. Nevertheless, calves with FPT suffered from greater mortality, which occurred mos tly in the first month of age (Figure 4 1), so these calves did not contribute to the measurement taken after the first 30 d of age. After 30 d of age FPT calves that survived may have had a greater ability to withstand inadequate passive transfer and had compensatory responses relative to nutrient intake and weight gain. Mortality in pre weaned calves occurs primarily in the first weeks of life and it is mostly associated with GIT problems (NAHMS, 2007) although respiratory problems are also important B erge et al. (2009) showed that calves with serum IgG < 1.0 g/dL had more days with diarrhea than those with APT in the preweaning period Incidence of diarrhea was evaluated in the current study, but not the duration, and no association was observed betwee n passive transfer of IgG and risk of diarrhea Most dairy calves are diagnosed at least once with diarrhea in the preweaning period (Magalhes et al., 2008), which was also observed in the current study (74.8%) Therefore, it is possible that APT is more critical to minimize the intensity of diarrhea, but not necessarily to prevent it. In fact, Parreo et al. (2004) showed that the number of days that calves experi enced diarrhea was greater in colostrum deprived calves than colostrum fed calves, but incide nce was the same, 100%. Antigen specificity of absorbed Ig is important in determining the specific protective ability of colostrum (Nechvatalova et al., 2011). Deficiency of specific antibod ies might also explain the lack of association between incidence of diarrhea and passive immunity. On the other hand, it is well

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118 know n that colostral IgA block the adhesion of microbial pathogens onto the intestinal epithelial surface ( Murphy et al., 2008 ), thereby contributing to mucosal defense. The lack of significan t relationship between passive transfer and diarrhea in the present study should not be interpreted as a sign that transfer of colostral IgG is not an important determinant of preweaning diarrhea as duration and severity might be more critical than inciden ce per se The second cause of calf morbidity and mortality in the preweaning period of dairy calves are diseases that affect the r espiratory tract. According to surveys by NAHMS (2007), 12.4% of dairy calves are diagnosed and treated for respiratory disea ses before weaning and diseases that affect the respiratory tract are reported to be responsible for 22.5% of the deaths in preweaning dairy calves (NAHMS, 2007). Calves with APT tended ( P = 0.08) to have less incidence of pneumonia in the current study, which is similar to the finding of Virtala et al. (1999), who reported that calves with serum IgG > 1.2 g /dL had a smaller risk of pneumonia than those with IgG < 1.2 g/dL. T he study was conducted on different farms, thus management, environment and diseas e pressure varied across animals. T he results of this study likely represent the potential impact of inadequate passive transfer on health, growth, efficiency, and survival of calves in dairy farms. Although calves were classified as having APT when serum Berge et al., 2005; Tyler et al., 1996) it is clear that increases in serum IgG beyond 1.0 g/dL reduced the risk of mortality (Figure 4 2). Based on these results it is evident that if mortality is to be reduced, an initial step is to ensure that most calves receive adequate colostrum to maximize IgG transfer. Clearly, the chosen cut off of 1.0 g/dL has impacts

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119 on health, growth and survival, but to further minimize the probability of m ortality greater IgG concentrations shou ld be achieved. The typical pre weaning mortality of dairy heifers is approximately 7.8% in the US (NAHMS, 2007), which is slightly higher than the 6.3% observed in the 7 farms studied. Based on Figure 4 2, if mortal ity is to be reduced to less than 4%, serum IgG has to be greater than 3.0 g/dL This is important as it indicates that additional colostrum feeding or better timing of feeding and/ or management of colostrum quality might be needed to optimize IgG transfer to the newborn to achieve concentrations of serum IgG greater than 3 g/dL in most calves. Although serum IgG is the reference measurement to determine passive transfer, most producers use indirect measures of Ig concentration in serum. Total protein mea surement using a refractometer is often recommended as a proxy for serum IgG. In fact, serum IgG increased by 1.19 g/dL for each 1.0 g/dL increase in STP (r 2 = 0.53). However, the pattern of probability of mortality according to STP differed from that of serum IgG. Initially, as STP increased, the probability of death declined until approximately 5. 5 g/dL. It then reached a nadir that was maintained until STP of 7. 0 g/dL, after which the probability of death increased again. In fact, 23% of the calves with STP > 7.0 g/dL died during the first 11 d of life and 15% of them died during the first 5 day s of age High STP reflects increased concentrations of albumin and globulins; however, the concentration of STP can be also falsely increased when the calf is de hydrated ( Tyler et al., 1999 b ). Dehydration as a consequence of diarrhea is a major cause of death in dairy calves (Davis and Drackley, 1998). The increase in probability of mortality in calves with STP above 7. 0 g/dL might indicate dehydration at 48 h of age, thereby compromising the ability to survive in the preweaning period. An important

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120 finding of this study is that when serum IgG, STP, and the percent of STP as IgG were all included in the statistical model to predict risk of death, only the percent o f IgG as STP influenced mortality as shown in Figure 4 4. These data suggest that increases in STP per se can be detrimental to survival, unless it is the result of increased concentrations of IgG. Therefore, measuring STP only is an indicator of passive t ransfer, but it is less than adequate when concentrations are high. On the other hand, if concentrations of STP are high, but as a result of increased serum IgG, then risk of mortality is reduced. Heifer rearing represents the second largest expense on a dairy operation, and producing high quality replacement heifers at minimum cost will be one of the many challenges facing the dairy producers of the 21 st century (Heinrichs, 1993). Researchers have examined association s between mortality, serum STP and IgG concentrations but have not fully examined the economic implications. In this study, the daily rearing cost per calf did not differ between groups, but the total rearing costs in the preweaning period increased in APT calves compared to FPT calves. Despit e increased rearing costs in calves with APT, the value of a weaned calf and the total income per calf were both greater for calves with APT than those with FPT. This discrepancy was caused by improved survival that led to a longer period of feeding of mil k and grain to calves. Indeed, the improved survival resulted in 12.5 kg more weaning weight because more calves contributed with the weaning population in APT than FPT. Martin s and Wiggins (1973) examined the impact of calf death on income from sales of c alves in the first 5 of this period. They found that a 20% calf mortality rate reduce d the profitability of the calf

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121 rearing operation by 38% compared with a mortality of zero. The results in this study indicated that in spite of the increased r earing costs, APT calves result in major economic advantages compared with calves having FPT. Overall, the income per male and female calf increased 35% and 55% respectively, for APT com pared with FPT. Conclusions The results of this study rev ealed that transfer of Ig from the dam to the neonate is important in the first two month s of life of calves. Assuring that calves attain serum IgG concentrat improve growth, feed ef ficiency, health, and reduce mortality in dairy calves during the preweaning period, which increase s profitability of the calf rearing operations Although APT was classified based on serum dL, the probability of death within the first 75 d of age decreased as IgG concentration in serum increased Th e result s suggested that to reduce mortality in preweaning dairy calves to less than 4%, the first step must be to ensure s erum IgG greater than 3.0 g/dL which will require better management of colostrum feeding Interestingly, t he measurement of STP by refractometry as an estimate of serum Ig concentration needs to be taken with precaution whe n concentrations are high (> 7.0 g/dL) or when the con tribution of STP from IgG is low When STP increased as a result of increases in serum IgG, it reduced risk of mortality. Although high STP was closely related to serum IgG, when concentrations were either < 5. 5 or > 7. 0 g/dL, probability of mortality incr eased. R esults from this study indicate that IgG as percent of STP i s a better predictor of survival of preweaned calves than STP or serum IgG concentration s alone.

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122 Table 4 1. Descriptive statistics according to farm (mean SD or proportion) Farm 1 2 3 4 5 6 7 All Farm Male, n 44 77 52 48 17 7 72 317 Female, n 0 282 0 76 72 60 440 930 Serum protein, g/dL Total 5.58 0.47 6.08 0.59 5.12 0.58 5.72 0.63 5.41 0.56 6.40 0.58 6.12 0.67 5.97 0.68 IgG 2.42 0.83 2.89 1.18 1.5 3 0.73 1.98 0.74 1.88 1.12 3.16 1.05 2.64 1.03 2.56 1.12 FPT, 1 % (n/n) 9.1 (4/44) 4.7 (17/359) 32.7 (17/52) 9.7 (12/124) 25.9 (23/89) 0 (0/67) 4.3 (22/512) 7.6 (95/1247) BW 2 d 1, kg 44. 2 5.2 43.9 5.2 43.9 4.2 40.0 5.0 41.4 5.6 40.3 5.1 40.2 5.2 41.6 5.5 Morbidity, % (n/n) 75.0 (33/44) 35.9 (129/359) 46.2 (24/52) 95.2 (118/124) 86.5 (77/89) 89.6 (60/67) 96.7 (495/512) 75.1 (936/1247) Mortality, % (n/n) 6.8 (3/44) 2.5 (9/359) 9.6 (5/52) 2.4 (3/124) 7.9 (7/89) 1.5 (1/67) 9.8 (5 0/512) 6.3 (78/1247) 1 FPT = failure of passive transfer based on serum IgG < 1.0 g/dL. 2 BW = body weight.

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123 Table 4 2. Association between passive transfer and performance of Holstein calves in the pre weaning period Passive transfer 1 APT FPT P Bo dy weight, kg Day 1 42.1 0.3 42.6 0.6 0.37 Day 30 51.1 0.2 49.8 0.4 < 0.01 Day 64 74.6 0.4 73.6 1.0 0.33 Average daily gain, g/d 1 to 30 d 298 8 213 17 < 0.01 31 to 64 d 727 10 718 23 0.71 G rain DM intake, g/d 1 to 30 d 156 4 126 9 < 0.01 31 to 64 d 1,134 18 1,133 39 0.98 Total grain DM intake, Kg 1 to 30 d 4.6 0.1 3.8 0.3 < 0.01 31 to 64 d 39.4 0.9 33.2 1.9 < 0.01 Total milk DM intake, Kg 1 to 30 d 15.6 0.1 14.6 0.2 < 0.01 31 to 64 d 11.2 0.2 10.1 0.3 < 0.01 DM conversion ratio 2 1 to 30 d 0.45 0.01 0.34 0.02 < 0.01 31 to 64 d 0.45 0.01 0.43 0.01 0.04 1 transfer, serum IgG < 1.0 g/dL. 2 Kg of body weight change per kg of DM consumed (milk + grain).

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124 Table 4 3. Association between passive transfer and health of Holstein calves i n the pre weaning period Passive transfer 1 Item 2 APT FPT AOR (95% CI) 2 P Serum protein, g/dL Total 5.89 0.11 5.07 0.13 --< 0.01 IgG 2.58 0.13 0.80 0.16 --< 0.01 IgG as % total protein 43.0 1.7 15.5 2.1 --< 0.01 Morbidity, 3 % (n/n) 77.8 (896/1152) 84.2 (80/95) 0.50 (0.24 1.04) 0.06 Diseases, % (n/n) Fever 39.2 (452/11 52) 43.2 (41/95) 0.92 (0.59 1.45) 0.72 Diarrhea 74.8 (808/1152) 74.7 (71/95) 0.61 (0.29 1.30) 0.20 Pneumonia 16.2 (187/1152) 25.3 (24/95) 0.63 (0.38 1.06) 0.08 Multiple diseases, 4 % (n/n) 40.5 (467/1152) 54.7 (52/95) 0.61 (0.39 0.96) 0.03 Disease cases /calf 1.33 0.05 1.62 0.12 --0.01 Death, % (n/n) 5.0 (57/1152) 22.1 (21/95) 0.16 (0.08 0.29) < 0.01 1 transfer, serum IgG < 1.0 g/dL. 2 AOR = adjusted odds ratio; CI = c onfidence interval. 3 Morbidity = calf diagnosed of any disease in the first 75 d of age. 4 Multiple diseases = calf diagnosed with more than one disease in the first 75 d of age.

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125 Table 4 4. Association between passive transfer and rearing cos t of Hol stein calves in the pre weaning period Passive transfer 1 APT FPT P Daily rearing costs, $/day Milk 0.54 0.002 0.56 0.003 < 0.01 Grain 0.24 0.01 0.20 0.01 < 0.01 Health treatment 0.12 0.04 0.14 0.05 0.50 Total rearing costs, 2 $ /day 1.36 0.01 1.35 0.02 0.68 Rearing costs, $/calf Milk 32.9 0.4 31.0 0.5 < 0.01 Grain 16.4 0.4 13.2 0.8 < 0.01 Labor 28.5 0.3 25.0 0.6 < 0.01 Health treatment 6.11 1.97 5.93 2.05 0.78 Total rearing costs, 2 $/calf 84.6 1.0 74.5 2.0 < 0.01 Kg of weaned calf 3 71.6 1.6 59.2 2.5 < 0.01 Value of a calf, 4 $ Male 270.4 10.8 209.1 19.2 < 0.01 Female 716.7 17.9 484.0 40.5 < 0.01 Income per calf, 5 $ Male 186.5 8.9 137.7 15.7 < 0.01 Female 631 .8 17.0 408.0 38.5 < 0.01 1 transfer, serum IgG < 1.0 g/dL. 2 Rearing costs include the costs with milk and grain feeding, labor, and health treatments. 3 Kg of body weight at weaning for all calves in the study. Calves that died before weaning resulted in 0 kg of weaned calf. 4 Value of a calf calculated based on the revenue value of a weaned calf (male = $300; female = $750), lost value of a dead calf (male = $100; female = $500). 5 Value of a weaned calf calculated based on the value of a calf minus the rearing cost.

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126 Figu re 4 1. Survival curves for time to death in dairy calves according to passive FPT = failure of passive transfer based on serum IgG < 1.0 g/dL. The adjusted hazard ratio was 0.28 (95% CI = 0.17 0.46).

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127 Figure 4 2. Probability of death within 75 d of age according to serum immunoglobulin (Ig) G concentrations in dairy calves calculated from the logistic function. Each dot represents one or more of the 1247 calves. Probability of dea th decreased ( P < 0.001) as serum IgG increased. The adjusted odds ratio was 0.66 (95% CI = 0.51 0.84).

