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Nutraceutical and Hormonal Regulation of Immunity, Uterine Health, Fertility, and Milk Production of Postpartum Dairy Cows

Permanent Link: http://ufdc.ufl.edu/UFE0022826/00001

Material Information

Title: Nutraceutical and Hormonal Regulation of Immunity, Uterine Health, Fertility, and Milk Production of Postpartum Dairy Cows
Physical Description: 1 online resource (316 p.)
Language: english
Creator: Silvestre, Flavio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A series of experiments aimed to improve rate of uterine involution, immunity, uterine health, fertility and milk production during the postpartum period of dairy cows by hormonal and dietary means. In the first study cows received increasing doses (1 or 2 implants of 2.1 mg) of a GnRH agonist degradable implant within 1.5 days after parturition for comparison with non-treated control. Both doses of implants increased rates of uterine involution and suppressed ovarian follicular activity in the period before initial and during the synchronization of ovulation for artificial insemination. This later effect makes the use of the degradable implant at parturition impractical within a reproductive management. In the second study, cows were supplemented with either sodium selenite or selenium-yeast in the diet, begun at pre-partum and continuing until a minimum of 80 days postpartum during the summer. Supplementation of selenium-yeast increased plasma concentrations of selenium and seleno-enzyme (i.e., type I 5? deiodinase) activity, reduced incidence of fever and purulent cervical discharge in the early postpartum period associated with improved blood neutrophil bactericidal activity and serum humoral response to an inert antigen. Also supplementation with selenium-yeast increased milk yield associated with reduction in milk somatic cell count and increased pregnancy per artificial insemination after second insemination. In the last study, diets supplemented with a fatty acid source rich in linoleic acid during the transition period improved innate immunity (i.e., neutrophil adhesion molecules expression, bactericidal activity and cytokine production) and milk yield. Following the transition period, feeding a diet enriched with n-3 fatty acids increased accumulated pregnancy per artificial insemination particularly when cows were fed linoleic fatty acid during the transition period. In summary, we propose a dietary supplementation program for cows with selenium-yeast and linoleic acid enriched supplements, begun pre-partum, to improve innate immunity (i.e., neutrophil function), uterine health and overall cow health leading to greater milk yield. Following the transition period, the continuation of selenium-yeast supplementation with n-3 fatty acids can benefit fertility of dairy cows. More studies for better manipulation of Deslorelin delivery systems may confer this technology suitable for the early postpartum cow.
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 Flavio Silvestre.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Thatcher, William W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022826:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022826/00001

Material Information

Title: Nutraceutical and Hormonal Regulation of Immunity, Uterine Health, Fertility, and Milk Production of Postpartum Dairy Cows
Physical Description: 1 online resource (316 p.)
Language: english
Creator: Silvestre, Flavio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Animal Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: A series of experiments aimed to improve rate of uterine involution, immunity, uterine health, fertility and milk production during the postpartum period of dairy cows by hormonal and dietary means. In the first study cows received increasing doses (1 or 2 implants of 2.1 mg) of a GnRH agonist degradable implant within 1.5 days after parturition for comparison with non-treated control. Both doses of implants increased rates of uterine involution and suppressed ovarian follicular activity in the period before initial and during the synchronization of ovulation for artificial insemination. This later effect makes the use of the degradable implant at parturition impractical within a reproductive management. In the second study, cows were supplemented with either sodium selenite or selenium-yeast in the diet, begun at pre-partum and continuing until a minimum of 80 days postpartum during the summer. Supplementation of selenium-yeast increased plasma concentrations of selenium and seleno-enzyme (i.e., type I 5? deiodinase) activity, reduced incidence of fever and purulent cervical discharge in the early postpartum period associated with improved blood neutrophil bactericidal activity and serum humoral response to an inert antigen. Also supplementation with selenium-yeast increased milk yield associated with reduction in milk somatic cell count and increased pregnancy per artificial insemination after second insemination. In the last study, diets supplemented with a fatty acid source rich in linoleic acid during the transition period improved innate immunity (i.e., neutrophil adhesion molecules expression, bactericidal activity and cytokine production) and milk yield. Following the transition period, feeding a diet enriched with n-3 fatty acids increased accumulated pregnancy per artificial insemination particularly when cows were fed linoleic fatty acid during the transition period. In summary, we propose a dietary supplementation program for cows with selenium-yeast and linoleic acid enriched supplements, begun pre-partum, to improve innate immunity (i.e., neutrophil function), uterine health and overall cow health leading to greater milk yield. Following the transition period, the continuation of selenium-yeast supplementation with n-3 fatty acids can benefit fertility of dairy cows. More studies for better manipulation of Deslorelin delivery systems may confer this technology suitable for the early postpartum cow.
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 Flavio Silvestre.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Thatcher, William W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022826:00001


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1 NUTRACEUTICAL AND HORMONAL REGULATION OF IMMUNITY, UTERINE HEALTH, FERTILITY AND MILK PRODUCTION OF POSTPARTUM DAIRY COWS By FLVIO TEIXEIRA SILVESTRE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA

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2 F lvio T eixeira S ilvestre

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3 To all dairy cows and producers of the world

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4 ACKNOWLEDGMENTS I am deeply proud to be a graduate student of Dr. William W. Thatcher, my supervisory committee chair. In my early days in the College of Veterinary Medicine, Brazil, I had the th r ough scient ific publications. The experience of reading cow and in science. The importance of his work has inspired many students like me to pursue experimental science and the discover y of the unknown. Dr. Thatcher provided me with all the tools necessary for my accomplishments during these seven fruitful years of my graduate car e er and he will be forever a friend. I would like to extend my appreciation to Dr. Jos E. P. Santos who acce pted to be o n my committee, even while he was not part of our department. Dr. San tos made all the efforts to be involved in my PhD program which allowed a great deal of collaboration between laboratories. I would like to thank Dr. Peter J. Hansen, who has been contributing to my graduate ca r e his insights on immunology and the scientific method have been paramount during these years. I thank Dr. Carlos Risco for his collaborations in my studies regarding management of the postpar tum dairy cow and for his great friendship. Also, I would like to thank Dr. Joel V. Yelich for sharing his laboratory, technician and equipment. I greatly appreciate Mrs. Marie Joelle Thatcher for all the help with hormonal assays, excel techniques and dat a handling; Marie Joelle is the most altruistic person I have encounter ed at the University of Florida. S he spent countless hours helping me, even when it was not part of her responsibilities. Marie Joelle teachings were of great importance to m e during my graduate car e er and therefore in my future life. I also thank Dr. Charles Staples for his contributions in the area of dairy cattle nutrition, hormonal and feed analysis and data interpretation. Dr. Staples, although not part of my committee, has been c losely involved in my PhD program and his

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5 friendship will not be forgotten. I am very grateful to Dr. Cynda Crawford, Dr. Tom Jenkins and Evanne Thies for their collaboration with sample analys e s. I also would like to thank Dr. Max Paape for welcoming me d uring a productive week at the USDA Beltsville and provi di ng guidance regarding immune studies. I owe special thanks to intern students for their endless help with all aspects of my projects. Daniel T. Silvestre and Thiago S. M. Carvalho spent long days wo rking with me in the farms or enduring late hours in the laboratory T hese brave men made possible to achieve the bold accomplishments of our projects. I thank Miriam Lopez, Jill Gregorieff Edson M. Mattiello and Mayra Crupe for helping with cattle handli ng, data acquisition and sample analys e s. I want to express a great appreciation to Mr. Don Bennink, Mr. Ron Saint John and Peter Gelber for allowing the use of dairy cows. I also thank the staff of North Florida Holsteins (David Temple, Dr. Gina Temple, J ohn Karanja, Saide, Angel, Mario, Jos and Raul) and Alliance Dairies (Nilo, Amadeo, June, Ktia, Junia, Antnio, Eraldo, Melvin, Fabr cio, Franklin and Leonel) for their help managing cows, professionalism and camaraderie during the projects. I would like to thank laboratory technicians (Idania Alvarez, Sergei Sennikov, Dr. Douglas Bates, Brad Austin, Jim Moss, Nancy Wilkson, Shelly Lanhart and Jennifer Bilheimer) for helping with techniques, assays and equipment support. I also extend my appreciation to t he ladies in the business office (Toyuna Grant, Linda Blackson, and Debra Sykes) who were always very helpful in the hiring process of trainee students, time cards and ordering laboratory supplies. Additionally, I would like to thank Joan n M. Fischer (stud ent advisor) and the secretaries (Sabrina Robinson, Glenda Tucker) for always helping with miscellaneous issues. I appreciate the friendship and camaradie of colleagues Dr. Todd Bilby, Dr. Ayd i n Guzeloglu, Dr. Jeremy Block, Dr. Dean Jousan, Cristina Caldar i, L lian Oliveira, Davi Arajo,

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6 Fabio Lima and Brbara Loureiro. I also extend my appreciation to all friends in the Tanglewood football group (Evandro, Carlos and Cristovan, Valrio, Cl udio, Andr s, Pato, Pepe, Federico, Alejandro, Srgio and Sebastian brothers, Marcelo, Geraldo, Pablo, Thakadu, Bernard and everyone else who came to play) for the evenings of great enterta iment. Special thanks are extended to members of my family (Pai, me, Gustavo/Roberta/ Arthur and Joo Lucas, Paula and Daniel) for all their encouragement and belief in me. To all member of the Silvestre family (primos: Eduardo/Tatiana/Luis Gabriel, Cristina/Srgio/Camila, Lo, Henrique, Ricardo, Claudine, Ronaldinho, Andr and Dulce; Tios: Ronald Luizinho Jos Roberto; Tias: Jlia, L gia and Luclia) I appreciate their support and friendship. Special appreciation also is extended to V Cinira and Tereza for their endless support and positive thinking of me. Last but not least, I extend my most sincere recognition and appreciation t o Maria Juliana Silvestre de S. H er never ending unconditional support brought me where I am today W ithout Juliana nothing in the past seven years could have been accomplished. To Juliana I give all my heartfelt thanks.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 ABSTRACT ................................ ................................ ................................ ................................ ... 17 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 19 2 REVIEW OF LITERATURE ................................ ................................ ................................ 22 GnRH Agonists ................................ ................................ ................................ ....................... 22 Deslorelin ................................ ................................ ................................ ........................ 22 GnRH Agonist Therapy ................................ ................................ ................................ ... 22 Delaying First Ovulation Postpartum ................................ ................................ .............. 24 Direct Effects of Gonadotrophins on the Reproductive Tract ................................ ......... 25 Uterine Histological Responses to Chronic Exposure of GnRH Agonists ...................... 27 Selenium ................................ ................................ ................................ ................................ 28 Absorption and Excretion ................................ ................................ ................................ 28 Selenium Deficiency and Toxicity ................................ ................................ .................. 30 Selenoproteins ................................ ................................ ................................ ................. 31 Role of Selenium in Immunity and Health ................................ ................................ ...... 34 Fatty Acids ................................ ................................ ................................ .............................. 35 Enzymes and Biohydrogenation ................................ ................................ ...................... 35 Tran sport and Metabolism ................................ ................................ ............................... 39 Fatty Acids for Cows ................................ ................................ ................................ ....... 40 Fatty Acids Incorporation in Tissues ................................ ................................ ............... 44 Effects of Linoleic Acid in the Postpartum Period ................................ .......................... 46 Effects of n 3 Fatty Acid on Fertility ................................ ................................ .............. 51 Fatty Acids Profile in Peripheral Blood Cells ................................ ................................ 54 Fatty Acids Nuclear Receptors ................................ ................................ ........................ 57 Ac ute Phase Response and Proteins ................................ ................................ ....................... 61 Haptoglobin ................................ ................................ ................................ ..................... 61 Fibrinogen ................................ ................................ ................................ ........................ 62 Ceruloplasmin ................................ ................................ ................................ .................. 63 The Neutrophil ................................ ................................ ................................ ........................ 64 Cytokines ................................ ................................ ................................ ......................... 67 Adhesion Molecules ................................ ................................ ................................ ........ 68 Oxidative Burs t ................................ ................................ ................................ ................ 70

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8 3 USE OF INCREASING DOSES OF A DEGRADABLE DESLORELIN IMPLANT TO ENHANCE UTERINE INVOLUTION IN POSTPARTUM LACTATING DAIRY COW S ................................ ................................ ................................ ................................ ..... 76 Introduction ................................ ................................ ................................ ............................. 76 Materials and Methods ................................ ................................ ................................ ........... 77 Ovarian St ructures ................................ ................................ ................................ ........... 78 Uterine and Cervical Diameters ................................ ................................ ...................... 79 Health Monitoring ................................ ................................ ................................ ........... 80 Estrous Synchronization ................................ ................................ ................................ .. 81 Blood Sa mples and Hormone Assays ................................ ................................ .............. 82 Statistical Analyses ................................ ................................ ................................ .......... 84 Results ................................ ................................ ................................ ................................ ..... 85 Ovarian Responses ................................ ................................ ................................ .......... 86 Hormonal Respo nses ................................ ................................ ................................ ....... 86 Involution of Uterus and Cervix ................................ ................................ ...................... 87 Vaginoscopy ................................ ................................ ................................ .................... 90 Estrous Synchronization and Breeding ................................ ................................ ........... 91 Milk Yield ................................ ................................ ................................ ....................... 92 Discussion ................................ ................................ ................................ ............................... 92 4 EFFECT OF SOURCE OF SUPPLEMENTAL SELENIUM ON UTERINE HEALTH, REPRODUCTION AND LACTATION IN DAIRY COWS DURING SUMMER IN FLORIDA ................................ ................................ ................................ ............................. 111 Introduction ................................ ................................ ................................ ........................... 111 Materials and Me thods ................................ ................................ ................................ ......... 114 Animals, Experimental Design and Feeding ................................ ................................ 114 Health Monitoring ................................ ................................ ................................ ......... 116 Estrous Synchronization and Re synchronization Protocols ................................ ......... 118 Blood Samples and Progesterone Assay ................................ ................................ ....... 119 Plasma Concentration of Selenium and T3:T4 Molar Ratio ................................ ......... 120 Milk Yield, Milk Composition and Feed Samples ................................ ........................ 121 Statistical analyses ................................ ................................ ................................ ......... 122 Results ................................ ................................ ................................ ................................ ... 124 Animals, THI, Diets and DMI ................................ ................................ ....................... 124 Plasma Concentration of Se and T3:T4 Molar Ratio ................................ .................... 126 Postpartum Health ................................ ................................ ................................ ......... 126 First and Second Services Pregnancies per AI ................................ .............................. 128 Milk Yield and Composition ................................ ................................ ......................... 129 Discussion ................................ ................................ ................................ ............................. 130

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9 5 EFFECT OF SOURCE OF SUPPLEMENTAL SELENIUM ON NEUTROPHIL ACTIVITY AND HUMORAL IMMUNE RESPONSES IN THE PERIPARTUM PERIOD OF DA IRY COWS IN FLORIDA ................................ ................................ ........ 155 Introduction ................................ ................................ ................................ ........................... 155 Material and Methods ................................ ................................ ................................ ........... 158 Animals, Experimental Design and Feeding ................................ ................................ 158 Health Monitoring ................................ ................................ ................................ ......... 159 Body Condition Score (BCS) ................................ ................................ ........................ 159 Neutrophil Phagocytic and Oxidative Burst Activity ................................ .................... 160 Serum Anti Ovalbumin Antibody ................................ ................................ ................. 162 St atistical Analyses ................................ ................................ ................................ ........ 164 Results ................................ ................................ ................................ ................................ ... 165 Colostrum Quality ................................ ................................ ................................ ......... 165 Neutrophil Phagocytic and Oxidative Burst Activity ................................ .................... 165 Serum Anti Ovalbumin Antibody ................................ ................................ ................. 167 Discussion ................................ ................................ ................................ ............................. 168 6 EFFECT OF DIFFERENTIAL SUPPLEMENTATION OF FATTY ACIDS DURING THE PERIPARTUM AND BREEDING PERIOD ON UTERINE AND METABOLIC STATUSES, REPRODUCTION AND LACTATION IN HIGH PRODUCTION DAIRY COWS ................................ ................................ ................................ ..................... 181 Introduction ................................ ................................ ................................ ........................... 181 Material and Methods ................................ ................................ ................................ ........... 183 Animals, Experimental Design and Feeding ................................ ................................ 183 Placentome Collection ................................ ................................ ................................ ... 185 Vaginoscopy ................................ ................................ ................................ .................. 186 Estrous Synchronizat ion and Re synchronization Protocols ................................ ......... 186 Blood Samples and Progesterone Assay ................................ ................................ ....... 187 Body Condition Score ................................ ................................ ................................ ... 188 Milk Yield and Feed Samples ................................ ................................ ....................... 188 PGFM and Blood Metabolites ................................ ................................ ....................... 189 Statistical Analyses ................................ ................................ ................................ ........ 191 Results ................................ ................................ ................................ ................................ ... 193 Animals, Diets, DMI, BCS, Vaginoscopy ................................ ................................ ..... 193 Fatty Acid Profiles in Cotiledon and Caruncle Tissues ................................ ................. 195 First Service Pregnancy per AI ................................ ................................ ...................... 196 Second Service Pregnancy per AI ................................ ................................ ................. 197 Accumulated Proportion Pregnant ................................ ................................ ................ 198 Milk Yield ................................ ................................ ................................ ..................... 198 PGFM and Blood Metabolites ................................ ................................ ....................... 199 Discussion ................................ ................................ ................................ ............................. 20 0

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10 7 EFFECT OF DIFFERENTIAL SUPPLEMENTATION OF FATTY ACIDS DURING THE PERIPARTUM AND BREEDING PERIOD ON INNATE IMMUNITY: NEUTROPHIL FUNCTION AND ACUTE PHASE PROTE IN ................................ ........ 230 Introduction ................................ ................................ ................................ ........................... 230 Material and Methods ................................ ................................ ................................ ........... 232 Animals, Experimental Design and Feeding ................................ ................................ 232 Body Condition Score and Vaginoscopy ................................ ................................ ...... 233 Acute Phase Proteins ................................ ................................ ................................ ..... 233 Neutrophil Phagocytic and Oxidative Burst Activity ................................ .................... 234 Neutrophil and Mononuclear Blood Cells Adhesion Molecules ................................ ... 236 Neutrophil Isolation, Culture and Fatty Acids Analysis ................................ ............... 238 Enzyme Linked Immunosorbent Assays ( ELISA) ................................ ........................ 239 Statistical Analyses ................................ ................................ ................................ ........ 241 Results ................................ ................................ ................................ ................................ ... 242 Acute Phase Proteins ................................ ................................ ................................ ..... 242 Neutrophil Phagocytic and Oxidative Burst ................................ ................................ .. 243 Neutrophil Adhesion Molecules: CD62L and CD18 ................................ .................... 245 Neutrophil Cytokines ................................ ................................ ................................ ..... 247 Neutrophil Fatty Acid Composition ................................ ................................ .............. 247 Discussion ................................ ................................ ................................ ............................. 248 8 GENERAL DISCUSSION AND CONCLUSIONS ................................ ............................ 273 LIST OF REFERENCES ................................ ................................ ................................ ............. 286 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 316

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11 LIST OF TABLES Table page 2 1. Gas chromatography analysis of the fatty acid chain in human erythrocytes phospholipids ................................ ................................ ................................ ..................... 73 3 1. Frequency distribution of cows among uterine tone scores at days 8, 15, 22, 29 an d 36 postpartum in CON, 1DESL and 2DESL groups ................................ .............................. 99 3 2. Frequency distribution of cows among cervical discharge scores at days 8, 15, 22, 29 and 36 postpartum in CON, 1DESL and 2DESL groups ................................ ................. 100 3 3. Proportion of cows cyclic and that ovulated based upon plasma concentrations of progesterone and presence of corpus luteum at CIDR removal and first GnRH of the Ovsynch ................................ ................................ ................................ ........................... 101 4 1. Ingredients and chemical composition of pooled experimental diets during the pre and postpartum periods for groups supplemented with sodium selenite and selenium yeast ................................ ................................ ................................ ................................ 142 4 during the first 10 days postpartum ................................ ................................ ................. 143 4 3. Frequency distribution of cows among cervical discharge categories diagnosed at days 5 and 10 postpartum ................................ ................................ ................................ ............ 144 4 4. Frequency distribution of cows with subclinical endometritis obtained by uterine flushing at day 37 postpartum ................................ ................................ .......................... 145 4 5. P roportion of cyclic cows, adjusted odds ratio, 95% confidence interval and levels of significance ................................ ................................ ................................ ...................... 146 4 6. First and second services pregnancies per AI at days 27 to 30 and 55 after insemination and first service pregnancy loss ................................ ................................ ....................... 147 4 7. First service pregnancy per AI at 27 to 30 days after insemination diagnosed by per rectum ultrasonography ................................ ................................ ................................ ... 148 4 8. Least squares means and pooled standard errors for milk parameters obtained monthly during the first 8 months postpartum ................................ ................................ ............... 149 6 1. Ingredient and chemical composition of diets fed to pre partum cows ................................ 211 6 2. Ingredient and chemical composition of diets fed to lactating cows ................................ .... 212 6 3. Fatty acid profiles of fat supplements ................................ ................................ ................... 213 6 4. Fatty acid profiles of diets ................................ ................................ ................................ .... 214

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12 6 5. Frequency distribution of cows among body condition scores quartiles at close up, parturition, 43 days postpartum and at timed artifi cial insemination .............................. 215 6 6. Frequency distribution of cows among cervical discharge categories diagnosed between days 8 to 10 postpartum ................................ ................................ ................................ ... 216 6 7. Least square means and pooled SE of the cotyledon and caruncle fatty acid profiles (% of the total fatty acid; g/100 g of fatty acids) collected at the time of parturition ........... 217 6 8. Least squares means and pooled SE for total fatty acids (g/10 0 g of freeze dried tissue) and different fatty acid percentages (% of the total fatty acid; g/100 g of fatty acids) in the cotyledon and caruncle tissues collected at the time of parturition ....................... 218 6 9. Proportion of cyclic cows, adjusted odds ratio, 95% confidence interval and levels of significance ................................ ................................ ................................ ...................... 219 6 10. First service pregnancy per AI at 32 and 60 days after insemination and pregnancy loss for experimental diets ................................ ................................ ................................ ...... 220 6 11. First service pregnancy per AI, adjusted odds ratio, 95% confidence interval and levels of significance for the risk of pregnancy for cows inseminated at 81 days postpartum and evaluated at 32 and 60 days after insemination ................................ ........................ 221 6 12. Second service pregnancy rates at 32 and 60 days of pregnancy and pregnancy loss for experimental diets ................................ ................................ ................................ ............ 222 6 13. Second service pregnancy per AI, adjusted odds ratio, 95% confidence i nterval and levels of significance for the risk of pregnancy for cows inseminated at 126 days postpartum and evaluated at 32 and 60 days after insemination ................................ ..... 223 6 14. Second service pregnancy per AI, adjusted odds ratio, 95% confidence interval and levels of significance for the risk of pregnancy for cows inseminated at 126 days postpartum and evaluated at 32 and 60 days after insemina tion ................................ ..... 224 6 15. Accumulated proportion pregnant and pregnancy losses after the first and second services, adjusted odds ratio, 95% c onfidence interval and levels of significance for the risk of pregnancy for cows inseminated at 81 and 126 days postpartum and evaluated at 32 and 60 days after insemination ................................ ............................... 225 7 1. Least squares means ( S.E.) percent of mononuclear cells, percent mononuclear cells adjusted by percent of neutrophils and log number of mononuclear cells positive per L of blood for L Selectin (CD62L) a 2 Integrin (CD18) ................................ ......... 259 7 2. Least squares means ( S.E.) percent of neutrophils, log number of neutrophils per L of blood positive and mean fluorescence intensity for L 2 Integrin (CD18) ................................ ................................ ................................ ................ 260

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13 7 3. Least squares means and pooled SE o f the neutrophil fatty acid profiles (g/100 g of fatty acids) collected at the time of initiation of diets, 30 days postpartum for cows supplemented with palm oil or safflower oil and at 80 days postpartum for cows supplemented with palm oil or fish oil ................................ ................................ ............ 261 7 4. Least squares means and pooled SE for total fatty acids (g/100 g of freeze dried tissue) and different fatty acid percentages (% of the total fatty acid; g/100 g of fatty acids) in neutrophils collected at the time of initiation of diets, 30 days postpartum for cows supplemented with palm oil or safflower oil and at 80 days postpartum for cows supplemented with palm oil or fish oil ................................ ................................ ............ 262

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14 LIST OF FIGURES Figure page 2 1. Routes of dietary selenium metabolism in animals ................................ ................................ 74 2 2. Phospholipid generation of inflammatory mediators. ................................ ............................ 75 3 1. Least squares means ( S.E.) number of class 1 follicles in CON, 1DESL and 2DESL groups during the postpartum period measured by per rectum ultrasonography ............ 102 3 2. Least squares means ( S.E.) number of classes 2 and 3 follicles in CON, 1 DESL and 2DESL groups as measured by per rectum ultrasonography at days 8, 15, 22, 29 and 36 postpartum. ................................ ................................ ................................ .................. 103 3 3. Least squares means ( S.E.) accumulated plasma concentrations of progesterone (ng/mL) in CON 1DESL and 2DESL groups during the postpartum period before initiation of pre synchronization ................................ ................................ ..................... 104 3 4. Least squares means ( S.E. ) plasma concentrations of PGFM (pg/mL) for CON 1DESL and 2DESL groups during the postpartum period. ................................ ............. 105 3 5. Least squares means ( S.E.) plasma concentrations of PGFM (pg/mL) for cows with metritis and no metritis during the postpartum period and before initial systemic antibiotic treatment. ................................ ................................ ................................ ......... 106 3 6. Least squares means plasma concentrations of PGFM (pg/mL) profiles for two cows diagnosed with metritis, treated with systemic antibiotics at 5 days postpartum and continued for 4 consecutive days. ................................ ................................ .................... 107 3 7. Least squares means diameter (cm) of previous pregnant uterine horn and previous non pregnant uterine horn for CON, 1DESL a nd 2DESL groups during the postpartum period. ................................ ................................ ................................ .............................. 108 3 8. Least squares means ( S.E.) diameter (cm) of the uterine horn lumen of the p revious pregnant uterine horn and previous non pregnant uterine horn for CON, 1DESL and 2DESL groups during the postpartum period. ................................ ................................ 109 3 9. Least squares means ( S.E.) diameter (cm) of cervix for CON, 1DESL and 2DESL groups during the postpartum period. ................................ ................................ .............. 110 4 1. Least squares means ( S.E.) hourly vaginal temperatures for cows in the sodium selenite and selenium yeast diets. ................................ ................................ .................... 150 4 primiparous and multiparous cows fed diets containing sodium selenite or selenium yeast in the pre partum and postpartum periods. ................................ ............................. 151

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15 4 3. Least squares means ( S.E) plasma triiodothironine (T3) to thyroxine (T4) molar ratios (x10 2 ) for primiparous and multiparous cows fed diets containing sodium selenite or selenium yeast in the pre partum and postpartum periods. ................................ ............. 152 4 4. Least squares means ( S.E.) milk yields for co ws fed diets containing sodium selenite or selenium yeast in the pre partum and postpartum periods. ................................ ......... 153 4 5. Least squares means ( S.E.) milk somatic cell score for cows fed diets containing sodium selenite or selenium yeast in the pre partum and postpartum periods. ............... 154 5 diets containing sodium selenite or selenium yeast in the pre partum and post partum periods. ................................ ................................ ................................ ............................. 176 5 2. Least squares means ( S.E.) percentage of neutrophils with phagocytotic and oxidative burst activity upon exposure to heat killed Escherichia coli in whole blood collected for all experimental cows at pre partum a nd post partum periods ................................ .. 177 5 3. Least squares means ( S.E.) perc entage of neutrophils with phagocytotic and oxidative burst activity upon exposure to heat killed Escherichia coli in whole blood for primiparous and multiparous cows fed diets containing sodium selenite or selenium yeast in the pre partum and post partum periods. ................................ ............................ 178 5 4. Least squares means ( S.E.) neutrophil mean fluorescence intensity (MFI) of the green ( indicator of intensity of ROS produced/neutrophil ; lines) and red (indicator of number of bacteria/neutrophil; bars) wave lengths of fluorescence following exposure to heat killed Escherichia coli in whole blood for primiparous and multiparous cows fed diets containing sodium sele nite or selenium yeast in the pre partum and post partum periods ................................ ................................ ..................... 179 5 5. Least squares means ( S.E.) optical density for serum an ti ovalbumin antibody in primiparous and multiparous cows fed diets containing sodium selenite or selenium yeast in the pre partum and postpartum periods. ................................ ............................. 180 6 1. Interaction of transition and breeding diets by season for second service pregnancy per AI examined at 32 days after timed artificial insemination ................................ ............. 226 6 2. Interaction of transition and breeding diets by season interaction for second service pregnancy per AI examined at 60 days after timed artificial insemination (TAI) ........... 227 6 3. Milk yield regression curves for transition and breeding diets were not parallel based on tests for homogeneity of regression ................................ ................................ ................. 228 6 4. Least squares means ( S.E.) for plasma concentrations of 15 keto 13,14 dihydro prostaglandin F (PGFM) for the first 14 days postpartum in a subsampl e of cows fed calcium salts of palm oil or safflower oil during the pre partum period (at least 20 days) to 35 days postpartum. ................................ ................................ ...................... 229