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128 Figure 4 3. Probability of death within 75 d of age according to serum total protein concentrations in dairy calves calculated from the logistic f unction. Each dot represents one or more of the 1247 calves. The linear and quadratic effects of serum total protein influenced ( P < 0.001) the probability of death in the pre weaning period. 0 10 20 30 40 50 60 70 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Probability of death, % Serum total protein, g/dL

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129 Figure 4 4. Probability of death within 75 d of age accor ding to serum IgG as percent of total protein in dairy calves. The probability was calculated from the logistic function. Each dot represents one or more of the 1247 calves. The probability of dying in the pre weaning period decreased ( P < 0.001) as the per cent of serum total protein increased (AOR = 0.96; 95% CI = 0.95 0.98). 0 2 4 6 8 10 12 14 16 18 20 0 10 20 30 40 50 60 70 80 90 100 Probability of death, % IgG as percent of serum total protein

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130 CHAPTER 5 EFFECT OF FEEDING POMEGRANATE EXTRACT ON GROWTH, HEALTH, AND IMMUNE RESPONSES OF HOLSTEIN CALVES Introduction Calves are born with a competent, but naive immune system, a nd specific immunoresponse s develop over time. During the pre weaning period calves are highly susceptib le to pathogens because their immune system is unprimed. A common management strategy for rearing calves is to add a subtherapeutic amount of anti microb ials, particularly antibiotics, to the calf milk replacer to limit microbial growth in the digestive tract in an attempt to minimize diarrhea and other health problems. However, there have been associations between use of medicated milk replacer and change s in patterns of antimicrobial resistance in fecal E. coli of calves (Pereira et al., 2011). Farms that use antibiotics in milk had E. coli with increased resistance patterns compared with farms that did not administer antibiotics in milk of calves (Pereir a et al., 2011). Although these results do not establish cause and effect, they suggest that the proph y la c tic use of antimicrobials in milk might be a risk factor for increased resistance to antibiotics in pathogenic and commensal bacteria. Because of scru tiny on prudent use of antimicrobials, alternative methods to limit morbidity and mortality in calves by supplementing the diet with bioactive compounds that might improve gut and overall health have been studie d ( Heinrichs et al., 2003). Pomegranate juice contains acids, sugars, vitamins, polysaccharides, minerals and polyphenol ic compounds ( Melgarejo et al., 2000) Polyphenols present in pomegranate juice include flavonoids, condensed tannins and hydroly z able tannins. The main antioxidant compounds in pom egranate juice are hydrolysable tannins (Gil et al., 2000) These tannins are mainl y represented by ellagitannins. Punicalagins are the

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131 major ellagitannins in the whole fruit, which are unique to pomegranates (Gil et al., 2000) Punicalagins can be hydrol yzed to ellagic acid and other smaller polyphenols in vivo ( Landete, 2011; Seeram et al. 2004). The flavonoids are mainly represented by anthocyanins which constitute 10% of total polyphenols and have potent antioxidant properties (Gil et al., 2000). Pome granate is well known for its antimicrobial, antioxidant, antiinflammatory, and anticancer properties (Guo et al., 2008; Reddy et al., 2007; Chidambara Murthy et al., 2002). The antimicrobial activity of pomegranate is likely due to its chemical constituen ts. Ellagic acid, gallagic acid, punicalins, and punicalagins isolated from pomegranate juice had antimicrobial activity against several microbes (Reddy et al., 2007). However, the antimicrobial effect detected in vitro contrast with those detected in in v ivo studies. In relation to antioxidant capacity, feeding rats with extract of pomegranate peel caused preservation of catalase, peroxidase, and superoxide dismutase values comparable with control values; in addition lipid peroxidation was reduced when co mpared to controls (Chidambara Murthy et al., 2002). In humans, supplementation with pomegranate juice increased plasma antioxid ant capacity of glutathione peroxidase (Guo et al., 2008) and reduce d the malondialdehyde concentration ( Guo et al., 2008; Heber et al., 2007). There are contradictory results related to immunomodulatory properties of pomegranate. Some studies showed that pomegranate improved the immune response, whereas others showed immunosuppression. In fishes, peritoneal administration of the leaf extracts of P unica granatum enhanced the innate immune responses su ch as neutrophil and complement activity (Harikrishnan et al., 2010). Neutrophil phagocytic

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132 and killing activities were not affect ed by pomegranate extract supplementation in calves (O liveira et al., 2010). However, in this study pomegranate juice extract improved the adaptive immune response, increasing the synthesis of INF 4 by PBMC and improved total IgG responses to ovalbumin vaccination. In rabbits, the pomegranate fed gro up showed a delayed hypersensitivity reaction and an increased antibody titer response to thymus dependent antigen (Gracious et al., 2001). The effect of feeding pomegranate on animal performance has been evaluated in goats and calves. Feeding pomegranate seed pulp or fresh pomegranate peels had no effect on DMI, or ADG (Modar esi et al., 2011 ; Shabtay et al., 2008). In Holstein calves, supplementation with 5 or 10 g /d pomegranate juice extract had no effect on DMI or ADG in the first 30 d of age, but after 30 d of age, DMI and ADG decreased with increasing addition of pomegranate juice extract and no positive effect s on health were reported (Oliveira et al., 2010). There has been very limited research studying the effects of pomegranate on the performance and immune system of calves. Knowing the benefi cial properties of pomegranate, we hypothesized that pomegranate extract could reduce the incidence of diarrhea thus improving health status and grow th performance of calves. Therefore, the objective of these studies were to determine the effects of feeding liquid pomegranate extract on grain intake, growth, health response s incidence duration and severity of diarrhea, measures of immun e response and antioxidant defense during the first 63 d of life in Holst ein calves

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133 Materials and Methods Calves and Treatments All study procedures were approved by the University of Florida Institutional Animal Care and Use Committee. The study was conducted at the University of Florida Dairy Unit. Eighty eight Holstein cal ves on the day following birth (birth = day 0) were assigned to 1 of 3 treatments in a completely randomized design after stratifying by gender Treatments were c ontrol no additive added to milk (n = 26 calves); antibiotic a mixture of 200 mg of oxytetr acycline (Agrilabs, St. Joseph, MO) and 200 mg of neomycin (Durvet, Blue Springs, MO) added to milk once daily (n = 32 calves); and POM a p omegranate extract (POM Wonderful, Los Angeles, CA) provided orally once daily before milk feeding at a daily dose of 15 mg of gallic acid equivalent (GAE) /kg of body weight (n = 30 ). The dos e chosen for POM w as based on intakes of GAE/kg of body weight extrapolated from studies with human subjects in which clinical benefits to consuming polyphenols from pome granate ju ice were reported (He ber et al., 2007; Pantuck et al., 2006). The pomegranate juice extract formulation contained approximately 11% GAE (110 mg/mL) and resulted in daily mean intakes of GAE of 650 or 975 mg in the first and second months in the study resp ectively Calves received 3.8 L of frozen and thawed colostrum containing at least 80 g of total IgG/L within the first 2 h after birth, and 1.9 L in the following 12 h. Calves housed in individual hutches. On the day following birth, calves were weighed and a blood sample collected for determination of hematocrit, STP, and total IgG Calves were fed 3.8 L /day of nonsaleable pasteurized hospital milk (milk derived from cows with mastitis or receiving systemic antimicrobials because of other diseases) divid ed into two feedings daily during the first 49 d of age. From d 50 to 56 milk was provided at 1.9 L/d

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134 once daily. Calves were weaned on d 5 7 of age and the study was completed on d 63 of age. Calves had ad li bitum access to a starter grain (PURINA, St. Lo uis, MO) throughout the study. Dry matter content of calf grain was monitored weekly to determine grain DM intake. Grains offered and refused were measured daily A mounts offered were adjusted to allow for 10% orts (minimum of 200 g) in the first 63 d of a ge. Dry matter intake from grain and milk were calculated daily for each calf. Milk and Grain Sampling Nutrient Analyses and Quantification of Bacteria in Milk Samples of pasteurized milk were collected four time weekly throughout the study and analyzed for concentrations of total solids, lactose, fat and true protein at the DHIA Laboratory ( Belleview Fl) using a Bently 2000 NIR analyzer. Solids not fat were calculated by difference between total solids and fat As h was calculated by difference from tot al solids minus the sum of fat, true protein and lactose. Composition of pasteurized milk (Table 5 1) was used to estimate the energy concentration in milk using NRC (2001) equation s. Although amount of milk consumed by each calf was fixed at 3.8 L/d, comp osition of milk varied throughout the study, which resulted in calves of different ages consuming different quantities of milk solids at different time points in the study. Samples of milk post pasteurization from individual bucket were taken three times p er week, every week, immediately stored in ice and transported to the Microbiology Laboratory of Department of Animal Sciences. Microbiological analysis was performed according to Hogan et al. ( 1999). Fifty microliter s of milk were plated on blood and MacC onkey agars (Hardy Diagnostics, Santa Maria, CA), and incubated for 24 h at 35 o C. After incubation, the number of CFU was counted to determine number of total bacteria, coliforms and E. coli per mL of milk (Table 5 1).

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135 Grain was sampled once a week, dried at 55 C for 48 h and moisture content recorded. Dried samples were ground to pass a 1 mm screen ( Wi ley mill, Philadelphia, PA) and samples were then composited month ly Samples were analyzed at the Dairyland Laboratory (Arcadia, WI) for DM at 105 o C, N by combustion (crude protein = N x 6.25), fat (AOAC, 2000), NDF, ADF (Van Soest et al., 1991), and minerals using an inductively coupled plasma emission spectrometer (Thermo Garrell Ash, Franklin, MA) Composition of starter grain is in Table 5 2. Body W eigh t, Height and Scoring Body weight was measured before the morning feeding o n 2 consecutive days at 1, 14, 28, 56, and 63 d ay of study and averaged for each measurement Body weight gain for specific intervals was determined by difference. Wither and hip h eight of calves were evaluated o n at 1, 56 and 63 d of age. Attitude score w as evaluated daily as follows: 1 alert and r esponsive ; 2 n on active ; 3 d epressed ; and 4 m oribund ( Magalh es et al., 2008) Fec es were also scored based on consistency as an in dicator of presence and severity of diarrhea. Feces were scored according to the following scale: 1 firm consistency, no diarrhea; 2 moderate consistency, soft, no diarrhea; 3 r unny feces, mild diarrhea ; and 4 w atery feces, diarrhea (Magalh es et al., 2008). Fecal scores > 2 were categorized as diarrhea for purposes of statistical analysis for risk and duration of diarrhea. A fecal score of 4 was categorized as severe diarrhea. Evaluation of Incidence of Health Disorders Incidence of health disorders w as recorded daily for individual calves. Rectal temperature was measured daily during the first 14 d of age, and on days when display ed clinical signs of disease such as diarrhea, bloat, coughing, increased

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136 respiratory frequency, depress ion or lack of app etite. Calves with 39.5 o C were cat e g o rized as febrile. Day when disease was first diagnosed was recorded and duration of each illness event was determined. Number of episodes of fever, diarrhea, and pneumonia was determined. To distinguish between differ ent episodes, an interval of 4, 4, and 10 d between diagnoses of fever, diarrhea and pneumonia, respectively, had to elapse to characterize a new event. Calves with digestive and respiratory problems were treated by the farm personnel according to protocol s established by the herd veterinarian. Medication used (antibiotics, anti inflammatory and anti diarrheic products), dosage, and duration of treatments were recorded for individual calves. Costs of health treatments were calculated based on currents costs for each product, daily dosage for each medication for individual calves, and duration of treatments. Blood Sampling Blood samples were taken 30 min after the morning feeding of milk on the day of study enrollment and then every 14 d until d 56. Blood wa s sampled by puncture of the jugular vein using an 18 gauge 2.5 mm needle (Air Tite Virgina Beach, VA), and collected into specific evacuated blood collecting tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ). Immediately after bleeding, K 2 EDTA t ubes were placed in ice Tube s for serum separation and tubes containing heparin were kept at ambient temperature P lasma or serum was separated by centrifugation at 3,000 rpm at 4C for 20 min ( Allegra X 15R Centrifuge, Beckman Coulter). Plasma was stored at or 80 C for subsequent analys e s of metabolites and an tioxidant enzymes, respectively Blood was also sampled using evacuated tubes containing heparin for neutrophil phagocytic and killing assays.

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137 Hematocrit, Serum Total Protein and Total Immunoglobu lin G Quantification Hematocrit and STP were measured in duplicate on the same day of blood collection. Hematocrit was measured by placing whole blood in microcapillary tubes and centrifug ing for 4 min using Microspin 24 (Vulcon Technologies, Grandview, M O) T he percentage of packed blood cell s was measured using micro hematocrit tube reader Model CR (International Equipment Co., Needham Heights, MA). Serum samples were analyzed for concentration of total protein using a clinical refractometer (Reichert Ju ng, Buffalo, NY). Total IgG concentration in serum was quantified by a bovine IgG radial immunodiffusion kit ( VMRD Pullman, WA). Ser a were diluted with DPBS at a ratio of 1 to 5 when the concentrations of IgG were greater than 2.8 g/dL The intra and int erassay s coefficient of variations were 2.5 and 5.4 %, respectively. Assays for Plasma Metabolites Plasma concentrations of glucose, urea N, NEFA, and BHBA were measured in samples collected on 7, 14, 28, 42 and 56 d of age Plasma concentration s of gluco se and urea N were measured using AutoAnalyzer (Technicon Instruments Corp., Chauncey, NY). The autoanalyzer method for plasma glucose was a modification of Gochman and Schmitz (1972), whereas th e method used for urea N was a modification of Coulombe and F avreau (1963). Plasma concentrations of NEFA and BHBA were measured using enzymatic methods from Wako Diagnostic (Richmond, VA) in a microplate reader SpectraMax 340PC384 (Mo lecular Devices, Sunnyvale, CA) The intra and interassays coefficients of variat ion for NEFA were, respectively, 4.5 and 10.3%. The intra and interassay s coefficients of variation for BHBA were 3. 3 and 9. 4 % respectively.

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138 Antioxidant Enzyme s and Quantification of Unstable Lipid Hydroperoxides Plasma samples collected on days 7, 28, and 56 were analyzed for activity of SOD and GPx and concentrations of TBARS. The assays were performed following the manufacture protocols ( Cayman Chemical Co, Ann Arbor, MI). Plasma samples were diluted at 1 to 5 ratio with sample buffer before assayi ng for SOD and GPx activit ies Superoxide dismutase activity was determined by using the xanthine/xanthine oxidase method to generate superoxide radicals, which reacted with 2,4 iodiphenyl 3,4 nitrophenol 5 phenyltetrazolium chloride to form a red formaza n dye, which was determined at 450 nm. Superoxide activity was then measured by the degree of inhibition of this reaction. One unit of SOD was defined as the amount of enzyme needed to in hibit 50% dismutation of the superoxide radical. The sensitivity of t he SOD kit was 0.025 unit/mL of SOD and the intra and interassay s coefficient s of variation were 3.8 and 9.6% respectively. Glutathione peroxidase activity was measured based on the method by which GPx catalyzes the oxidation of glutathione by cumene hyd roperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione was immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP + and the corresponding decrease in absorbance at 340 nm was measured. The intra and interassay s coefficients of variation were 6.8 and 8.4% respectively. Concentration of TBARS was measured based on the method by which malondialdehyde (MDA), a naturally occurring product of lipid peroxidation which reacts with thiobarbituri c acid (TBA) producing MDA TBA adduct which is measured calorimetrically at 540 nm. The intra and interassay s coefficients of variation were 3. 4 and 3.2% respectively

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139 Immunization with Ovalbumin and Assay for Anti OVA IgG Ovalbumin (OVA) solution was prepared by dissolving 1 mg of OVA (Sigma Aldrich St. Louis, MO ) in 1 mL of sterile PBS and emulsified in 0.5 mg of adjuvant Quil A (Accurate Chemical ,Westbury, NY). Calves were immunized with 1 mg of OVA at 2, 14, and 28 d of age. Blood was sampled pr ior to each immunization and again at 42 and 56 d for measurement of anti OVA IgG in serum. Immunoglobulin G anti Ova was quantified using an enzyme linked immunosorbent assay (ELISA). Briefly, flat bottom 96 well polystyrene plates (Immulon 2, Dynex Tech ., Chantilly, VA) were coated with a solution of OVA dissolved in carbonate bicarbonate coating buffer (1.4 mg OVA/mL of carbonate bicarbonate buffer). Plates were incubated at 4C for 4 8 h, then washed 4 times using a multiwash III microplater washer (Tri continent, Grass Valley, CA ) with washing solution (PBS w ith Tween 0.05%, pH = 7.4). Plates were blocked with a PBS 3% Tween and bovine serum albumin (Sigma Chemical, St. Louis, MO) solution and incubated at room temperature for 1 h. Plates were washed fo ur times and diluted sera samples and control sera (1 to 50 and 1 to 200) were added in duplicate using a quadrant system. Positive and negative control sera to anti OVA IgG were obtained from a pool of sera of calves with known high (after 3 immunizations with OVA) and low (pre OVA immunization) concentrations, respectively. Plates were incubated at room temperature for 2 h and washed with washing buffer solution. After washing, alkaline phosphatase conjugate rabbit anti bovine IgG (Sigma Chemical, St. Lou is, MO) was dissolved in Tris buffer solution and added to the plates and incubated for 1 h at room temperature. After incubation, plates were washed 4 times and 80 L of substrate solution (P nitrophenyl phosphate disodium; Sigma Chemical, St. Louis, MO) was added and the plate was

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140 incubated at room temperature for 30 min. Plates were read using 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 Results were calculated from the sum of the average of each duplicated sample dilution multiplied by the correction factor of each plate. The intra and interassays coefficients of variation were 6.1 and 12.3%, respectively. Leukocyte Population Quantifi cation H eparinized blood samples collected at 7, 28, and 56 d of age were sent at Veterinary Clinical Laboratory from University of Florida Quantification of total and individual leukocyte population was performed using ADVIA 120 Hematology System (Siemen s, Deerfield, IL). Phagocytosis and Oxidative Burst Assay s Neutrophil phagocytosis and oxidative burst were measured using a dual color flow cytometry assay, methodology modified from Smit s et al. (1997). Blood sampled on days 7, 14, and 56 using evacuated tubes containing heparin were transported to the laboratory Whole blood (100 L ) was pipetted into each of 3 polystyrene round bottom tubes (12 x 75 mm) and 10 L of 50 M dihydrorhodamine 123 (DHR) (Sigma Aldrich, Saint Louis, MO) was added to all tubes. Tubes were slowly vortexed and incubated at 37 o C for 10 min with constant rotation using nutator (BD, San Jose, CA). Ten microliter s of 20 g/mL solution of phorbol myristate acetate ( PMA Sigma Aldrich) was added to tube numbe r 2 (positive control for oxidative burst). A pathogenic E coli bacterial suspension (10 6 CFU /mL) isolated from a case of bovine mastitis and labeled with propidium iodide (Sigma Aldrich) was added to tube number 3 to establish a ratio of bacteria to neut rophil of 40 to 1, using the concentration of neutrophil in blood

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14 1 provided by the hematology results. Tubes were slowly vortexed and incubated at 37 o C for 30 min with constant rotation using a nutator. After incubation, tubes were placed immediately on ice to stop neutrophil activity. Tubes were processed in a Q Prep Epics immunology workstation on the 35 sec cycle using three reagents ( s olution A: 88% formic a cid, red blood lysis solution; s olution B: b uffer solution constituted for s odium c arbonate, s odiu m c hloride and s odium s ulfate; and s olution C: 1% paraformaldehyde, cell fixative solutio n). Cold distilled water (500 L ) for completion of hemolys is, and 0.4% tryphan blue (10 L ) for quenching extracellular oxidized DHR was added to each tube. Tubes wer e slowly vortexed, kept on ice, and 10,000 total white blood cells were read at the Facsort flow cytometer (BD biosciences, San Jose, CA). Experimental Design and Statistical Analyses The experimental design was a complete randomized. Calves were stratifi ed by gender and randomly assigned to 1 of 3 treatments on the day after birth The number of experimental units/treatment w ere power = 0.80) to detect a 200 g/d difference in grain intake when the SD for grain intake between 3 and 10 weeks of age is 250 g. Similarly, the sample size was calculated to provide enough experimental units to detect an 80 g/d difference in BW gain when daily changes in BW in the first 80 d of age range from 500 to 700 g/d C ontinuous data were tested for normality of residuals using the Shapiro Wilk test of SAS ver. 9.2 (SAS Inst. Inc., Cary, NC). Non normally distributed data were transformed as suggest ed using the guided data analysis of SAS, and then back transformed using the ilink function to generate least square means and SEM. Continuous variables were analyzed by ANOVA using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). Variables with a single measurement during the study

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142 were analyzed with the fixed effects of treatment and gender of calf Variables with repeated measurements within the same calf were analyzed with the fixed effects of treatment, time of measurement (day or week), interaction between treatment and time, treatment and gender of calf, and the r andom effect of calf nested within treatment. The repeated statement was included and the covariance structure was chosen based on Daily fecal and attitude scores were analyzed by the GLIMMIX procedure of SAS (S AS Inst. Inc., Cary, NC) using weekly means. When scores averaged for the week did not fit normal distribution, they were then analyzed fitting a Poisson distribution with link log function. The model included the effects of treatment, week, interaction be tween treatment and week, and gender of calf, with calf nested within treatment as the random error. The covar iance structure that best fitted the data was chosen based on the or high scores were analyzed by the GLM procedure of SAS (SAS Inst. Inc., Cary, NC) with the effects of treatment and gender of calf. Bin ary data were all analyzed by logistic regression using the LOGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). The m odels included the effects of treatment and gender of calf. Adjusted odds ratio and the 95% confidence interval (CI) were calculated. Number of cases of health disorders per calf was analyzed by the Kruskal Wallis nonparametric method to test equality of m edians between treatments. Medians and mean rank were generated using MINITAB (M initab Inc., State College, PA) Survival time measured as age of calf at leave the study, wa s evaluated using the product limit method of the Kaplan Meier model by the LIFETEST procedure of SAS

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143 (SAS Inst. Inc., Cary, NC). Calves that survived the entire study period were censored at 63 d of age. Serum IgG and BW at enroll were used as covariates in all statistical analysis. Treatment differences with 0.05 were considered significant and 0.05 < P 0.10 were designated as tendency Results Milk fed to cal ves had high somatic cell count but low bacterial load (Table 5 1). The high somatic cell count was the result of most milk to be originated from cows having clinical mastitis. In general, calves received 437 g of milk solids/d and approximately 2.4 Mcal/d of ME in the first 49 d of age On the day of study enrollment, serum total protein and IgG did not differ among treatments (Table 5 3); however, calves assigned to POM were heavier ( P < 0.01) than those assigned to antibiotic. No differen ce was detected for control and other treatments. Because of the difference in BW at enrollment among tre atments, body weight on day 1 was used as a covariate in all statistical analyses. Twenty four percent of calves had IgG < 1.0 g/dL. Serum IgG at enrolment was also included as a covariate in all statistical analysis, because 7 calves per treatment (27, 2 2 and 2 3 % calves from control, antibiotic and POM treatment, respectively) did not receive adequate transfer of colostral antibodies, indicating failure of passive transfer (Berge et al., 2005; Weaver et al., 2000; Tyler et al., 1996). Grain DM intake di d not differ among treatments (Table 5 3). Intake was negligible in the first 2 weeks of age, and only after 4 weeks did calves consumed considerable amounts of grain DM (Figure 5 1). Despite the low grain DM intake, average daily gain averaged 360 and 775 g/d in the first 28 d and between 28 and 63 d of age, respectively. Nevertheless, treatment had no effect on average BW at 28 and 63 d of

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144 age, average daily gain, and efficiency of conversion of DM intake into BW The 2.4 Mcal of metabolizable energy cons umed as milk coupled with the low grain intake in the first 2 weeks of age resulted in little or no change in BW in the first 14 d of age (Figure 5 2). In fact, average daily gain in the first 14 d of age was less than 200 g/d (Figure 5 3) Average daily g ain increased up to 850 g/d by 56 d of age, when calves were weaned from milk, and then declined to approximately 650 g/d in the final week of the study. In spite of the moderate grain DM intake, feed conversion ratio was high, and averaged 0.70 kg of weig ht gain for every kg of DM consumed as milk and grain (Table 5 3). Plasma concentration s of glucose and BHBA did not differ among treatments (Figure 5 4). The mean values for glucose were 102.0 1. 3 10 1.0 1. 3 and 99.5 1. 3 mg/dL for control, antibio tic and POM, respectively w hereas, the respective values of BHBA were 1.13 0.09, 1.28 0.09, and 1.11 0.09 mg/dL. No significant interactions among treatment and age of calves were observed for glucose or BHBA Concentration s of glucose increased ( P < 0.01) with age until 42 d but those of BHBA increased ( P < 0.01) throughout the study. Concentrations of NEFA declined ( P < 0.01) with age, but they were not influenced by treatment (Figure 5 5). Non esterified fatty acid concentrations averaged 128.3 9.1, 127. 6 9.2, and 124. 5 9.0 M for control, antibiotic and POM respectively. Plasma concentration s of urea N tended ( P = 0.08) to be affected by treatment because calves fed antibiotic had lower concentration than control and POM on d 14 (Figure 5 5). Concentrations averaged 9.2 0. 3 8.4 0.3, and 9.0 0. 3 mg/dL for control antibiotic and POM, respectively. Supplementing calves with antibiotic and POM did not affect hematocrit and serum total protein through ou t the study (Figure 5 6) Hematocr it slightly declined ( P < 0.01) with age of

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145 calves, whereas concentrations of total protein declined in the first 14 d, but then increased ( P < 0.01) until day 56 F ecal scores sharply increased ( P < 0.01) in the first 2 weeks of age, and then declined to basal values by week 4 (Figure 5 7). A tendency ( P = 0.08) for interaction between treatment and age was observed for fecal score because control calves had a slower decline in fecal score after week 2 of age whereas antibiotic calves experienced a greate r increase in fecal score in the last 2 weeks of the study. At week 3 of age, control calves had a higher ( P < 0.01) fecal score than antibiotic and POM calves ( 1.8 vs. 1.3 and 1.5 respectively). Attitude score worsen ed at week 2 (Figure 5 7), which coinc ided with calves developing diarrhea (Table 5 4 ), but it promptly improved. Treatment had no influence on attitude scores. The mean age at the diagnosis of the first case of diarrhea was not different among treatments (Table 5 4). Approximately 20% of the days in the study, calves had fecal score > 2, whereas 14% of the days fecal score was 4. Nevertheless, treatment did not influence the number of days with high fecal scores. Every calf was diagnosed with diarrhea in the study (Table 5 5). Fever affected 4 8% of the calves in the study (Table 5 5), but incidence did not differ with treatments. The number of days with fever per calf was less than 1.0 and it was unaffected by treatment (Table 5 4). In fact, rectal temperature of calves increased ( P < 0.01) in the first few days in the study, but this response was not affected by feeding antibiotic or POM (Figure 5 8). In general, more control calves were diagnosed with pneumonia and medication costs per calf because of pneumonia tended ( P = 0.06) to be greater for control than antibiotic or POM (Ta ble 5 4). Approximately 58% of calves were diagnosed with more than one disease. This high morbidity resulted in 5. 6 % of the calves dying before completing the 63 d experiment. Survival time, age of calves at

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146 leave of the study, of calves fed antibiotic tended ( P = 0.10) to be less than those of control and POM calves (Figure 5 9). Concentration s of total leukocytes in blood of calves decreased ( P = 0.03) with age, but they were not affected by treatment ( Table 5 6 ). Si milar to total leukocytes, individual white blood cell populations were influenced by age, but not by treatment. Although the concentration of individual leukocytes in blood was not affected by treatment, the proportion of the total white blood cells repre sented by each type of leukocyte differed with treatment (Table 5 7). The interaction treatment and age tended ( P = 0.09) affect the proportion of neutrophil. C alves fed antibiotic tended ( P = 0.0 9 ) to had a smaller proportion of neutrophils than those fe d control at 7 d of age, whereas POM calves had greater ( P < 0.05) proportion of neutrophil than control and antibiotic fed calves at 56 d of age Overall, the proportion s of neutrophils were 46.6 1.8, 42.1 1.8 and 47.5 1.8% for control, antibiotic and POM, respectively. On the other hand, calves fed antibiotic had a greater ( P < 0.05) proportion of lymphocytes than control and POM ( c ontrol = 43.5 1.8, antibiotic = 48.2 1.8, POM = 43.1 1.8%). The proportions of other leukocyte populations were not affected by treatment. The percentage of neutrophils phagocytizing E. coli was lower ( P = 0.02) on day 28 compared with days 7 and 56, but it was not influenced by treatment (Figure 5 10A). The same pattern was observed for the proportion of neutrophil s with oxidative burst (Figure 5 10B). Effect of supplementation of pomegranate extract or antibiotic on neutrophil phagocytosis mean fluorescence intensity (MFI, indicator of number bacteria phagocytized per neutrophil) and oxidative burst MFI (as indicat or of intensity of reactive oxygen species produced per neutrophil) were also not affected by dietary treatment (Figure 5 11A,B). Treatment had no effect on the response to immunization with OVA

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147 (Figure 5 1 2 ). Serum titers did not change after the initial immunization on d 2 of age, but increased sharply following the subsequent immunizations on days 14 and 28 of age. The plasma SOD activity was unaffected by treatment (Table 5 8) In general, plasma SOD activity remained somewhat constant throughout the f irst 56 days of life in calves. A similar response was observed for GPx. Activity remained mostly unaltered with age of calves and no treatment effect was observed. On the other hand, concentrations of TBARS in plasma were highest on day 7 and decreased ( P < 0.01) with age, but treatment did not affect these responses. Discussion Antimicrobials are often added to milk or milk replacers fed to calves in an attempt to minimize the risk of diarrheal disease in the preweaning period. However, increasing restr ictions on the use of antibiotic s have driven a search for non antibiotic alternatives in animal production. Pomegranate extracts contain polyphenolic compounds, which have antimicrobial activity and immunomodulatory properties that might favor health of y oung calves Pomegranate contains primarily punicalagin and ellatannins, which have been shown to pos s e s s antimicrobial, antioxidative, and immunodulatory properties both in vivo and in vitro (Van Parys et al., 2010; Heber et al., 2007; Rosenblat and Avira m, 2006). Despite the potential benefits of pomegranate to mammals, growth performance and measures of health and immune responses were unaltered i n preweaning calves fed POM during first 63 d of age in the current study Tannins often are seen only in ter ms of their negative impacts on intake and production: decrease d nutrient utilization, reduce d palatability, and reduced digestibility. In fact, Oliveira et al. (2010) reported reduction s i n crude protein and fat digestibilit ies by

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148 calves fed 10 g /d of a d ried pomegranate extract topdressed onto grain However, results from the current study indicate the pomegranate extract used did not negatively affect performance of preweaned dairy calves. Oliveira et al. (2010) offered dry POM extract top dressed onto t he grain, whereas in this study POM was administ e re d orally as a bolus before the morning milk feeding. The different methods of providing PO M to calves in these studies likely explain the difference in the grain intake and BW found here and th o se found b y Oliveira and co workers The productive performance found in the current study is in agreement with the result s reported by Shabtay et al (2008) in Holstein Friesian calves with an average BW of 328 kg fed fresh pomegranate peels, and in goat s fed pomeg ranate seed pulp ( M odaresi et al., 2011) A ntimicrobial and immunomodulatory properties of pomegranate were not observed in this study, although several in vitro studies showed the potential benefits of pomegranate. The lack of effect might be attributed to the difference i n dose used in in vivo and in vitro studies, low bioavailability of polyphenol as well as the conversion of ellagic acid to the metabolite Van Parys et li et al. (2009) reported that pomegranate extract inhibited parasite growth, including Salmonella spp, in vitro but not in vivo In the current study oral administration of POM did not affect neutrophil function, confirming the result found by Oliveira et al. (2010). However, Harikrishnan et al. (2010) observed increased phagocytic and respiratory burst activit ies in infected fish after 8 week s of intraperitoneal administration of pomegranate leaf extract. B ioavailability of polyphenols was likely not lim ited in the Harikrishnan et al. (2010) study, as the extract was intraperitoneally administ ered. The absence of effect of POM supplementation on humoral response found in the current study contrast s with the result s found in calves by Oliveira et al.