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16 7 1. Least squares means ( S.E.) for plasma concentration of haptoglobin, adjusted for cervical discharge score, for cows fed calcium salts of palm oil or safflower oil during the pre partum period (at least 20 days) to 35 days postpartum. .......................... 263 7 2. Least squares means ( S.E.) for plasma concentration of fibrinogen, adjusted for cervical discharge score, for cows fed calcium salts of palm oil or safflower oil during the pre partum period (at least 20 days) to 35 days postpartum. .......................... 264 7 3. Least squares means ( S. E.) for plasma concentration of ceruloplasmin for cows diagnosed with cervical discharge of clear, flacks or lochia and mucopurulent or purulent. ................................ ................................ ................................ ........................... 265 7 4. Least squares means ( S.E.) number of white blood cells, neutrophils and mononuclear cells in whole blood from all cows sampled ................................ ................................ .... 266 7 5. Least squares means ( S.E.) n umber of neutrophils (log transformed) in whole blood from cows supplemented with palm oil or safflower oil during the transition period. .... 267 7 6. Least squares means ( S.E.) percentage of neutrophils with phagocytic and oxidative burst activity in whole blood stimulated with E. coli or S. aureus from cows supplem ented with palm oil or safflower oil during the transition period. ...................... 268 7 7. Effects of number of neutrophils per L of whole blood on the least square means ( S.E.) percentage of neutrophils with phagocytic and oxidative burst act ivity when stimulated with E. coli or S. aureus during the transition period ................................ .... 269 7 8. Least squares means ( S.E.) of neutrophil me an fluorescence intensity (MFI) of the red (indicator of number of bacteria phagocytised per neutrophil; bars) and green ( indicator of intensity of reactive oxygen species produced per neutrophil ; lines) wave lengths in neutrophils from whole blood stimula ted with E. coli or S. aureus from cows supplemented with palm oil or safflower oil during the transition period. .... 270 7 9. Least squares means ( S.E.) for TNF neutrophils cultured for 18 hours in RPMI 1640 stimulated or not with lypo polysaccharide (LPS) incubated at 37 o C and 5% CO 2 at 30 days postpartum (dpp) for cows supplemented with palm oil or safflower oil during the transition period or at 80 dpp for cows supplemented with palm oil or fish oil from 30 to 80 dpp. ................................ ................................ ................................ ................................ ... 271 7 10. Least squares means ( S.E.) IL neutrophils cultured for 18 hours in RPMI 1640 stimulated or not with lypopolysaccharide (LPS) incubated at 37 o C and 5% CO 2 at 30 days postpartum (dpp) for cows supplemented with palm oil or safflower oil during the transition period or at 80 dpp for cows supplemented with palm oil or fish oil from 30 to 80 dpp. ................................ ................................ ................................ ................................ ... 2 72

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17 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the D egree of Doctor of Philosophy NUTRACEUTICAL AND HORMONAL REGULATION OF IMMUNITY, UTERINE HEALTH, FERTILITY, AND MILK PRODUCTION OF POSTPARTUM DAIRY COW By Flvio T. Silvestre December 2008 Chair: William W. Thatcher Major: Animal Molecular and Cellular Biology A series of experiments aimed to improve rate of uterine involution, immunity, uterine health, fertility and milk production during the postpartum period of dairy cows by hormonal and dietary means. In the first study cows received increasing doses (1 or 2 implants of 2.1 mg) of a GnRH agonist degradable implant within 1.5 days after parturition for comparison with non treated control. Both doses of implants increased rates of uterine involution and suppressed ovarian follicular activity in the period before initial and during the synchronization of ovulation for artificial insemination. This later effect makes the use of the degradable implant at parturition impractical within a reproductive management. In t he second study cows were supplement ed with either sodium selenite or selenium yeast in the diet, begun at pre partum and continuing until a minimum of 80 days postpartum during the summer. Supplementation of selenium yeast increased plasma concentrations of selenium and seleno enzyme (i.e., purulent cervical discharge in the early postpartum period associated with improved blood neutrophil bactericidal activity and serum humoral response to an inert antigen. Also supplementation with selenium yeast increased milk yield associated with reduction in milk somatic cell count and increased pregnancy per artificial insemination after second insemination.

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18 In the last study, diets supplemented with a fatty acid source rich in linoleic aci d during the transition period improved innate immunity (i.e neutrophil adhesion molecules expression, bactericidal activity and cytokine production) and milk yield. Following the transition period, feeding a diet enriched with n 3 fatty acids increased accumulated pregnancy per artificial insemination particularly when cows were fed linoleic fatty acid during the transition period. In summary, we propose a dietary supplementation program for cows with selenium yeast and linoleic acid enriched supplements begu n pre partum, to improve innate immunity (i.e., neutrophil function), uterine health and overall cow health leading to greater milk yield. Following the transition period, the continuation of selenium yeast supplementation with n 3 fatty acids can be nefit fertility of dairy cows. More studies for better manipulation of Deslorelin delivery systems may confer this technology suitable for the early postpartum cow

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19 CHAPTER 1 INTRODUCTION The l ifetime milk production of cows depends on re occurring pregnancy because pregnancy initiates and renews the lactation cycle. Increase in herd size and milk yield per cow, severity of negative energy balance, greater use of free stall housing, postpartum disorders, and a reduction in estrus detection are reported as risk factors for a decrease in pregnancy rates (Lucy, 2001). D ata collected from 15,320 Holstein cows during a period of 3 years were used to dete rmine the risk factors for conception; cows with retained placenta, metritis or ovarian cysts had 14%, 15% and 21% reduced likelihood s of conceiving than normal cows, re spectively (Grohn and Rajala Schultz, 2000). After parturition, the reproductive tract of the cow needs to undergo a series of modifications over a period of 4 to 8 weeks, such as uterine involution and resumption of cyclic ovarian activity before a new pregnancy can be established During the early postpartum period, lactating dairy cows undergo a negative energy balance whose magnitude is associated with extended anovulatory periods and decreased fertility (Beam and Butler, 1997; Beam and Butler, 1998). Furthermore, impaired neutrop hil function during the peri partum period increases the risk of retained fetal membranes (Kimura et al., 2006), metritis and subclinical endometrtitis (Hammon et al., 2002) in dairy cows. Metabolic disorders, hypocalcemia, stress of parturition and minera l deficiencies can reduce neutrophil bactericidal activity during the peri partum period. Approximately 90% of cows have some degree of bacterial contamination during the first 10 days postpartum (Morrow et al., 1986). Metritis accounted for 6.1% of cows t o be culled (Bartlett et al., 1 986) and t he incidence rate of metritis has been reported by Erb and Martin (1980) Dohoo et al (1983), Martin et al (1983), and Bartlett et al (1986) to be 13.8 18.2, 11.1, and 18, respectively. Postpartum metritis has a major economic impact for dairy operations

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20 because of increased treatment expenses, milk loss, prolonged days open and culling rates (Esslemont and Peeler, 1993). Moreover, incidence of endometritis is still high at 50 days postpartum and has marked negat ive effects on fertility (Gilbert et al., 1998; Hammon, 2001). The incidence of severe endometritis, which often progresses to pyometra (Arthur et al., 1989), is increased at approximately the same time that cows experience their first postovulatory period (i.e., 15 to 28 days postpartum) with increased progesterone concentrations. Pre disposing factors for metritis involves dystocia, retained fetal membranes, poor hygiene and metabolic imbalances occurring in the peri parturient period (Grhn et al., 1990) The risk factors for uterine infections are well established, but the incidence of uterine infection has not changed appreciably over the last 30 years (Griffin et al., 1974; Sheldon et al., 2002 ) which implies the lack of programs for its incidence redu ction A health monitoring program has been developed for dairy cows in the postpartum period (Upham, 1996). In this program, rectal temperature, attitude, appetite and evaluation of manure are considered to determine whether or not cows appear sick. Treat ment is based on the assumption that the major cause of fever early postpartum is related to uterine infections and that sick cows have metabolic problems such as ketosis and hypocalcemia. Several recent retrospective studies that analyzed milk production and reproduction in dairy herds in the United States reported that increasing milk production is usually associated with deteriorating fertility (Lucy, 2001). Genetic improvement has been the most important driver of increased milk production, and it now a ppears that further increases, especially during early lactation (the first 8 weeks), are constrained by the ability of the cow to ingest enough feed to supply the required nutrients to support lactation. The integration of the disciplines of ruminant nutr ition reproductive physiology immunology and clinic al medicine has the potential

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21 to provide useful alternatives to improve postpartum health and therefore fertility in dairy cows in a scenario of increasing milk production. Collectively, low fertility in lactating dairy cows results from a complex array of events and interactions between energy balance, ovarian function, uterine health and overall health status of the cow. Postpartum status of the cow sets the stage for subsequent reproductive performance The objectives of the literature review (Chapter 2) are to summarize: 1) the mechanisms regulating postpartum uterine involution an d uterine ovarian interactions; 2) aspects of neutrophil physiology and its role in the postpartum period of dairy cows and 3) nutritional relevance of selenium and fatty acids for health and fertility in dairy cows. In the subsequent research chapter, ovarian follicular activity was suppressed with the use of increasing doses of a degradable GnRH agonist (Deslorelin) implant for evaluation of uterine involution and recrudescence of ovarian follicular activity in dairy cows (Chapter 3). Subsequently, the effects of a source of selenium supplementation on postpartum health, milk yield and fertility (Chapter 4 ) and on neutrophil function and humoral antibody response s (Chapter 5) were evaluated in dairy cows Additionally, the effects of a combination of fatty acids supplemented during the transition and breeding periods on postpartum health, milk yield and fertility (Chapter 6) a nd several aspects of neutrophil function and fatty acid composition (Chapter 7) were evaluated in dairy cows. Finally, i n Chapter 8 implications of the research will be addressed in a general discussion

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22 CHAPTER 2 REVIEW OF LITERATURE GnRH A gon ist s Deslorelin Deslorelin is a synthetic GnRH agonist, formed by nine aminoacids : pyroGlu1 His2 Trp3 Ser4 Tyr5 D Trp6 Leu7 Arg8 Pro9 NHethylamide, and it is abbreviated as [D Trp6, Pro9 NEt] GnRH. In this molecule glycine at position 6 of the pepti de is substituted with a D amino acid such as D tryptophan. This feature increases the half life of Deslorelin in the circulation. Another characteristic is the removal of glycine at the amino terminus, which increases the affinity of the GnRH agonist (i.e ., Deslorelin) to its receptor (Karten and Rivier, 1986). GnRH A gonist Therapy The effects of continuous treatment with a GnRH agonist are characterized by an acute stimulatory phase followed by a chronic phase of depress ed gonadotrophin release. Cows impl anted with Deslorelin on day 7 of the estrous cycle had an acute increase in plasma lutein i zing hormone ( LH ) with maximum concentrations detected at 6 h ours Most of the cows had associated increases in concentrations of plasma progesterone, possible due to the induction of an accessory corpus luteum ( CL ) 1995) Plasma concentrations of both LH and FSH increased within 1 h our of Buserelin infusion in heifers treated at day 5 of the estrous cycle, and concentrations at 2 h ours were consistent with a preovulatory surge (Gong et al., 1996). In this study the LH surge induced ovulation of dominant follicles in all heifers. Furthermore, total CL life span was equal to t hat of control

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23 animals. A Deslorelin biodegradable implant (2.1 mg) inserted at day 7 postpartum induced ovulation in five of eight cows (Mattos et al., 2001) as a consequence of an induced LH surge. In another study pre pubertal heifers also responded with an acute increase in imm unoreactive LH after insertion of a Deslorelin implant (Bergfeld et al, 1996). Cyclic Holstein cows also responded with an acute release of LH within 1 h our after GnRH a gonist treatment injection. The LH response was greater to the Deslorelin implant than to an injection of Buserelin (Rajamahendran et al., 1998). The effect of a chronic GnRH agonist on gonadotrophin responses was well described by Gong et al. (1996). Cyclic heifers were treated continuously with Buserelin for 48 days. After a surge at the b eginning of infusions using osmotic mini pumps, LH concentrations fell at day 8 of infusion to basal concentrations until the termination of GnRH agonist treatment. The concentrations of FSH remained elevated for 3 days following the start of infusions and returned to normal basal levels characteristic of the control group. However, FSH decreased to concentrations below th ose observed for controls after 28 days of treatment. In this study suppression of pulsatile secretion of LH was achieved earlier than FS H. Somewhat similar results were obtained when heifers were injected with Buserelin twice a day, although the day of LH suppression was different due to twice a day injection as opposed to continuous infusion. In the later study, LH suppression was achieve d after 16 17 days of treatment and FSH remained at constant higher concentration in plasma than the control group (Gong et al., 1995). In summary, these studies have shown that complete suppression of gonadotrophs is achieved within 28 days of chronic tre atment with a GnRH agonist Suppression in LH secretion was achieved earlier because it is necessary to have a higher number of GnRH receptors to

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24 release LH (Turzillo et al., 1994), whereas FSH secretion can still occur with a lower number of GnRH receptor s. Delaying First Ovulation Postpartum Delayed first ovulation in suckled cows has been well reviewed (Yavas and Walton, 2000). The interval from calving to first ovulation in non suckled beef cows was 10.2 days compared with 34.7 days for suckled cows (Ca rter et al., 1980). Delayed first ovulation in suckled cows was associated with increased rate of uterine involution in dairy (Riesen et al., 1968) and beef cows (Lauderdale et al., 1968). Others (Wagner and Hansel, 1969) have found no effect of suckling o n the rate of uterine involution. Presence of the calf throughout the postpartum period may represent a natural defense mechanism to prevent early ovulation. Also, frequent suckling by the calf is related with increased secretion of oxytocin in comparison to machine mi lking (Bar Peled et al., 1995). Cows ovariectomized between 3 and 5 days postpartum had a significantly shorte r interval to uterine regression compared with untreated controls and ovariectomized cows treated with progesterone (Marion et al., 1 968). In contrast, interval between parturition and occurrence of uterine involution in cows was not affected by ovariectomy (Oxenreider, 1968). Perhaps the absen ce of gonadal hormones enhanced the physical involutionary process of the uterus. Moreover, pr esence of the calf after parturition may represent a natural phenomenon to protect the involuting uterus against the relaxation and immunosuppressive effects of progesterone and stimulate s a h igher secretion of oxytocin. Chronic treatment with GnRH agonist s can induce downregulation of GnRH receptors on the gonadotroph cells, desensitize the anterior pituitary gland to endogenous GnRH, and abolish GnRH agonist s could be used to block ovulation during the period of uterine involution, such

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25 that this involutionary process could be concluded prior to the first ovulation postpartum. The GnRH agonist attributable to presence of the calf. High doses of estradiol during the early postpartum period could be another approach for delaying the onset of ovulatory cycles. A single intramuscular injection of 10 mg of estradiol cypionate (ECP) at 7 days postpartum delayed the resumption of ovu lation until at least 40 days postpartum in association with a suppression of follicle development (< 10 mm) until 29 days postpartum (Haughian et al., 2002) However, there appeared to be considerable variation in the interval to resumption o f ovulation a fter ECP treatment; for example, only 35% of the cows had ovulated by day 50 postpartum and 94% by day 90 postpartum (Haughian et al., 2002). Moreover, in the same study, a reduction in conception rate for cows injected with 4 mg of ECP was of potential co ncern Direct Effects of Gonadotrophins on the Reproductive Tract Receptors of LH (LHr) are present in the myometrium of pigs (Ziecik et al., 1986), women (Reshef et al., 1990), rats (Bonnamy et al., 1990), rabbits (Jensen and Odell, 1988), and cows (Shemesh et al., 2001). Expression of LHr is dependent on stage of the cycle. Expression of LHr is high du ring the luteal phase and expressed weakly during the follicular phase. The LHr may have a role in regulating uterine motility. Binding of LH to its receptor activates signaling pathways that increase expression of cyclooxygenase and production of PGE 2 in the myometrium that results in relaxation (Shemesh et al., 2001). Uterine relaxation seen during early pregnancy in pigs may be regulated in part by pituitary LH (Flowers et al., 1991). Therefore, increased receptor binding of LH and increased cAMP may ser ve to maintain quiescence of the uterus during the luteal phase. Also, the relaxing effect of hCG on uterine motility was observed in women (Eta et al., 1994) and the action of hCG is tissue specific and

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26 appears to be mediated by decreasing intracellular free calcium concentrations in myometrial smooth muscle cells (Eta et al., 1994). In the cervix, LH binding initiates signaling pathways that increase expression of cyclooxygenase and the production of PGE 2 which could be related to the softening of the c ervix during the proestrous and estrous stages (Shemesh, 2001). Studies in gilts revealed that LH caused relaxation of the oviduct, especially during the peri ovulatory stage of the estrous cycle (Gawronska et al., 1999). Thus LH action may facilitate the passage of embryos through the isthmus towards the uterus. In summary, it is proposed that activation of adenylate cyclase is associated with relaxation; whereas, activation of COX 2 can lead to either a release of PGE 2 to relax the muscles, or of PGF which may contract them. Therefore, suppression of gonadotrophins may eliminate some of their relaxation effects on the reproductive tract, such that increase d uterine tone is a default. Studies using reverse transcription PCR revealed that myometrium an d isolated myometrial smooth muscle cells express GnRH receptor mRNA in rats (Chegini et al., 1996) and in endometrium of humans (Raga et al., 1998; Imai et al., 1994). However the actions of GnRH on these tissues still are not clear GnRH agonist s are use d widely in the treatment of women with symptomatic leiomyomas (Broekmans, 1996), endometriosis (Rumore and Rumore, 1989) and endometrial carcinoma (Dessole et al., 2000). These conditions are considered to be estrogen dependent. Thus, GnRH agonist treatme nt is an efficient and reversible pharmacological method to achieve hypoestrogenism, which leads to reduction in tumor volume. Also hysterectomy is preceded by

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27 GnRH agonist treatment in order to decrease uterine volume and facilitate either abdominal or va ginal procedures (Broekmans, 1996). Uterine Histolog ical Responses to Chronic Exposure of GnRH Agonists Ultrasound measurement of endometrial volume and thickness is used in medical practice as a predictor of pituitary suppression following chronic adminis tration of a GnRH agonist The relationship between estradiol status and endometrial thickness has been documented (Child et al., 2 002; Nakamura et al., 1996). Ultrasound measurement of three dimensional volume is used for predicting pituitary down regulat ion in human IVF embryo transfer programs. It is a tool for diagnosing relative hypo estrogenism or down regulation after chronic treatment with GnRH agonist s, and it eliminates the need for blood sample collection and estradiol assays. U ltrasound studies reported a decrease in myometrial volume of 40% in response to GnRH agonist therapy (Carr et al., 1993). Doppler ultrasound of the uterine artery indicated that patients treated with GnRH agonist s underwent a uterine vasoconstrict ion (Matta et al., 1988). In one study, premenopausal women were treated with a GnRH agonist for 8 weeks before hysterectomy. Post operatively, two independent pathologists examined the myometrium. The uterine volume of women treated with GnRH agonist decreased by 28%, as opposed to no changes of volume in the control group. The cellularity (cell/mm 2 ) of the GnRH agonist treated myometrium was higher than the controls with less stromal edema. The arteries in the GnRH agonist treated uteri underwent atr ophy of the tunica media and had significantly more perivascular fibrosis. The number of vessels per 100 myocytes also was decreased. Therefore, the authors concluded that the hypoestrogenism as a secondary response to GnRH agonist treatment led to myocyte atrophy, decreased stromal edema, atrophy of the arcuate arteries, and decreased myometrial vascularity (Weeks et al., 1999).

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28 Selenium Absorption and Excretion Selenium (Se) is an essential nutrient for human and animal health. It is a component of a numb er of important selenoproteins and enzymes required for biological functions including antioxidant defense and thyroid hormone production. Therefore, adequate dietary intakes of Se are essential. The most common forms of Se consumed by ruminants are seleno methionine ( SeMet ), selenocysteina (SeCys), selenite ( SeO 3 ), and selenate ( SeO 4 ). Feedstuffs and selenized yeast ( Se yeast ) provide Se mostly as Se containing amino acids, whereas inorganic supplements provide selenite and selenate. Plants can also contain a substantial proportion of their total Se as selenate (Whanger, 2002). The seleno amino acids are identical to their counterparts (i.e., methionine and cysteine) except that an atom of Se replaces one of S in the amino acid molecule. Synthesis of SeMet o ccurs with the incorporation of Se either from selenite or selenate (Schrauzer, 2000). Selenium is absorbed mainly in the duodenum and no absorption occurs from the rumen or abomasum of sheep or the stomach of pigs. Absorption of orally administered Se was only 35% in sheep compared to 85% in swine (Wright and Bell; 1966) In dairy cows, apparent absorption of Se varied with level in the diet, but ranged from 28 to 48% (Harrison and Conrad, 1984). Lower absorption for ruminants may be due to selenite being reduced to insoluble (i.e., elemental Se and selenides) compounds in the rumen. Most research in Se absorption comes from monogastric models, particularly the rat. Vendeland et al. (1992) used 75 Se from SeO 3 SeO 4 and SeMet to determine Se absorption in ligated loops of duodenum, jejunum, and ileum of rats that received a diet either deficient or adequate in Se from SeO 3 They demonstrated that SeMet was absorbed most rapidly from all segments of the small intestine. Both inorganic forms, SeO 3 and SeO 4 w ere absorbed more

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29 efficiently from the ileum than either the duodenum or the jejunum. Because inorganic forms might have less diffusibility during digestion and increased binding to vesicles in the brush border membrane of intestinal loops, the inorganic f orms are thought to be less absorbed than Se containing amino acids. In fact, the transfer to the body of 75 Se as Se Met was greater than 60% of the total absorbed, whereas only 20 to 65% was absorbed as SeO 3 or SeO 4 form (Vendeland et al., 1992). In a revi ew paper by Weiss (2003) 18% and 90% increase s in whole blood and milk Se concentration w ere found, respectively, when Se yeast was fed compared with selenite in ten studies using beef or dairy cattle. Statistical differences were detected for whole blood Se in five studies and in the majority of the studies milk concentration s of Se increased in response to Se yeast The glutathione peroxidase (GPX) activity was increased by 18% (11 studies) with variable statistical differences detected depending on stud y design. There is no significant difference in Se retention associated with different sources of Se when fed at levels of less than 0.10 ppm. Beyond this level, organic forms of Se result in higher tissue levels than inorganic forms (Cary et al., 1973). O ne advantage of administering Se in the amino acid form is that it can be absorbed in conjunction with the amino acid. Both methionine and cysteine are absorbed by specific amino acid carrier or transport systems in the small intestine. If Se is present as part of these amino acids, it would simultaneously be absorbed with the methionine and cysteine. Consequently, for efficiency o f absorption seleno amino acids should resist microbial deamination and degradation of the carbon chain in the rumen. On the other hand, SeO 4 appears to be absorbed by an active transport system and SeO 3 possibly by passive diffusion. After absorption of SeMet, the Se

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30 moiety can be removed, reduced to selenide and enter in to the inorganic pool of Se for later synthesis of SeCys, or SeMet to be used for protein synthesis ( Figure 2 1; Jacques 2003). Selenium is e xcreted via feces, urine and exhalation and secreted via milk In ruminants the main route is feces and urine in monogastrics. As Se increases in the diet, urinary excret ion is increased. The retention of Se is dependent on tissue demands and Se is retained more efficiently in Se deficient than Se adequate animals The concentration of Se in milk ranged from 0.01 to 0.025 mg/kg (Conrad and Moxon, 1979) Urinary excretion i n lactating dairy cows consuming approximately 2.5 mg of Se/day was approximately 0.5 mg/day (Ivancic and Weiss, 2001). Fecal losses in dairy cows ranged from 0.011 to 0.019 mg/kg of dry matter intake ( DMI; Harrison and Conrad, 1984). Selenium Deficiency a nd Toxicity Clinical signs of severe Se deficiency in ruminants include white muscle disease (nutritional muscular dystrophy) in newborn or young lambs and calves poor growth rates, which may occur in the absence of any other recognizable disease, and inf ertility. In general, older cattle do not develop clinical signs. A m yopathic condition in yearling cattle has been reported (Allen et al., 1975, Buergelt et al., 1996) Research has indicated clearly that Se deficiency in cattle reduces the ability of blo od and milk neutrophils to kill yeast and bacteria (Boyne and Arthur, 1979; Gyang et al., 1989; Grasso et al 1990; Hogan et al 1990). Reduced neutrophil killing activity in Se deficient steers was associated with non detectable GPx activity in neutrophils (Boyne and Arthur, 1979). Davis et al (2006) reported that ewes were able to tolerate up to 20 ppm of dietary Se a s sodium selenite for 72 weeks without signs of Se toxicosis based on histopathological evaluation of liver, kidney, diaphragm, heart, and psoas major.

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31 Pigs supplemented with either s odium selenite or Se yeast at 5, 10, 15, or 20 ppm appeared to display signs of toxicity when dietary concentrations were above 5 ppm However SeMet from yeast was less toxic than the same level of Se from selenite (Kim and Mahan, 2001). Symptoms of selenosis include a garlic odor on the breath, gastrointestinal disorders, hair loss, sloughing of nails, fatigue, irritability and neurological damage. Extreme cases of selenosis can result in c irrhosis of the liver, pulmonary edema and death. Selenoproteins Glutathione Peroxidases (GPx) The generation of reactive oxygen species ( ROS ; i.e., O 2 and H 2 O 2 ) is unavoidable as products of normal metabolic processes. These compounds are involved physiologically in the chemistry of several enzymes and are used by phagocytic cells to kill bacteria. The excess of ROS, however, can initiate oxidative chain reactions and lipid peroxidation, thereby damaging cellular and organelle membranes resulting in cell death. Cells contain enzymatic systems such as GPx, glutathione reductase, and glutathione transferase capable of protecti on against oxidative stress and thus play a crucial role to alleviate oxidative injuries. There are two main types of GPx Se dependent and Se independent. The Se dependent GPx ( Se GPx ) was the first class of selenoenzymes to be identified (Flohe et al., 1973). Using the peptide glutathione as a cofactor, Se GPx catalyzes the reduction of H 2 O 2 to water and organic hydroperoxides to their correspondent alcohols (Mezzetti et al., 1990) These reaction s lead to conversion of Se GPx into its oxidized form (glutathione disulfite). In the presence of NADPH, glutathione reductase recycles the oxidized glutathione into its reduced active form. Five SeCys containing GPx s have been identified: GPx1, GPx2, GPx3, GPx4 and snGPx. Glutathione peroxidase type 1 ( GPx1 ) was the first GPx identified (Flohe et al., 1973), and it is

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32 comp rised of four identical SeCys containing subunits. It is present in the cytosol of nearly all tissues ; therefore it is also known as cytosolic or cellular GPx. Glutathione peroxidase type 1 metabolizes H 2 O 2 and a wide range of organic hydroperoxides. The GPx2 also called gastrointestinal GPx, is similar to GPx1 in com position, substrate specificity and function. Glutathione peroxidase type 3 ( GPx3 ) is found in plasma and extracellular fluids. It is composed by four identical SeCys containing subunits and it is glycosylated to improve stability (Takahashi et al., 1987). In contrast to GPx1 and GPx2, GPx3 has antioxidant activity against phospholipid hydroperoxides, giving it a more direct role in protection of membranes (Takahashi et al., 1987). Glutathione peroxidase type 4 ( GPx4 ), also called phospholipid hydroperoxide glutathione peroxidase, is a monomer found in cytosolic or membrane associated forms (Roveri et al., 1992). This enzyme can directly reduce phospholipid and cholesterol hydroperoxides (Thomas et al., 1990; Ursini et al., 1985), thereby protecting the cell and organelle membranes against oxidative destruction. Glutathione peroxidase type 4 is also present in the mitochondrial capsule in its inactive form, the oxidative crosslinked form. Spermatozoid nuclei glutathione peroxidase ( snGPx ) is an enzyme localiz ed in the nucleus of spermatozoa and late spermatids and it is important for proper maturation and fertilizing capacity of sperm cells (Behne et al., 1997). Iodothyronine Deiodinases The group of iodothyronine deiodinases catalyzes the activation and inact ivation of thyroid hormones that regulate metabolic processes. There are three types of iodothyronine deiodinases: type 1 (Arthur et al., 19 90), type 2 (Salbe et al., 1990 ) and type 3 (Croteau et al., 1995) deiodinase.