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149 (2010 ) and in rabbits by Gracious et al. (2001). The reason of this discrepancy is unclear, because the selected dose used in the current study (15 mg GAE/kg BW) was similar to the low dose of used by Oliveira and co workers. It is likely that the different tim es of OVA vaccination influence ed the different response s Oliveira and co workers (2010) injected OVA at 4, 21, and 42 d of age of calves, whereas in this study the OVA vaccination s were performed at 2, 14, and 21 d of age. Kampen et al. (2006) indicated that the proportion of B lymphocytes increases rapidly during the first weeks of life of calves. Antioxidants can slow down the process of lipid peroxidation by blocking the generation of a free radical chain reaction and by raising the levels of endoge nous defenses by up regulating the expression of genes encoding key enzymes such as SOD, catalase, or GPx (Chidambara Murthy et al., 2002). Reduction in plasma TBARS was found i n overweight adult humans supplement ed with 610 mg of GAE /day (H eber et al., 20 07). A decline in MDA concentration and increase in GPx activity, but not in SOD activity were reported in elderly subjects after daily intake of 250 mL of pomegranate juice for 4 week s (Guo et al., 2008). However, POM fed to calves did not cause the anti cipated increase in antioxidant enzymes or reduction in TBARS. The doses of POM provided 650 or 975 mg of GAE/day during the first 30 d, and from 31 to 63 d of age, respectively and were similar to th ose of Heber et al. (2007) in human subjects The condi tion of the calves and the conversion of ellagic acid to the metabolites urolithins could explain the lack of antioxidant effect of POM It is likely that bioavailability of ellagitannins and ellagic acid might have been limited. Pomegranate phenolic metab olites, urolithins, are not as potent antioxidants as ellagitannins (Bialonska et al., 2009 b ). M etabolism of pomegranate phenolic compounds by young

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150 calves is unknown Unfortunately, we did not measure changes in plasma contents of these compounds after co nsumption of pomegranate in the present study. Gonzlez Barrio et al. (2010) observed a large person to person variation in the timing, quantity and types of urolithins excreted in urine, this variation appeared to be dependent on colonic microflora compos ition. This seems likely th at a difference in microflora composition between adult human and neonatal calves will determine the bioavailability of ellagic acid. Oxidative damage is age dependent, and it is also increased in people with excess abdominal fat and in condition s of intense exercise and environmental stress. It is likely that young calves during the preweaning period are not subject to severe stress conditions, and the potential antioxidant activity of POM could not be observed. The literature i s scarce regarding the mode of action of antibiotic s such as tetracyclines as growth promotants The most widely accepted hypothesis is that these antibiotics modulate the intestinal microbiota, reducing the microbial competition for nutrient s suppress es opportunist ic pathogens and decrease s growth depressing metabolites (Niewold, 2007). The most recent hypothesis postulates that some antibiotics have antiinflammatory and immunomodulatory effects which in consequence, decrease the immunologic stress on th e host (Costa et al., 2011). In absence of antibiotic protection in intestinal mucosa, proinflammatory cytokines are released at the site of pathogen invasion s and consequen tly the vascular system and inflammatory cells are activated. These cytokines pro mote changes characterized by fever, anorexia, and catabolism of muscle protein resulting in a negative N balance (Gruys et al., 2006). For dairy calves, antibiotics are added to the diet as an attempt to reduce the incidence of diarrheal diseases, which a re known to compromise nutrient

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151 absorption and reduce BW gain. In the current study, antibiotic fed calves tended to have low er concentrations of urea N I t seems that the use of oxytetracycline and neomycin tended to improve N utilization perhaps by modul ation of the im mune system which may have reduce d muscle catabolism or improve d protein synthesis. However, effects o n growth were not observed confirming the results found by Berge et al. (2009), who reported no effect on grain intake and ADG in calves r eceiving antimicrobial s in milk (54 mg of neomycin sulfate and 108 mg of tetracycline HCl). In this study fecal score s in antibiotic fed calves tended to be high during and after weaning. It is likely that antibiotic supplementation affected the commensal enteric flora and this effect was evident when calves increased grain intake The lack of effect of antimicrobial s on days of diarrhea found in the current study confirmed those reported by Berge et al. (2009). Mortality affected 5. 6 % of the calves, and those fed antibiotics tended to survive less time than control and POM calves This result was unexpected and unclear. This may suggest that antimicrobials reduced the normal enteric flora, allowing the growth of pathogenic microorganism. In fact, 80% of a ntibiotic fed calves that did not finish the study were diagnosed and treated for an infection. Conclusion s Daily s upplementation of Holstein calves with pomegranate extract at 15 mg of GAE/kg BW or addition of antibiotics to the milk did not affect perfor mance during the preweaning period. Because grain and total DM intakes were not influenced by treatments, no changes in concentrations of metabolites in plasma were observed with feeding either antibiotics or pomegranate extract. Feeding antibiotic s or pom egranate extract did not influence fecal and attitude scores of calves, neutrophil function, humoral response to immunization with o valbumin, and antioxidant activity in plasma Mortality

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152 affected 5.6% of the calves, and those fed antibiotics tended to sur vive less time than control and POM calves. In conclusion, feeding 15 mg of GAE/kg BW or subtherapeutic doses of antibiotics to preweaned calves did not benefit growth, feed efficiency, measures of health and immune response, and antioxidant activity of bl ood.

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153 Table 5 1. Nutrient composition and microbial contamination of pasteurized milk fed to calves1. Nutrient composition As is DM basis Mean SD Median Mean SD Median DM,% 11.5 0.5 11.6 --Fat, % 3.04 0.43 3.10 26.4 3.1 26.8 True protein, % 3.07 0.15 3.07 26.8 1.2 26.5 Lactose, % 4.51 0.16 4.53 39.2 1.9 39.0 Solids no n fat, % 8.46 0.29 8.48 73.6 3.1 73.2 Ash, % 0.88 0.06 0.88 7.6 0.6 7.6 ME, 2 Mcal/kg 0.63 0.04 0.64 5.5 0.2 5.5 SCC, x 10 3 /mL 3 1060 478 1051 ----SCS 4 6.13 1.16 6.39 ----Bacteria, log 10 CFU/mL 5 Total 3.50 0.56 3.51 ----Coliforms 2.79 0.71 2.70 ----E scherichia coli 2.30 0.58 2.30 ----1 Milk samples were taken 4 times weekly for chemical analysis an d 3 times weekly for bacterial analysis throughout the study. 2 Metabolizable energy calculated according to NRC (2001) based on the nutrient composition of milk. 3 SCC = somatic cell counting. 4 SCS = somatic cell score. 5 CFU = colony forming units.

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154 Table 5 2. Nutrient composition (mean SD) of the starter grain (DM basis) 1 DM, % 85.7 0.5 ME, Mcal/kg 3.09 0.05 OM, % 92.0 0.02 CP, % 21.6 0.1 NDF, % 18.3 1.1 ADF, % 8.8 0.5 Nonfibrous carbohydrates, % 49.6 1.0 Fat, % 3.6 0.1 Ca, % 1.51 0.01 P, % 0.79 0.02 Mg, % 0.32 0.00 K, % 1.45 0.01 S, % 0.36 0.01 Na, % 0.53 0.04 Cl, % 0.82 0.07 Zn, mg/kg 288 38 Cu, mg/kg 80 1.4 Mn, mg/kg 169 9 Decoquinate, mg/kg 26 1 Each kg contains 0.33 mg of Se, 0.5 mg of Fe, 0.3 mg of Co, 15,000 IU of vitamin A, 6,250 IU of vitamin D 3 30 IU of vitamin E according to manufacturer (PURINA). 2 Calculated based on the nutrient content (NRC, 2001)

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155 Table 5 3. Effect of feeding pomegranate extract or antibiotic s on p erformance and growth of preweaned calves Treatment 1 Control Antibiotic POM P Study d ay 1 STP 2 g/dL 5.32 0.11 5.28 0.11 5.45 0.11 0.48 Total IgG, g/dL 1.59 0.23 1.60 0.23 1.82 0.24 0.56 Body weight, kg 42.2 1.1 ab 39.5 1.1 b 44. 1 1.2 a 0.01 Grain DM intake, g/d 1 to 28 d 90.8 12.3 90.8 13.2 85.0 12.6 0.90 28 to 63 d 742.1 55.3 675.7 55.9 667.4 57.5 0.53 Body weight, kg 28 d 51.2 0.7 51.3 0.8 52.0 0.8 0.66 63 d 78.5 1.7 78.3 1.8 79.5 1.8 0. 86 Body weight change, kg 1 to 28 d 9.8 0.7 10.1 0.7 10.6 0.8 0.69 28 to 63 d 27.3 1.4 26.6 1.4 27.2 1.5 0.94 ADG, kg/d 1 to 28 d 0.3 5 0.03 0.36 0.03 0.38 0.03 0.55 28 to 63 d 0.78 0.04 0.76 0.04 0.78 0.04 0.94 Feed efficiency 3 1 to 28 d 0.66 0.05 0.69 0.05 0.73 0.05 0.56 28 to 63 d 0.68 0.03 0.70 0.03 0.73 0.03 0.48 Height, cm Wither s at 1 d 75. 6 0.5 75.5 0.5 76.0 0.5 0.68 Hip at 1 d 79.7 0.6 79.5 0.6 80.2 0.6 0.64 Wither s at 63 d 86.0 0.6 86.1 0.6 87.0 0.6 0.41 Hip at 63 d 92.0 0.7 92.1 0.7 92.0 0.7 0.98 Growth, cm Withers, 1 to 63 d 10.2 0.6 10.3 0.6 11.0 0.6 0.54 Hip, 1 to 63 d 12.2 0.7 12.3 0.7 12.1 07 0.98 Different s uperscripts in the same r ow indicate statistical differences at P < 0.0 1 1 Control, no additive added to milk; A ntibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW 2 STP = serum total protein. 3 kg of BW change/kg of total DM intake (grain + milk).

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156 Table 5 4 Effects of feeding pomegranate extract or antibiotic s on health of preweaned calves Treatment 1 Control Antibiotic POM P Days to first diarrhea 2 7.7 1.3 6.0 1. 3 4.9 1. 2 0.14 Duration, d D iarrhea 12. 1 1.0 12. 5 1.0 12. 1 1.0 0.94 Severe d iarrhea 3 8.4 0. 8 9. 3 0. 8 8.5 0.8 0.66 Percent of study days, % Diarrhea 19.3 1.8 20. 7 1.8 19.6 1.8 0.83 S evere d iarrhea 13. 6 1.3 15. 3 1.3 13. 7 1.3 0.53 Number of d ays with fever 4 0.73 0.31 0.90 0.31 0.51 0.32 0.66 Treatment cost, $/calf 5 3.26 0.68 2.21 0.40 2.20 0.71 0.40 Diarrhea 2.03 0.39 2.17 0.39 2.46 0.40 0.69 Pneumonia 1.22 0.49 0.05 0.49 0.00 0.51 0.06 1 Co ntrol, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW 2 Days in the study to first diarrhea event 3 Fecal s core = 4 4 Days wi in the first 63 d of age. 5 Cost of medication

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157 Table 5 5 Effect of feeding pomegranate extract or antibiotic s on incidence of diseases in preweaned calves Item Treatment 1 %(n/n) AOR 2 95% CI P Diarrhea C ontrol 100 ( 26/26) Antibiotic 100 ( 31 / 31 ) POM 100 ( 30 / 30 ) Fever 3 C ontrol 57. 7 (15/26) Ref. Antibiotic 4 5.5 (1 5 / 33 ) 0.5 3 0.18 1. 56 0. 25 POM 4 3.3 (1 3 / 30 ) 0. 51 0.1 7 1. 55 0. 24 Pneumonia C ontrol 15. 4 ( 4/26) Ref. Antibiotic 6.7 (2/ 30 ) 0. 39 0.07 2. 35 0.3 0 POM 3. 5 (1/2 9 ) 0. 20 0.02 1.89 0.1 6 Multiple diseases 4 C ontrol 61. 5 (16/26) Ref. Antibiotic 6 1.3 ( 19 / 31 ) 0.9 9 0.3 4 2.8 9 0. 9 8 POM 51.7 (1 5 /2 9 ) 0. 67 0. 23 1. 9 6 0. 47 Mortality 5 Control 0 (0/26) An tibiotic 12.5 (4/32) POM 3.3 (1/30) 1 Control, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW 2 Adjusted odd s ratio, control is the reference (Ref.). 3 4 More than one disease. 5 antibiotic tended ( P = 0.09) to have greater mortality than control calves.

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158 Table 5 6 Effect of feeding pomegranate extract or antibiotic s on concen tration of white blood cells of preweaned calves, 103/ L S EM Treatment 1 P 2 Age, d Control Antibiotic POM TRT D ay TRT*Day Leukocytes 7 10.93 0.69 10.03 0.65 9.53 065 0.36 0.03 0.75 28 10.25 0.66 9.11 0.64 8.77 0.64 56 10.00 0 .66 8.83 0.64 9.44 0.66 Neutrophils 7 6.95 0.60 5.83 0.55 5.67 0.56 0.32 <0.01 0.41 28 4.37 0.55 3.82 0.52 3.93 0.54 56 3.96 0.54 3.20 0.53 4.53 0.55 Lymphocytes 7 3.12 0.27 3.48 0.26 3.07 0.26 0.19 <0 .01 0.21 28 4.02 0.27 4.40 0.26 4.06 0.27 56 4.99 0.26 4.80 0.27 4.09 0.27 Monocytes 7 0.40 0.07 0.39 0.07 0.41 0.07 0.64 0.03 0.80 28 0.44 0.07 0.44 0.07 0.41 0.07 56 0.64 0.07 0.53 0.07 0.47 0.07 E osinophils 7 0.25 0.04 0.16 0.04 0.16 0.04 0.11 0.34 0.41 28 0.23 0.04 0.26 0.04 0.21 0.04 56 0.26 0.04 0.17 0.04 0.19 0.04 Basophils 7 0.09 0.01 0.09 0.01 0.07 0.01 0.45 <0.01 0.34 28 0.11 0.01 0.12 0.01 0 .11 0.01 56 0.13 0.01 0.11 0.01 0.12 0.01 1 Control, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW 2 TRT = eff ect of treatment; TRT*Day = interaction between TRT and day.

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159 Table 5 7 Effect of feeding pomegranate extract or antibiotic s on the proportion of individual leukocytes in blood of preweaned calves % S EM Treatment 1 P 2 Age, d Control Anti biotic POM TRT D ay TRT*Day Neutrophils 7 59.7 2.6 53. 9 2. 5 56.1 2.5 0.05 <0.01 0.09 28 43.6 2.5 39. 1 2.4 41. 8 2. 5 56 37.7 2.5 34. 6 2.6 45. 2 2.5 Lymphocytes 7 32.02 2.59 38.3 2.5 35.0 2.5 0.05 <0.01 0.26 28 4 9.93 2.50 50.8 2.5 48.6 2.5 56 51.39 2.51 55.4 2.5 45.7 2.5 Monocytes 7 3.71 0.63 4.19 0.60 4.85 0.61 0.92 0.02 0.40 28 4.56 0.61 4.87 0.60 4.93 0.61 56 6.16 0.61 5.87 0.61 4.95 0.62 Eosinophils 7 2.55 0.46 1.56 0.43 1.71 0.43 0.13 0.08 0.58 28 2.74 0.44 2.90 0.44 2.52 0.44 56 2.81 0.44 1.89 0.44 2.15 0.44 Basophils 7 0.90 0.08 0.99 0.07 0.86 0.07 0.54 0.34 0.61 28 1.29 0.07 1.32 0.07 1.28 0.07 56 1.35 0.07 1.24 0.07 1.24 0.07 1 Control, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW 2 TRT = effect of treatment; TRT*Day = interaction between TRT and day.

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160 Table 5 8 Effect of feeding pomegranate extract or antibiotic s on superoxide dismutase (SOD), glutathione peroxidase (GPx), and thiobarbituric acid reactive substances (TBARS) of plasma of pre wean ed calves Treatment 1 P 2 Age d Control Antibiotic POM TRT D ay TRT*Day SOD, unit/mL 7 11.8 0.9 13. 7 0. 9 12. 2 0. 9 0.25 0.35 0.83 28 12. 7 0.9 13. 8 0. 9 13.5 0.9 56 12.6 0.9 13.8 0. 9 13. 6 0.9 GPx nM /min/mL 7 122. 6 5.9 109. 2 6. 2 112. 9 6.0 0.40 0.16 0.15 28 106. 2 6.1 112.5 6.0 117.4 6. 1 56 119.5 6. 2 11 3.0 6.2 127. 7 6. 3 TBARS, MDA, M 3 7 1.90 0.09 1.85 0.09 2.03 0.09 0.21 <0.01 0.85 28 1.92 0.09 1.82 0.09 1.88 0.09 56 1.69 0.09 1.6 1 0.09 1.78 0.09 1 Control, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW 2 TRT = effect of treatment; TRT*Day = inter action between TRT and day. 3 MDA = malondialdehyde

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161 Figure 5 1. Effect of feeding pomegranate extract (POM) or antibiotic s on grain intake of preweaned calves CON, no additive added to milk ; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW Effect s of treatment ( P = 0.35), age ( P < 0.0 1), and interaction between treatment and age ( P = 0.55)

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162 Figure 5 2. Effect of feeding pomegranate ext ract (POM) or antibiotic s on body weight of preweaned calves CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW Effects of tr eatment ( P = 0.97), age ( P < 0.01), and interaction between treatment and age ( P = 0.63).

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163 Figure 5 3. Effect feeding pomegranate extract (POM) or antibiotic s on average daily gain of preweaned calves CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW Effect s of treatment ( P = 0.70), age ( P < 0. 01), and interaction between treatment and age ( P = 0.19)

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164 Figure 5 4. Effect of feeding pomegranate extract (POM) or antibiotic s on plasma concentration of glucose (A) and BHBA (B) in preweaned calves. C ON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extrac t dosed orally at 15 mg of gallic acid equivalent/kg BW Glucose : Effect s of treatment ( P = 0.33 ), age ( P < 0.0 1), and interaction between treatment and age ( P = 0.53) BHBA : Effect s of treatment ( P = 0.36), age ( P < 0. 01), and interaction between treatmen t and age ( P = 0.46)

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165 Figure 5 5. Effect of feeding pomegranate extract (POM) or antibiotic s on plasma concentration of NEFA (A) and urea N (B) in preweaned calves. CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomy cin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW NEFA : Effects of t reatment ( P = 0. 94 ), age ( P < 0. 01), and interaction between treatment and age ( P = 0. 97 ). Urea N: Effects of t reatment ( P = 0.08), age ( P < 0. 01), and interaction between treatment and age ( P = 0.11).

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166 Figure 5 6 Effect of feeding pomegranate extract (POM) or antibiotic s on hematocrit (A) and s erum total protein (B) in preweaned calves. CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW Cov = covariate. Hematocrit: Effects of t reatment ( P = 0.68 ), age ( P < 0.0 1), and interaction between treatment and age ( P = 0. 6 1 ). Serum total protein: Effects of t reatment ( P = 0.30), age ( P < 0.0 1), and interaction between treatment and age ( P > 0.56).

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167 Figure 5 7 Effect of feeding pomegranate extract (POM) or antibiotic s on fecal score ( A) and attitude score (B) of preweaned calves CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg of body weight. Fec al score: 1 f irm feces no diarrhea; 2, soft feces no diarrhea ; 3, mild diarrhea ; and 4 w atery feces and diarrhea. Attitude s core: 1 r esponsive ; 2 n on active ; 3 d epressed ; and 4 Moribund. Fecal score: Effect s of treatment ( P = 0.90 ), age ( P < 0.01 ), and interaction between treatment and age ( P = 0.0 8) Attitude s core: Effects of t reatment ( P = 0.91), age ( P < 0. 01), and interaction between treatment and age ( P = 0.99).

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168 Figure 5 8 Effect of feeding pomegranate extract (POM) or antibiotic s on rectal temperature during first 14 d of age. CON, no add itive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW Effect s of treatment ( P = 0.13), age ( P < 0.0 1) and interaction between treatment and age ( P = 0.62)

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169 Figure 5 9. Survival curves for the effect of feeding pomegranate extract (POM) or antibiotic s to dairy calves in the preweaning period. CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in m ilk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW The mean survival times were 63.7 2.5, 58.7 3.4, and 62.1 2.7 d for control, antibiotic and POM, respectively. Calves receiving antibiotic tended ( P = 0.10) to surv ive less time than calves in control and POM S urvival time is expressed as age of cal f at leave of the study.

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170 Figure 5 10 Effect of feeding pomegranate extract (POM) or antibiotic s on neutrophil phagocytosis (A) and o xidative b urst (B) in preweaned calves. CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg BW Phagocytosis: Effect s of treatment ( P = 0.74) age ( P = 0.02 ) and i nteraction between treatment and age ( P = 0. 59 ) Oxidative burst: Effect s of treatment ( P = 0.76), age ( P = 0.03) and i nteraction between treatment and age ( P = 0.55)

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171 Figure 5 1 1 Effect of feeding pomegranate extract (POM) or antibi otic s on neutrophil phagocytosis mean fluorescence intensity (MFI, indicator of number bacteria phagocytized per neutrophil) (A), oxidative burst mean fluorescence intensity (MFI, indicator of intensity of reactive oxygen species produced per neutrophil) (B ) in preweaned calves CON no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegranate extract dosed orally at 15 mg of gallic acid equivalent/kg of body weight. Neutrophil p hagocytosis MFI : Effects of treatment ( P = 0. 92 ), age ( P = 0.0 4 ), and interaction between treatment and age ( P = 0. 1 9). Neutrophil o xidative burst MFI : Effects of treatment ( P = 0.7 3 ), age ( P < 0.0 1 ), and interaction between treatment and age ( P = 0. 63 ).

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172 Figure 5 1 2 Effect of fe eding pomegranate extract (POM) or antibiotic s on anti ovalbumin IgG serum titers in response to ovalbumin immunization in pre wean ed calves. CON, no additive added to milk; Antibiotic : 200 mg of oxytetracycline and 200 mg neomycin/d in milk; POM : pomegrana te extract dosed orally at 15 mg of gallic acid equivalent/kg BW Cov = covariate. Effect s of treatment ( P = 0.49), age ( P < 0.0 1) and interaction between treatment and age ( P = 0. 79 )

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173 CHAPTER 6 IMPACT OF FEEDING DIFFERENT AMOUNTS OF SACCHAROMYCES CER EVISIAE FERMENTATION PRODUCT S ON IMMUNE STATUS, HEALTH AND PERFORMANCE OF HOLSTEIN CALVES Introduction The use of subtherapeutic doses of antibiotics in feed is a common practice in raising dairy calves in an attempt to reduce the incidence of diarrhea and morbidity in neonatal calves caused by bacterial infection. In 2007, the National Animal Health Monitoring System reported that 57.5 % of dairy farm s use medicated milk replacer and 18.2 % of operations use d antibiotics in weaned heifer rations t o prevent disease or promote growth (NAHMS, 2007) Nevertheless, the use of anti microbials in animal agriculture is under increased scrutiny because of concerns of potential development of antimicrobial resistance by pathogenic bacteria common to dairy calves ( Perei ra et al., 2011). Saccharomyces cerevisiae either as live yeast or as yeast culture often is used as an additive in cattle diets because of its properties to potentially alter rumen function promote fiber digestion reduce the risk of milk fat depression and minimize rum inal pH fluctuations ( Longuski et al., 2009; Martin and Nisbet, 1992). When fed as a culture, the fermentation products contain B vitamins, organic acids, and other products of fermentation ( NRC 19 98 ). The cell wall of y easts is also rich in mannan oligosaccharides and glucans. These compounds may prevent the interaction between pathogenic bacteria and intestinal cells, as well as strengthen the im mune system (Shen et al., 2009; Gao et al., 2008). Studies have evaluated the impact of supplementing S. cerevis i ae to cal ves as an alternative to antimicrobial s and growth promot ants However, t he effects of yeast s and yeast cultures on growth and performance of calves in the preweaning period remain contradictory. Some studies

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174 showed increased growth performance ( Magalhes et al., 2008; Galv o et al., 2005; Lesmeister et al., 2004 ), whereas others reported minor effects ( Kim et al., 2011; Hill et al., 2009 ; Pinos Rodri guez et al. 2008 ) The effects of feeding yeast and yeast culture on the are inconclusive A d dition of yeast culture at 2% of the grain reduced the incidence and duration of diarrhea, d ecreased the incidence of fever and mortality ( Magalhes et al., 2008). Addition of 1% brewer's yeast to the dry feed reduced the incidence of fever and the number of associated antibiotic treatments during the preweaning period (Seymo u r et al., 1995). H owever, others showed no effect of feeding yeast culture on the incidenc e and duration of diarrhea and incidence of pneumonia in dairy calves ( Pinos Rodriguez et al. 2008; Lesmeister et al., 2004 ) glucans and mannan present in the yeast cell wall have been shown to stimulate immune responses ( Volman et al., 2008 glucans stimulate d phagocytosis and production of inflammatory cytokines by mammalian macrophages ( Brown and Gordon, 200 3 ), and stimulate d production of reactive oxygen species by huma n n eutrophils and monocytes (Rubin Bejerano et al., 2007). In calves, supplementation of yeast culture to the grain tended to increase the number of phagocytized pathogeni c bacteria and killing of phagocytized bacteria but did not influence humoral immune response ( Magalhes et al., 2008) Saccharomyces cerevisiae fermentation products (SFP) is produced during fermentation of a rumen specific strain of S cerevisiae and i nclude the by products of fermentation, yeast cells, yeast cell wall fragments, and the media utilized during fermentation (Shen et al., 2011). Studies of the effects of live yeast and yeast culture on immunity in rodents are abundant, but few experiments have addressed the effects of yeast on immune modulation in calves (Kim et al., 2011; Magalhes et al., 2008).

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175 Thus, it was hypothesized that yeast cell wall and metabolites present in SFP might improve measures of immune response, health and growth of da iry calves. The objective of this study was to evaluate the effect of feeding increasing amounts of SFP on immune status, health and performance of preweaned Holstein calves Materials and Methods Calves Housing and C olostrum Feeding All study procedures were approved by the University of Florida Institutional Animal Care and Use Committee. The study was perform ed at the Dairy Unit of the University of Florida. One hundred and twenty eight (n = 128 ) Holstein calves on the day following birth (birth = day 0) were used in the study. Calves received 3.8 L of frozen and thawed colostrum containing at least 80 g of total IgG/L within the first 2 h after birth, and 1.9 L in the following 12 h. Calves were housed in individual hutches. At enroll ment calves were weighed and blood was sample d Treatments, Feeding and Measurement s Calves were blocked by gender and, within each block, randomly assigned to 1 of 4 treatments (n = 32/treatment) Treatments were T0, c ontrol no supplemental SFP; T1, 1 g/calf/d of SFP; T2, 2 g/calf/d of SFP; and T4, 4 g/calf/d of SFP. Once daily and immediately before the morning feeding, SFP was incorporated and mixed i n individual bucket s containing pasteurized milk according to the specific treatment. The SFP was produced by Diamond V Mills, Inc. ( Cedar Rapids, IA ) to contain a culture of a strain of S. cerevisiae with additional antioxidant properties by incorporating hydroly z able tannins in the fermentation process The SFP was provided in a soluble form to facilitate incorporation i nto milk.

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176 Milk was fed twice daily at 6 and 14 h C alves were offered 1.9 L of pasteurized nonsal e able milk (milk from cows in the hospital pen because of mastitis or other diseases) at each feeding during the first 49 d of age, and then once a day until 56 d of age, when calves were weaned from milk. Calves had ad libitum access to a starter grain (P urina Mills St. Louis, MO) throughout the study. Dry matter content of calf grain was monitored weekly to determine grain DM intake. Grain intake and weigh b acks were measured daily after the morning milking, and amounts offered were adjusted to allow for 10% orts (minimum of 200 g) in the first 61 d of age. Dry matter intake from grain and milk were calculated daily for each calf. Body weight was measured o n 2 consecutive days at 1 and 2 30 and 31 and 60 and 61 d of age, and averaged for each measurement Calves were weighed consistently at the same time before the morning milk feeding Body weight gain s for specific intervals w ere determined by difference. Wither and hip height of calves were evaluated at 1, 30 and 60 d of age. Grain and Milk Sampling, and Nutrient Analyses Samples of pasteurized milk were collected thrice weekly throughout the study and analyzed for concentrations of total solids, fat, tru e protein, and lactose at the Southeast DHIA Laboratory ( Belleview FL) using a Bently 2000 NIR analyzer. Solids not fat were calculated by difference between total solids and fat Ash was calculated by difference from total solids minus the sum of fat, tr ue protein and lactose. Composition of pasteurized milk (Table 6 1) was used to estimate the energy concentration in milk using NRC (2001) equation s. Although amount of milk consumed by each calf was fixed at 3.8 L/d in the first 49 d of age, followed by 1 .9 L from 50 to 56 d of age, composition

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177 of milk varied throughout the study, which resulted in calves of different ages consuming different quantities of milk solids at different time points in the study. Samples of milk post pasteurization from individu al calf buckets were taken thrice weekly, immediately stored in ice and transported to the Microbiology Laboratory of Department of Animal Sciences A 50 L milk sample was plated o n blood and MacConkey agars to determine number of total bacteria, coliform s, and E. col i (Hogan et al., 1999) Grain was sampled once a week, dried at 55C for 48 h and moisture content was recorded. Dried samples were ground to pass a 1 mm screen (Wiley mill, Philadelphia, PA) and samples were then composited for two month peri ods Samples of grain and SFP were analyzed at the Dairyland Laboratory (Arcadia, WI) for DM at 105 o C, N by combustion (crude protein = N x 6.25), fat (AOAC, 2000), NDF, ADF (Van Soest et al., 1991), and minerals using an inductively coupled plasma emissi on spectrometer (Thermo Garrell Ash, Franklin, MA) Composition of starter grain and SFP are provided in Table 6 2. Scoring, Incidence of Health Disorders, and Costs Associated with Treatments Calves were scored daily using the calf health score chart from University of Wisconsin. Attitude fecal consistency nasal discharge, ocular discharge and cough were scored daily during the morning milk feeding using a 0 to 3 scale. For attitude, calves were categorized as 0 when alert and responsive 1 when non acti ve, 2 when depressed, and 3 when moribund. Fecal consistency was scored as 0 when firm, 1 when soft or of moderate consistency, 2 when runny or mild diarrhea, and 3 when watery and profuse diarrhea. For nasal score, 0 was normal serous discharge, 1 when a small amount of unilateral cloudy discharge was present, 2 when bilateral cloudy or