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33 Deiodinase type 1 was first indentified in the thyroid gland, liver, kidney, and pituitary gland of rats (Behne et al., 1988) and it contains a Se atom bound to its active site (Behne et al., 1990). Deiodinase type I catalyzes the monodeiodination of the iodothyronines at t position of the phenolic ring of the inactive thyroxine ( T4 ) This process results in the formation of triiodothyronine ( T3 ) which is the active form of the thyroid hormone. The deiodinase type 2 is mainly expressed in the brain, brown adipose tissu e, pituitary gland, and placenta (Leonard et al., 2000). This enzyme is responsible for the intracellular production of T3 from plasmatic T4. It catalyze s monodeiodination of T4 and T3, producing T3 and T2, respectively (Leonard et al., 2000). The role of Se on the enzymatic activity of deiodinase type 2 is still unclear. Deiodinase type 3 is expressed in the central nervous system, placenta and skin (Croteau et al. 1995). It functions to inactivate the thyroid hormones by forming reverse T3 from T4 and T2 from T3 by deiodination of the tyrosyl ring (Kaplan et al., 1986). Se lenium status and activity of deiodinases may be evaluated by measuring T 3 and T 4 ratios. The peripheral concentration of T3 was reduced and concentration of T4 increased in calves fe d a synthetic diet that was deficient in Se compared with calves fed the same diet supplemented with Se (Arthur et al., 1988). Similarly, supplementation of calves with intra ruminal Se pellets increased the basal plasma concentration of T3 and reduced the basal plasma concentration of T4 (Wichtel et a l ., 1996). Thioredoxin Reductases The thioredoxin reductases are homodimeric flavoenzymes present in various tissues. They contain SeCys residues that are indispensable for enzymatic activity (Gromer et al., 1 998). These enzymes catalyze the NADPH dependent reduction of oxidized thioredoxin. Reduced thioredoxin provides reducing equivalents for redox dependent systems and has important functions in regulating cell growth and inhibition of apoptosis (Mustacich a nd Powis, 2000).

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34 Role of Selenium in Immunity and Health Cows fed during the transition period a diet supplemented with 2 mg of Se daily as sodium selenite had greater blood Se concentration and GPx activity, greater neutrophils bactericidal activity and increased viability of neutrophils when incubated with S. aureus compared with cows fed a diet not supplemented with Se ( Grasso et al 1990) Intracellular kill of S aureus, but not of E coli was greater in neutrophils isolated from Se supplemented cows than in neutrophils from cows not fed supplemental Se However, d iets did not change the ability of neutrophils to phagocytize either S. aureus or E. coli (Hogan et al., 1990). Selenium supplementation (i.e., intra muscular injections) pre partum was associated with postpartum reduction of uterine size and the incidence of metritis (Harrison et al., 1986). Selenium supplementation with vitamin E as a single intramuscular injection at 3 weeks before expected parturition reduced the incidence of ret aine d fetal membranes from 10% t o 3% (Archiga et al., 1994) Also, a relationship between the intake of Se and vitamin E on the incidence of retained placenta was observed in dairy cows (Trinder et al., 1973). When cows received diets either deficient or su pplemented with Se (0.01 and 0.1 ppm in the dry matter respectively) the number of neutrophils killing phagocytized Candida albicans was greater in the supplemented than the deficient animals (Boyne and Arthur, 1979). However, the number of neutrophils p hagocytizing C. albicans and number of C. albicans phagocytized per neutrophil did not differ between the two dietary treatments. In a study in which nine well managed dairy herds were monitored for 1 year b ulk tank somatic cell count (SC C) decreased sign ificantly as Se concentration in plasma increased in cows sampled during the study period Also, plasma GPx activity was correlated positively to Se intake but negatively to SCC ( Erskine e t al., 1887)

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35 Rate of clinical mastitis was negatively related to plasma Se concentration and concentration of vitamin E in th e diet (Weiss et al., 1990). Supplementation of vitamin E and Se decreased the number of mammary gland infected at calving reduced occurrence o f clinical mastitis, reduced incidence of mammary gland infection throughout the lactation, and shortened the duration of intramammary infections (Smith et al., 1984). Moreover, Erskine et al. (1987) reported a negative correlation of plasma GPx activity a nd percentage of mammary quarters infecte d with major mastitis pathogens and mean herd SCC with a positive correlation of mean herd GPx activity with concentration of Se in whole blood ( Erskine et al. 1987) More specifically, c oncentrations of Se above 0 L in blood were associated with a decreased proportion of mammary gland infections by A rcanobacterium pyogenes and Corynebacterium spp (Jukola et al., 1996). Feeding pre partum beef cows with a supplement containing 60 or 120 ppm of Se as selenite and 60 ppm as selenomethionine increased the T3 / T4 ratio in plasma and IgG and IgM in colostrums compared with 20 ppm of selenite ( Awadeh et al., 1998). More recently, the effec ts of feeding Se yeast or selenate diets (0.3 ppm of dry matter) to dry and ea rly lactation dairy cows increased the serum Se concentrations at calving and at 28 days postpartum but neutrophil phagocytic and bactericidal activity was not altered at 28 dpp, which is a time beyond the immunossupressed period of parturition (Weiss and Hogan, 2005) Fatty Acids Enzymes and Biohydrogenation Fatty acids (FAs) are classified as lipids, which are biological compounds that are soluble in organic solvents. Lipids include cholesterol and fats such as triacylglycerols and phospholipids. Phospho lipids are major components of cellular membranes, and are a source of FAs for the synthesis of a variety of effector molecules such as the eicosanoids, a group of

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36 compounds that includes prostaglandins, thromboxanes and leukotrienes. Cholesterol is anothe r component of the cellular membrane and is the precursor for the synthesis of steroid hormones. The structure of FAs in particular the length of the acyl chain, the number of double bonds in the chain, and the types of isomer formed by each double bon d largely determines their function. Saturated FAs do not have double bonds in the acyl chain. Unsaturated FAs are classified into different families according to the position of the first double bond relative to the methyl end of the molecule. For example linoleic acid (LN) has 18 carbon atoms and two double bonds (C18:2n 6), with its first double bond at the sixth position from the methyl end, and is therefore a member of the n 6 family. In contrast, linolenic acid (C18:3n 3) belongs to the n 3 family be cause the first of its double bonds is at the third carbon position. Processing of FAs in one family can only generate FAs of the same family, i.e. a FA of the n 3 family cannot be converted into a member of the n 6 family and vice versa. For lactating dairy cows, most diets without supplemental fat contain approximately 2% long chain FAs that are predominantly polyunsaturated. The major FA in most seed li pids (e.g., corn and soybean meal) is LN. Linolenic acid predominates in most forage lipid. Fatty acids from any source may undergo elongation and desaturation to generate FAs with different biochemical properties. Elongation involves the addition of two c arbon units to a carbon chain through the action of elongases. Fatty acid desaturases are non heme iron containing enzymes that introduce a double bond between defined carbons of the fatty acyl chains. Delta desaturases create a double bond at a fixed posi tion counted from the carboxyl end of FAs 9 desaturase) catalyze synthesis of monounsaturated FAs (MU FAs ) from saturated FAs (SFA;

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37 9 desaturase to form the unsat urated FA, oleic acid (C18:1). The desaturases are classified according to the position of insertion of the double bond. 9 desaturase enzymes introduce the first cis double bond between 9 and 10 carbon 5 desaturases are required for the synthesis of highly unsaturated FAs 6 desaturases are membrane bound desaturases that catalyze the synthesis of polyunsaturated FAs (PU FAs 6 desatur 5 desaturases are classified as front end desaturases because they introduce a double bond between the pre existing double bond (i.e., n 3 and n 6) and the carboxyl (front) end of the FA (Nakamura and Nara, 2004). 6 desaturase inserts the double bond between the sixth and 5 desaturase inserts the double bond between the 5 desaturases are the conversion of C24:5 into C24:6 and C20 :3 into C20:4, respectively. In animals, desaturation of FAs 9, therefore FAs of the n 6 and n 3 families are considered essential. Membranes require unsaturated FAs to maintain their structure, fluidity and functio n; therefore, a mechanism for the introduction of double bonds (i.e. desaturation) exists. The introduction of a single double bond between carbon 9 and carbon 10 is catalyzed by the enzyme 9 desaturase, which is universally present in both plants and a nimals. This enzyme results in the conversion of stearic acid ( C 18:0) to oleic acid ( C 18: 1 n 9). Plants, unlike animals, can insert additional double bonds into oleic acid between the existing double bond at the carbon 9 position and the methyl terminus of 12 desaturase converts oleic acid into LN linolenic acid. Because animal tissues are unable to

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38 linolenic acids, these FAs must be consumed in the diet and so are ter med essential FAs linolenic acid into eicosapentaenoic acid ( C 20:5n 3) and docosahexaenoic acid ( C 22:6n 3); by a similar series of reactions dietary LN is converted linolenic ( C 18:3n 6) and dihomo linolenic ( C 20: 3n 6) acids to arachidonic acid ( C 20:4n 6). Again, the n 9, n 6 and n 3 families of PU FAs are not metabolically interconvertible in mammals. Many marine plants, especially the unicellular algae in phytoplankton, also carry out chain elongation and further linolenic acid to yield the long chain n 3 PU FAs eicosapentaenoic and docosahexaenoic. It is the formation of these long chain n 3 PU FAs by marine algae and their transfer through the food chain to fish that accounts for their abundance i n some marine fish oils. The precursor PU FAs are released from membrane phosphatidylcholine by the action of phospholipase A 2 or from membrane phosphatidylinositol 4, 5 bisphosphate by the actions of phospholipase C and a diacylglycerol (DAG) lipase. The p athways of eicosanoid synthesis begin with cyclooxygenase, which yields the prostaglandins and thromboxanes, or with the 5 12 or 15 lipoxygenases, which yield the leukotrienes, hydroperoxy eicosatetraenoic acids, hydroxyl eicosatetraenoic acids and lipo xins. The n 3 PU FAs eicosapentaenoic and docosahexaenoic, competitively inhibit the oxygenation of arachidonic acid by cyclooxygenase. In addition, eicosapentaenoic acid (but not docosahexaenoic acid) is able to act as a substrate for both cyclooxygenase and 5 lipoxygenase. Thus, ingestion of fish oils which contain n 3 PU FAs results in a decrease in membrane arachidonic acid concentrations and a concomitant decrease in the capacity to synthesize eicosanoids from arachidonic acid; eicosapentaenoic acid giv es rise to the 3 series prostaglandins

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39 and thromboxanes and the 5 series leukotrienes. The eicosanoids produced from eicosapentaenoic acid do not always have the same biological properties as the analogues produced from arachidonic acid. Transport and Meta bolism Diets for dairy cows are composed mainly of forages and concentrates. Forages are rich source s of glycolipids and phospholipids, which are predominantly composed of LN and linolenic FAs In contrast, the lipid composition of seed oils used in the concentrate portion of the diet is predominantly triglycerides containing LN and oleic acid s (National Research Council [NRC], 2001). When these dietary lipids are consumed by ruminants they under go hydrolysis of the ester linkages catalyzed by microbial lipases (Dawson and Kem p, 1970; Dawson et al., 1977). The anaerobic bacteria found in the rumen secrete lipases which rapidly hydrolyze triacylglycerols and galactolipids to release the FA and gala ctose from their glycerol backbone. The glycerol and galactose released are fermented by the bacteria to volatile FAs Biohydrogenation by ruminal bacteria is attained through the addition of a hydrogen ion at the point of the double bond. The h ydrogenatio n results in the conversion of unsaturated FAs into saturated FAs An example of this would be the conversions of C18:3 C18: 2 and C18:1 into C18:0. As a result, the proportion of saturated FAs reaching the small intestine is greater than that entering th e rumen. This increased amount of saturated FAs occurs at the expense of unsaturated FAs such as the essential FAs LN and linolenic acid (Corl et al., 2001). Dietary fat enters the circulation from the gut via the lymph in the form of chylomicrons. Hepati c very low density lipoproteins (VLDL) production from dietary FAs and FAs synthesized de novo contribute to the serum lipid level in the postprandial state. The average concentration s of free FAs and triglycerides in the post absorptive state are lesser t han during the absorptive phase following ingestion of a fatty meal. More important, the composition of plasma

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40 FA (chain length and degree of unsaturation) is determined by the type of fat ingested (Palmquist and Jenkins, 1980) Cellular uptake of lipid s f rom chylomicrons and VLDL is similar. The majority of complex lipid is hydrolyzed by two related lipases, hepatic lipase and lipoprotein lipase. This hydrolysis enables free FAs to enter cells through membrane associated FA transporters. At least five plas ma membrane proteins have been identified as potential FA transporters, including: a ) FA binding protein (FABP), b ) FA translocase, c ) caveolin, d ) 56 kDa kidney FA binding protein, and e ) FA transport protein. A likely candidate for fatty acid transport in adipose and liver cells is fatty acid transport protein (Vance and Vance, 1996) Once in the cell, most FAs are non covalently bound to proteins, like FABP. Several FABP subtypes have been described, including liver, adipocyte, and intestinal. A requisite step for fatty acid entry into several metabolic pathways is the formation of fatty acyl CoA thioesters (FA CoA), catalyzed by FA CoA synthetases. Several FA CoA synthetases subtypes displa y fatty acid chain length specificity. The FA CoA products of these enzymes also are bound to FABP. The FABP exists within both the cytosol and nucleus, which suggests that FA or FA CoA may be in the nucleus and serve as ligands that regulate the activity of specific transcription factors. The total intracellular FA CoA level is highly variable and depends on the cell examined. Although the intracellular concentration of fatty acid and FA CoA that is not bound to protein is low, < 10 M, it is the unbound o r FA CoA in this fraction that is likely to serve as regulatory ligands for specific transcription factors (Vance and Vance, 1996) Fatty Acids for C ows The transition from non lactating (pregnancy) to lactation in the dairy cow is a critical phase of the animal cycle. The transition period, typically considered the time 3 weeks pre partum until 3 weeks postpartum, is marked by declining DMI as the cow approaches parturition

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41 and negative energy status when lactation initiates Total energy intake in early lactation is usually less than what is required for maintenance and milk production (Staples et al., 1990); therefore the cow must mobilize ener gy substrates primarily from adipose stores in the form of non esterified FAs ( NE FAs ) to support lactation Non esterified FAs are removed from the circulation by the liver. Once in the liver, NE FAs can be utilized for energy; however, ruminants have reduced ability to export triglycerides from the liver and lipids can accumulate causing a metabolic disorder k nown as fatty liver (Drackley, 1999). Fat supplementation is commonly used to increase the energy density of the diet of lactating dairy cows. During the first few weeks of lactation, dairy cows are limited by nutrient intake by which to meet the demands o f lactation. Early postpartum DMI is limited by appetite of the cow and does not peak until 10 to 14 wk postpartum, while milk production usually peaks at 4 to 8 w ee k s postpartum (NRC, 2001). In early lactation reduction in appetite is likely cause d by liv er oxidation of lipids increased lipolytic activity more ATP generated by oxidation of FA in the liver and more ketones p roduced. It is thought that oxidation in the liver sends signals to centers in the brain that regulate hunger and satiety through the vagus nerve. Many different types of supplemental fat have been fed to lactating cows under experimental conditions. Some of these include blends of animal vegetable fat, tallow, yellow grease, oil in fish meal, whole oilseeds (cottonseeds, soybeans, canola seeds, peanut hearts, safflower seeds, sunflower seeds), flaked fat, prilled fat, hydrogenated fat, calcium soaps of fat, medium chain triglycerides, and saturated free FAs Thus, the availability o f fat sources is diverse (Staples et al., 1998). Supplemental fats can increase the energy density of the diet and reduce an energy deficit in early lactation. However, milk production often increases when fats are added to the diet,

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42 resulting in no improv ement of energy status. In addition, fat feeding can often result in a depression of DMI (Allen, 2000). The mechanisms by which supplemental fat depresses DMI are not apparent but could involve negative effects on ruminal fermentation and gut motility, rel ease of gut hormones, oxidation of fat in the liver, and palatability of diets containing added fat (Allen, 2000). Feeding supplemental fat in the form of calcium salts of FAs ( CS FAs ) makes the f at partially inert in the rumen The use CS FAs allow s feeding of more unsaturated FAs in the diets of cows in an attempt to increase the duodenal flow of specific FAs for absorption and utilization. Diets with 3 to 5% if most composited with saturated fats, can increase energy intake without having a major negative effect on fiber digestion or milk fat (Palmquist and Jenkins, 1980). The extent of ruminal biohydrogenation is variable and dependent upon many factors including the diet and ruminal conditions However it has been estimated by Chilliard et al. (2000) tha t 80% of dietary LN is biohydrogenated Klusmeyer and Clark (1991) calculated the biohydrogenation of unprote cted unsaturated FA to be approximately 70% using ruminally and duodenally cannulated lactating dairy cows. Therefore, microbial biohydrogenation i s not 100% efficient. Approximately 25% of the consumed unprotected unsaturated FAs may be available for absorption at the small intestine of the lactating dairy cow for delivery to tissues for metabolism. M anufacturing CS FAs can make them partially inert, especially if composited mainly of monounsaturated fats. P reformed soaps of FA should not dissociate in the rumen, but they dissociate in the abomasum where pH is acid. The FAs are then available in the small intestine for absorption and utilization by th e body (Jenkins and Palmquist, 1984). Recently, protection of FAs using whey proteins gels has been studied. Basically, the processes involve heating an emulsion of oil, water, and whey proteins to induce gelation. Once gelation has

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43 occurred, small lipid d roplets are encapsulated by a whey protein matrix (Rosenberg and DePeters, 2006). Carrol and colleagues (2006) fed soybean oil as a whey protein isolated gels linolenic acid by 122%, respectively, compared w ith a diet containing unprotected oil. Also, feeding a mixture of soybean and lindseed linolenic acid of 60 and 70% compared with 75 and 85% for cows fed CS FAs (Junchem et al., 2007) Using the fat sub model of the Cornell Penn Miner (CPM) Dairy model, the amount of FA required for a modern dairy cow c an be calculated. For example, LN acid excreted in 45.5 kg of milk daily exceeds the post ruminal uptake from typical die ts (Sanchez and Block 2002). Calculation of LN balance of the lactating dairy cow would be the following: LN acid balance = LN acid absorbed ( LN acid for maintenance + LN acid for milk production). Therefore, a cow consuming 25 kg of DM/day of a typical diet (no added fat source) would consume 225 g/day of LN acid. In t he 225 g consumed, only 20% will escape biohydrogenation (45 g), and of that, approximately 82% (37 g) will be absorbed in the small intestine. The LN acid requirement for maintenance of th e mature lactating ruminant has not been defined. However, a calculation based on metabolic body weight using the non lactating rat (Mattos and Palmquist, 1977) yields a maintenance requirement of 10.7 g/day for a cow weighing 607 kg. Milk output of LN aci d of a cow producing 45 kg milk/day would be 54 g/day (3.4% milk fat containing 3.5% LN acid). In this situation, the LN balance would be 27.7 g/day (37 g/day absorbed from the diet [10.7 g/day for maintenance + 54 g/day for milk production]). Thus in o rder to get this animal out of a deficient situation, a fat rich in LN acid should be supplemented.

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44 Fatty Acids Incorporation in Tissues Feeding fish oil ( FO ) increased concentrations of the n 3 PUFA, including EPA ( C20:5 ) and DHA ( C22:6 ) in the milk fat and caruncles (Mattos et al., 2004) Bilby et al (2006) supplemented lactating dairy cows with FO containing 2.0% of EPA and 2.3% of DHA at 1.9% of DM as CS FAs Ingestion was calculated to be approximately 15 g/cow per day of EPA and DHA combined. Proport ions of FA distribution in the endometrium, liver, mammary, milk fat, muscle, subcutaneous and internal adipose tissues were analyzed. In endometrium, concentrations of EPA and DHA were increased in FO fed cows with a concurrent decrease in arachidonic aci d The lesser arachidonic acid available for PGF 2 production and other products of the PG 2 series could be beneficial for reducing secretion of PGF 2 upon oxytocin stimulus maintaining the CL and possibly pregnancy. In liver feeding FO increased proportions of linolenic EPA and DHA but decreased di homo linolenic ( C20:3n 6 ) In mammary tissue, feeding FO decreased the proportion of C14:1, but increased the proportions of C18:3; C20:0; EPA and DHA In milk feedin g FO increased C4:0; C16:0; C18:3; cis 9, trans 11 CLA; and DHA The n 6/n 3 ratio was reduced in all tissues, except the adipose tissue. These results confirmed that dietary supplementation of a FO enriched lipid can alter FA proportions and distribution in reproductive and other tissues. In eukaryotic membranes the glycerol based phospholipids are predominant, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phophatidylinnositol (PI) and cardiolipin. Cholesterol is also a major component of eukaryotic membranes, particularly in mammalian plasma membranes, where it may be present in equimolar proportions with phospholipids. Furthermore, each lipid species exhibits a characteristic FA composition. In the case of gly cerol based phospholipids, for example, it is usual to find saturated fatty acid esterified at the 1 position of the glycerol backbone and an

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45 unsaturated FA at the 2 position. Also, in eukaryotic membranes it is usual that PE and PS, for example, are more unsaturated than other phospholipids. A list of FA composition of phospholipids found in human e rythrocytes is shown in Table 2 1. The inner and outer leaflets of membrane bilayers may exhibit different lipid composition (Devaux, 1991). The plasma membrane of human erythrocytes is the most thoroughly investigated. The results demonstrated that most of the membranes display some degree of lipid asymmetry. In normal erythrocytes al l the PS is located in the inner monolayer, whereas approximately 20% of the PE can be detected at the outer surface, with 80% confined to the inner monolayer. The outer monolayer consists of predominantly PC, sphingomyelin and glycolipds. It is interesting that the information available for organelle membranes suggests that PE and P S are also oriented towards the cytosol. The FA composition of PE and PS is highly enriched (~24%) with arachidonic acid and found in the inner monolayer because of phospholipid asymmetry in plasma membranes (Vance and Vance, 1996). This could have physiol ogical importance because arachidonic acid is a substrate for synthesis of eicosanoids by enzymes present in the cytoplasm. FAs The major precursor of these compounds is arachidonic acid and the pathways leading to the eicosanoids cascade, including the cyclooxygenase, lipoxygenase, and epoxygenase pathways. In each case, these pathways a re named after the enzyme that catalyzes the first committed step. The prostanoids, which include the prostaglandins and thromboxanes, are formed via the cyclooxygenase pathway. Eicosanoids are not stored by cells, but rather are synthesized and released r apidly. Prostanoid formation occurs in three steps: a) release of arachidonate from the

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46 membrane phopholipid; b) conversion of arachidonate to the prostaglandin endoperoxide PGH 2 ; and c) cell specific conversion of PGH 2 to one of the major prostanoids. Pro staglandin synthesis is initiated by the interaction of various hormones with their cognate cell surface receptor. Hormonal activation results in the activation of one or more lipases. There are a variety of lipases that could participate in the mobilizing arachidonate from cellular lipids (phopholipase A 2 and phopholipase C). Arachidonic acid can be processed by PGHS complex PGHS1 that is constitutively present in cell and PGHS2 that is inducible; both have similar activities of catalyzing the oxidation a nd peroxidation of arachidonate into PGH 2 and further acted upon by PGF synthetase, PGE 2 synthetase, TXA 2 syntheta se to generate prostaglandins F PGE 2 and tromboxane A 2 Also arachidonate can be processed by epoxygenase and lipoxygenase to generate e poxyeicosatrienoic acids leukotrienes an d hydroxyeicosatetraenoic acids respectively Eicosapentaenoic acid is processed by PGHS to generate prostaglandins of the 3 series (Figure 2 2 ) Effects of Linoleic Acid in the Postpartum Period During the puerperium, a very intense secretion of uterine PGF 2 occurs as evidenced by dramatic increases in concentration of plasma 13, 14 dihydro, 15 keto PGF 2 metabolite ( PGFM ) (Guilbault et al., 1984) in cows Plasma concentrations of PGFM peak at concentratio ns >1 ng/ml within 1 to 4 days after parturition and decreased progressively until 15 days postpartum (Guibault et al., 1984, 1985) as the uterine size decreases and caruncular tissue (i.e., main source of uterine PGF 2 ) is sloughed. Also the magnitude of this secretion is correlated positively with the resumption of ovarian activity or follicular growth (Guilbault et al., 1987a; Guilbault et al., 1987b).

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47 Seals et al (2002) showed that concentrations of PGFM in jugular plasma were higher during the first 6 days postpartum for cows that did not develop uterine infections (2160 pg/ml) in comparison to cows that developed endometritis (1450 pg/ml) between 15 to 21 days postpartum. C oncentrations of PGFM only increased subsequently at the time of onset of uteri ne infection, which mostly likely reflected a uterine inflammatory response to the proliferation of bacteria and release of endotoxins. Linoleic acid is an 18 carbon essential FA and is in mammals the precursor of the two series prostaglandins. Therefore, dietary LN may be an ideal feed additive for the peri parturient period of cow s because of its potential effects to elevate PGF 2 secretion during the puerperium Duration of increased uterine synthesis of PGF is negatively correlated with number of days to complete uterine involution and length of the interval between parturition and resumption of normal ovarian activity (Madej et al., 1984) Prostaglandins are derived from the membrane phospholipid stores of a rachidonic acid which are synthes ized from dietary LN However, there is some indication of immediate use of linoleate or its direct effect on the PGF production pathways in lipid infused estrous cycling heifers. Intravenous infusion of soybean oil emu lsion (50% LN ) on days 9 to 13 of the estrous cycle of heifers increased basal plasma concentration of PG F M after each infusion compared with saline in heifers (Lucy et al., 1990). Same results were ob served when oil emulsion (~50% LN ) was infused intraven ously to beef heifer daily from 7 to 11 days postpartum with PGFM peaking 4 hours after initiation of treatments on days 7 and 11 (Filley et al., 1999). Additionally, in this study, heifers infused with lipids had a greater capacity to synthesize and/or se crete PGF as indicated by plasma concentration of PGFM, when challenge with oxytocin at 12 days postpartum.