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178 excessive mucus discharge was present, and 3 when copious bilateral mucopurulent discharge was present. Ocular discharge was scored as 0 when normal, 1 when a small amount of ocular discharge was present, 2 when moderate amount of bilateral discharge was present, and 3 when heavy ocular discharge was present. Cough was scored after pressing the trachea as 0 when absent, 1 when induced a single cough, 2 when induced repeated cough or occasional spontaneous cough, and 3 when repeated spontaneous cough was detected. Weekly averages of all scores were generated for individual calves for statistical analyses. Calves with fecal score > 1 were used for analysis of incidence of diarr hea. Incidence of health disorders was recorded daily for individual calves. Rectal temperature was measured daily during the first 14 d of age, and on days when display ed clinical signs of disease such as diarrhea, bloat, coughing, increased respiratory frequency, depress ion or lack of appetite. Calves with rectal temperature 39.5 o C were categorized as febrile Day when disease was first diagnosed was recorded and duration of each illness event was determined. Number of episodes of fever, diarrhea, an d pneumonia was determined. To distinguish between different episodes, an interval of 4, 4, and 10 d between diagnoses of fever, diarrhea and pneumonia, respectively, had to elapse to characterize a new event. Calves with digestive and respiratory problems were treated by farm personnel according to protocols established by the herd veterinarian. Medication used, dosage, and duration of treatments was recorded for individual calves. Costs of health treatments were calculated based on currents costs for each product, daily dosage for each medication for individual calves

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179 Fecal samples were collected from calves on the second or third sequential day of fecal score > 1. Samples were kept on ice until transported to the microbiology laboratory. For calves that d id not develop diarrhea until d 14 1 of age, a sample w as collected on d 14 1, so every calf ha d a representative sample for statistical analyses of risk of fecal bacteria l shedding and intensity of shedding. Blood Sampling Blood samples were taken 30 min after the morning feeding of milk on the day of study enrollment and then every 7 d until d 42. Blood was sampled by puncture of the jugular vein using an 18 gauge 2.5 mm needle (Air Tite, Virgina Beach, VA), and collected into specific evacuated b lood collecting tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ). Immediately after bleeding, K2 EDTA tubes were placed in ice. Tubes for serum separation and tubes containing heparin were kept at ambient temperature. Plasma or serum was separated by centrifugation at 3,000 rpm at 4C for 20 min (Allegra X 15R Centrifuge, Beckman Coulter). Plasma was stored at 20C for subsequent analyses of metabolites. Blood was also sampled using evacuated tubes containing heparin for neutrophil phagocytic and k illing assays. Hematocrit, Serum T otal P rotein and Total I mmunoglobulin Q uantification Hematocrit va lues were measured in duplicate Briefly, whole blood was p laced in micro capillary tubes and centrifuged for 4 min using Microspin 24 (Vulcon Technologies, Grandview, MO). The amount of packed blood cell s was measured using a micro hematocrit tube reader Model CR (International Equipment Co., Needham Heights, MA). Sera were analyzed for STP using a clinical refractometer (Reichert Jung, Buffalo, NY). Serum c ollected on the day of study enrollment was frozen and later analy zed for

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180 concentrations of total IgG The determination of total IgG in serum was performed using agar gel immunodiffusion specific for bovine IgG (Triple J Farms, Bellingham, WA ) and followi ng Ser a were diluted with DPBS (1:5) when the concentrations of IgG were greater than 2. 5 g/d L T he intra and interassay s coefficients of variation were 2. 0 and 8.2 %, respectively. Assay s for Plasma Metabolites Plasma concentr ations of glucose, urea N, NEFA, and BHBA were measured in samples collected weekly until 42 d. Plasma concentration of glucose and urea N were measured using AutoAnalyzer (Technicon Instruments Corp., Chauncey, NY). The AutoAnalyzer method for plasma gluc ose was a modification of Gochman and Schmitz (1972), whereas that method used for urea N was a modification of Coulombe and Favreau (1963). The glucose intra and interassay s coefficients of variation were 0.6 and 2. 5 %. Whereas the respective value s for u rea N assay s were 1.2 and 1.7 %. Plasma NEFA and BHBA were measured using enzymatic methods from Wako Diagnostic (Richmond, VA ) with a microplate reader SpectraMax 340PC 384 (Molecular Devices, Sunnyvale, CA) .The BHBA intra and interassay s coefficients of variation were 4. 2 and 7.3 %. Whereas the respective value for NEFA assay were 3.2 and 10. 5 %. Immunization with Ovalbumin and Assay for Anti OVA IgG Ovalbumin (OVA) solution was prepared by dissolving 1 mg of OVA (Sigma Aldrich, St. Louis, MO) in 1 mL of st erile PBS and emulsified in 0.5 mg of adjuvant Quil A (Accurate Chemical, Westbury, NY). Calves were immunized with 1 mg of Ova (1 mg/mL) at 2, 14, and 28 d of age and blood was sampled prior to each immunization and again at 42 d for measurement of anti O VA IgG in serum.

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181 Immunoglobulin G anti O VA was quantified using an enzyme linked immunosorbent assay (ELISA). Briefly, flat bottom 96 well polystyrene plates ( Immulon 2, Dynex Tech., Chantilly, VA) were coated with a solution of OVA dissolved in carbonate bicarbonate coating buffer (1.4 mg OVA/mL of carbonate bicarbonate buffer). Plates were incubated at 4C for 48 h, then washed 4 times using a multiwash III microplater washer (Tricontinent, Grass Valley, CA ) with washing solution (PBS with Tween 0.05 %, pH = 7.4). Plates were blocked with a PBS 3% Tween and bovine serum albumin (Sigma Chemical, St. Louis, MO) solution and incubated at room temperature for 1 h. Plates were washed four times and diluted sera samples and control sera ( 1 to 50 and 1 to 20 0) were added in du plicate using a quadrant system. Positive and negative control sera to anti OVA IgG were obtained from a pool of sera of calves with known high (after 3 immunization s with OVA ) and low (pre OVA immunization ) concentrations, respectively. Plates were incubated at room temperature for 2 h and washed with washing buffer solution. After washing, alkaline phosphatase conjugate rabbit anti bovine IgG (Sigma Chemical, St. Louis, MO) was dissolved in Tris buffer solution and added to the plates a nd incubated for 1 h at room temperature. After incubation, plates were washed 4 times and 80 L of substrate solution ( P n itrophenyl p hosphate d isodium ; Sigma Chemical, St. Louis, MO) was added and the plate was incubated at room temperature for 30 min. P lates were read using 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. Results were calculated from the sum of the average of each duplicate d sample dilution multiplied by the correction factor of each plate. The intra and interassay s coefficients of variation were 6. 4 and 13. 9 %, respectively.

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182 Leukocyte Population Quantification Blood was collected from puncture of the jugular vein into hepar inized tubes at 14 28, and 42 2 d of age Q uantification of total and individual leukocyte population s was performed using ProCyte Dx h ematology a nalyzer (IDEXX L aboratories, Inc., Westbrook, M E ). Phagocytosis and Oxidative Burst Assay Neutrophil phagoc ytosis and oxidative burst were measured using a dual color flow cytometry assay methodol ogy modified from Smit s et al. (1997). Blood was collected from the jugular vein in evacuated tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) containing hepar in on d 7, 14 and 56 1 of age. Approximately 100 L of whole blood was pipetted into each of 3 polystyrene round bottom tubes (12 x 75 mm) and 10 L of 50 M dihydrorhodamine 123 (DHR ; Sigma Aldrich, St Louis, MO) was added to all tubes. Tubes were slowl y vortexed and incubated at 37 o C for 10 min with constant rotation using nutator (BD, San Jose, CA). Ten microliter s of 20 g/mL solution of PMA (Sigma Aldr ich) was added to tube number 2 (positive control for oxidative burst). A pathogenic E coli bacter ial suspension (10 6 CFU /mL) isolated from a case of mastitis in a dairy cow and labeled with propidium iodide (Sigma Aldrich) was added to tube number 3 to establish a ration of bacteri a to neutrophil of 40:1 according to concentration of neutrophil in blo od provided by hematology results. Tubes were slowly vortexed and incubated at 37 o C for 30 min with constant rotation using nutator. After incubation, tubes were placed immediately on ice to stop neutrophil activity. Tubes were processed in a Q Prep Epics immunology workstation on the 35 sec cycle using three reagents (Solutio n A: 88% formic acid, red blood lysis solution; Solution B: b uffer solution constituted for s odium c arbonate, s odium c hloride and s odium s ulfate; and

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183 s olution C: 1% paraformaldehyde, cell fixative solution). Cold distilled water (500 L ) for completion of hemolysis and 0.4% tryphan blue (10 L ) for quenching extracellular oxidized DHR w ere added to each tube. Tubes were slowly vortexed, kept on ice, and 10,000 leukocytes were read at t he Facsort flow cytometer (BD B iosciences, San Jose, CA). Fecal Shedding of E. coli and Salmonella spp One f ecal sample per calf was collected within the first 14 d of age Fecal samples were cultured to quantify presence and shedding of E. coli and Salm onella spp During defecation, about 10 g of feces were collected from individual calves in a sterile tube. After homogenization, 100 mg of feces were resuspended in 5 0 mL of sterile PBS The suspension was shaken vigorously and incubated for 15 min at roo m temperature Then 1 L of the dilution was plat ed o n M a cConkey agar (Hardy Diagnostics, Santa Maria, CA) and incubated for 24 h at 35 o C. The number of CFU of E. coli was quantified. The colony count was calculated on the basis of fecal weight. Qua nti tat ive detection of Salmonella spp. was performed using the same sample by culturing 1 g of feces in tetrat h ionate broth with iodine iodide solution (Hard y Diagnostics, Santa Maria, CA) for enrichment for 24 h. After incubation, 1 L of this suspension was pl ated o n x ylose l ysine t ergitol 4 a gar (Hardy Diagnostics, Santa Maria, CA) After 48 h of incubation at 3 5 o C s uspicious Salmonella colon ies were quantified, and one suspicious colony was p la ted o n lysine iron agar to confirm Salmonella after 24 h of incu bation. Experimental Design and Statistical Analyses The experimental design was a randomized complete block design. Calves on the day after birth were blocked according to gender and, within each block, randomly

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184 assigned to one of four treatments. Pre pla nned single degrees of freedom orthogonal polynomial contrasts were evaluated for each outcome to determine linear and quadratic responses to addition of SFP. The number of experimental units/treatment was calculated to be sufficient to provide sufficient detect a 140 g/d difference in grain intake when the SD for grain intake between 3 and 9 weeks of age is 2 0 0 g /d Similarly, the sample size was calculated to provide enough experimental units to detect 10 0 g/d differ ence in BW gain when daily changes in BW in the first 60 d of age range from 4 00 to 600 g/d Continuous data were tested for normality of residuals using the Shapiro Wilk test of SAS ver. 9.2 (SAS Inst. Inc., Cary, NC) N on normally distributed data were t ransformed as suggest using the guided data analysis of SAS and then back transformed using the ilink function to generate least square means and SEM. Continuous variables were analyzed by ANOVA using the GLIMMIX procedure of SAS ver. 9.2 fitting a normal distribution. Variables with a single measurement during the study were analyzed with the fixed effects of treatment, season (winter or spring) gender of calf and the interaction between treatment and season, and treatment and gender of calf Variables with repeated measurements within the same calf were analyzed with the fixed effects of treatment, time of measurement (day or week), season, gender of calf and the interactions between treatment and time, treatment and season, treatment and gender of calf and the random effect of calf nested within Bayesian criterion. The BW and concentration of total IgG in serum on the day of study enrollment were used as covariates for all statistical analysis performed

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185 Binomial data were analyzed by logistic regression using the GLIMMIX procedure of SAS with a link logit function and fitting a binomial distribution. The models included the effects of treatment, gender of calf season and interactions between treatment and gender and treatment and season For all statistical analysis total IgG at enrollment was used a covariate. Treatment differences with 0.05 were considered significant and 0.05 < 0.10 were designated as tendency Results The body weight on the day of study enrollment was not different ( P = 0.54) among treatments and averaged 41.2, 40.0, 41. 2, and 39.8 kg for T0, T1, T2 and T4, respectively. Serum total protein concentration s on day 1 did not differ ( P = 0.49) am ong treatments and averaged 5.7 5.8, 5.6 and 5.7 g/dL for T0, T1, T2 and T4 respectively. Similarly, serum total IgG ( 1. 8 2.1 1.9 and 1.9 g/dL, respectively) and hematocrit (3 3 1 3 3 1, 32.9 and 31. 5 %, respectively) did not differ for T0, T1, T2 and T4 Eight percent of calves had serum total IgG < 1.0 g/dL, meaning FPT ( Berge et al., 2005; Weaver et al., 2000; Tyler et al., 1996). These calves were distributed in T0 (16%), T1 (0%), T2 (6%), and T4 ( 9%). Grain DM intake was not different among treatment s ( Table 6 3; Figure 6 1) Overall, calves consumed an average of 628 g grain DM /d in the first 6 0 d of age. Total DM intake (Figure 6 2) was not different among treatment s and averaged 1,000, 985, 987 and 10 10 g/d for T0, T1, T2 and T4, respectively an d th e lack of difference was observed for the first 30 d and also from 31 to 61 d of age (Table 6 3). There were not differences among treatments in ADG during the first and second month of age and calves gained on average 38 3 and 796 g/d, respectively I n fact, BW at 30 and 60 d of

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186 age were not affect ed by treatment, with mean s of 51.8 and 75.7 kg, respectively. C onsequen tly supplementation with SFP did not affect feed conversion ( ADG / total DM intake ), which averaged 0.68 and 0.6 1 for the first and seco nd month of age respectively. Wither and hip hei ghts at 30 and 60 d of age and skeletal growth based on change in height were not influenced by treatment (Table 6 3). The mean g lucose c oncentrations did not differ among treatments Concentrations were af fected ( P = 0.02) by age of calves, but no interaction was found between treatment and age of calves (Figure 6 3). Concentrations of BHBA tended ( P = 0.10) to be affected quadratically by level of SFP ( Figure 6 4 ) because it increased with feeding T1 and r eturn to T0 values when calves were fed 2 or 4 g/d of SFP. As anticipated, c oncentrations of BHBA increased ( P < 0.01) with age of calves, but n o interaction was observed between treatment and age of calves The mean NEFA concentrations did not differ amon g treatments but c oncentrations decreased ( P < 0.01) with age of calves (Figure 6 5) A quadratic response to SFP tended ( P = 0.1) to influence the plasma urea N. Concentrations increased with feeding 1 or 2 g/d, but decreased in calves fed 4 g/d. Concent rations of urea N decreased ( P < 0.01) with age of calves (Figure 6 6) Supplementing calves with SFP tended ( P = 0.09) to result in a linear decrease in rectal temperature of calves in the first 14 d of age (Figure 6 7) but this decline was only 0.1 o C. Incidence s of fever or pneumonia throughout the study w ere not affected by feeding SFP (Table 6 4 ) The age at first day of diarrhea, t he percent of day s that calves were all similar among treatments As expected, c osts for medication per calf did not differ among treatments. Feeding SFP had no impact on nasal, ocular, cough and a ttitude