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48 Caruncular and intercaruncular areas of the bovine uterus have chemo attractant properties towards neutrophil s regardless of whether the cells w ere from the donor of the tissue or from other non pregnant cows T his effect is seen consistently from 255 days of gestation to parturition and it is more effective than other tissues, such as the skelet al muscle (Hoedemaker et al., 1992 a ). Moreover, cow s in which the neutrophil chemotaxis response to caruncular supernatants is reduced are at increased risk to develop retained fetal membranes (Gunnink, 1984; Kimura et al., 2002). Elmes et al (2004) fed pregnant ewes a LN enriched diet from 96 to 136 days of gestation (normal gestation length of 145 days). Treatment increased the concentration s of longer chain, more unsaturated metabolites of linoleic in the maternal and fetal circulations (i.e. arachidonic acid) and selectively altered the phospholipid F A composition of membrane phospholipids of the endometrium and allantochorion Supplementation significantly increas ed the proportion of arachidonic acid, and non significantly the metabolite intermediates of LN (i.e. 18:3n 6 and 20:3n 6). These changes in tissue n 6 PU FAs concentrations constitutively increased secretion of PGF and PGE 2 in the cultured placental explants. It was reported that plasma concentration of PGFM at calving were lower in cows with retained placenta (i.e., lack of shedding of fe tal membranes within 12 hours after calving ) compared with n o retained placenta cows ( Chassagne and Barnouin 1992). Shorter gestation length (279 vs. 283 days) also was associated with retained or no retained placenta, respectively. In this same study l ower plasma PGFM concentrations in grass silage fed cows (6/10, retained placenta) compared with corn silage fed cows (8/44, no retain placenta) could have resulted from the low and high LN contents of grass silage and corn silage respectively

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49 Indeed, ca runcular tissues taken via the vagina about 6 hours postpartum and incubated for 6 hours in minimum essential medium containing arachidonic acid, resulted in less thromboxane B 2 and more 6 keto PGF in tissue from cows with retained fetal membranes than i n the controls. The metabolism of arachidonic acid in the maternal components of the placenta is impaired in cows with retained placenta (Slama et al., 1993). Kemp et al (1998) fed 48 pre partum cows (i.e. 4 weeks to due date) diets different in LN /linol enic acid ratio by adding flaked sunflower seed (65% LN ) to one treatment group and flaked linseed (16% LN and 54% linolenic acid) to the other treatment group. G reater linoleic/linolenic acid ratio s for the sunflower seed compared with linseed diets were detected in f eed (2.22 vs. 0.35) and plasma (2.0 [ 30 to 24 dpp] to 4.41 [ 23 to 1.5 dpp] vs. 1.07), respectively. Mean duration of pregnancy was 280 d (s d = 5) and 282 days (s d = 5) in the sunflower and linseed diet groups, respectively. Plasma concentr ations of PGFM were greater at pre partum days 23 to 17, 16 to 10, 0.5 and parturition in cows experiencing placental expulsion within 6 h ours after parturition compared with those requiring mo re than 6 hours. No dietary effects were detected for t ime of placental expulsion. Plasma concentration of PGFM was higher between pre partum days 9 and 3 in sunflower diet and numerically greater in samples collected from calving to 1.5 dpp ; however greater variation among cows prevented statistical significa nce. Cullens (2005) supplemented cows begun 24 day pre partum with CS FAs at 2% of DM. Linoleic acid was 30.5% of total fat supplemented Plasma concentrations of PGFM were greater in the fat supplemented cows compared with no fat. Difference is possibly ca used by the increased intake of the direct precursors to the 2 series prostaglandins. Similarly, Junchem (2007) observed greater plasma concentrations of PGFM at day 1 postpartum when primiparous

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50 cows were supplemented pre partum with CS of linoleic and tr ans octadecenoic acids compared with palm oil. Also, in the same study the incidence of severe uterine infection (i.e metritis CS of linoleic and trans octadecenoic acids. U terine involution, based on the absence of intrauterine fluid, tended to be 3.2 days shorte r and pregnancy rates greater (27 [37.9% vs. 28.6%] and 41 [35.5% vs. 25.8%] days of pregnancy) in cows supplemented with CS of linoleic and trans octadecenoic acids compared with palm oil, respectively. In cows with normal puerperium, those showing a relatively longer duration of elevated plasma concentrations of PGFM needed a shorter period for postpartum uterine involution than the cows showing a shorter duration o f elevated PGFM; however, no such relationship was observed in cows with an abnormal puerperium in which PGFM concentrations were elevate d concurrently with an uterine bacterial infection (Lindell et al., 1982; Nakao et al., 1997). Prostaglandin F 2 had ut erotonic effects when measured during the estrous cycle (Eiler et al., 1984) or post partum (Gajewski et al., 1999). Ex vivo bovine neutrophils had greater direct chemotaxis responses to arachadonic metabolites (i.e. PGF leikotriene B 4 5 and 15 hydroxyeicosatetraenoic) and improved phagocytic capacity towards labeled S. aureus in the presence of PGF (Hoedemaker et al., 1992 b ). Also, PGF increased bactericidal activity of neutrophils from ovariectomized mares (Watson, 1988). These aspects of neutrophil function are important for the first line of defense against pathogens. Therefore, feeding supplements enriched with the LN acid may increas e uterine production of PGF and perhaps other immunostimulatory eicosanoids, and enhance in nate immune functions to improve uterine health.

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51 Effects of n 3 Fatty Acid on Fertility Staples et al (1998) reviewed 20 studies in which cows were supplemented with some form of FA In 11 of these studies there was an improvement ( P < 0.10 or 15 percent age unit differences between means) either in first AI service conception rate or in the overall rate of conception or pregnancy. Mean improvement in rate of conception or pregnancy of studies reporting a positive response was 17 percentage units. Of the f our studies in which a source of n 3 FAs were used two studies reported improvement in pregnancy rates. Bruckental et al (1989) observed higher pregnancy rates (72%, 47/65) for lactating dairy cows fed a diet containing 7.3% of fish meal in the DM compared with cows receiving a diet higher in soy bean meal (13.2% in the DM [52%, 35/67]). Difference in pregnancy rate was les s (10%) when the fish meal diet was compared with a third concurrent diet containing lesser amounts of soy bean meal (4.2% in the DM [65%, 47/72]). Greater pregnancy rates for the fish meal diet may represent the anti luteol y tic effects of n 3 FAs or bette r aminoacid content in fish meal compared with soy bean meal. Armstrong et al (1990) replaced or not the concentrate with fish meal and offered it at increasing quantities (0.8, 4.0 and 7.2 Kg) to lactating dairy cows in a 3x2 factorial design Pregnancy rates were not affected by quantity of concentrate ; however pregnancy rate was higher for fish meal treated cows (64%, 25/39) compared to control (42%, 18/41). Carrol et al (1994) fed fish meal at 3.5% of dry matter in the total mixed ration for lactating cows in diets that were isocaloric and isonitrogenous. No difference in DMI was detected for cows individually fed the control or fish meal diets. During the first 6 weeks of lactation, a trend occurred toward higher daily milk production for cows supplem ented with fish meal (37.6 kg 1.1) than those fed the control diet (34.7 kg 1.1). Conception rate to first AI

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52 was increased from 68% to 89% when cows were fed the control and fish meal diets, respectively. Burk e et al (1997) fed lactating dairy cows M enhaden fish meal at 2.7% of DM to compare with an isocaloric and isonitrogenous control diet. Pregnancy rate by 120 days post partum tended to improve ( P < 0.06; interaction of diet by dairy) when cows were fed fish meal than control diets at one dairy (4 1.3% vs. 31.9%), but not in a second dairy (60.2% vs. 65.4%), respectively. In this study at 2 days after an injection of PGF (58 dpp ), the proportion of cows with plasma concentrations of progesterone that were >1 ng/ml was greater when fish meal was f ed than when the control diet was fed (29% vs. 4%) F ish meal appeared to alter the dynamics of CL regression that was induced by injection of PGF As suggested by Mattos et al. (2000) for fish meal, n 3 FAs could have reduced the sensitivity of the CL to PGF 2 or reduced the uterine secretion of PGF 2 that delayed the completion of functional luteolysis. Although the FAs in fresh grass can contain a high proportion of linolenic acid, flaxseeds are the only c oncentrated source of lin olenic acid (~20% of DM ) available. Flaxseeds have been evaluated on reproductive performance of lactating dairy cows with mixed results. Petit et al (2001) fed dairy cows formaldehyde treated flaxseed (n = 16) which is a source linolenic acid a precursor to the FAs EPA and DHA or Megalac (n = 14), a CS of palm oil, from 9 to 19 weeks of lactation. Cows in the flaxseed diet had a greater first service conception rate than those fed CS of palm oil (87% vs. 50 %). Small sampl e sizes in this study prevent a more definite conclusion. More recently Petit and Twagiramungu (2006) reported a lower pregnancy loss from 30 to 50 days of pregnancy over 2 services for cows supplemented wit h whole flaxseed (0%, 0/23) at 10% of DM compared with a predominantly saturated fat supplement (Megalac, 15.4% [4/26]) at

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53 ~4% of DM However total conception rate (~64%, 45/70) was not different because conception to first service was numerically lower (10%) in the whole flaxseed group. Results of this study must be interpreted carefully because of the small samples sizes. In a Canadian study (Ambrose et al., 2006a) 121 Holstein cows were fed isocaloric diets containing either coarsely rolled flaxseed or rolled sunflower seed at 9% of DM. Diets were fe d for 28 days prior to insemination using a timed AI protocol and continued for 32 days after AI. First service conception rate tended (P < 0.07) to be greater for the flaxseed (48.4%) compared with th e sunflower seed diet (32.2%). Although the overall pr egnancy rates, after 2 services, were not different between the two groups (67.7% vs. 59.3%), the proportion of pregnant cows that delivered a calf favored those fed flaxseed (90.2% vs. 72.7%), indicating that early and late pregnancy loss was less for cow s fed flaxseed. Conclusions draw from this study must be taken in account that sunflower seeds are enriched in LN acid, which is known to be a precursor of the 2 series of prostaglandins Such a diet may be unfavorable to CL life spa n and not the best cont rol diet. Indeed, the percent ages of LN and linolenic FAs in milk w ere greater and lesser, respectively, in the sunflower seed diet. Fuentes et al. (2008) on a commercial dairy in Spain fed Holstein cows (n = 356) diets of either 5.5% extruded whole flaxseed or 4.9% extruded soybeans plus 1% CS of palm oil bet ween 4 to 20 weeks postpartum. Cows were detected in estrus using visual obser vation and the Afimilk pedometer system. First service (39 vs. 39%) and overall conception rates (40 vs. 34%) did not differ between soybean and flaxseed groups, respectively. Lastly, lactating dairy cows fed rolled flaxseed (8% of diet DM) had a similar c onception rate (43.3%; n = 141) to those fed a mixture of tallow and C S of palm oil distillate (41.6%; n =

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54 125) at 35 days post AI (Ambrose et al., 2006b). Although not significantly different, embryo loss was 8% and 16% for cows fed flaxseed or control fa t, respectively. Mean serum concentrations of P 4 from AI to 20 days after AI were higher for cows fed wh ole flaxseed (57% of C18:3) at 10% in the DM compared to those cows fed (47% of C16:0) at 4% of the DM (Lessard et a l 2003) These re sults are in agreement with the greater P 4 concentration reported by Petit et al (2001) for cows fed flaxseed. Similarly, the proportion of cows with plasma P 4 concentration greater than 1 ng/ml at AI was larger when fish meal was included in the diet tha n when a control diet was fed (Burke et al., 1997). Suppression of PGF 2 secretion and maintenance of the CL are obligatory steps for establishment of pregnancy of cows (Thatcher et al., 1994) ; thus feeding anti luteolytic diets may contribute to an increa se in embryo survival. Fatty Acids Profile in Peripheral Blood Cell s Calder et al (1994) investigated the FA composition of the neutral lipid fraction of lymphocytes from the rat. The neutral lipid fraction includes acylglycerol, cholesterol esters non e sterified FAs and triacylglycerol the major fatty acid containing component of this fraction. These lipids largely represent an intracellular store of FAs The neutral lipids of freshly prepared cells contained high proportions of the saturated FA (~50% palmitic and stearic acids) and low proportion of PU FAs which accounted for less than 20% (~12% L N and arachidonic acids and ~1% EPA and DHA). Great changes of the FAs profile were observed after 48 h our s of culture in the presence of Con A (mitogen) The proportion of stearic, L N and arachidonic acid were decreased compared with those in fresh cells, whereas that of oleic acid was increased. The phospholipid fraction of freshly prepared lymphocytes contained a higher proportion of PU FAs (~30%) than did the neutral lipid fraction, with arachidonic acid comprising over 20% of the phospholipid fatty acid (i.e., more than three times its proportion in the neutral lipid

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55 fraction ) These differences resulted in a significantly lower saturated/unsaturated ratio of FAs in the phospholipids than in the neutral lipids fractions The changes in the FA profile after a 48 h our incubation with Con A were even more pronounced in the phospholipids fraction, with reduced proportions of palmitic, stearic, LN and arachidonic a cids and increased proportion of oleic acid. These changes in the FA composition resulted in a decrease d proportion of PU FAs in the phospholipids and a marked increase (~ 40%) in the saturated/unsaturated fatty acid ratio in comparison to non stimulated ce lls. Incorporation of L N arachidonic oleic and palmitic acids into lipids of lymphocytes was of the same low rate when no mitogen stimulation was performed H owever the rate of LN and arachidonic acid incorporation was the highest when cells were stimul ated with Con A. Palmitic, L N and arachidonic acid were incorporated mainly in to the phospholipid fraction and oleic being incorporated to the mono and di acylglycerol fraction s ( Calder et al ., 1994) When lymphocytes were incubated with different FAs the FA profile changed in the neutral and phospholipids fractions. For example, the incubation of l y mphocytes with either L N or arachidonic acid resulted in an increase of these FAs in the phospholipids fraction from 4% and 16.8% to 29% and 27.5%, respec tively. Likewise, the incubation of cells with either EPA or DHA resulted in an increase of these FAs in the phospholipids fraction from 0.5% and 1.3% to 20% and 23%, respectively ( Calder et al ., 1994) In healthy adult human supplemented with different FA s linolenic acid, arachidonic acid and DHA [0.7g], and FO containing 1 g of EPA and DHA) the FA compositions in phospholipids of plasma and peripheral blood mononuclear cells ( PBMC ) were not affected by t linolenic acid treatments. li nolenic acid resulted in an increase in the

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56 proportion of di homo linolenic acid ( 63% increase in plasma phospholipids and 45% increase in PBMC phospholipids at week 12) (Thies et al., 2001). Supplementation with arachidonic acid resulted in an 85% increase in the proportion of arachidonic acid in plasma phospholipids and a 15% increase in the proportion of arachidonic acid in PBMC phospholipids. Supplementation with DHA resulted in an increase in the proportion of DHA in phospholipids of plasma and PBMC (91 and 80% increases at week 12, respectively) (Thies et al., 2001). Finally, supplementation with FO caused increases in the proportions of EPA (230% increase at wee k 12) and DHA (33% increase at week 12) in plasma phospholipids. Likewise, FO supplementation increased the proportions of EPA and DHA in PBMC phospholipids (220 % and 16% increases, respectively) (Thies et al., 2001). W hen humans ingest fish or fish oil, the EPA and DHA FAs in fish or fish oil led to: 1) decreased production of prostaglandin E 2 metabolites ; 2) decrease d in thromboxane A 2 a potent platelet aggregator and vasoconstrictor ; 3) decrease d in leukotriene B 4 formation, an inducer of inflammation and a powerful inducer of leuko cyte chemotaxis and adherence ; 4) increase d in thromboxane A 3 a weak platelet aggregat or and a weak vasoconstrictor ; 5) increase d in prostacyclin PGI 3 leading to an overall increase d i n total prostacyclin by increasing PGI 3 without a decrease d in PGI 2 (both PGI 2 and PGI 3 are active vasodilators and inhibitors of platele t aggregation) ; and 6) increase d in leukotriene B 5 a weak inducer of inflammati on and a weak chemotactic agent (Simopo ulos, 2002). Cytokines such as IL 1 and IL 6 are the most important cytokines produced by monocytes and macrophages. They induce fever and the synthesis of acute phase proteins by the liver, activate T and B lymphocytes and endothelial cells and are involved in many other aspects of the acute phase response. Fatty acids of the n 3

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57 family supplementation to healthy humans suppresse d the capacity of monocytes to synthesize IL 1 and and IL 1 mRNA Thanasak et al (2004) fed male goats diets enriched with LN (corn oil) and oleic (olive oil). Linoleic acid was significantly increased in plasma and in erythrocyte membranes of the corn oil supplemented group. Olive oil supplementation increased oleic acid in plasma an d erythrocyte membranes of the control group. However, no changes were observed in lymphocyte proliferation responses to Con A and PHA, expression of lymphocyte subset markers (CD2, major histocompatibility com plex II ( MHCII ) percentage of monocytes expressing (CD14) and CD49d. Fatty Acids Nuclear Receptor s Changes in the dietary fats ingested can impact cellular processes affecting many physiological systems. Many of these effects can be linked to changes in m embrane lipid composition, affecting membrane fluidity or eicosanoid signaling. More than a decade ago, however, a family of FA regulated nuclear receptors was discovered ( peroxisome proliferator activated receptors [ PPARs ] ). This discovery refocused much of the research in this area around FA effects on gene expression. In the fatty acid regulated nuclear receptors, PPARs are the most extensively characterized. Fatty acid regulation of PPARs was first reported by Gottlicher et al (1992) using a chimeric re ceptor containing the putative PPAR ligand binding domain fused to the glucocorticoid receptor DNA binding domain in transactivation assays. At 100 M, the relative potency for PPAR activation was WY14, 643 ( ) > LN > arachidonic acid > oleic a cid > stearic acid > lauric = caproic acid. There are three subtypes of PPAR s upon ligand binding, heterodimerize with the retinoid receptor ( RXR ) and interact with specific PPAR response

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58 elements in the promoter region of target g enes to affect transcription. isoforms are expressed in the bovine endometrium, although whether they are expressed in the stroma is not known (MacLaren et al., 2006). Prostanoids arise from the oxidative conversion of PU FAs released from t he plasma membrane. PU FAs (n 6 and n 3) released from phospholipids are further metabolized to form bioactive lipid mediators. Arachidonic acid metabolites ( i.e. PGF PGD 2 PGE 2 PGI 2 and TXA 2 ) act in an autocrine or paracrine fashion at nanomolar leve ls through G proteins. Some effects of eicosanoids are acute and represent rapid changes in the activity of pre existing proteins, whereas other effects are attributed to changes in gene expression. For example, the cyclooxygenase product of LN hydroperoxy octadecadienoic acids, induces c Fos, c Jun and c myc mRNA levels in aortic smooth muscle cells (Rao et al., 1995). In Swiss 3T3 fibroblast cells, arachidonic acid conversion to PGE 2 stimulates DNA synthesis and mitosis by activating expressi on of c Fos and Egr 1 through a protein kinase C regulatory pathway (Danesch et al., 1994). Many genes involved in pro inflammatory responses, such as COX 2 cytokines, and elements in their pro moters. The specific response elements and induce the transcription of specific ge nes. One mechanism by which PU FAs (n 6 and n 3) could affect gene expression is through changes in the signal transduction pathways which link cell surface receptors to the activation of nuclear transcription factors. Alternatively, PU FAs or their derivati ves (indeed derivatives of

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59 PUFA in general) might bind directly to nuclear transcription factors thereby altering their activity. Arachidonate, an FA present in vegetable oils or as a product of LN metabolism mon ocytic cell line U937 ; however EPA does not (Camandola et al ., 1996). The eicosanoids of arachidonic acid oxidation are, at least in part, responsible for arachidonic acid induced NF kB activation. Camandola et al. (1996) observed that simultaneous cell t reatment with arachidonic acid and indomethacin or nordihydroguaiaretic acid, inhibitors of the cyclooxygenase and lipoxygenase dependent routes respectively, resulted in significant down modulation of the arachidonic acid induced level of NFkB binding ac tivity P rostaglandin E 2 markedly increased the DNA binding activity of NFkB not only in the human promonocytic line U937 but also in murine macrophages J774. Eicosapentaenoic acid enrichment of membrane phospholipids arises from ingestion of n 3 PU FAs su ch as those in FO Because EPA is a competitor substrate for COX 1 and COX 2 its (1997) provided support for the concept that arachidonate metabolism was requi translocation. Their studies showed that ETYA (5, 8, 11, 14 eicosatetraynoic acid), an inhibitor of arachidonate metabolism, blocked the TNF regulation of E selectin, intercellular adhesion molecule 1, and IL 8 through inhibitio phosphorylation and degradation. Moreover, the n 3 PU FAs inhibition of cytokine induced VCAM 1 (De Caterine et al., 1998) in cultured endothelial cells may also involve inhibition of Treatment of human 643) inhibits IL 1 induced production of IL 6 and COX 2 This inhibition of COX 2 induction occurs

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60 transcriptionally as a result of PPAR repression of NF k B signaling (Staels et al., 1998). Thus, the anti inflammatory effects of EPA previously attributed to its interference with prostanoid These studies indicate that FAs can potentia lly affect the production of inflammatory cytokines and cell surface adhesion molecules through effects on two signaling pathways i.e. PPAR and cyclooxygenase dependent pathways These regulatory pathways converge to mediated signali ng. Thus, the effects of highly unsaturated n 3 FAs may be mediated transactivation. Xu et al (1999) showed in competition binding assays that saturate d FAs containing 12 carbon atoms or fewer were unable to interact with the three PPAR FAs FAs fail Saturated FAs with a chain length of 20 or more carbon units failed to bind well with any of the PPAR s. Unsaturated FAs bound to all three PPAR sub types. Of the molecules tested, the PU FAs linolenic (18:3), eicosa trienoic acid (C20:3), dihomo linolenic ( C 20:3), arachidonic acid (C20:4), and EPA T hese PU FAs FAs of corresponding carbon length. Thus, PPAR unsaturated FAs FAs albeit less efficiently than ficiently with PU FAs and only weakly with monounsaturated FAs

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61 Acute Phase Response and Proteins The acute phase protein response is induced by protein hormones called cytokines acting as messengers between the local site of injury and the hepatocytes synt hesizing the acute phase proteins. The pro inflammatory cytokines can be divided into two major groups with respect to acute phase protein induction, namely IL 1 type cytokines (including IL 1 and TNF ) and IL 6 type cytokines (including IL 6) acting through different receptors located on the membrane of the hepatocytes (Petersen et al., 2004). Pro inflammatory cytokines (IL 1, IL 6 and TNF secreted initially by monocytes and macrophages that are activated by bacterial toxins or in r esponse to l ocal tissue injury. The acute phase response is now considered to be a dynamic process involving systemic and metabolic changes providing an early nonspecific defense mechanism against insult before specific immunity is achieved Acute phase protein respon ses occur differently among species of veterinary importance. In cattle, an acute phase reaction induces increased concentrations of 1 acid glycoprotein, fibrinogen and serum amyloid A (Petersen et al., 2004). Haptoglobin Bovine h aptoglobin w as found to consist of monomers of 16 to 23 kDa ( chains) and 35 to 40 kDa ( chains) and to exist as a polymer in association with albumin with a molecular weight above 1 000 kDa in serum ( Eckersall and Conner, 1990 ) Numerous functions of haptoglobin have been proposed, but the primary function is to prevent loss of iron by the formation of very stable complexes with free hemoglobin in the blood. H aptoglobin is thus believed to have a bacteriostatic effect by restricting the availability of iron necess ary for bacterial growth (Eaton et al., 1982) For example, human haptoglobin was shown to inhibit the growth of Streptococcus pyogenes in vitro (Delange et al., 1998), and i n rats inoculated

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62 intraperitoneally with E. coli l ethality was decreased by simul taneous administration of haptoglobin (Eaton et al., 1982) In cattle, increased serum or plasma haptoglobin concentration was found after experimentally induced inflammation (Conner and Eckersall, 1988) trauma (Fisher et al., 2001) and also after inflamm atory diseases (Alsemgeest et al., 1995). Indeed, haptoglobin has been associated with bacterial contamination of the uterus and delayed uterine involution ( Sheldon et al., 2001 ) However, others have reported that haptoglobin concentrations remain low in acute postpartum metritis and high concentrations are seen only in cases of severe metritis ( Hirvonen et al., 1999 ) In dairy cows with toxic puerperal metritis, antimicrobial therapy is associated with a decrease in serum haptoglobin concentration ( Smith et al., 1998 ) Fibrinogen Fibrinogen, a classic acute phase reactant, is a complex dimeric protein with each subunit composed of three non which has a well documented role in he mostasis. However, it is becoming increasingly clear that hemostatic and inflammatory pathways are highly integrated with each being activated by the same spectrum of challenges (i.e., mechanical, chemical, or toxic injury and microbial infection). Notably fibrin appears to locally regulate inflammatory responses. In vitro studies ( Rubel et al., 2002, Shi et al., 2005 ) have shown that fibrinogen can profoundly alter leukocyte function, leading to changes in cell migration, phagocytosis, NF B mediated trans cription, production of chemokines and cytokines, degranulation, and other processes. Many of the effects of fibrinogen on leukocyte activity appear to be mediated by a specific receptor on leukocytes, the integrin receptor M 2 (Flick et al., 2004). Mice genetically engineered gave rise to a fibrinogen that retains its homeostatic properties, but failed to interact with the M 2 domain of the integrin receptor in cells such as

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63 neutrophils and macrophages. The mutation resulted in mice that, unlike the wild type exhibited a remarkable impediment in the elimination of the microbial pathogen S aureus using an acute peritonitis model (Flick et al., 2004). In smooth muscle cells IL 1 alone increased IL 6 secretion more than fivefold. Addition of PPAR activato rs (gemfibrozil, fenofibra t e or W Y 14 643) by themselves did n ot influence production of IL 6. H owever, these activators prevented the IL 1 induced secretion of IL 6 in a dose dependent manner. The in vitro results led to studies in which human patients wi th documented coronary artery disease s were treated with fenofibrate. The treatment induced lower concentration of plasma IL 6, further lowering fibrinogen, a known risk factor of coronary artery disease, and C reactiv e peptide (Staels et al., 1998). Thus FAs can regulate systemic levels of acute phase proteins by lowering the pro inflammatory state of patients. Ceruloplasmin Ceruloplasmin is a member of the multicopper oxidase family of enzymes and, a lthough the liver is the predominant source of serum ceruloplasmin, extra hepatic ceruloplasmin gene expression has been demonstrated in many tissues including spleen, lung, testis, and brain ( Alfred et al., 1997; Fleming and Gitlin 1990; Klomp et al., 1996; Yang et al., 1996 ). Serum ceruloplasmin has a hal f life of 5.5 days, and studies with radioactive copper demonstrate little or no copper exchange of ceruloplasmin bound copper following synthesis Although copper has no effect on the rate of synthesis or secretion of ceruloplasmin, failure to incorporate this metal during synthesis results in the secretion of an unstable apoceruloplasmin moiety that is devoid of ferroxidase activity and that has a half life of 5 hours ( Gitlin et al., 1992; Holtzman and Gaumnitz, 1970a; Holtzman and Gaumnitz, 1970b ). Cerul oplasmin plays an essential role in determining the rate of iron efflux via oxidation, which is required for binding to transferrin, an iron transporter. In a series of elegant nutritional

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64 studies in pigs, Cartwright and collegues demonstrated that copper deficiency results in a marked decrease in circulating ceruloplasmin with concomitant iron accumulation in the liver and other tissues. The administration of oxidase active ceruloplasmin to these animals resulted in the prompt release of iron into the circ ulation, which was detectable in circulating transferri n ( Lee et al., 1968; Roeser et al., 1970 ). Superoxide anion radicals have been implicated as mediators of inflammation and tissue injury. Protection from superoxide anion radicals is provided primarily by a copper containing, intracellular enzyme, superoxide dismutase, which catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen. Goldstein et al (review, 1979) identified that the action of cytoplasmic superoxide dismutase was to scaven ge superoxide and thereby to inhibit superoxide mediated reactions which were mimicked by ceruloplasmin. Ceruloplasmin, at concentrations present in normal plasma, inhibited reduction of both cytochrome c and nitroblue tetrazolium mediated by the aerobic a ction of xanthine oxidase on hypoxanthine (a superoxide generating system). Also, purified human ceruloplasmin inhibited the oxidation of tissue lipids extracts, liposomal membranes, PU FAs and phospholipids. The Neutrophil Neutrophil s are part of the innat e immune system acting upon antigens in a non specific manner as the first line of defense against pathogens. The migration of neutrophils from the vasculature is regulated by at least three distinct steps. The first step involves a capture and rolling adhesion mediated by the selectins. After this initial step, the neutrophil must be activated by chemoattractants and the third step is firm adhesion or arrest of the cells to the endothelium by the activated integrins. An essential feature is that these steps act in sequence, not in parallel. The inhibition of any one of these steps gives complete, rather than part ial, inhibition of neutrophil migration.

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65 Neutrophils are phagocytes, capable of ingesting pathogens. They can internalize and kill many microbes E ach phagocytic event result s in the formation of a phagosome into which ROS and hydrolytic enzymes are secret ed. The consumption of oxygen during the generation of ROS has been termed the oxidative burst (Paape et al., 2003). The oxidative burst involves the assembly and activation of the enzyme NADPH oxidase upon encountering bacteria The NADPH oxidase facilita tes the shuttling of electrons from cytosolic NADPH to oxygen present in the phagosome or in the proximal extracellular environment which produces large quantities of superoxide anion. Superoxide anion is the most proximally generated ROS by NADPH oxidase and forms as a result of one molecule of oxygen acting as an acceptor for a single donated electron. Superoxide is dismutate d spontaneously or through catalysis via an enzyme known as superoxide dismutase to hydrogen peroxide, which is then converted to hypochlorous acid by the enzyme myeloperoxidase (Paape et al., 2003) R eactive oxygen species are harmful to the neutrophil and surrounding tissues. g lutathione peroxidase, a selenoprotein, and cat alase, an iron containing enzyme, are important enzymes to reduce the excessive amounts of tissues (Paape et al., 2003) Stress, when it occurs, is an undesirable aspect of livestock production as it results in immunosuppression and increase likelihood of an infection. Mechanisms of immunosuppression at parturition are related to the release of glucocorticoids (i.e., cortisol) during this time (Preisler et al., 2000) and reduced levels of neutro phil L selectin (Weber et al., 2001). Loss of L selectin

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66 stressed imunosuppressed animal has a reduced ability to monitor endothelial cell lining for sites of infect ion and to subsequently attack and sequester pathogens. Reduced neutrophil function as measured by chemotaxis activity and killing ability (myeloperoxidase activity) in neutrophils isolated prepartum may account for increased incidence of retained placen ta (Kimura et al., 2002). Cytokines released by neutrophils, macrophages and endothelial cells can regulate in autocrine and paracrine manner s neutrophil function and in a n endocrine m ode for global integration of tissues via induc tion of an acute phase protein response in the liver A cute phase response s are also part of the innate immune system. Neutrophils secrete small amounts of an array of cytokines including IL 1, IL 6, IL 8, TNF and granulocyte macrophage colony stimulating factor ( GM CSF ) The pyrogenic cytokines IL 1, TNF and IL 6 prime various pathways that contribute to the activation of NADPH oxidase that is important to facilitate the shuttling of electron s from the extracellular environment into the phagosom e for generation of superox ide anion. The p ro inflammatory cytokine IL 8, which is also known as neutrophil activating factor, is also a potent chemoattractant; it synergizes with interferon ( IFN ) TNF CSF, and granulocyte macrophage colony stimulating factor ( G CSF ) to am plify various neutrophil cytotoxic functions. Cytokines also increase the microbiostatic and killing capacities of neutrophils against bacteria, protozoa and fungi. IFN CSF independently amplify neutrophil antibody dependent cytotoxicity. The a nti inflammatory cytokines IL 4 and IL 10 inhibit the production of IL 8 and the release of TNF 1 which contribute to a blockade of neutrophil activation.

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67 Cytokines Cytokines are small proteins (~ 25 KDa) that are released by various cells in the b ody, usually in response to an activating stimulus, and they induce response s through binding to specific receptors. They can act in a n autocrine manner, a paracrine manner to affect behavior of adjacent cells, and some cytokines are stable enough to act in an endocrine manner to regulate behavior of distant cells However an endocrine action depend s on the ability to enter the circulation and its half life in the blood (Janeway at al., 2005 ). Caughey et al. (1996) demonstrated that a diet enr iched with flaxseed oil can inhibit the ex vivo monocyte production of cytokines by 30% in four weeks, whereas 9 g of FO for another four weeks inhibited IL 1 by 80% and TNF Interleukine 6 is a 26 kDa cytokine, produced by many different cells i n the body, including lymphocytes, monocytes, fibroblasts and endothelial cells Interleukine 6 stimulate s the synthesis of all the acute phase proteins involved in the inflammatory response: C chymotrypsin and haptoglobin (Castell et al., 1986). Bacterial cell wall components such as lipopolysaccharide (LPS) stimulate the production of cytokines by monocytes and macrophages. These cytokines include TNF 1, and IL 6, which act to induce up regulation of adhesion molecules on vascular endothelial cells and on inflammatory cells (i.e. neutrophils) Adhesion molecules facilitat e the adhesion accumulation and activation of neutrophils and monocytes at the sites of infection to allow migration The inflammat ory cytokines also provide one link between inflammatory cells and specific immunity because they can stimulate T and B lymphocytes. A rachidonic acid increased IL and by human monocytes (Sinha et al., 1991). In contrast, EPA and DHA inhibited production of IL

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68 Docosahexaenoic acid ingestion (6 g/d) by young healthy men for a 90 day period increased its concentration f rom 2.3 to 7.4% in peripheral blood mononuclear cells total lipids, and decreased arachidonic acid concentration from 19.8 to 10.7%. It also lowered prostaglandin E 2 (PGE 2 ) and leukotriene B 4 production in response to lipopolysaccharide by 60 75% with further effects on lo wering the production of TNF IL When PBMC were cultured ex vivo, from humans supplemented with moderate quantities linolenic acid, arachidonic acid DHA and FO), there was no effect of supplementation on production of TNF 6 (Thies et al., 2001) Adhesion M olecules Increased adherence of circulating neutrophils to vascular endothelium is an essential earl y event of an acute inflammatory response that prece d e s neutrophil migration through vessel walls and accumulation in tissues. The adhesion of neutrophils to endothelial cells results from interactions between adhesion molecules on both cell types. A major group of such molecules on the surface of neutrophils is the integrin family of adhesive glycoproteins (CD11/CD18 complex) consisting of a common chain defined by the CD 18 antigen and three different chains specified as CD11a (LFA l), CD11b (Mac 1), and CD11c (p150 95) antigens. Among the adhesion molecules up regulated on the endothelial cell surface in response to cytokines and other inflammatory stimuli, such as LPS and ROS are vascular cell adhesion molecule 1 (VCAM 1), E selectin, and intercellu lar adhesion molecule 1 (ICAM 1) In addition ICAM 1 is also expressed on monocytes, macrophages, and lymphocytes. L Selectin (CD62L) expression on the surface of blood neutrophils is critical for animal health because this leukocyte adhesion molecule medi ate s the initial contact of circulating cells with blood vessels as the cells survey peripheral tissue for signs of infection. The emigration of neutrophils from the blood involves a capture and rolling adhesion mediated by the selectins

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69 followed by a fir m adhesion or arrest of the cells to the endothelium by the activated integrins. Down regulation of L selectin at parturition may render neutrophils unable to egress the vascular system despite up regulation of 2 integrin expression at calving (Kimura et al., 1999). The acute decline in L selectin expression observed in cows at parturition is largely due to the effect of increased plasma cortisol concentrations at pa rturition (Weber et al., 2001), resulting in a typical neutrophilia at this time. A dditiona l strong evidence for the essential role of neutrophil adherence in host defense mechanisms comes from the understanding of bovine leukocyte adhesion deficiency (BLAD) in Holstein cattle (Nagahata, 2004). The molecular basis of BLAD is a single point mutation in the gene for CD18, which results in impaired neutrophil recruitment to sites of infection This result in recurrent bacterial infections, delayed wound healing and stunted growth, and is also assoc iated with a persistent marked neutrophilia. Adherence of human neutrophils to 96 well plates pre coated with autologous serum was measured for neutrophils pre incubated (30 min) with DHA, EPA, arachidonic acid and dipalmitoyl phosphatidylcholine (DPC). Each FA was solubili z ed in DPC. At the 10 M concentration and 5 x 10 5 cell/ml all FAs increased adherence compared to DPC The FA, arachidonic acid ha d a greater effect than the n 3 FAs The incubation time s w ere test ed from 0 to 50 minutes at 10 minute increment s. C o incubation of 80 M of FA with neutrophils demonstrated that the adhesion response obtained with arachidonic acid was similar to TNF well known pro inflammatory cytokine. E icosapenatenoic and DHA had lesser effect s being intermedia te between arachidonic acid and DPC and arachidic acid ( C 20:0) and Hanks balanced salt solution indicative that the omega family of FA induces differential responses on neutrophil adhesion (Bates et al., 1993). Differential responses of n 3 FA could be because of an indirect

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70 decrease in leukotriene B 4 formation, an inducer of inflammation and a powerful inducer of leukocyte chemotaxis and adherence associated with an increase in leukotriene B 5 a weak inducer of inflammation and a weak chemotactic agent. However, the effects of arachidonic acid may be direct and independent of conversion into its derivatives because: 1) neutrophils incubated with arachidonic acid peroxidation derivatives hydroperoxy C 20:4 ( C 20:4 HP), hydroxy 20:4 ( C 20:4 H) did not increase adherence of cultured neutrophils compared with vehicle only and had a lesser effect than arachidonic acid and 2) i nhibition of lipoxigenase with caffeic acid or cyclooxygenase with indomethacin with neutrophil s before the addition of arachidonic acid did not prevent effects of arachidonic acid on neutrophil adherence (Bates et al., 1995) Interestingly, human neutrophils incubated with arachidonic acid induced greater detachment of human umbilical vein cells from plates and induce d greater degranulation of neutrophils, measured by azurophilic granule release, than when incubated with vehicle alone, C 18:0, C 18:1n 9, C 18:2n 6 C18:3 n 6, C 18:3n 3, C 18:4n 3 The effects of EPA and DHA were not tested (Bates et al., 1995). These findings could be related to the lower risk of retained fetal membranes (Kimura et al., 2002) and metritis (Seals et al., 2002) in postpartum cows when pre partum neutrophils have gre ater function al activity and when PGFM, by product of arachidonic acid oxidation, is high in the early postpartum period (Seals et al., 2002) Oxidative Burst There is abundant evidence in human s for differential effects of n 6 and n 3 PU FAs in cell cultur e and/or ex vivo studies. Linoleic acid and arachidonic acid enhanced superoxide release from neutrophils and macrophages (Badwey et al., 1981; Badwey et al., 1994; Hardy et al., 1991; Poulos et al., 1991), whereas EPA and DHA either inhibited superoxide p roduction (Chen et al., 1994) or were less potent stimulators than arachidonic acid (Poulos et al., 1991).

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71 Supplementation with LN as phosphatidylcholine for 3 days increased incorporation of arachidonic acid in neutrophil, resulting in greater release of PGF and leukotriene B 4 and greater in vitro phagocytosis/killing of C albicans in health adult humans (Jannace et al., 1992). Feeding FO, which contains EPA and DHA, to laboratory animals and pigs decreased superoxide production by neutrophils (Fletcher and Ziboh, 1991; Sassen et al., 1993) and decreased hydrogen peroxide production by macrophages (Joe and Lokesh, 1994). A number of studies have investigated the influence of supplementing the diet of healthy human volunteers with encapsulated FO on inflammatory cell functions. F ish oil decreased superoxide production by human neutrophils (Thompson et al., 1991; Luostarinen and Saldeen, 1996; Varming et al., 1995), and decreased neutrophil binding to endothelial cells (Lee et al., 1985). These stu dies provided between 3.1 and 9.6 g EPA and DHA per day and were of 3 to 6 w eeks in duration. Most often the idea that n 3 PU FAs diminish inflammatory cell functions is interpreted in a favorable way, with the conclusion that n 3 PU FAs are anti inflammator y and so will be beneficial to health of humans consuming a typical western dietary regime However, because inflammatory cells are key components of the early host response to invading pathogens and because they produce mediators that promote the specific immune response, a reduction in inflammatory cell functions could compromise host defense. Indeed murine macrophages pre treated with EPA and DHA decreased the phagocytic ability of this cells compared with arachidonic acid pre treatment ( Calder et al., 1990) Moreover, dietary FO diminished rat macrophage phagocytosis (Joe and Lokesh, 1994) In contrast, degree of neutrophil phagocytic activity and oxidative burst did not differ among adults humans supplemented with moderate

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72 lino linolenic acid, arachidonic acid DHA, and FO in a 12 week period (Thies et al., 2001).

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73 Table 2 1. Gas chromatography analysis of the fatty acid chain in human erythrocytes phospholipids Fatty Acid Total Phospholipids SPM PC PE PS 16:0 a 20.1 23.6 31.2 12.9 2.7 18:0 17.0 5.7 11.8 11.5 37.5 18:1 13.3 + 18.9 18.1 8.1 18:2 8.6 + 22.8 7.1 3.1 20:0 + 1.9 + + + 20:3 1.3 1.9 1.5 2.6 22:0 1.9 9.5 1.9 1.5 2.6 20:4 12.6 1.4 6.7 23.7 24.2 23:0 + 2.0 + + + 24:0 4.7 22.8 + + + 22:4 3.1 + 7.5 4.0 24:1 4.8 24.0 + + + 22:5 2.0 + 4.3 3.4 22:6 4.2 2.1 8.2 10.1 The data are expressed as weight % of the total. SPM, sphingomyelin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine. + denotes that the concentration did not exceed 1% of the total. a Indicates the number of carbon atoms in t he chain and the number of double bonds.

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74 Figure 2 1 Routes of dietary selenium metabolism in animals (Jacques, 2003).

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75 Figure 2 2. Phospholipid generation of inflammatory mediators.

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76 CHAPTER 3 USE OF INCREASING DO SES OF A DEGRADABLE DESLORELIN IMPLA N T TO ENHANCE UTERINE INVO LUTION IN POSTPARTUM LACTATING DAIRY COWS Introduction Complete gross involution of the uterus occurs over a wide range of 25 to 30 days postpartum (dpp) during which uterine size approximates the pre gravid state (Hussain and Daniel, 1991). Major changes are occurring within a few days after parturition (Gier et al., 1968). Uterine involution involves contraction of uterine musculature for sloughing of excessive caruncular tissue (Olson et al., 1986). This caruncul ar tissue loss is initiated around days 6 and 7 postpartum and is accompanied with a granular degeneration of the sarcoplasm, vacuolization and atrophy of the myometrial cell nucleus without cell necrosis (Archbald et al., 1972). Delayed first ovulation in suckled cows was associated with increased rates of uterine involution in dairy (Riesen et al., 1968) and beef cows (Lauderdale et al., 1968). Presence of the calf may represent a natural phenomenon to protect the involuting uterus from relaxation (Rodrig uez Martinez et al., 1987; Bonafos and Ginther, 1995) and the immunosuppressive effects of progesterone after an ovulation (Chacin et al., 1990; Subandrio et al., 2000). Furthermore, if first ovulation occurs in the presence of a heavy contaminated uterus then prolonged luteal phases may occur (Smith and Wallace, 1998; Opsomer et al., 2000; Royal et al., 2000). Prolonged luteal phases are associated with pyometra (Farin et al., 1989) and lower fertility (Smith et al., 1998; Royal et al., 2000; Opsomer et a l., 2000). Indeed, a side effect of an early induced ovulation in response to injections of GnRH (i.e., at day 15 postpartum) was an increase in the incidence of pyometra and pre breeding anestrus (Etherington et al., 1984). Contin u o us treatment of cattle with GnRH agonists can induce down regulation o f GnRH receptors on gonadotroph desensitization of the anterior pituitary gland to endogenous GnRH, and abolishment of pulsatile release Continuo us treatment with a

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77 non degradab le GnRH agonist (Deslorelin, 5 mg) during the period of uterine involuti on had a multiplicity of effects such as follicular development limited to 2 3 mm in diameter, enhanced physical involution of the uterus and cervix, i ncreased tonicity of the uterine wall and reduced frequency of abnormal cervical discharges (Silvestre et al., 2008). However, a prolonged anovulatory condition after withdrawal of treatment imposed a barrier to normal fertility (Silvestre et al., 2008). Therefore, a lower dose of Deslor elin (DESL) may have positive effects on uterine involution without a prolonged anovulatory condition. Additionally, cows inserted with a degradable DESL implant (2.1 mg) at day 7 postpartum responded to a synchronization protocol with synchronized ovulations at 69 dpp (Mattos et al., 2001). Therefore, it is proposed that d ose effects of the DESL implant and practical routes of treatment need ed further inv estigation to optimize potential complementary effects of enhancing uterine involution and synchronized restoration of ovarian activity in lactating dairy cows. The objectives of this study were to investigate the effects of increasing doses of a degradabl e implant containing a GnRH agonist (1 and 2 DESL implants of 2.1 mg) on uterine involution and ovarian follicular activity during the period before initial synchronization of ovulation, and to determine whether treatment with increasing doses of a DESL im plant would alter responsiveness to a synchronization of ovulation protocol initiated at approximately 45 dpp. The hypothesis was that a lower dose of a degradable implant containing a GnRH agonist ( DESL 2.1 mg) will suppress follicular development and en hance uterine involution early postpartum, which will be followed by resumption of follicular activity to permit a normal synchronization of ovulation during the breeding period. Material s and Methods The experiment was conducted at the University of Flori da Dairy Research Unit (Hague, FL) from January 200 3 through July 2003. Cows were managed according to the guidelines

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78 approved by the University of Florida Animal Care and Use Committee. Holstein cows in late gestation were housed in sod based pens beginni ng approximately 3 weeks before expected parturition After parturition cows were moved to a free stall facility having grooved concrete floors with fans and sprinklers that operated when the temperature exceeded 25C. Cows were milked thrice daily and in dividual milk weights recorded at each milking for the duration of lactation Cows were offered ad libitum amounts of a total mixed ration to allow 5 to 10% feed refusals twice daily. Only clinically normal (i.e., no milk fever, dystocia and stillborns) po stpartum cows with a body condition score (BCS) equal to or greater than 2.75 were enrolled. Recording of BCS occurred on the day of parturition. Body condition score was given by one veterinarian following a quarter point system of 1 (thin) to 5 (obese) scale (Edmonson et al., 1989). After parturition, c ows that had not shed their fetal membranes within 12 hours based upon visual inspection of the vulva and perineum were considered to have a retained fetal membrane (RFM) and were maintained in the expe r iment. M ultiparous (n = 31) and primiparous (n = 20) cows were assigned randomly into one of the three experimental groups within 12 to 4 6 h ours postpartum. Cows received one (1DESL; n = 16) or two (2DESL; n = 15) degradable implant s containing 2.1 mg of t he GnRH agonist DESL (Peptech Animal Health, North Ride, Australia). Implants were placed subcutaneously in the neck region by pulling up a fold of skin and insert ing the needle into the space just beneath the fold of skin using an implanter device. Contro l (CON ; n = 20 ) cows did not receive a DESL implant. Ovarian Structures A real time ultrasound Aloka 500 scanner (Aloka Co., Ltd, Tokyo, Japan) equipped with a 5.0 MHz linear rectal transducer was used to record ovarian structures at days 8, 15, 22 29, and

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79 36 postpartum (S.E. = 1 day). Size, location and number of follicles were recorded onto ovarian maps and categorized into follicular classes 1 (2 5 mm), 2 (6 et al., 1 992) as well as presence or absence of a corpus luteum (CL). Size of CL was determined by measurements of length and width, and analyzed as the average. Uterine and Cervical Diameters At day 8 postpartum per rectal palpation of the reproductive tract was p erformed to identify the previous pregnant uterine horn (PPH) as the longer horn with the greater diameter compared to the previous non pregnant uterine horn (PNPH). The ultrasound Aloka 500 scanner equipped with a 5.0 MHz linear rectal transducer was used to determine uterine horn and cervical diameters at days 8, 15, 22, 29, and 36 postpartum (S.E. = 1 day). The diameters were obtained by placing the transducer in a transverse position in relation to the uterine horns, at approximately 4 cm past the bifur cation of the uterine horns. When the transducer was positioned and the horns could be seen clearly, the image was fixed. Pressure with the transducer on the uterine horns was avoided in order to obtain a circular cross section image. Built in machine cali pers were activated such that a vertical line was extended from serosa to serosa of the uterine horn cross section. Furthermore, utilizing the same fixed image of uterine horn cross section, measurements of the largest width of the myometrial and endometri al layers were obtained for each uterine horn. Diameter of the lumen was obtained by subtracting a doubled width of myometrial and endometrial measurements from the diameter of the cross section of the respective uterine horn. Cervical diameter was measure d by placing the transducer in a transversal position in relation to the cervix at its middle section; distance between two points was obtained as described above. Immediately prior to each ultrasound scanning, uterine tonus was estimated by

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80 per rectal pal pation and classified as no tone, moderate tone, and intense tone. Uterine tone was assessed without knowledge of treatment. Health Monitoring Trained farm employees monitored pre partum cows for signs of parturition daily. Only animals with calving assist ance categories 1 (no assistance), 2 (minor assistance ) and 3 (mild assistance) were enrolled in the experiment. During the first 14 dpp, all cows were monitored daily before the morning milking (i.e., at approximately 7:00 am) for rectal temperature using a digital thermometer (GLA M525/550, GLA Agricultural Electronics, San Luis Obispo, CA ). C were considered to have a fever. Ketosis was determined by a pink to purple color on the Ketostik urine strip (Bayer Corp., Shawnee Mission, KS). Mastitis was determined by presence of flecks or clots in milk, or a serum like secretion from an affected quarter and/or swelling in an affected quarter with systemic signs including fever. A new case of mastitis was d efined for the same cow when a different quarter was affected or a period of 21 days had elapsed since the previous diagnosis. Displacement of the abomasum (DA) was determined when a tympanic sound was heard over the left or right paralumbar fossa region d uring simultaneous percussion and auscultation of the left or right flank. Examinations for ketosis, mastitis and DA were made at 4 and 9 dpp or any time milk production depression or increased milk conductivity was observed. Additionally, cows were evalua ted for cervical discharge on days 8, 15, 22, 29, and 36 postpartum (S.E. = 1 day). The vulva was first cleaned using a povidone iodine scrub (0.75% titratable iodine and 1% povidone solution, Agripharm, Memphis, TN, USA) and dried off with a clean paper t owel. A glass vaginal speculum was inserted into the vagina until the external cervical os could be seen; visualization was performed using illumination from a penlight. Appearance of cervical discharge was categorized into clear mucus (or absence of disch arge),

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81 mucopurulent (50% clear mucus and approximately 50% of pus) or purulent (> 50% pus to brown and foul smell). Color of the external cervical os was classified as pink (no inflammation), intermediary (pink with scattered red regions) and red (inflamma tory). Metritis was defined as the presence of an abnormally enlarged uterus in the presence of a purulent cervical discharge within 22 dpp. Cows were treated with antibiotics when systemic signs of illness (i.e., fever, decreased milk yield, inappetance a nd dullness) occurred for two consecutive days. Two veterinarians performed and agreed with the procedures described above, and were unaware of the experimental groups. Estrous S ynchronization A pre synch ronization protocol was initiated a t day 44 postpart um (S.E. = 2) with an injection of GnRH (100 g; g onadorelin diacetate t etrahydrate, Cystorelin Merial Ltd., Athens, GA ) and insertion of an intra vaginal controlled internal drug releasing (CIDR, EAZI BREED; Pfizer Animal Health, New York, NY ) containin g 1.38 g of progesterone (P 4 ) followed 7 days later by CIDR (a.m.) removal and injection of PGF ( 25 mg dinoprost tromethamine i.m., Lutalyse ; Sterile Solution; Pfizer Animal Health, New York, NY ) 8 hours later (p.m.). The Ovsynch protocol was initiated 10 days after CIDR removal at 61 dpp with a GnRH injection followed 7 days later with an injection of PGF GnRH injected 48 hours later, and a timed artificial insemination (TAI) for first service at 16 hours after the second GnR H injection when cows were 70 dpp Ultrasound scanning of the ovaries was performed at the time of CIDR removal and 10 days later (i.e., first GnRH of the Ovsynch). Additional scanning s w ere performed at the PGF and last GnRH of the Ovsynch to determine recruitment of a new follicular wave and presence of pre ovulatory follicle, and 7 days after TAI to monitor formation of a CL. Occurrence of ovulation was determined by observation of luteal tissue at the site

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82 previously occupied by a dominant follicle. C ows without follicular activity during the synchronization protocol were not inseminated and were examined weekly until appearance of a class 3 follicle (> 10 mm) at which time a second Ovsynch protocol was initiated for insemination. Pregnancy diagnosis w as done by per rectum ultraso nography scanning of the uterine horns at 2 8 days after insemination. Observation of amniotic fluid, appearance of the embryo, and embryonic heart beat were used as determinants of pregnancy. Blood S amples and H ormone A ssays B l ood samples were collected by puncture of coccygeal vessels into evacuated tubes containing K 2 EDTA (Vacutainer BD, Franklin Lakes, NJ, USA). Samples were placed immediately in to an ice bath until centrifugation for 20 minutes at 2 619 x g After centrifugation, plasma was harvested and stored frozen at 20 C until assayed for concentrations of 15 keto 13, 14 dihydro prostaglandin F 2 (PGFM) and P 4 Blood samples collected daily from parturition to 14 dpp were used for measurement of plasma concentr ations of PGFM as an index of uterine PGF secretion. Experimental plasma samples were analyzed for concentrations of PGFM using a modification of the radioimmunoassay procedure described by Mattos et al (2004). The PGFM standard solutions were made by se rial dilutions in a buffer of a stock solution (1 g/ mL in 10% ethanol and 90% PBS buffer) of authentic PGFM (Sigma, St. Louis, MO). Standards (100 ) were run in duplicates at the following concentrations: 31.2, 62.5, 125, 250, 500, 1000, 2000, 4000, and 8000 pg/ mL The standard curve included 100 of prostaglandin free plasma, which was obtained from a cycl ic non lactating beef cow pre treated twice with flunixin meglumine ( 1 g/i.m.; Banamine, Intervet/ Schering Plough Animal Health Kenilworth, NJ) a p rostaglandin endoperoxide synthase inhibitor. Samples of prostaglandin free plasma had undetectable concentrations (< 0.01 ng/ mL ) of PGFM when assayed utilizing

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83 standard curves in 0.05 M PBS buffer. The PBS buffer contained 2.3 g/ L NaH 2 PO 4 H 2 O, 4.76 g/ L Na 2 HPO 4 7H 2 O, 1 g/ L sodium azide, and 8.41 g/ L NaCl. The buffer pH was adjusted to 7.5 with NaOH. Activity and volume of radioactively labeled PGFM (Amersham Pharmacia Biotech, Piscataway, NJ) used were 10,000 cpm and 100 respectively. For unknown sample s, final assay volume was 400 comprised of sample 100 100 of rabbit antiserum to PGFM (1:10,000; Meyer et al., 1995), 100 of buffer, and 100 of labeled PGFM. After a 12 h our incubation at 4C, free PGFM was separated using 750 of a solut ion of charcoal coated dextran (1.25% dextran [Sigma, St. Louis, MO] and 12.5% activated charcoal [Sigma, St. Louis, MO] ) in PBS buffer). After centrifugat ion for 20 minutes at 3565 x g the supernatant was transferred to scintillation vials and mixed with 4.5 mL of scintillation fluid (Scintiverse II, Fisher Scientific, Pittsburgh, PA). Activity was measured using a liquid scintillation counter (model LKB 1219, Wallac Inc., Gaithersburg, MD). Intra and interassay coefficients of variation for one referenc e sample (276 pg/ mL ) w ere 7.5% and 15.75%, respectively. Intra and interassay coefficients of variation for a second reference samples (1 335 pg/ mL ) w ere 10.6% and 5.8%, respectively Blood samples for determination of plasma concentration of P 4 were colle cted thrice weekly from the day of parturition until the initiation of the pre synchronization protocol. Additionally, a set of blood samples was collected just before the injection of PGF of the pre synchronization protocol and just before the first in jection of GnRH of the Ovsynch protocol. With this set of blood samples, cows were considered to be cyclic if they had plasma concentration of P 4 greater than 1 ng/mL in at least one of the two samples (cyclic) or anovulatory if both samples were below 1 n g/mL (non cyclic). Another set of blood samples was collected at the day of TAI and at 7 days after TAI; cows were considered to have ovulated a

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84 synchronized follicle if plasma concentration of P 4 was below 1 ng/mL at the TAI and greater than 1 ng/mL 7 day s later (ovulated), other combinations of plasma concentrations of P 4 were considered to be cows that failed to ovulate (non ovulated). Concentrations of plasma P 4 were determined by radioimmunoassay using a Coat A Count Kit (DPC Diagnostic Products Incorporation, CA, USA) designed for the quantitative measurement of P 4 in plasma. Plain tubes were used in duplicate for total counts and non specific of increa sing concentrations of calibrators (P 4 ), 0.1, 0.25, 0.5, 2, 5, 10, 20 and 40 ng/mL were added to the tubes. Reference samples (100 L) containing high (7.0 ng/mL) concentrations of P 4 es and 1 mL of [ 125 I] P 4 to all tubes. In every 10 th sample, duplication was performed. Reference samples had intra and interassay coefficients of variation of 2.13% and 9.8 % respectively Sensitivity of the assay was 0.1 ng/ mL Statistical A nalyses Fo llicle numbers, CL numbers, diameter of uterine horns, widths of the myometrial and endometrial layers of the uterine horns, diameter of cervix, plasma concentrations of P4 and PGFM as well as daily milk yield for the first 5 months were analyzed using rep eated measures of the mixed model procedure of SAS (SAS Institute Inc.; 2001; Version 8). Plasma concentrations of progesterone before synchronization protocol were converted to accumulate d P4 and analyzed as repeated measures. The accumulated value of a P 4 on a given experimental day for a cow is the sum of all sample concentrations from the first experimental sample collected to that given experimental day. For each dependent variable, the covariance structure that had the best relative goodness of fit ba sed upon penalty criteria (Bayesian criterion) was

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85 used. The mathematical model contained group, day, parity and higher order interactions with cow, the random variable, nested within group and parity. The GENMOD procedure of SAS was used to analyze categ orical responses such as uterine tone, cervical discharge, and proportion of cows cyclic, ovulated and with a CL during the synchronization protocol. For uterine tone and cervical discharge scores a Generalized Estimating Equations (GEE) with an independen t covariance structure, and multinomial distribution of response and a logit link function were used. The mathematical model included group, day, parity and higher order interactions with cow as a repeated subject. For the proportion of cows found to be cy clic, ovulated and with a CL during the synchronization protocol a binomial distribution of response and a logit link function were used. For all responses, pre determined contrasts were used to test the effects of GnRH agonist treatment (CON vs. 1DESL + 2 DESL) and dose of GnRH agonist (1DESL vs. 2DESL). Results During the period of January 2003 to April 2003, primiparous (n = 9, 6, 5) and multiparous (n = 11, 10, 10) cows were assigned to CON, 1DESL and 2DESL treatment groups, respectively. Data collection was discontinued in cows among the CON (n = 2), 1DESL (n = 1) and 2DESL (n = 3) groups during the period before initiation of pre synchronization because of health related circumstances. Therefore, 18, 15, and 12 CON, 1DESL, and 2DESL cows were used for s tatistical analyses, respectively. Frequency distribution of cows in the calving assistance category of no assistance (57.7% [30]) was greater (P < 0.01) than minor assistance (23% [12]) and mild assistance (19.2% [10]) and were not different among experim ental groups. A total of 7 cows (CON [n = 2], 1DESL [n = 3] and 2DESL [n = 3]) had their fetal membranes retained. Median BCS at parturition was 3.0 and ranged from 2.75 to 3.5 for all experimental groups.