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187 scores through ou t the study. Supplementing the milk of calves with SFP did not affect hematocrit and STP during the study (Figure s 6 8 and 6 9). Feeding SFP resulted in a quadratic ( P = 0.07) increase in the number of leukocytes per L of blood of calves (Figure 6 10), although the concentrations of individual leukocytes remained unaffected by treatments (Figures 6 11 and 6 12). Phagocytic activit y of neutrophils against E. coli did not differ among treatments (Figure 6 1 3 A ). Phagocytosis MFI, as indicator of the amount of bacteria phagocytized per neutrophil was also not affected by dietary supplemen tation ( Figure 6 1 4 A ) The age of calve s or interaction between treatment and age did not affect the percentage of neutrophil s perform ing phagocytosi s or MFI However, neutrophil oxidative burst increased linearly ( P = 0.03) with increasing amounts of SFP and tended ( P = 0.06) to be quadratically affected by level of SFP Age of calves did not affect oxidative burst. Neutrophil oxidative burst MFI, as indicator of intensity of reactive oxygen species produced per neutrophil was not affected by SFP supplementation, age of calves or interaction between treatment and age of calves (Table 6 1 4 B) Supplementing milk of calves with SFP did not affect antibod y titers in responses to immunization with OVA (Figure 6 1 5 ). Concentrations of anti OVA IgG in serum differed increased ( P < 0.01) with sequential immunizations of calves, but no interaction was observed between treatment and age Discussion Prophylactic antimicrobials often are added to milk or milk replacers in an attempt to minimize the negative impact of preweaning diarrheal diseases on calf growth performance and survival Nevertheless, the benefits to feeding antimicrobials seem to not be consistent or even deleterious. Calves receiving 54 mg of neomycin sulfate and

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188 108 mg of tetracycline HCl per day in milk for the first 2 w eeks of life had 31% more days with diarrhea compared with control calves (Berge et al., 2009). Because of the increased scrutin y on use of antim icrobial growth promotants, supplements that might improve calf health and performance have been studied as potential alternatives to antimicrobials. In fact, the use of substances able to modulate immune functions has gained increasing in terest in animal research (Gallois et al., 2009). Magalhes et al. (2008) suggested that yeast cells and compounds produced during fermentation activity of S. cerevisiae might be responsible for the positive effects of yeast culture on performance and hea lth of young animals. In fact, the cell wall glucans and mannans) have been recognized as modulators of immune responses in mammals through specific interactions with different immunocompetent cells (Kogan and Kocher, 2007). Part of thi s effect is thought to be glucans coming in contact with the mucosal immune system (Volman et al., 2008). In addition, yeast and its compounds contain a high amount of soluble bioactive particles that could activat e immune cells (Jensen et al ., 2007). Despite the potential benefits of feeding yeast and yeast cultures to mammals, growth performance and measures of health and immune responses of dairy calves to feeding SFP in the current study were mostly unaltered. Interestingly, calves fed SF P had neutrophils with improved killing activity. Magalhes et al. (2008) reported some improve ment in neutrophil function when they were incubated with a pathogenic strain of E. coli a response similar to the increment in oxidative when calves were fed S FP I t appears that the effect of yeast, or its compounds or derivates, on neutrophil s is specific to improve the killing activity but not the ir phagocyt ic ability and this effect could be dose dependent. The reasons for this

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189 finding are unclear. It is un know n whether dietary administration of SFP might be able to reac h the immune cells and activate them. However, the result s found here w ere similar to those reported by Sauerwein et al. (2007), who reported a greater neutrophil oxidative burst in piglet s f ed with 0.03% of yeast wall extract compared with unsupplemented control s but no effect on neutrophil phagocytosis were found Oral glucans could modulate t he mucosal immune response th rough the interaction with cells of the Peyer's pa tches (Suzuki et al., 1990). It ha s been suggested glucans are taken up by intestinal macrophages and then transported to lymph nodes, spleen and bone marrow (Volman et al. 2008). In an i n vitro study glucans altered macrophage and neutrophil function via their activation of specific receptors (Williams, 1997). However, the data o n the glucans on immune cell s are based primarily on in vitro studies, which might not always be directly comparable wit h in vivo responses. As a glucans role on neutrophil burst activity remains speculative. Adding yeast products to milk guarantees its intake from the first day of age, and facilitates the fast arrival to the intestinal tract Studies evaluating ye ast supplementation through milk are scar c e, but no effect on performance has been reported (Hill et al., 2009; Galvo et al., 2005). Some studies reported improve ment in daily grain intake when supplemented 0.5 g /d of live S. cerevisiae added to the grain for 84 d, or when 2% of yeast culture is offered with starter ( Galv o et al., 2005 ; Lesmeister et al., 2004). On the other hand, data on benefits to feeding yeast and yeast culture on health are also limited and contrasting in prewean ed calves. The curre nt study revealed no effect of SFP on incidence of fever, pneumonia or percentage of days with diarrhea. Th ese results were similar to those reported by Pinos Rodriguez et al. (2008) and Lesmeister

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190 et al. (2004), but different from those found by Magalhes et al. (2008), Galvo et al. (2005), and Seymour et al. (1995). Magalhes et al. (2008) found that feeding 2 % yeast culture during the first 70 d of age improved fecal scores, reduce d number of days with watery feces, and reduced the incidence of fever an d diarrhea. Galvo et al. (2005) reported that numbers of days with diarrhea were reduced by feeding live yeast to calves before weaning. In both studies, calves were either challenged with high incidence of diseases and mortality (Magalhes et al., 2008) or had failure of passive transfer (Galvo et al., 2005). Seymour et al. (1995) found a reduction i n percentage of days with fever at 2 week of age, and on percentage of days with scours at 3 week of age when calves were fed It is possib le that the benefits of supplementing yeast culture or SFP might only be observed when calves are under increased risk of morbidity and mortality. Another possibility to explain the differences in health and growth performance reponses amo n g studies may b e related with difference s in composition of yeast products, type of yeast, disease challenge and/or calf passive immunity. In most studies, detailed description of yeasts and yeast products (i.e. yeas t cell components and fermented products such as organi c acids, vitamins and nucleotides) and purity are usually not characterized, and information is usually proprietary of the manufacturing companies. Galvo et al. (2005) reported improve d performance when a live strain of Saccharomyces cerevisiae was f ed bu t not with S. cerevisiae spp. boulardii. Because response to supplementing calves with live yeast or yeast culture have been observed under conditions of increased disease (Magalhes et al., 2008; Galvo et al., 2005), it is possible that in the current st udy disease challenge s might not have been sufficient for the potential health benefits of SFP to be observed. In fact, only 8% of the calves had

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191 failure of passive transfer (serum IgG < 1.0 g/dL) and, although 25% of the days calves had fecal score compat ible with mild diarrhea, mortality was absent in the current study. because microbial challenge was low. R esults from Chapter 4 showed that calves with APT have reduce d incidence of diseases and risk of mortality, and improve growth performance Therefore, it is suggested that potential positive effect s of SFP on health and growth might have not been observed because of the lack of a greater disease challenge in the pre sent study. Conclusion s Supplementing dairy calves with SFP added to the milk up to 4 g/d in the first 2 months of life did not affect growth performance, grain DM intake, and feed conversion into BW gain Calves fed SFP had an increased proportion of neut rophils with capacity for oxidative burst, but this effect did not influence measures of health based on daily fecal, nasal, ocular, cough and attitude scores, and rectal temperature. Concentrations of metabolites in plasma remained mostly unaltered by fee ding SFP, likely because of the lack of impact on DM intake.

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192 Table 6 1. Nutrient composition and microbial contamination of pasteurized milk fed to calves 1 As is DM basis Mean SD Median Mean SD Median DM, % 11.6 0.9 11.8 ----Fat, % 3.02 0.68 3.11 25. 9 4. 6 26. 8 True protein,% 3.15 0.23 3.14 27.2 2.0 26.7 Lactose, % 4.55 0.32 4.60 39. 3 2.4 39.0 Solids not fat, % 8.58 0.57 8.66 74. 2 4. 6 73. 3 Ash, % 0.89 0.10 0.89 7. 7 0. 9 7.6 ME, 2 Mcal/kg 0.64 0.07 0.65 5. 5 0.2 5.5 SCC, x 10 3 /mL 3 525 316 490 ----SCS 4 4.97 1.43 5.29 ----Bacteria, log 10 CFU/mL 5 Total 2.70 0.55 2.70 ----Coliforms 1.82 1.13 2.15 ----E scherichia coli 1.24 1.02 1.30 1 Milk samples were taken thr ice weekly throughout the study. 2 Metabolizable energy calculated according to NRC (2001) based on the nutrient composition of milk. 3 SCC = Somatic cell counting. 4 SCS = Somatic cell score. 5 CFU = colony forming units.

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193 Table 6 2. Nutrient composition (mean SD) of the starter grain and Saccharomyces cerevisiae fermentation products (SFP) (DM basis) Starter grain 1 SFP DM, % 83.8 0.01 92.9 0.1 ME, 2 Mcal/kg 3.02 0.08 -OM, % 92.0 0.02 86.2 0.0 CP, % 21.2 0.7 32.7 0.1 NDF, % 23.8 0.8 0.01 0.0 ADF, % 8.7 0.3 --Nonfibrous carbohydrates, % 43.3 1.4 52.9 0.2 Fat, % 3.7 0.04 0.6 0.1 Ca, % 1.61 0.09 1.02 0.01 P, % 0.78 0.04 1.25 0.01 Mg, % 0.35 0.02 0.80 0.01 K, % 1.40 0.01 7.2 5 0.04 S, % 0.38 0.01 1.62 0.01 Na, % 0.51 0.04 0.27 0.02 Cl, % 0.75 0.04 1.42 0.04 Zn, mg/kg 206 13 63 0.1 Cu, mg/kg 43 8 15 0.1 Mn, mg/kg 150 43 35 1.4 Decoquinate, mg/kg 26 --Polyphenols as hydroly z able tannins, % --4.0 1 Each kg contains 0.33 mg of Se, 0.5 mg of Fe 0.3 mg of Co, 15,000 IU of vitamin A, 6,250 IU of vitamin D 3 30 IU of vitamin E according to manufacturer (PURINA) 2 Calculated based o n the nutrient content (NRC, 2001).

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194 Table 6 3. Effect of feedin g Saccharomyces cerevisiae fermentation products (SFP) on growth parameters of preweaned calves Treatment 1 P 2 T0 T1 T2 T4 SEM SFP L Q DM intake, g/d Grain 1 to 30 137 135 126 133 15 0.75 0.75 0.78 Total 1 to 30 574 572 563 570 15 0.72 0.73 0.78 Grain 31 to 60 1010 982 1028 1071 64 0.82 0.43 0.58 Total 31 to 60 1346 1319 1363 1407 64 0.81 0.42 0.58 ADG, 3 g/d Day 1 to 30 378 386 385 382 23 0.81 0.92 0.80 Day 31 to 60 784 790 798 813 34 0.94 0.54 0.89 ADG/DM intake Day 1 to 30 0.675 0.682 0.694 0.682 0.038 0.81 0.84 0.82 Day 31 to 60 0.611 0.620 0.606 0.592 0.015 0.81 0.31 0.43 Body weight, kg Day 30 51.7 51.9 51.9 51.8 0.7 0.82 0.93 0.81 Day 60 75.2 75.6 75.9 76.2 1.4 0.67 0.61 0.97 Growth, cm/d With er 0.175 0.176 0.178 0.180 0.007 0.74 0.63 0.95 Hip 0.190 0.191 0.197 0.200 0.008 0.48 0.28 0.91 Height, cm Day 30 withers 78.7 79.5 79.1 78.9 0.33 0.28 0.98 0.14 Day 30 h ip 83.8 84.2 84.8 84.1 0.38 0.25 0.45 0.15 Day 60 withers 84.8 84.9 85.0 85.1 0.41 0.74 0.63 0.95 Day 60 h ip 90.1 90.2 90.5 90.5 0.44 0.63 0.44 0.99 1 T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP Daily in the morning feeding, SFP was added to the milk until 56 d of age. 2 SFP = effect of feeding Saccharomyces cerevisiae fermentation product s (T0 vs. T1 + T2 + T4); L = linear effect of amount of SFP; Q = quadratic effect of amount of SFP. 3 ADG = average daily gain.

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195 Table 6 4. Effect of feeding Saccharomyces cerevisiae fermentation pr oducts (SFP) on health of preweaned calves Treatment 1 P 2 T0 T1 T2 T4 SEM SFP L Q Fever 3 Calves, % 53.1 (17/32) 71.9 (23/32) 56.3 (18/32) 62.5 (20/32) 0.28 0.70 0.42 Calf days, % 4.8 8.5 5.8 6.6 1.5 0.24 0.61 0.35 Fecal score 4 25.2 25.4 28.2 24.1 1.9 0.79 0.92 0.28 = 3, % days 8.3 9.1 9.2 7.2 1.0 0.90 0.44 0.15 Age at diarrhea 7.6 7.3 7.5 8.3 0.8 0.93 0.54 0.50 Nasal score 5 > 0, % days 10.0 11.1 13.3 9.6 2.0 0.57 0.94 0.24 2 or 3, % days 2.3 1.2 1.9 1.5 0.8 0.35 0.69 0.67 Ocular score 6 > 0, % days 8.0 9.1 14.0 7.9 1.6 0.30 0.64 0.05 2 or 3, % days 1.7 1.6 2.6 0.5 0.6 0.62 0.27 0.12 Cough score 7 > 0, % days 5.2 5.3 5.3 3.8 1.1 0.74 0.39 0.46 Attitude score 8 > 0, % days 4.0 2. 9 4.4 5.3 0.9 0.94 0.18 0.26 Pneumonia, % 25.0 (8/32) 28.1 (9/32) 28.1 (9/32) 12.5 (4/32) --0.60 0.27 0.27 Medication, $/calf 4.49 3.97 5.02 2.94 0.88 0.61 0.36 0.38 1 T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP Daily in the morning feeding, SFP was added to the milk until 56 d of age. 2 SFP = effect of feeding SFP (T0 vs. T1 + T2 + T4); L = linear effect of amount of SFP; Q = quadratic effect of amount of SFP. 3 o C. 4 Feces score scale: 0 : f irm feces, no diarrhea; 1: soft feces, no diarrhea, 2: mild diarrhea, and 3: w atery diarrhea. 5 Nasal score scale: 0: normal serous discharge, 1: small amount of unilateral cloudy discharge, 2: b ilateral cloudy or excessive mucus discharge, 3: copious bilateral mucopurulent discharge. 6 Ocular score scale: 0: normal, 1: small amount of ocular discharge, 2: m oderate amount of bilateral discharge, 3: h eavy ocular discharge. 7 Cough score scale: 0 : none, 1: induce d single cough, 3: induced repeated cough or occasional spontaneous cough, 3: repeated spontaneous cough. 8 Attitude score scale: 0: r esponsive, 1: n on active, 2: d epressed, and 3: m oribund.