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86 Ovarian Responses Cows in t he DESL implant group s had increased number of c lass 1 follicles ( P < 0.01 ; Figure 3 1 ) during the period before initiation of pre synchronization (i.e., weekly from 8 to 36 dpp) and only follicles of 2 3 mm diameter were detected in the DESL treated cows. Mean numbers of c lass 2 and class 3 follicles was less (P < 0.01) in DESL implant treated cows compared with CON (Figure 3 2) during the same postpartum period (i.e., weekly from 8 to 36 dpp) First detection of CL by weekly per rectum ultrasonography from 8 to 36 dpp was at days 22 (n = 1), 29 (n = 4), and 36 (n = 3) postpartum for CON (n = 18) cows resulting in an ovulation rate of 44.4% (8/18). In contrast, ovulation was suppressed in the 1 and 2 DESL groups resulting in a complete absence of visible CL by per rectum ult rasonography during the per iod before pre synchronization. Hormonal R esponses P lasma concentrations of P 4 were measured thrice weekly and first rise of P 4 above 1 ng/mL, indicative of ovulation, occurred in CON (55.5%, 10/18) cows at 28 dpp (S.E. = 9 days) and in two 1DESL (13.3%, 2/15) cows at 43 dpp (S.E. = 3). Plasma concentrations of P 4 were suppressed completely in all 2DESL cows during the sampling period before initiation of pre synchronization. Accumulated plasma concentrations of P 4 was greater (P < 0.01) in CON compared with DESL implant treated cows beginning at approximately 29 dpp (Fig ure 3 3 ). Mean plasma concentrations of PGFM decreased (P < 0.01) from the highest values at the time of parturition to low basal values after 10 dpp. Mean plasma concentrations of PGFM did not differ between the CON and 1DESL and 2DESL groups of cows ( Figure 3 4 ) or between 1DESL and 2DESL groups ( Figure 3 4). Cows that were diagnosed with metritis had lower (P < 0.01) mean plasma concentrations of PGFM prior to in itiation of systemic antibiotic treatments compared to cows without metritis ( Figure 3 5). PGFM values were not included in statistical

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87 analyses for cows with metritis after initiation of systemic antibiotic treatment. Systemic antibiotic treatment was ini tiated in average at 5 dpp (range: 2 to 9 dpp). Figure 3 6 shows the profiles of plasma PGFM for two cow s that received initial systemic antibiotic treatments at 5 dpp and continued for four consecutive days Patterns of the selected cows were representative of their corresponding herd mates treated with antibiotics In these cow s PGFM decreased to low basal values after initiation of antibiotic treatments (i.e., 5 dpp) and remained low until sampling termination (i.e., 14 dpp). Involution of U terus and C ervix Mean diameter of the PPH was less (P < 0.01) in the 1DESL and 2DESL groups compared to CON ( Figure 3 7A ). The PPH was smaller in diameter on days 15 and 22 postpartum ( P < 0.01; Figure 3 7A ). A significant day effect ( P < 0.01) and a non s ignificant group by day interaction indicated that the pattern s of PPH regression in size were parallel for the groups. Orthogonal contrasts determined the time in which physical involution was completed for the PPH for the groups. A single day of measurem ent was compared with subsequent days, and involution was considered complete when no significant differences were detected in a sequential contrast. Diameter of PPH at 1 5 dpp (4.21 0.2 cm) was greater (P < 0.01) than subsequent days (2.3 0.2 cm; 2 2 2 9 and 3 6 dpp ) of measurement for all groups. Diameters of PPH at 22 dpp (2.1 0.1 cm) were no longer differ ent than the subsequent days (1.92 0.1 cm; 29 and 3 6 dpp) for both 1DESL and 2DESL groups. However, the CON group had a larger (P < 0.06) diameter PPH at 22 dpp (2.45 0.1 cm) compared with the subsequent days (2.2 0.1 cm; 29 and 3 6 dpp) and only at 29 dpp (2.2 0.1 cm) was diameter of PPH no longer different than the 36 dpp (2.1 0.1 cm) measurement This indicated that complete physical invol ution of the PPH was earlier in DESL treated (i.e., 22 dpp) than CON (i.e., 29 dpp) cows.

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88 Diameter of PNPH was smaller (P < 0.01) in the DESL implant groups in comparison to CON ( Figure 3 7 B) at 22, 29 and 36 d pp. A significant day effect ( P < 0.01) and a non significant group by day interaction indicated that the pattern of P N PH regression in size was the same for both groups. Orthogonal contrasts of day of measurement for diameter of PNPH were performed as described for PPH. Diameter of PNPH at 15 dpp (2.2 0.1 cm) was greater (P < 0.09) than subsequent days (1.89 0.1 cm; 22, 29 and 36 dpp), and only at 22 dpp (1.9 0.1 cm) were diameters equal to subsequent days of measurement (1.8 0.1 cm; 29 and 36 dpp). This indicated that complete ph ysical involution of the PNPH occurred simultaneously for all groups at 22 dpp although uterine diameter at the time of completed involution was less for the DESL groups. No significant effects of DESL implant groups were detected f or the ratio of the uter ine horns (i.e., PPH/PNPH). The group by day interaction also was not affected by DESL implant groups Thus, the smaller uterine horns (PPH and PNPH) for the DESL implant group had a relative proportional difference between horns that was the same for both groups. Myometrial width of the PPH (S.E. = 0.02 cm) and PNPH (S.E. = 0.02 cm) did not differ between CON (0.31 and 0.28 cm), 1DESL (0.31 and 0.29 cm) and 2DESL (0.32 and 0.29 cm), respectively. However, myometrial widths of the PPH and PNPH were smaller (P < 0.01) for primiparous (0.28 and 0.25 cm) compared with multiparous (0.35 and 0.32 cm) cows, respectively. Also, a continuous decrease (P < 0.01) in width of the myometrium of the PPH occurred at 8 (0.44 cm), 15 (0.37 cm), 22 (0.29 cm) and 29 (0.24 cm) dpp, which was of the same width at 36 (0.24 cm) dpp. Myometrial width of PNPH at 8 dpp decreased by day 15 (0.27 cm) pospartum to the same width at 22 to 36 (0.26 cm) dpp. A significant day effect ( P < 0.01)

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89 and non significant group and group by day int eraction effects indicated that reduction in myometrial layer width of PPH and PNPH was the same for all experimental groups. Endometrial width of the PPH (S.E. = 0.02 cm) and PNPH (S.E. = 0.02 cm) did not differ between CON (0.45 and 0.42 cm), 1DESL (0.43 and 0.38 cm) and 2DESL (0.41 and 0.40 cm) groups and was not affected by parity. Also, a continuous decrease (P < 0.01) in width of the endometrium of the PPH and PNPH occurred at 8 (0.63 and 0.57 cm), 15 (0.43 and 0.41 cm) to 22 (0.38 and 0.35 cm) dpp, w hich was of the same width at 29 (0.35 and 0.33 cm) and 36 (0.35 and 0.33 cm) dpp. A significant day effect ( P < 0.01) and non significant group and group by day interaction s indicated that reduction in width of the endometrial layer of PPH was the same fo r the experimental groups. Diameter s of the uterine lumen for the PPH (Figure 3 8 A) and PNPH (Figure 3 8 B) were lesser (P < 0.01) in the 1DESL and 2DESL groups compared to CON. The uterine lumens of the PPH and PNPH w ere smaller (P < 0.01) in diameter on days 15 to 29 and 22 to 36 postpartum, respectively ( Figure 3 8 A and B ). A significant day effect ( P < 0.01) and a non significant group by day interaction indicated that the pattern in regression of the uterine luminal diameter for the PPH was the same for all th e groups although diameter of the uterine lumen at the time of completed involution was less for the DESL groups. Mean diameters of the cervix were not affected by DESL implant groups ( Figure 3 9 ). A significant day effect ( P < 0 .01) and a non significant group by day interaction determined that the pattern of cervical reduction was the same for all groups. Frequency distribution of cows in the category of intense uterine ton e was lowest at first day of measurement (i.e., 8 dpp), followed by an increase at 15 and 22 dpp (Table 3 1). Frequency of intense uterine tonus decreased throughout the remaining days of measurement

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90 (i.e., 29 and 36 dpp) (Table 3 1). The changes in the frequency of intense uterine tonus during the postpartum d ays of measurement were accompanied by inverse changes in the frequency of moderate uterine tonus since frequency of no uterine tonus were approximately the same throughout the days postpartum (Table 3 1). Frequency distribution of cows in the category of intense tonus was greater (P < 0.01) in the DESL groups compared with CON (Table 3 1), but there were no differences in distributions between 1DESL and 2DESL groups of cows (Table 3 1). N o interaction of group s and day was detected. Therefore tonus was c onsistently increased during all days of data collection for 1 DESL and 2DESL groups of cows. Vaginoscopy Purulent cervical discharge was diagnosed at a greater frequency (P < 0.05) at 8 dpp and frequency decreased to an absence of purulent cervical dischar ge at 29 and 36 dpp that was associated with increased frequencies of mucopurulent and clear discharges (Table 3 2). Interactions between day of cervical discharge examination with group, parity and RFM were not significant. Therefore, all observations at examination days (i.e., 8, 15, 22, 29 and 36 dpp) were pooled for the main effects. Frequencies of purulent and mucopurulent cervical discharges were not different between CON compared with 1DESL and 2DESL (Table 3 2). However, frequency distribution of co ws with purulent and mucopurulent discharges tended to be greater (P < 0.07) in 1DESL compared with 2DESL groups (Table 3 2). A greater (P < 0.01) proportion of cows with a purulent cervical discharge were diagnosed in primiparous cows and cows that experi enced RFM than multiparous cows and cows that released the placenta within 12 hours postpartum, respectively (Table 3 2).

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91 Estrous S ynchronization and Breeding Proportion of cyclic and ovulated cows were less (P < 0.01) in those treated with DESL implants compared with CON cows (Table 3 3). However, suppression of cyclicity and ovulation was to a greater (P < 0.01) extent in the 2DESL (Table 3 3). Similarly, proportion of cows with presence of CL at CIDR removal an d at the initiation of the Ovsynch protocol was less (P < 0.01) in DESL implant groups that were even to a greater (P < 0.01) extent in 2DESL cows (Table 3 3). A total of 18 cows in the CON group were bred following pre synchronization and Ovsynch protocol s. Based upon per rectal ultrasonographic scanning, all CON cows had pre ovulatory follicles at the time of the last GnRH injection that average 18.5 mm in diameter. All cows of the CON group but one ovulated and formed a corpus luteum 7 days after insemin ation. The one cow had a luteinized follicle. Pregnancy per AI for CON cows was 33.3% (6/18). A total of 14 cows in the 1DESL group were submitted to pre synchronization and Ovsynch protocol. However, only 71.4% (10/14) of the cows had a synchronized pre o vulatory follicle that average 20 mm in diameter at the time of the last GnRH injection, and only these 10 cows were inseminated. At 7 days after insemination, 40% (4/10) of cows responded to the last GnRH determined by the presence of corpus luteum and on e became pregnant. Conversely, 60% (6/10) of cows did not respond to the last GnRH determined by the absence of corpus luteum 7 days after insemination. The remaining cows (4/14) that were not inseminated at the first Ovsynch in the 1DESL, underwent a seco nd Ovsynch program when a class 3 follicle (> 10 mm) was visualized in a weekly per rectum ultrasonographic scanning and were inseminated at approximately 90 dpp with a pregnancy per AI occurrence of 50% (2/4). A total of 13 cows in the 2DESL group were su bmitted to pre synchronization and Ovsynch protocols, only one cow had a synchronized pre ovulatory follicle at the time of the last

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92 GnRH injection and when inseminated did not conceived. The remaining 12 cows were begun second Ovsynch program when a clas s 3 follicle (> 10 mm) was visualized in a weekly per rectum ultrasonographic scanning. These cows were inseminated at approximately 94 dpp and only one cow became pregnant. Milk Yield Milk yield over a 150 day period was not different between CON (37.0 1.4 Kg; n = 17) and DESL treated cows (35.6 1.7 Kg; n = 25) or between the 1DESL (35.0 1.7 Kg) and 2DESL (36.2 1.7 Kg) groups. There was no interaction of groups by day in the yield of milk, but multiparous (39.2 1.1 Kg; n = 27) cows produced more milk (P < 0.01) compared with primiparous cows (33.0 1.5 Kg; n = 15). Discussion Treatment with DESL during the postpartum period limited follicular development to a follicular class size of 2 3 mm in diameter improve d reduction in uterine sizes and in creased tonicity of the uterine wall These responses were similar between a low (2.1 mg) and higher (4.2 mg) doses of DESL implants. However, DESL treatment did not affect frequency of abnormal cervical discharge occurrences and delayed ovulatory activity with the greatest delay at the higher DESL implant dose (i.e., 4.2 mg). Absence of an induced ovulation in cows treated with DESL implant s within 1.5 dpp reflects the absence of LH stores in the anterior pit uitary (Saiduddin et al., 1968), a minimal relea se of LH upon exogenous stimulation with GnRH at this stage (Fernandes et al., 1978) and no induced ovulation (Silvestre et al., 2008a) Insertion of the DESL implant appeared to result in a long term pituitary desensitization with a hypo secretion of gon adotrophin s that led to a sustained suppression of ovarian follicular growth ovulation and attenuation in plasma concentrations of P 4 for both DESL treated cows Postpartum suppression of follicular growth to

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93 class 1 follicles in the postpartum period ha ve been reported ( Mattos et al., 2001; Silvestre et al., 2008 b ) which is li kely associated with low basal plasma concentrations of FSH necessary for follicular recruitment (Gong et al., 1996). The absence o f follicular growth in the DESL implant treat ed c ows resulted i n no ovulations during the period before pre synchronization except for 2 cows in the 1DESL group. In contrast, CON cows had normal follicular growth and ovulations characteristic of the postpartum period (Beam and Butler, 1997; Beam and But ler, 1998). A DESL dose dependent suppression of follicular growth and ovulation was evident in that the 2DESL group had a greater suppression of ovulation during the period of synchronization compared with 1DESL, but the later group also had responses bel ow that of normal CON cows. Paradoxically, when cows were treated with a degradable 2.1 mg DESL implant at day 7 postpartum, 62.5% (5/8) of cows had spontaneous ovulations between days 46 to 60 postpartum (Mattos et al., 2001). The uterus of cattle, mainly the caruncular endometrium, is a primary source of F series prostaglandins during the early postpartum period with secretion increasing from day of parturition to approximately 4 dpp followed by a decline to basal concentrations at 14 dpp (Guilbault et al ., 1984). P lasma concentrations of PGFM were similar among experimental groups and were representative of a normal postpartum period (Guilbault et al., 1984). Absence of effects of DESL treatment on PGFM responses in plasma is in agreement with Mattos et a l (2001). Silvestre et al (2008b) reported lower plasma concentrations of PGFM in the postpartum period in cows treated with 5 mg DESL implant, which was associated with a lower frequency distribution of cows with purulent cervical discharge. Either a s pon taneous uterine infection (Del Vecchio et al., 1994) or induction of a uterine infection by bacterial inoculation (Del Vecchio et al., 1992) is associated with elevated plasma concentrations of PGFM in postpartum dairy cows.

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94 In the present study, frequency distribution of cows among cervical discharge categories was similar among the groups, except for a slightly greater proportion of cows with purulent cervical discharge in the 1DESL group compared with the 2DESL groups. Also, the rate of involution/slough ing of the endometrial tissue were the same among all groups based upon measurements of endometrial width. Collectively, differences in plasma concentrations of PGFM were unlikely to occur. Cows that developed metritis had lower plasma concentrations of PG FM, during the period before initiation of antibiotic treatment, compared with cows without metritis. Seals et al (2002) reported that concentrations of PGFM in jugular plasma were lower, during the first 6 days postpartum for cows that did develop uterin e metritis compared to cows without metritis. Indeed, PG F 2 has tonic effects on the uterus during the estrous cycle (Eiler et al., 1984) or postpartum period (Gajewski et al., 1999), and ex vivo bovine neutrophils incubated with PGF had increased chemotaxis and phagocytic activity (Hoedemaker et al., 1992). Thus cows with a higher PGFM concentration would be less prone to develop metritis. Additionally, systemic treatment with antibiotics eliminates bacteria, thus inflammation, resul ting in a considerable decrease of plasma PGFM to low basal values as depicted in Figure 3 6. Treatment with DESL implants resulted in smaller uterine horns and no differences in cervical diameter, regardless of dose. In a previous study, the use of a 5 mg DESL treatment resulted in similar reduction responses in size of the uterine horns with tendencies for a smaller cervix (Silvestre et al., 2008b). In the present study, the diameter of a cross section of the uterine horn was spatially comprised of widths of the myometrial and endometrial layers and diameter of the uterine lumen. The widths of both tissue layers were not different among groups, but diameter of the uterine lumen was smaller when cows were treated with either of the DESL

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95 implant doses. Addit ionally, if diameter of the uterine lumen was used in the statistical model as a covariate then the DESL implant treatment effects on diameter of uterine horns were no longer significant. In contrast effects of DESL treatments were significant even after c ovariate adjustments for widths of both myometrial and endometrial layers. Therefore, we concluded that the reduction of uterine horn diameters were not due to greater sloughing or atresia of endometrium and muscular tissues, respectively. Reduction in dia meters of uterine cross section and lumen were the same for both DESL treatment doses. Furthermore, the reduction in diameter for PPH and PNPH was proportionally the same in both treatments. This indicated an effect of the DESL implant treatment s on the en tire uterus. The smaller size of the uterine horns and uterine lumens as well as increased uterine tone at such early stages of the postpartum period c ould reflect greater uterine contractility and expulsion of uterine contents that would h ave an important impact on reducing bacterial contamination and inflammation within the uterine environment. In the present experiment, such an increase in expulsion of uterine contents was not associated with a greater occurrence of these materials being classified as m ucopurulent or purulent. Uterine involution involves contraction of uterine musculature for sloughing of excessive caruncular tissue (Olson et al., 1986) that is initiated around days 6 and 7 postpartum, and a granular degeneration of the sarcoplasm, vacuo lization and atrophy of the myometrial cells nucleus without cell necrosis (Archbald et al., 1972) The reduction in uterine size after chronic GnRH a gonist treatment in women is a result of induced atrophy of the myocyte and arcuate arteries, and a decrea se in stromal edema (Weeks et al., 1999). In addition endometrial volume and thickness were reduced during GnRH a gonist treatment in women (Child et al., 2002; Nakamura et al., 1996). In the present study with postpartum cows endometrial and myometrial

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96 widths were not affected by DESL treatment with the greatest changes in width occurr ing from 8 to 15 d ays postpartum and minimal and stable sizes achieved by days 22 to 36 postpartum The smaller lumen diameter associated with increased uterine tonicity in DESL treated cows could be a result of a greater muscular contraction. Binding of FSH and LH to its receptors is associated with a direct activation of adenylate cyclase to produce cAMP and activation of signaling pathways that increase expression of COX 2 and production of PGE 2 in the myometrium and cervix (Shemesh et al., 2001). Both cAMP and PGE 2 induce relaxation of these tissues. Although plasma FSH and LH concentration were not measured in this study, the DESL treatment most likely suppressed secre tion of both FSH and LH in the DESL treated cows based on suppression of follicle development (Mattos et al., 2001). Consequently, treatment with a DESL implant that reduces LH secretion would lead to an increase in contractility or tonus as a default. Thi s could contribute to an expulsion in uterine luminal content that accounted for the reduction in size of the uterine lumen in cows treated with the DESL implant. The absence of follicles beyond the 3 mm sizes in the DESL treated cows is associated with low basal plasma concentrations of estradiol and support s recent concepts that estradiol may not play a major role on uterine involution (Silvestre et al., 2008 b ; Sheldon et al., 2003a, b). Additionally, the relaxation (Rodriguez Martin ez et al., 1987; Bonafos and Ginther, 1995) and immunosuppressive effects of progesterone follow ing an ovulation (Chacin et al., 1990; Subandrio et al., 2000) were not a confounding effect, because accumulated plasma concentrations of progesterone were the same among the three experimental groups, and only became greater in cows of the CO N group after 27 d pp. Only two cows had a spontaneous o vulation during the period before the pre synchronization in the 1 DESL treated group and none in the 2DESL treated group There were a

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97 greater proportion of cows classified as cycl ic in the 1DESL group compared with the 2DESL group; however th ese proportions were to values unacceptable for implementation of a synchronization of ovulation program to achieve acceptable pregnancies rates. The positive effects of the smaller dose of DESL (i.e., 2.1 mg) on uterine sizes were comparable to the highest (4.2 mg) dose used in this study and a previous report (Silvestre et al., 2008 b ). The low ovulation rate after the last GnRH injection of the Ovsynch in the 1DESL treated c ows disagrees with Mattos et al (2001), who reported that all cows treated at day 7 postpartum with a degradable 2.1 mg DESL implant ovulated a synchronized follicle upon a GnRH injection at day 69 postpartum. Additionally, previous results indicated appearance of class 3 follicles 22 and 37 days after insertion (i.e., day 5 of estrous cycle) of DES L implants containing 0. 7 mg and 2.1 mg, respectively (Rajamahendran et al., 1998). The longer period of desensitization observed in the present study raises the possibility of differential carry over effects or sensit ivities to DESL treatment when applied at parturition. The mechanism by which continuous administration of GnRH agonist reduces LH secretion in cattle may involve a decrease in the concentration of GnRH receptors and GnRH receptor mRNA of gonadotropes (Vi zcarra et al., 1997). Growth of c lass 1 follicles (up to 5 mm diameter) can occur in the absence of gonadotrophin stimulation, whereas growth of c lass 2 follicles (6 to 9 mm) requires FSH stimulation (Gong et al., 1996). Expression of LH receptors and depe ndence on LH stimulation for growth and ovulation occurs c lass 3 follicles ) (Xu et al., 1995) T he absence of both class 2 and 3 follicles in the majority of cows treated with DESL indicates the lack of both FSH and LH secretion. The pattern of DESL release from the DESL implant in vivo is not known. A prolonged release of the DESL in vivo associated with a long half life could explain the

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98 extended period of suppression of follicular growth. Alternatively, long term e ffects of the DESL on the gonadotropes could last after clearance of the GnRH agonist molecule. The beneficial effects of DESL treatment on uterine involution during the postpartum period is overshadowed by the reduction in ovarian activity during the peri od of synchronization of ovulation in cows receiving either doses of DESL treatment. This later effect makes the use of the DESL degradable implant impractical within a reproductive management system when cows receive the implant at parturition. Future res earch should focus in GnRH agonist delivery systems in which initiation and termination of treatments could be manipulated practically into a restricted time frame through 22 days postpartum to optimize uterine involution. This may avoid a prolonged period of anovulation.

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99 Table 3 1. Frequency distribution of cows among uterine tone scores at days 8, 15, 22, 29 and 36 postpartum in CON, 1DESL and 2DESL groups 1 Variable (n) No t on e % (n) Moderate t on e % (n) Intense t on e % (n) P value Day postpartum D8 (47) 23.4 (11) 66.0 (31) 10.6 (5) < 0.06 D15 (47) 4.3 (2) 34.0 (16) 61.7 (29) < 0.01 D22 (46) 19.6 (9) 23.9 (11) 56.5 (26) < 0.05 D29 (46) 17.4 (8) 37.0 (17) 45.6 (21) NS D36 (46) 19.6 (9) 50.0 (23) 30.4 (14) Ref. Group Contrast 1 CON (90) 26.7 (24) 46.7 (42) 26.6 (24) Ref. 1DESL + 2DESL (142) 10.6 (15) 39.4 (56) 50.0 (71) < 0.01 Contrast 2 1DESL (75) 12.0 (9) 34.7 (26) 53.3 (40) Ref. 2DESL (67) 9.0 (6) 44.8 (30) 46.2 (31) NS 1 CON (no implant), 1DESL and 2DESL (1 and 2 degradable Deslorelin implant, 2.1 mg, Peptech Animal Health, North Ride, Australia ). DESL implants were inserted subcutaneously in the neck region at 0.5 to 1.5 days postpartum. Ref. = Reference. Group by day postpartum: non significant.

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100 Table 3 2. Frequency distribution of cows among cervical discharge scores at days 8, 15, 22, 29 and 36 postpartum in CON, 1DESL and 2DESL groups 1 Variable (n) Clear or f lecks % (n) Mucopurulent % (n) Purulent % (n) P value Day postpartum D8 (47) 21.3 (10) 36.2 (17) 42.5 (20) < 0.05 D15 (47) 14.9 (7) 55.3 (26) 29.8 (14) < 0.05 D22 (46) 41.3 (19) 50.0 (23) 8.7 (4) < 0.08 D29 (46) 60.9 (28) 39.1 (18) 0.0 (0) NS D36 (46) 73.9 (34) 26.1 (12) 0.0 (0) Ref. Group Contrast 1 CON (90) 42.2 (38) 43.3 (39) 14.5 (13) Ref. 1DESL + 2DESL (142) 42.3 (60) 40.1 (57) 17.6 (25) NS Contrast 2 1DESL (75) 34.7 (26) 44.0 (33) 21.3 (16) Ref. 2DESL (67) 50.8 (34) 35.8 (24) 13.4 (9) < 0.07 Parity Primiparous (80) 33.7 (27) 41.3 (33) 25.0 (20) Ref. Multiparous (152) 46.7 (71) 41.5 (63) 11.8 (18) < 0.01 RFM 2 No (197) 45.7 (90) 41.1 (81) 13.2 (26) Ref. Yes (35) 22.8 (8) 42.9 (15) 34.3 (12) < 0.01 1 CON (no implant), 1DESL and 2DESL (1 and 2 degradable Deslorelin implant, 2.1 mg, Peptech Animal Health, North Ride, Australia ). 2 Retained fetal membranes. DESL implants were inserted subcutaneously in the neck region at 0.5 to 1.5 days postpartum. Ref. = Reference. Group by parity: non significant.

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101 Table 3 3 Proportion of cows cyclic and that ovulated based upon plasma concentrations of progesterone and presence of corpus luteum at CIDR removal (CL1) and first GnRH of the Ovsynch (CL2) based upon per rectum ultrasonography in control (CON), 1 (1DESL) and 2 (2DESL) Deslorelin implants t reated cows Groups Group c ontrast 1 ( P value ) CON (n = 18) 1DESL (n = 14) 2DESL (n = 13) C1 C2 Cyclic % (n=) 94.4 (17) 28.6 (4) 0.0 (0) < 0.01 < 0.01 Ovulated % (n=) 88.9 (16) 57.2 (8) 0.0 (0) < 0.01 < 0.01 CL1 % (n=) 77.7 (14) 7.1 (1) 0.0 (0) < 0.01 < 0.01 CL2 % (n=) 88.9 (16) 21.4 (3) 0.0 (0) < 0.01 < 0.01 1 C1: CON vs. 1DESL + 2 DESL; C2: 1DESL vs. 2 DESL.

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102 Fi gure 3 1. Least squares means ( S.E.) number of class 1 follicles in CON ( ), 1DESL ( ) postpartum period measured by per rectum ultrasonography CON vs. 1DESL and 2DESL (P < 0.01); 1DESL vs. 2DESL (non significant). 4 6 8 10 12 14 16 8 15 22 29 36 Number of follicles Days postpartum

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103 Fi gure 3 2. Least squares means ( S.E.) groups as measured by per rectum ultrasonography at days 8, 15, 22, 29 and 36 postpartum. Class 2 and class 3 (CON vs. 1DESL and 2DESL [P < 0.01]); 1DESL vs. 2DESL (non significant). 0 0,2 0,4 0,6 0,8 1 1,2 class 2 class 3 Number of follicles Follicle classes

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104 Fi gure 3 3. Least squares means (S.E.) accumulated plasma concentrati ons of progesterone ( P 4 ng/ mL ) in CON ( ), 1DESL ( ) period before initiation of pre synchronization CON vs. 1DESL and 2DESL (P < 0.01); 1DESL vs. 2DESL (non significant). 0 2 4 6 8 10 12 2 5 9 13 17 21 25 29 33 41 45 P 4 (ng/mL) Days postpartum

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105 Fi gure 3 4. Least squares means (S.E.) plasma concentrations of PGFM ( p g/ mL ) for CON ( n = 18) 1DESL ( n = 15 ) period. Day (P < 0.01), group (non significant). 0 500 1000 1500 2000 2500 3000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PGFM (pg/mL) Days postpartum

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106 Fi gure 3 5. Least squares means (S.E.) plasma concentrations of PGFM ( p g/ mL ) for cows with metritis ( --, n = 18) and no metritis ( n = 1 3 ) during the postpartum period and before initial systemic antibiotic treatment. Day (P < 0.01), metritis (P < 0.01). 0 500 1000 1500 2000 2500 1 2 3 4 5 6 7 8 9 PGFM (pg/mL) Days postpartum

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107 Figure 3 6. Least squares means plasma concentrations of PGFM (pg/ mL ) profiles for two cows diagnosed with metritis, treated with systemic antibiotics at 5 days postpartum and continued for 4 consecutive days. Patterns of plasma concentrations of PGFM of the selected cows were repr esentative of their corresponding herd mates with metritis and treated with antibiotics 0 500 1000 1500 2000 2500 3000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PGFM (pg/mL) Days postpartum Antibiotics 0 200 400 600 800 1000 1200 1400 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PGFM (pg/mL) Days postpartum Antibiotics

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108 Fi gure 3 7. Least squares means (S.E.) diameter ( cm) of previous pregnant uterine horn (A) and previous non pregnant uterine horn (B) for CON ( n = 18), 1DESL ( n = 15 ) 0.01), CON vs. 1DESL and 2DESL (P < 0.01); 1DESL vs. 2DESL (non significant). 1,5 2,5 3,5 4,5 5,5 8 15 22 29 36 Diameter (cm) Days postpartum A 1,5 2,5 3,5 4,5 5,5 8 15 22 29 36 Diameter (cm) Days postpartum B

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109 Fi gure 3 8. Least squares means (S.E.) diameter ( cm) of the uterine horn l umen of the previous pregnant uterine horn (A) and previous non pregnant uterine horn (B) for CON ( n = 18), 1DESL ( n = 15 ) period. In A and B: Day (P < 0.01), CON vs. 1DESL and 2DESL (P < 0.01); 1DESL vs. 2DESL (non significant). 0 0,5 1 1,5 2 2,5 3 8 15 22 29 36 Diameter (cm) Days postpartum A 0 0,5 1 1,5 2 2,5 3 8 15 22 29 36 Diameter (cm) Days postpartum B

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110 Fi gure 3 9. Least squares means (S.E.) diameter ( cm) of cervix for CON ( n = 18), 1DESL ( n = 15 ) 0.01); CON vs. 1DESL and 2DESL, an d 1DESL vs. 2DESL (non significant). 1,5 2,5 3,5 4,5 5,5 9 15 22 29 36 Diameter (cm) Days postpartum

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111 CHAPTER 4 EFFECT OF SOURCE OF SUPPLEMENTAL SELENIU M ON UTERINE HEALTH, REPRODUCTION AND LAC TATION IN DAIRY COWS DURING SUMMER IN FLO RIDA Introduction Selenium (Se) is an important component of antioxidant enz ymes. Mammalian selenoproteins catalyze biochemical reactions associated with antioxidant defense systems, thyroid hormone metabolism and redox control of cell reactions (Arthur, 1999). For example, Se in the form of selenocysteine is an essential componen t o f enzyme s such as glutathione peroxidase, thioredoxin reductase and i odothyronine d eiodinases One mole of purified glutathione peroxidase contains four gram atoms of Se (Rotruck et al., 1973). Deiodinase type I is expressed in the thyroid gland, liver, kidney, and pituitary gland and it contains a Se atom bound to its active site in the form of selenocysteine (Behne et al., 1990). Deiodinase type I position of the phenolic ring of t hyroxin ( T4 ) to form triiodothyronine ( T3 ) which is the active form of the thyroid hormones. Thyroxine is the predominant thyroid hormone in the circulation and has little, if any, inherent biological activity ( Chopra et al., 1978). The T3:T4 molar rati o can refl ect the deiodinase type I activity (Kahl et al., 1995). Absorption of orally administered sodium selenite was 35% in sheep compared to 85% in swine (Wright and Bell; 1966). Lower absorption for sheep is likely caused by ruminal selenite reduction to insolu ble compounds such as elemental Se or selenides. In rats absorption of organic Se in the form of selenomethionine was 80% compared to 35% absorption when inorganic Se was fed as selenite (Venderland et al., 1992). Limited information is available on differ ential absorption rates of Se from organic and inorganic sources in ruminants. Extrapolation of data collected from non ruminants should be done with caution for application to ruminants because

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112 of extensive ruminal metabolism of Se. In ruminants excretion of Se occurs through feces, the main route, urin e and exhalation via the lungs. Since 1987 cattle supplementation of Se in the United States is regulated by the Food and Drug Administration (FDA USA) at a concentration of 0.3 mg/Kg of dry matter (DM). So dium selenite and sodium selenate are the major sources of supplemental Se for dairy and beef cattle in the USA. In 2003 an organic source of Se in the form of Se yeast (Sel P lex, Alltech Inc Nicholasville, KY) was approved for dairy and beef animal supplementation in the USA. The maximum allowed supplementation ra te was maintained at 0.3 mg/Kg of the DM. In a review by Weiss (2003), 18% and 90% increases in whole blood and milk Se concentrations were reported when Se yeast was fed compared with selenite or selenate in ten studies using beef or dairy cattle. Statistical differences were detected for whole blood concentrations of Se in five of these studies and increased milk concentration of Se in the majority of the studies. The relative activit y of glutathione peroxidase was increased 18% for 11 studies with variable statistical differences depending on study design. Selenium in feedstuffs occurs predominantly in the amino acids selenomethionine and selenocysteine (Whanger, 2002) whereas the maj ority of supplemental Se is fed in the selenite or selen a te forms. Numerous studies have shown the retention of the selenomethio nine form, as found in Se yeast, to be greater than the inorganic sources, both in tissues and in blood (Knowles et al., 1999; P ehrson et al., 1999). Additionally, the effects of Se on reproductive function of dairy cows have been inconsistent possibly associated with differences in routes of administration, doses and concurrent treatment with vitamin E. Several studies have shown that administration of Se vitamin E or the combination of both reduced incidence of retained fetal membranes (Julien et

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113 al., 1976a and 1976b; Arechiga et al., 1994), and metritis (Harrison et al., 1984), clinical mastitis (Smith et al., 1984), improved fe rtility (Segerson and Ganapathy, 1981; Arechiga et al., 1994) and increased ability of neutrophils to kill Staphyloccocus aureus (Hogan et al., 1990). Beneficial effects of Se supplementation for animal health could be explained by the increased neutrophil bactericidal activity. In contrast, other studies have shown little or no beneficial effect of administration of vitamin E and/or Se (Gwazdauskas et al., 1979; Hidiroglou et al., 1987; Stowe et al., 1988). Neutrophils generate reactive oxygen metabolites within the phagossome environment to kill phagocytized bacteria, but it is essential that neutrophils regulate the balance betwe en reactive oxygen metabolites (i.e., superoxide anion and hydrogen peroxide) in order not to damage cells, leading to cell death. Glutathione peroxidase is one of the selenoenzymes capable of protecting the cell against oxidative injury The objectives were to compare the effect of dietary organic Se ( Se yeast, Sel P lex, Alltech I nc Nicholasville, KY) with inorganic sodium selenite on responses such as: a) u terine health in the early postpartum; b) i ncidence o f health disorders; c) pregnancy per artificial insemination (AI) after two postpartum inseminations during the summer sea son; d) p regnancy loss after first postpartum insemination during the summer season; e) lactation performance and f) milk somatic cell score (SC S ). Also, the e ffects of pre synchronization using a controlled internal drug releasing (CIDR, EAZI BREED; Pfize r Animal Health, New York, NY ) containing 1.38 g of progesterone (P 4 ) followed by an Ovsynch was evaluated. Therefore, the experimental design was comprised of a 2X2 factorial design for evaluation of pregnancy per AI after first postpartum insemination.

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114 M aterials and Methods Animals Experimental D esign and Feeding The experiment was conducted at a north Florida dairy farm comprised of 3,000 Holstein cows that were milked thrice daily. Cows (n = 769) were enrolled during the period of March 2005 to August 2005. Cows were allocated randomly into two experimental diets begun at approximately 25 days prior to the expected date of parturition. Primiparous and multiparous cows were blocked when assigned to diets. Cows more than 100 days dry were not included in the experiment. Calving assistance categories were recorded ro utinely. Only animals with calving assistance categories 1 (no assistance), 2 (minor assistance) and 3 (mild assistance) remain ed in the experiment. Cows that had been fed for less than 14 days in the pre partum period and gestation lengths less than 265 d ays were excluded from the experiment. Cows fed either an inorganic source of Se (sodium selenite; SS) or an organic source of Se (Se yeast; SY; Sel Plex; Alltech Inc., Nicholasville KY) composed of approximat ly 60% selenomethionine and 40% other seleno a minoacids according to the supplier. Cows were fed a total mixed ration to meet or exceed the requirements of the NRC (2001) for lactating Holstein cows weighing 650 kg and producing > 40 kg/d of milk containing 3.5% fat. Pre partum diets were formulated t o provide supplemental Se at a concentration of 0.45 mg/Kg of DM according to the recommendation of the farm veterinarian, and 0.3 mg/Kg of DM in the postpartum diets. Ingredients and chemical composition of the diets were similar for SS and SY groups and pooled values are summarized in Table 4 1. Postpartum experimental diets were fed to at least 81 days postpartum (dpp). Primiparous and multiparous cows of both experimental diets were housed together during the pre partum (enrollment to parturition) and p ostpartum (0 to 30 dpp) periods. Pre and postpartum cows were housed in open dry lots of the same size that w ere separated solely by the

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115 feed bunk area (oriented face to face) and longitudinal fence. Pens were equipped with a shade structure, fans, and wa terers of the same size, orientation and location. Fans were equipped with high pressure nozzles and both fans and nozzles were activated once ambient temperature reached 23 o C. Pre and postpartum pens housed an average of 85 and 114 cows at any time durin g the study, respectively. After 30 dpp primiparous and multiparous cows were moved into free stall pens and grouped by parity. Multiparous and primiparous cows in each dietary group were housed in a separate free stall barn. Moreover, at the time in whic h approximately half of the total target population of experimental cows reached 40 dpp, primiparous cows with their respective diets interchanged pens and multiparous cows with their respective diets were moved into free stall pens located in a common bar n. All free stall pens were equipped with fans and high pressure nozzles immediately above the stalls and along the feed bunk area. Fans and nozzles were activated once ambient temperature reached 23 o C and nozzles inactivated once ambient relative humidity reached 91% inside the free stall barns. The temperature ( o C) and relative humidity (%) were recorded hourly in each pen and free stall barns by data recorders (HOBO H8 Pro Series Part No. H08 032 08, Onset Computer Corp., Bourne, MA), operated by a comp uter software program (BoxCar Pro 4.0 Starter Kit, Part No. BCP4.0 ON, Onset Computer Corp., Bourne, MA). One recorder was placed in each study pen at a height of 2.0 m from the floor and placed under the central shades in the pre and postpartum pens or i mmediately above the stalls in the fee stall pens. The probes recorded data during the entire experimental period. The temperature humidity index (THI) was calculated as: THI = td (0.55 0.55 RH/100) (td 58), where td is the dry bulb temperature in F and RH is relative humidity expressed as a percentage (NOAA, 1976). For each 24 hour period, average daily mean and maximum THI were determined and seasons defined as cool (THI < 72) and

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116 ubse t of SS (n = 11) and SY (n = 12) cows was fitted with a temperature data logger (HOBO Water Temp Pro v1, Onset Computer Co.) attached to a blank CIDR that was inserted into the vagina. The data logger recorded vaginal temperature at 15 min intervals fo r a period of 5 days per cow in three replicates. A new subset of cows was enrolled in each replicate so that vaginal temperatures were recorded from 12 August to 27 August. Dry matter intake (DMI) for each diet housing group was recorded. Dry matter intak e was obtained daily based on the difference in the amount of DM fed minus orts divided by the number of animals present in each diet housing group. All lactating cows had attached to their front leg a pedometer (S.A.E. Afikim) for individual identificatio n and daily activity. Therefore, to ensure correct number and location of animals in each pen the Afikim computerized dairy management system was used twice a week and at each experimental activity that required contact with the cows. Health M onitoring T ra ined farm employees monitored p re partum cows for signs of parturition daily in a 24 hour period. Cows that had not shed their fetal membranes within 12 hours based upon visual inspection of the vulva and pe rineum were considered to have retained fetal membrane s (RFM). During the first 10 dpp, all cows were monitored daily after the morning milking (i.e., at approximately 7:30 am) for rectal temperature using a digital thermometer (GLA M525/550, GLA Agricultural Electronics, San Luis Obispo, CA). Cows wi (103. 1 F) were considered to have fever Additionally, cows were evaluated for cervical discharge on days 5 and 10 postpartum using a disposable foil lined cardboard vaginal speculum (Milburn Distributors, Ocala, FL ) Appe arance of cervical discharge was categorized into clear

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117 mucus without flecks, clear mucus with flecks lochia, muc o purulent (50% clear mucus and approximately 50% of pus) and purulent (> 50% pus or watery red brown with foul smell). Ketosis was determined by a pink to purple color on the Ketostik urine strip (Bayer Corp., Shawnee Mission, KS). M astitis was determined by presence of fl ec ks or clots in milk or a serum like secretion from an affected quarter and/or swelling in an affected quarter with system ic signs including fever A new case of mastitis w as defined for the same cow when a different quarter was affected or a period of 21 d ays had elaps ed since the previous diagnosis. Displacement of the abomasum (DA) was determined when a tympanic sound was heard over the left or right paralumbar fossa region during simultaneous percussion and auscultation of the left or right flank Ketosis, mastitis and DA were examined at 4 and 9 dpp or any time of milk production depression or increased milk conductivity. T o further evaluate uterine health uterine cytology w as performed for all cow s at 37 dpp Briefly, t he vulva was cleaned with a p ovidone iodine s crub (0.75% titratable iodine and 1% povidone solution, Agripharm, Memphis, TN, USA) followed by c hlorhexidin e d iacetate (Nolvasan, Fort Dodge, WI) and dried off with clean paper towel. A silicon Foley catheter (i.e., 21 inches 18 Fr ench and 5 mL ) w as introduced through the cervix into the previous pregnant uterine horn determined as the horn with greater diamet er and length by per rectal palpation of the uterus. The balloon of the Foley catheter w as filled with air to a volume enough to occlude the horn cranially Furthermore, a 20 mL s terile saline (0.9% NaCl ) w as infused into the uterine horn, followed by brie f per rectal massage of the horn and aspirat ion of fluids using a syringe (60 mL) attached to the connector of the Foley catheter The aspirated fluid w as placed into a 6 0 mL container and upon visual inspection categorized into clean or translucent, clean with flecks of pus, cloudy, mucopurulent ( 50% clear mucus and approximately 50% of pus ) and purulent

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118 The aspirated fluid w as vortexed to obtain a uniform aliquot (20 L) and pipette d onto a glass slide and smeared Smear s w ere air dried and stained (Protocol Hema 3, Fisher Diagnostics, Middletown, VA) Slides were examined and number of total leukocytes epithelial endometrial cells and neutrophils were counted to complete 100 cells per slide to calculate percent neutrophils S ubclinical endometritis was de fined when the proportion of neutrophils > 18% ( Kasimanickam et al., 2004). Cows were evaluated for body condition score ( BCS ) at the day of enrollment, at parturition, 37 dpp, and at the first service pregnancy diagnosis (i.e ., 110 dpp) Scores were given by two veterinarian s based on a 1 (thin) to 5 (obese) scale using a quarter point system (Edmonson et al., 1989). Changes in BCS were obtained by subtracting BCS at parturition from BCS at enrollment, BCS at 37 dpp from BCS a t parturition, and BCS at first service pregnancy diagnosis from BCS at 37 dpp. Estrous S ynchronization and R e synchronization P rotocols To test pre synchronization protocols, cows within diets were allocated randomly into a Presynch (Moreira et al., 2001) begun at 45 dpp with two injections of PGF ( 25 mg dinoprost tromethamine i.m., Lutalyse Sterile Solution; Pfizer Animal Health, New York, NY ) injected 14 days apart or an insertion of a CIDR begun at 61 dpp for 7 days followed by an injection of PGF 8 hours after CIDR removal. At 12 days after Presynch and 3 days after CIDR removal, an Ovsynch protocol begun in both pre synchronization groups with a GnRH injection (100 g; gonadorelin diacetate tetrahydrate, i.m., Cystorelin Merial Ltd., Athens, GA ) followed 7 days later by an injection of PGF and a final injection of GnRH 56 hours later. Timed artificial insemination (TAI) for first service was performed 16 hours after the second GnRH injection of the Ovsynch protocol. Also, c ows were inseminat ed when observed in estrus after the second

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119 injection of PGF in the Presynch group or between the day of the last PGF and TAI for both groups. At 20 to 23 days after the first insemination, all cows received an injection of GnRH, followed 7 days later by pregnancy examination (i.e., at 27 to 30 days after insemination) using per rectum ultrasonograghy (Aloka 500 scanner, equipped with a 5 MHz linear rectal transducer) of the uterus to identify presence of an embryo and an embryonic heart beat Non pregn ant cows were injected with 25 mg of PGF and then injected with 100 of GnRH 56 h ours later. A TAI was performed 16 hours after the last GnRH for the second service. First service pregnant cows examined by ultrasonography (i.e., at 27 to 30 days after TAI) were re examined by per rectum palpation at 55 days after first service. Cows submitted for second service were only evaluated at 45 days after insemination by palpation per rectum Blood S amples and P rogesterone A ssay B lood samples were collected by puncture of coccygeal vessels into evacuated tubes containing K 2 EDTA (Vacutainer BD, Franklin Lakes, NJ, USA). Samples were placed immediately in to an ice bath until centrifugation for 20 minutes at 2619 x g After centrifugation, plasma was harvested a nd stored frozen at 20 C until assayed for concentrations of P 4 Blood samples were collected from all cows at time of uterine cytology (i.e., 37 dpp) to determine the presence of a functional corpus luteum. Furthermore, for the Presynch group, blood sam ples were collected at the second PGF of the Presynch and 12 days later at the initiation of the Ovsynch In the CIDR group, blood samples were collected at the time of insertion of CIDR and again 8 hours after CIDR removal just prior to PGF injection With this set of blood samples, cows were considered to have initiated estrous cycles if plasma concentration of P 4 was greater than 1 ng/ mL in at least one of the two samples (cyclic) or non cyclic or anovulatory if both samples were below 1 ng/m L A second set of blood samples was collected for all cows at the

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120 time of the first insemination and 7 days later. With this set of blood samples, cows were considered to have ovulated a synchronized follicle if plasma concentration of P 4 was below 1 ng/mL at AI and greater than 1 ng/mL 7 days later (ovulated), other combinations of plasma concentrations of P 4 were considered as a failure to ovulate (non ovulated). Plasma concentration of P 4 were determined using a Coat A Count Kit (DPC Diagnostic Products Incorporation, CA, USA) a solid phase 125 I radioimmunoassay designed for the quantitative measurement of P 4 in plasma. Plain tubes were used in duplicate for total counts and non specific binding. Standard curve dilution was prepared using coated tubes in duplicate. A 4 ), 0.1, 0.25, 0.5, 2, 5, 10, 20 and 40 ng/mL were added to the tubes. Reference samples (100 L) containing low (0.55 ng/mL), medium (1.39 ng/mL) and high (3.6 ng/mL) concentrations of P 4 were used. Experimental 125 I] P 4 to all tubes. In every 10 th sample, duplication was performed. An intra assay coefficient of variation was calculated from duplicated reference sample s obtained from all assays. Reference samples of low (0.54 ng/mL), medium (1.39 ng/mL) and high (3.6 ng/mL) concentration had intra assay coefficients of variation of 4.3%, 5.1% and 2.8% respectively. Inter assay coefficient of variation for low, medium a nd high samples were 10.4%, 13.3% and 8.2%, respectively. Sensitivity of the assay was 0.1 ng/mL. Pl asma C oncentration of Selenium and T3:T4 Molar Ratio Blood samples for analysis of plasma concentrations of Se, t hyroxine (T4) and triiodothyronine (T3) were collected processed and stored as described for P 4 samples. Blood s amples were collected at 25 days pre partum (8 primiparous and 10 multiparous cows per diet) and at parturition, 7, 14, 21 and 37 dpp (10 primiparous and 10 multiparous cows per diet ).