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196 Figur e 6 1. Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on grain dry matter intake of preweaned calves .T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk Effect of feeding SFP ( P = 0.92 ), age ( P < 0.01 ), and linear ( P = 0.90 ) and quadratic ( P = 0.73 ) effect s of amount of SFP.

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197 Figure 6 2. Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on total dry matter intake of preweaned calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk Effect of feed ing SFP ( P = 0.92), age ( P < 0.01), and linear ( P = 0.90) and quadratic ( P = 0.73) effects of amount of SFP.

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198 Figure 6 3. Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on plasma glucose concentrations of preweaned calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean concentrations were 100.3, 101.7, 100.6, and 100.6 mg/dL (SEM = 0.9) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.53), ag e ( P = 0.02), and linear ( P = 0.95) and quadratic ( P =0.44) effects of amount of SFP.

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199 Figure 6 4 Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on plasma h ydroxybutyric acid ( BHBA ) concentrations of preweaned calves. T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean concentrations were 0.77, 0.73, 0.98, and 0.74 mg/dL (SEM = 0.05) for T0, T1, T2 and T4, respectively Effect of feeding SFP ( P = 0.50), age ( P < 0.01), and linear ( P = 0.54) and quadratic ( P =0.10) effects of amount of SFP

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200 Figure 6 5 Effect of feeding Saccharomyces cerevisiae fermentation products (SFP) on concentrations of non esterified fatty acids (NEFA) in plasma of preweaned calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean concentrations were 105, 110, 111, and 114 M (SEM = 5.6) for T0, T1, T2, and T4, re spectively. Effect of feeding SFP ( P = 0.33), age ( P < 0.01), and linear ( P = 0.29) and quadratic ( P = 0.84) effects of amount of SFP.

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201 Figure 6 6 Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on concentrations of u rea N in plasm a of prewean ed calves. T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean concentrations were 9.5, 9.4, 10.2, and 9.1 mg/dL (SEM = 0.3) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.81), age ( P < 0.01), and linear ( P = 0.80) and quadratic ( P = 0.10) effects of amount of SFP.

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202 Figure 6 7. Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on rectal temperature during the first 14 d of age. T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean values were 38.8, 38.8, 38.7, and 38.7 o C (SEM = 0.03) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.19), age ( P < 0.01), and linear ( P = 0 .09) and quadratic ( P = 0.75) effects of amount of SFP.

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203 Figure 6 8. Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on hematocrit of prewean ed calves. T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g /d of SFP added to the milk. The mean values were 30.2, 29.5, 29.6, and 30.6 % (SEM = 0.6) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.61), age ( P < 0.01), and linear ( P = 0.62) and quadratic ( P = 0.14) effects of amount of SFP.

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204 Figure 6 9. Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on serum total protein concentration of prewean ed calves. T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean con centrations were 5.6 5.7 5.6 and 5.6 g/dL (SEM = 0. 04 ) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.73), age ( P < 0.01), and linear ( P = 0.22) and quadratic ( P =0.67) effects of amount of SFP.

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205 Figure 6 10. Effect of feeding Saccharomyces cerevisiae fermentation products (S FP) on white blood cell count of prewean ed calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean concentrations were 9.8, 11.0, 10.8, and 10.5 x 10 3 /L (SEM = 0.4) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.07), age ( P = 0.19), and linear ( P = 0.39) and quadratic ( P = 0.07) effects of amount of SFP.

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206 Figure 6 11. Effect of feeding Saccharomyces cerevisiae fe rmentation products (SFP) on concentrations of neutrophil (A) and lymphocyte (B) in blood of prewean ed calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. Neutrophil= mean concentrations were 4.3, 4 .8, 4.8, and 4.6 x 10 3 /L (SEM = 0.3) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.27), age ( P = 0.01), and linear ( P = 0.53) and quadratic ( P = 0.27) effects of amount of SFP. Lymphocyte = mean concentrations were 4.6 5 .0, 4.9 and 4.8 x 10 3 /L (SEM = 0. 2 ) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.99), age ( P = 0.0 2 ), and linear ( P = 0.94) and quadratic ( P = 0.99) effects of amount of SFP.

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207

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208 Figure 6 12 Effect of feeding Saccharomyces cerevisiae f ermentation products (SFP) on concentrations of basophil (A), eosinophil (B) and monocyte (C) in blood of prewean ed calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. Basophil concentrations were 0.05, 0.05, 0.04, and 0.05 x 10 3 /L (SEM = 0.008) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.87), age ( P < 0.01), and linear ( P = 0.92) and quadratic ( P = 0.56) effects of amount of SFP. Eosinophil concentrations were 0.03, 0.04, 0.03, and 0.03 x 10 3 /L (SEM = 0.005) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.8 63 ), age ( P < 0.01), and linear ( P = 0. 71 ) and quadratic ( P = 0. 28 ) effects of amount of SFP. Monocyte concentrations were 0.7, 1.0, 0.8, and 0.8 x 1 0 3 /L (SEM = 0.08) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.24), age ( P < 0.01), and linear ( P = 0.80) and quadratic ( P = 0.34) effects of amount of SFP.

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209 Figure 6 1 3 Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on neutrophil phagocytosis (A), and oxidative burst ( B) of prewean ed calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. Phagocytosis = mean values were 52.1 54.8 55.2 and 53.9 % (SEM = 1.6 ) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.18), age ( P = 0.20), and linear ( P = 0.42) and quadratic ( P = 0.22) effects of amount of SFP. Oxidative burst= mean concentrations were 38.1 41.3 44.2 and 41.9 % (SEM = 1.5 ) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.01), age ( P = 0.47), and linear ( P = 0.03) and quadratic ( P = 0.06) effects of amount of SFP.

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210 Figure 6 1 4 Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on neut rophil phagocytosis mean fluorescence intensity (MFI, indicator of number bacteria phagocytized per neutrophil) (A), neutrophil oxidative burst mean fluorescence intensity (MFI, indicator of intensity of rea ctive oxygen species produced per neutrophil) (B) in prewean ed calves T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. Phagocytosis MFI= mean values were 10.0, 9.8, 9.4, and 9.4 (SEM = 0.5) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.45), age ( P = 0.49), and linear ( P = 0.37) and quadratic ( P =0.84) effects of amount of SFP. Oxidative burst MFI= mean values were 107.8, 128.8, 98.4, and 105.4 (SEM = 9.4) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.75), age ( P = 0.33), and linear ( P = 0.33) and quadratic ( P = 0.42) effects of amount of SFP.

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211 Figure 6 1 5 Effect of feeding Saccharomyces cerevisiae fermentation product s (SFP) on anti ovalbumin IgG in prewean ed calves. T0 = control, no added SFP; T1 = 1 g/d of SFP; T2 = 2 g/d of SFP; T4 = 4 g/d of SFP added to the milk. The mean values were 0.24, 0.26, 0.24, and 0.25 (SEM = 0.01) for T0, T1, T2, and T4, respectively. Effect of feeding SFP ( P = 0.53), age ( P < 0.01), and linear ( P = 0.99) and quadratic ( P = 0.5 7) effects of amount of SFP.

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212 CHAPTER 7 GENERAL DISCUSION AND CONCLUSIONS We first set out to examine the effect of exogenous CLA and PPAR rosiglitazone, on LPS stimulated TNF Lipopolysaccharide stimulated PBMC proliferation and induced TNF whole bovine blood in a dose and time dependent manner. T he net effect of CLA on immune function of domestic mammals may vary depending on isomeric composition of CLA test products. In fact, t he isomer c 9, t 11 CLA or LA had negligible effect on LPS induced TNF whole bovine blood. Whereas, p retreatment of whole bovine blood with t 10, c 12 CLA isomer attenuated TNF This attenuation indicated that l ipolysaccharide may regulate TNF other pathways that do not involve th is fatty acid. I ncubation of whole bovine blood with a selective PPAR rosiglitazone, decreased LPS induced TNF by 29%. This attenuation was reversed when bovine blood was treated with both rosiglitazone and GW9662 a selective PPAR Rosiglitazone tended to decrease cytosolic and nuclear NF kBp65 formation. This would suggest that the PPAR induced TNF with activation and nuclear translocation of NF kB. We provide d evidence that LPS is a potent inducer of TNF t 10, c 12 CLA isomer and PPAR inflammatory response induced by LPS in growing Holstein heifers by activating PPAR ing the nuclear translocation of NF kBp65 (Figure 7 1 ). Since we did not evaluated the effect of t 10, c 12 CLA isomer on PPAR activation, studies are warranted to evaluate the effect of t 10, c 12 CLA on PPAR activation and NF kB translocation.

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213 Additional studies are also needed to fully establish the molecular evidence for CLA modulati on of immune function in cattle There are limited i nformation related to the efficacy of dietary supplementation of trans 10, cis 12 CLA on performance and health o f prewe aning calves. Studies are warrant ed to determine the impact on performance and health of preweaned calves fed with this isomer after challenge with LPS. The second experiment showed the importance of passive immunity to newborn calves. C alves with FPT ( IgG < 1.0 g/dL ) reduced daily grain intake, ADG and body weight at 30 d of age, and experienced increased incidence of multiple diseases and mortality during the preweaning period N o association was observed between passive transfer of IgG and risk of diarrh ea. Calves with APT had less incidence of pneumonia D aily rearing cost per calf did not differ between groups, but the total rearing cost in the preweaning period increased in APT calves compared to FPT calves. V alue of a weaned calf and the total inc ome per calf were both greater for calves with APT than those with FPT. A dditional colostrum management might be needed to optimize IgG transfer to the newborn calf to achieve concentrations of serum IgG great er than 3.0 g/dL in most calves which may reduc e the probability of death of preweaning calves. Evaluation of passive immunity through STP needs to be used with precaution w hen concentrations are high (> 7.0 g/dL) or when the contribution of STP from IgG is low. If STP inc reased as a result of increase s in serum Ig G, risk of mortality will be reduced Serum IgG as percent of STP is a better predictor of survival of preweaned calves than STP or serum IgG concentrations alone. In this study i ncidence of diarrhea was not associated with passive immunity. F urther studies are warrant ed to evaluate the association of passive immunity with duration and severity of diarrhea Studies that evaluate the effect of passive immunity

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214 on reproductive and lactation performance are also needed because the association of p assive transfer of IgG with subsequent production in dairy heifers had been scarcely evaluated. Despite the potential benefits of pomegranate to mammals, growth performance and measures of health and immune responses were unaltered i n preweaning calves fe d POM at dose of 15 mg GAE/kg of BW during first 63 d of age. In addition, s ubtherapeutic doses of antibiotics fed to preweaned calves did not affect growth, feed efficiency, and measures of health and immune response. Under the conditions of the current s tudy, the results do not support the use of these products in the diet of preweaned calves to improve health and growth. The lack of effect of feeding a combination of subt h erapeutic doses of tetracycline and neomycin demonstrated in Chapter 5 is supported by research conducted by others with preaweaning dairy calves (Berge et al., 2005) As oppose to initially anticipated, feeding POM did not improve the measures of innate and adaptive immunity evaluated in the current study, despite evidence of effects on calves from others (Oliveira et al., 2010). It is possible that the lack of effect of POM might be attributed to a reduced bioavailability of polyphenol s and/or the conversion of ellagic acid to less active metabolites such as urolithins by intestinal m T he metabolism of pomegranate phenolic compounds by young calves is unknown and ch anges in plasma contents of these compounds after consumption of POM should be evaluated in future studies to determine if the doses used result in changes in concentrations of active compounds in plasma or tissues. The lack of effect of POM on productive performance and health could a l so be related to the dose used in this study although treatments were based on previous research in which the dose of GAE was

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215 extrapolated based on BW Perhaps, the clear benefits observed in vitro or in vivo with monogastric subjects are only observed when calves are exposed to more stressful conditions. Future studies should attempt It could be possible th at this dose was not enough to promote health and performance of calves. Studies that evaluate the effect of feeding different amount s of pomegranate are also needed. It is also possible that young calves during the preweaning period are not subject to sev ere stress conditions, and the potential antioxidant activity of pomegranate not could be observed. Studies that evaluate the potential antioxidant properties of pomegranates should be conducted in cattle under stress conditions, such as fresh cows, or lac tating cows under heat stress. Supplementation of up to 4 g/d of SFP with 4% of hydroly z able tannins did not affect growth performance and measures of health and immune responses of dairy calves. N eutrophil killing activity was i mproved by SFP supplementat ion. This seems likely that the effect of yeast, or its compounds or derivates, on neutrophil could be specific to improve the killing activity but not the phagocytosis ability W hether dietary administration of SFP might be able to reach the immune cells and activated them is unknown Results do not support the use of SFP up to 4 g/d added to milk of preweaned calves to improve growth performance, immune response, and health Additional s tudies that evaluate the feeding SFP through grain are n eeded because it is likely that SFP promote the starter intake, and thus improve the productive performance of preweaning calves. In the chapter 4, we showed that calves with APT hav e reduced incidence of diseases It is likely that a positive effect of feeding pomegra nate or SFP on health could be more evident in FPT than APT calves Thus, additional s tudies that evaluate

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216 the effect of pomegranate or SFP supplementation on preweaning calves with failure passive transfer are desirable Categorical data require s greater sample size than continuous data to detect difference. Additional s tudies that evaluate the effect of pomegranate or SFP on health need to be conduct ed with greater sample size than those used in these studies.

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217 Figure 7 1 Proposed model for l ipopolysaccharide (LPS) and fatty acid signals in cultured blood cells. Trans 10, cis 12 conjugated linoleic acid ( t 10, c 12 CLA) and Rosiglitazone attenuate tu mor necrosis factor alpha (TNF by activating peroxisome proliferator activated rece ptor gamma (PPAR inhibiting the nuclear translocation of nuclear factor kappa B p65 (NF kBp65).

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239 Yu, Y., Z. Zhou, J. Xu, Z. Liu, and Y. Wang. 2002. Ketamine reduces NF kB activation and TNF production in rat mononuclear cells induced by lipopolysaccharide in vitro Ann. Clin. Lab. Sci. 32:292 298. Zambell K. L., N. L. Keim M. D. Van Loan B. Gale P. Benito D. S. Kelley and G. J. Nelson 2000. Conjugated linoleic acid supplementation i n humans: Effects on body composition and energy expenditure. Lipids 35:777 782.

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240 BIOGRAPHICAL SKETCH Milerky Cristina Perdomo Lozada was born in Barquisimeto, in the State of Lara, Venezuela. She is a daughter of Josefa Lozada de Perdomo and Victor Perdomo Rodriguez. She studied at the Universidad Centroccidental Lisando Alvarado where she received her Bachelor of Science degree in Agricultural Engineering in 1997. She started her Master of Science d egree in Animal Production at Universidad Central d e Venezuela in 1998 and was awarded the degree in 2001. The title of her thesis was: i ae and it by products in chicken instructor in a nimal n utrition. Her research emphasis was on nutritional evaluation of industrial by products for ration s of chicken s and pigs. In the f all of 2007, she was awarded a scholarship from University Lisandro Alvarado and moved with her family to Gainesville, Florida where she started her d octor ate degree in the Department of Animal Sciences at the University of Florida, under the guidance of Drs. Lokenga Badinga and Jos E. P. Santos. Her research at the University of Flor ida has focused on dietary strategies to modulate performance, health, and immune responses in Holstein calves. After completing the requirements for the doctoral degree, Milerky will continue her career as professor and research er after returning to Venez uela.