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121 Plasma concentrations of Se were analyzed using a fluorometric method described by Whetter and Ullrey (1978). All repeated samples of each animal were analyzed in the same assay that was balanced for numbers of animals for SS and SY diets. For each assa y, a linear regression of fluorescence emission was calculated from known duplicated Se standard solutions Also a 0.3 g of a certified biological reference (National Bureau of Standards Bovine Liver SRM 157 7a U.S. Department of Commerce, National Institute of Standards and Technology, Gaithersburg, MD) was used to ensure the reliability of the analytical method. Intra and interassay coefficients of variation for biological reference were 10.9% and 11. 5 %, r espectively. Concentrations of T4 and T3 were analyzed using commercially available radioimmunoassay kit s (MP Biomedicals, Diagnostic Division, NY) that have been validate d for bovine plasma (Kahl et al., 1995). S ample s w ere analyzed in duplication for bot h T 4 and T 3. I ntra and interassay coefficient of variation were for T4 ( 2.2 and 8%) and T3 (1.6% and 6 %), respectively. Assay s ensitivity was 5 .0 and 0.06 ng/mL for T4 and T3, respectively. Plasma T3:T4 molar ratio (T3/T4 1.1934) was used as an estimati a selenoprotein. Milk Yield, M ilk Composition and Feed Samples Milk yield and milk contents of protein, fat and SCC were monitored for individual cows monthly during official Dairy Herd Improvement (DHI) association (Raleigh, NC) test ing for a minimum of 4 months and a maximum of 8 months per cow. Somatic cell count (SCC) for individual cows were converted into SCS (SCS = Log 10 (SSC/12,500)/Log 10 2) Fat corrected milk (FCM) was calculated for 3.5% fat as milk (Kg) x (0.4255 + [ 16.425 x {fat (%)/100 }] ). Total mixed rations were sampled weekly at the time of feeding from respective feed bunks and immediately frozen in plastic bags Diets were composited (i.e., 4 samples/month) and

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122 dried (55 o C for 48 hours) to calculate DM and further composited into a 2 month sample Dry samples were ground in a Wiley Mill (Arthur H. Thomas Co., Philadelphia, PA) to pass a 2 mm screen, and analyzed for their contents of OM (AOAC, 2000), ADF and NDF (Van Soest et al., 1991), N using a N a nalyzer (FP 528 Nitrogen Determinator, LECO Corporation, St. Joseph, MI), with CP calculated as N percentage multiplied by 6.25, and lignin in sulfuric acid. Minerals were analyzed at the Dairyland Laboratory (Arcadia, WI) using an inductively coupled plasma emission spectrometer (Thermo Garrell Ash, Franklin, MA). Concentration of Se in total mixed rations was analyzed by the University of California Davis Agriculture and Natural Resources Analytical Laboratory according to the method of vapor generati on inductively coupled plasma emission spectrometer (Tracy and Moeller, 1990), with a minimum detection of 0.05 mg of Se/kg. I ngredient s and chemical composition of the pre and postpartum diets were similar for SS and SY groups and pooled values are shown in Table 4 1 Statistical a nalyses Proportion of cyclic cows, pregnancy per AI pregnancy losses and cumulative proportion of pregnan t cows after two services were analyzed by logistic regression (SAS Institute Inc; Version 9.1). The logistic regression stepwise selection procedure was used for all independent variables and possible interactions with pre determined significance levels set for a variable to be entered with a P P Mathematical model for cyclic responses included diet, pre synchronization protocol (i.e., Presynch vs. CIDR), parity, calving assistance categories (no assistance, minor as sistance and major assistance), cervical discharge scores (at 5 and 10 dpp), BCS, changes in BCS and all higher order interactions. Mathematical model for pregnancy responses included diet, pre synchronization protocol, parity, calving assistance categorie s, cyclic status (cyclic or non cyclic), ovulation status (ovulated or non ovulated), type of insemination (i.e., insemination upon estrus or TAI), sire, inseminator, BCS,

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123 changes in BCS and all higher order interactions. Body condition scores data were ca tegorized in to quartiles using the Univariate procedure of SAS. After identifying variables that were significant, the model was reduced and analyzed using the generalized linear model (G ENMOD ) procedure of SAS with a binomial distribution and a logit link function to obtain odds ratios, confidence intervals and probability values The GENMOD procedure of SAS was used to analyze categorical responses such as cervical discharge scores and subclinical endometritis For cervical discharge scores a Generalized Estimating Equations (GEE) with an independent covariance structure, and multinomial distribution of response and a logit link function were used. The mathematical model included diet day (i.e., days 5 and 10 postpartum), parity, calving assistance categories, RFM, BCS ( enrollment and parturition), changes in BCS and all higher order interactions. Cow was used as a repeated subject. For subclinical endometritis a binomial distribution of response and a logit li nk function were used. The mathematical model included diet, parity, calving assistance categories, cervical discharge scores at 5 and 10 dpp, RFM, BCS, changes in body condition scores and all higher order interactions. Binomially distributed health data (i.e., RFM, mastitis, ketosis, lameness, and displacement of abomasum) obtained from farm records were analyzed in GENMOD procedure of SAS with a binomial distribution of response and a logit link function. Continuous responses such as plasma concentration s of Se, T3:T4 mol ar ratio, monthly milk yield and SCS were analyzed using the mixed model procedure of SAS (Littell et al., 1996). The covariance structure that provided the best relative goodness of fit based upon penalty criteria (Bayesian criterion) wa s used. The statistical models consisted of diet, parity, day relative to parturition and diet by day interaction. Cow, the random variable was considered as the

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124 experimental unit nested within diet and parity Differences between means of the diet by day interaction were tested using the slice option of the mixed procedure. Results Animals THI, Diets and DMI A total of 768 cows were enrolled and calved during the period of March to August 2005. Cows (SS [n=51] and SY [n=57]) were excluded at parturition because the length of pre partum feeding was less than 14 days. Also, a total of 78 cows (SS [n=41] and SY [n=38]) were excluded during the experimental period because of movement inadvertently out of experimental lots, or for health, culling and death rea sons. Parturitions throughout the experimental period occurred at the same monthly rate for the two diets and parities. Therefore, 582 cows (primiparous [n=208] and multiparous [n=374]) remained in the experiment. Length of pre partum feeding was shorter ( P < 0.01) for multiparous (21.4 0.4 days) than primiparous (25.5 0.5 days) cows and did not differ between SS and SY diets (23.4 0.5 days ). Time period of postpartum feeding was shorter (P < 0.01) for primiparous (150 2 days) than multiparous (160 2 days) cows and did not differ between SS and SY diets (155 2 days ) Average BCS were not different between diets at each measurement time: enrollment (2.74 0.9), parturition (2.4 0.6), 37 dpp (2.25 0.8) and first service (2.3 0.6). Frequency distribution for all cows (n = 510) among calving assistance categories was not different between diets (no assistance [22%]; minor assistance [50%] and major assistance [28%]). P rimiparous (n = 185) compared with multiparous (n = 325) cows had a greater ( P < 0.01) frequency distribution of major assistance (48.1% and 16.2%) and less (P < 0.01) minor (46.5 % and 52 % ) to no (5.4% vs. 31.4%) assistance, respectively. W eekly mean and maximum THI were calculated from hourly measurements for the duration of the experiment. Data obtained from a thermometer located outside the barns

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125 indicated that mean THI was consistently below 72, a non heat stressed environment from February to end of May (64.2 1 .0) and above 72 from May to September (79.5 0.8). Based on the maximum THI, outside environment was not considered to be heat stressful from February to April (68.8 1.0) and was heat stressful from end of May to September (81.5 0.8). When daily THI inside of barns was considered, cows began to be exposed to stress from mid May to September (78.8 0.4) or from beginning of May to September (81.1 0.4) based upon mean and maximum THI, respectively. W eekly mean and maximum THI were similar a mong pre and postpartum pens. Additionally, hourly mean vaginal temperature collected in August was not different between cows located in separate pens that were supplemented with SS or SY (Figure 4 1). Vaginal temperatures were normalized for the day hour time for cows in both dietary pens. Vaginal temperatures oscillated (P < 0.01) from a minimum of 38.9 C to a maximum of 39.6 C during a 24 hour period (Figure 4 1). The lowest values of vaginal temperature were coincident with the time milking f or both housing g roups of cows. Chemical compositions of pre and postpartum diets were similar for SS and SY diets, and pooled values are presented (Table 4 1 ). Group DMI did not differ between diets during the pre partum period (11.8 0.8 Kg of DM/day) and during the fi rst 30 dpp (16.4 0.9 Kg of DM/day) After approximately 30 d pp, primiparous and multiparous cows were allocated to separate pens within each experimental diet. Multiparous cows of SS and SY groups were placed in lots located in different free stall barns from April 10 th to July 25 th During this period, group DMI did not differ between SS (28.7 1.6 Kg of DM/day) and SY (27.3 1.2 Kg of DM/day) diets. After July 25 th cows of each experimental diet were moved into separate lots located within the same f ree stall barn until the end of feeding (September 26 th ). During this second period, DMI did not differ between SS (23.0 1.2 Kg of DM/day) an SY (23.0 1.8 Kg of DM/day) diets.

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126 Primiparous cows of each diet were placed in lots located in different free stall barns from April 10 to August 3. On August 3, which was the time that half of the expected population of primiparous cows achieved approximately 40 dpp, primiparous cows and diets (i.e., SS and SY diets) were switched between locations until the end of experimental feeding (September 26). Overall group DMI for primiparous cows did not differ between the two periods (22.1 Kg of DM/day) H owever DMI for SS diet was less (21.4 0.2 Kg of DM/day) compared with SY (22.7 0.2 Kg of DM/day) during both pe riods (P < 0.01). Plasma C oncentration of Se and T3:T4 Molar Ratio Plasma concentrations of Se at the time of initiation of experimental diets (i.e., 25 days pre partum) did not differ between SY and SS cows (Figure 4 2). However, plasma concentrations of Se were greater (P < 0.01) in S Y compared with S S cows at parturition, 7, 14, 21 and 37 dpp (Figure 4 2) for both primiparous (n = 10/diet) and multiparous (n=10/diet) cows (diet by parity; non significant). Based on DMI and Se content of the TMR, intakes of Se in the pre partum and postpartum periods were approximately 5.24 and 4.91 mg/day, respectively and were the same for SS and SY cows. Plasma T3:T4 molar ratios tended (P = 0.10) to be greater for primiparous and were greater (P < 0.05) for multiparous cows in SY compared with SS diets at parturition (Figure 4 3, A and B). Overall T3:T4 molar ratio was less (P < 0.05) in primiparous (3.5 0.2) compared with multiparous (4.2 0.2) cows. Postpartum H ealth Frequency distribution of c ows with at l east o ne d ay of fever (r ectal t emperature 39.5 o C) with in the f irst 10 dpp was greater (P < 0.01) for primiparous (40.5%, 83/205) cows than for multiparous (19.2%, 71/369) cows. A diet by parity interaction (P < 0.05) was observed and a

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127 lower proportion of multip arous cows had fever in SY compar ed with SS diets (Table 4 2), while there was no difference between SS and SY for primiparous cows (Table 4 2). Frequency distribution of c ows with at l east o ne d ay of fever with in the f irst 10 dpp was greater (P < 0.01) fo r cows with lower BCS (2.75 3.25) compared with a greater BCS (3.5 3.75) at parturition and greater (P < 0.01) for cows with purulent cervical discharge score (Table 4 2). Furthermore, a tendency (P < 0.08) for a diet by discharge interaction was detec ted. For cows diagnosed with mucopurulent and purulent cervical discharges, there was a lower proportion of cows with fever in SY (20.9% [18/86] and 42.1% [8/19]) compared with SS (28.7% [25/87] and 54.4% [25/46]) cows, respectively. A similar proportion o f cows with fever in SS and SY diets (17.0% and 27/159) were detected when cows had clear discharge. Frequency distribution of cows with a purulent cervical discharge score was greater (P < 0.05) at 10 dpp compared with 5 dpp that was associated with a lower frequencies distribution of cows with mucopurulent and clear or no discharges (Table 4 3). Day of cervical discharge examination by diet, parity, RFM and calving assistance interactions were not significant. Therefor e, all observations at 5 and 10 dpp were pooled for the main effects. Frequency distributions of purulent and mucopurulent cervical discharges were less (P < 0.01) for SY compared with SS diets and for multiparous compared with primiparous cows (Table 4 3) Similarly, frequency distribution of cows with purulent and mucopurulent cervical discharges was lesser (P < 0.05) for cows with no calving assistance compared with cows in minor or major assistance categories (Table 4 3). The majority of cows that exper ienced RFM had purulent cervical discharge that was of greater (P < 0.01) proportion than cows that released the placenta within 12 hours postpartum (Table 4 3).

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128 and 2.4% [n = 7]), ketosis (4.2% [n = 12] and 5.9% [n = 17]) and mastitis (14.7% [n = 42] and 14.2% [n = 41]) did not differ between SS (n=285) and SY (n=289) cows, respectively. A total of 544 animals had their previous pregnant uterine horn successfully flushed w ith 20 mL of sterile saline. The average volume of saline recovered was 18.2 mL (S.E. = 6 mL; recover y rate of 91%). The overall incidence of subclinical endometritis was 31.2% (170/544) and was not affected by diet, parity or the interaction of both (Tabl e 4 4). Frequency distribution of cows with subclinical endometritis was greater (P < 0.05) for cows that had RFM and for cows diagnosed with purulent cervical discharge at 5 dpp, and tended (P < 0.10) to be greater for cows with mucopurulent cervical disc harge 10 dpp (Table 4 4). Moreover, frequency distribution of cows with subclinical endometritis was greater (P < 0.01) when visual inspection of the uterine flushes was categorized in flecks, mucopurulent and purulent compared with clean appearance (Table 4 4). F irst and S econd S ervices P regnancies per AI The p roportion of cows found cyclic was not affected by diet and pre synchronization protocols, but was less (P < 0.05) for primiparous cows, cows with BCS equal to or lower than 2.75 at 37 dpp and cows i n categories of minor or mild calving assistance (Table 4 5). Additionally, the proportion of cows ovulating to the Ovsynch program after the first insemination was not affected by diets (SS [82.3%; 205/249] and SY [82.7%; 187/226]) or pre synchronization protocols (Presynch [84.8%; 190/224] and CIDR [80.4%; 202/251]), but was greater (P < 0.05) in cows classified previously as cyclic (84.0%; 325/387) compared with anovulatory (75.6%; 65/86). All experimental cows were inseminated from May to October of 200 5 during the period in which maximum daily THI inside the barns indicated heat stress (THI = 81.1 0.4). A greater

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129 2 = 63.9, P < 0.01) proportion of cows was inseminated upon estrus in the Presynch (43%, 111/258) compared with CIDR (11.5%, 29/253) proto col. In the Presynch protocol, cows were inseminated at 4 days (S.E. = 2) after the second PGF of the Presynch (63 3 dpp) and in the CIDR protocol at 3 days (S.E. = 1) after CIDR removal and PGF injection (70 3 dpp). Pregnancy per AI were greater ( P < 0.05) for cows inseminated upon estrus (30.7%, 43/140) compared with TAI (21.8%, 81/371) regardless of pre synchronization protocol. First service pregnancy per AI at 27 to 30 and 55 days after first insemination and pregnancy loss were not affected b y diets or pre synchronization protocols (Table 4 6). First service pregnancy per AI at 27 to 30 days after insemination was affected by BCS at enrollment, BCS at 37 dpp and ovulation status (Table 4 7). Pregnancy loss w as less (P < 0.01) in cows with BCS at TAI equal or above 2.75 (36.4%, 40/110) compared with cows below 2.75 (58.3%, 5/12). 2 = 6.9, P < 0.01) proportion of cows were inseminated for second service upon estrus in the Presynch (35.9%, 75/209) compared with the CIDR (48.8%, 98/201) protocol s. Cows were inseminated in average at 19 days (S.E. = 4) after the first insemination, and interval between AI did not differ between Presynch and CIDR protocols. Second service pregnancy per AI were not different for cows inseminated upon estrus (16.2%, 28/173) compared with TAI (12.7%, 30/237) regardless of pre synchronization protocol. Second service pregnancy per AI was greater (P < 0.05) for cow s in SY compared with SS diets (Table 4 6). Milk Y ield and Composition Cows supplemented with SY (n = 249) had greater (P < 0.0 5 ) yields of milk, 3.5% FCM, milk fat and milk protein compared with SS (n = 262) during the first 8 months of lactation (Table 4 8). Source of Se did not affect milk fat and protein percentages (Table 4 8). Milk yield was greater (P < 0.05) for cows in SY compared with SS diets at the first month and during months 5 to 8 of lactation (Figure 4 4) and similar responses occu rred for 3.5% FCM yields.

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130 Milk yields, 3.5% FCM yield, percent of true protein content in milk and total protein content in milk were greater (P < 0.01) for multiparous (n = 325; 40.0 Kg/day, 39.0 Kg/day, 2.91%, 1157 g/day) compared with primiparous (n = 1 86; 33.3 Kg/day, 32.5 Kg/day, 2.86%, 948 g/day) cows, respectively. A diet by month interaction (P < 0.05) for SCS was observed (Figure 4 5). Somatic cell score was greater and less for cows in the SY compared with SS diets at month 2 and months 7 to 8, re spectively. Somatic cell score was greater (P < 0.01) in multiparous ( 2.4 0.0 5 ) compared with primiparous ( 2.77 0.0 3 ) cows. Discussion Supplementation of SY during the pre and postpartum periods of cows increased plasma concentrations of Se during the postpartum period and seleno enzyme (i.e., ) activity at parturition. Proportion of multiparous cows with fever and purulent cervical discharge were less in S Y diet, but there was no effect of diet for primiparous cows. The p roportion of cyclic cows, pregnancy per AI and pregnancy loss after first insemination w ere not affected by pre synchronization protocols and diets. However, pregnancy per AI was greater after second insemination for cows supplemented with SY Increased milk yield and milk components (fat and protein) for cows supplemented with SY occurred in later months of lactation that was associated with a less S CC in milk compared with cows in the SS diet. Concentration of Se in pre and postpartum diets was similar and equiva lent to what was added via the supplements as inorganic and organic Se sources in the two periods. The majority of the feedstuffs of the diets were from the s outheast ern USA. Chemical analyses of the dietary components indicated very low endogenous con cent rations of Se. Indeed, s outheast ern USA is considered a region in which soil is deficient in Se with approximately 80% of forages and grains containing less than 0.1 ppm of Se (Kubota et al., 1967). Kubota et al (1967) reported that in Florida, where corn crops were raised for silage, more than 80% of crops contained less than

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131 0.05 ppm of Se. Selenium content of feedstuffs is mainly as an organic form. Small portions of Se were provided by the feed ingredients of the diet and the majority of Se was provide d as supplements (i.e., SS and SY ). Thus the SS diet was enriched with an inorganic form of Se contrary to the SY supplement with the dietary Se provided as an organic form. The low concentration of Se in the feed ingredients allows for a direct comparison of the Se sources utilized in the two experimental diets regarding the absorption and retention of this mineral by lactating dairy cow s Supplementation of cows with SS above (pre partum) and at the maximum (postpartum) concentration allowed by the FDA was unable to meet the requirements of dairy cows in this experiment as compared to supplementation with SY C ows fed the diet with a SS had p lasma concentration s of Se during the postpartum sampling pe riod that were considered to be marginally deficient ( i.e., 0.069 g/ mL ) In contrast supplementation with SY resulted in adequate concentrations ( i.e., 0.087 g/ mL ) ( Dargatz and Ross 1996) Under the conditions of this experiment with a low background o f Se in the diets, supplementation with SY resulted in a 26% increase in plasma Se compared with feeding the same level of Se as an inorganic form of SS Kincaid (1999) stated that most investigators recommend that plasma concentrations of Se should exceed 0.07 g/ mL. Weiss et al (2003) examined 11 studies that compared organic versus and inorganic source s of Se when fed to beef and dairy cattle Feeding organic Se resulted in an average 18% increase in blood concentrations of Se. Lower Se absorption in ru minants is likely caused by ruminal reduction of selenite to insoluble compounds such as elemental Se or selenides (Wright and Bell, 1966). Additionally, urinary excretion of Se in lambs (Ehlig et al., 1967) and sheep (Peter et al., 1982) was greater when SS was used as a dietary supplement in comparison to selenomethionine. As a consequence, concentrations of Se were less in blood and various tissues

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132 (Ehlig et al., 1967) indicative that feeding SS caused a greater excretion and lower retention of Se (Peter et al., 1982). Indeed, various tissues contained greater levels of Se when young bulls were supplemented with Se derived from plants compared with SS at the same concentrations (Ekholm et al., 1991) The difference in the physiological value of dietary SS versus SY lies in the ability of selenomethionine to be incorporated non specifically into body proteins because tRNA does not discriminate between methionine and selenomethionine (Daniel, 1996). Selenomethionine incorporation into body proteins provides a tissue storage pool that is available during periods when the demand for Se is high (i.e stress, disease and low feed intake). Although the metabolic fate of dietary inorganic and organic Se forms differ, both must first be converted to selenide and hy drogen selenite before formation of selenocysteine and its incorporation (including dietary selenocysteine) into selenoproteins (Daniels, 1996). Selenomethionine is activated initially by adenosilation, undergoes demethylation and then is converted to sele nocysteine through the intermediates of selenohomocysteine and selenocystathione in a manner analogous to formation of methionine (Schrauzer, 2000). Activity of seleno enzymes can indicate the level of Se adequacy. Indeed, the greater concentrations of Se in plasma of cows fed the SY were associated with a greater activity of the Triiodothyronine (T3) is mainly derived from deiodination of the pro hormone T4 in non thyroidal tissues and plays important roles by controlling growth, development, differentiation and metabolism in the body (Arthur and Beckett, 1994). S upplementation of calves with Se, compared with Se inadequate diets, increased the basal plasma concentr ation of T3 concurrently with a reduc tion of T4 (Wichtel et a l ., 1996 ; Arthur et al., 1988 ) Also, f eeding pre partum cows a supplement containing 60 or 120 ppm Se as selenite and 60 ppm as selenomethionine increased

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133 the ratio of T3 : T4 in plasma compared with 20 ppm of selenite ( Awadeh et al., 1998). A decrease in deiodenase activity is caused by the absence of a Se dependent component (Behne et al., 1990). The mRNA of the deiodenase contains an UGA codon for selenocysteine, which is nec essary for maximal activity of the enzyme (Berry et al., 1991). Se lenium supplementation can reduce the incidence of retained placenta in dairy cows fed diets low in Se (reviewed in Allison and Laven, 2000). Julien and other (1976) found that oral Se suppl ementation was as effective as intramuscular administration of Se and vitamin E in preventing retained placenta in cows fed Se deficient diets. Also, cows with retained fetal membranes had less glutathione peroxidase activity in maternal and placental tiss ues than cows without retained placenta (Kankofer et al., 1996). In the present study, the incidence of retained fetal membranes was not affected by diets. Frequency of purulent cervical discharge was greater for primiparous compared with multiparous cows, and lower for both parities when cows were supplemented with SY Approximately 90% of cows have some degree of bacterial contamination during the first 10 days postpartum (Morrow et al., 198 6 ) The innate immune system, comprised of neutrophils, play an i mportant role to contain growth and eliminate bacteria from the uterus. T he reduction in neutrophil activity at parturition is a physiological event that has been reported previously (Kehrli et al., 1989; Cai et al., 1994) Impaired neutrophil function dur ing the purperium has been studied as the cause of RFM (Gunnink, 1984; Kimura et al., 2002), puerperal metritis (Cai et al., 1994), subclinical endometritis (Hammon et al., 2006) and mastitis (Heyneman et al., 1990; Vandeputte Van Messom et al., 1993) P resence of a purulent content in the uterus, with or without fever, is one the signs that define puerperal metritis (Sheldon et al., 2006), and the vi sualization and scoring of cervical discharge using vaginoscopy is associated strongly with a

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134 uterine bact erial infection (Dohmen et al., 1995). The uterus of postpartum cows is contaminated with a range of bacteria, but this is not consistently associated with clinical disease. Infection implies adherence of pathogenic organisms to the mucosa, colonization or penetration of the epithelium, and/or release of bacterial toxins that lead to establishment of uterine disease (Sheldon et al., 2006). The developmen t of uterine disease depends on the inherent uterine defense mechanisms as well as the species virulence and number (load or challenge) of bacteria present in the uterus (Hussain, 1989 ; Cohen et al., 1995; Sheldon et al., 2006). Number of pathogenic bacteria in the uterus of postpartum cows may amplify enough to overwhelm uterine defense mechanisms and cause life threatening infection (Sheldon and Dobson, 2004). Frequency of fever was greater for primiparous compared with m ultiparous cows. Multiparous cows supplemented with SY had a 47% reduction in the frequency of fever compared with their counterparts fed SS Multiparous cows diagnosed with a purulent cervical discharge in the puerperium and supplemented with SY had a lower risk of a septicemia based upon fever measures. However, beneficial effects of organic Se in the diet on responses such as fever were n ot observed in primiparous cows. Parity differences and differential effects of the diet on the incidence of fever may be a reflection of a greater proportion of major calving assistance category observed in primiparous (48.1%) compared with multiparous (1 6.2%) cows. Primiparous cows appeared to experience a greater degree of stress at first parturition and perhaps a greater suppression in immune competence. Cows experiencing dystocia have decreased activity of uterine phagocytes (Stevenson, 1997 ). There we re no effects of organic Se in the diet on neutrophil phagocytic and oxidative burst activity at parturition for primiparous cows (C hapter 5 ). However, activity was greater in subsequent weeks ( C hapter 5 ). Also, injury of genital tissues during dystocia o bstetric al manipulation and some traditional practice s of

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135 farmers and herdsmen such as inserting the hand or apparatus in the vagina can facilitate bacterial invasion and multiplication within the uterus. Additionally, stress relative to a difficult deliv ery can result in reduced uterine contractility leading to accumulation of bacteria Sheldon et al (2004) indicated that days postpartum was not a good indicator of the number of bacteria in the uterus. However, feve r, was correlated with the presence of specific uterine pathogens Fever is the result of a complex communication between the peripheral immune system and the brain in respons e to infection and inflammation, trauma or both (Leon, 2002). Fever can be initiated by bacterial lypopolysaccharide acting directly as exogenous pyrogen or indirectly by infections that activate liver macrophages (Steiner et al., 2006) to produce endogenous pyrogens such as IL 6, IL 1, and TNF (Macko wiak et al., 1997). Beneficial effects of SY supplementation on puerperal health (i.e., frequency proportion of purulent cervical discharge and fever) are likely to be associated with greater bactericidal activity of neutrophils during the postpartum perio d of cows supplemented with SY ( C hapter 5 ). The main function of neutrophils is the phagocytosis and killing of invading bacteria (Dhaliwal et al., 2001). These include many stages such as cellular endothelial adherence, chemotaxis, opsoniza tion, ingestion and digestion. Approximately 48 h ours after a normal unassisted parturition leukocyte accumulat ion in the uterine lumen mark s the beginning of a normal cleansing and uterine involution process (Dhaliwal et al., 2001). Greater neutrophil bactericidal acti vity may have contained the growth of bacteria within the uterus that prevented or reduced the occurrence of fever and/or s epticemia (or bacteremia ). The presence of neutrophils in the uterine lumen is an excellent indication of an active inflammatory proc ess and, therefore, presence of bacterial contamination. Once the offending contaminations are removed, inflammation subsides and neutrophils no longer migrate to the

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136 lumen. The 31% incidence of subclinical endometritis was in the range of previous reports (Kasimanickam et al., 2004; Gilbert et al., 2005) and there was a carryover effect in that cows with RFM and mucopurulent /purulent cervical discharges at days 5 and 10 postpartum had a greater occurrence of subclinical endometritis. These associations em phasize the importance of a normal uterine involution in the early postpartum because subclinical endometritis is associated with reduced fertility ( Kasimanickam et al., 2004; Gilbert et al., 2005). Interestingly, visual inspection of uterine flushes was a ssociated with the disease, which could eliminate the need for counting neutrophil and serve as a management tool for treatment decisions. Dietary and parity effects on the incidence of subclinical endometritis at day 37 postpartum were not detected even t hough occurrence of adverse cervical discharge scores at 5 and 10 dpp were less in cows supplemented with S Y and in multiparous cows. Perhaps supplementation with SY improved neutrophil bactericidal activity and reduced occurrence of abnormal cervical disc harges in the stressful puerperal period. This is evident by a reduction in occurrence of cows with a mucopurulent /purulent cervical discharge score from 65% to 53% when fed SY Clearly uterine health had improved by 37 dpp since only 31% of cows were diag nosed with subclinical endometritis. The restoration of uterine health is quite profound with a reduction in diagnosis of poor uterine health of 60% at 10 dpp to a 31% incidence of subclinical endometrit i s at day 37 postpartum It is interesting that the o ccurrence of subclinical endometritis was 24% in cows with clean cervical discharge at day 10 postpartum Thus it is clear that subclinical endometritis is not strictly associated with cows that had a p rior severe uterine infection. It is important to reco gnize that the similar distribution of neutrophil percentage in uterine flushings at 37 dpp in SS and SY diets also reflected the intensive health management conducted during the postpartum period allowing successful detection and treatment of diseased cow s. Antibiotic treatment of

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137 cows detected with metritis may have masked the possible carry over effects of increased mucopurulent/purulent cervical discharges during the first 10 dpp in the occurr ence of subclinical endometritis for SS supplemented cows Th e possibility of not treating cows with metritis during the early postpartum is virtually impossible when large field experiments are conducted on a commercial dairy farm. The association of subclinical endometritis and fertility was not adequately tested because by design all cows were inseminated during the hot season of the year when fertility is low. The p roportion of cyclic cows was not affected by sources of Se or by pre synchronization protocols. A greater proportion of multiparous cows were cyclic compared with primiparous cows. The greater proportion of minor to mild calving assistance and purulent cer vical discharge in primiparous cows may have delayed resumption of ovarian activity. The first postpartum dominant follicle grows slower and produces less estradiol in cows with high numbers of bacteria present in the uterine lumen in the puerperium (Sheld on et al., 2002). The relationship between increased prevalence of cyclic cows with increased BCS in the present study has been well documented (Santos et al., 2008). Body condition is correlated with body reserves, in particular body fat ( Ferguson et al., 1994) A greater degree of body fatness indicates improved energy reserves that m ay contribute to metabolic hormonal signal s that program an earlier resumption of ovarian activity. Pregnancy per AI and pregnancy loss after first insemination w as not affec ted by source s of Se or pre synchronization protocol s Low fertility and high pregnancy loss were more likely caused by high body temperatures associated with heat stress in which the cows were exposed during the experimental breeding periods (Armstrong, 1 994 ; Cartmill et al., 2001) Supplementation with SY raised plasma concentrations of Se but did not attenuate the effects of

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138 heat stress on first service pregnancy per AI A similar absence of Se supplementation effect s on pregnancy per AI has been report ed in beef cattle (Awadeh et al., 1998; Spears et al, 1986), dairy (Wichtel et al., 1994) and ewes (Kott et al., 1983). Similarly, second inseminations were performed during the period of heat stress and pregnancy per AI although low, w as increased in cow s supplemented with SY T he greater differences in pregnancy per AI for SY fed cows were observed in cows that were pregnan t at 27 to 30 days after first insemination but experienced pregnancy loss from this time to the time of per rectum reexamination of pregnancy (i.e., 55 days after first insemination) For this category of cows those supplemented with SS had lower second pregnanc y per AI (4.2%, 1/24) compared with cows supplemented with SY (22.7%, 5/22) diet. A dying fetus older than 27 days that h as been attached to the uterus must induce uterine immune response s similar to the early postpartum period in order to eliminate fetal contents. Perhaps, cows in the SY diets may have had a greater ability to restore a favorable uterine environment to supp ort a pregnancy following the second insemination. Even though cows were inseminated during a heat stress period, effects of B CS were detected. The relationship between increased pregnancy per AI for cows with increased BCS in the present study has been re ported before (Moreira et a l ., 2000 ; Santos et al., 2008 ). Also, w hen heat stressed lactating dairy cows received a timed embryo transfer, a 37 percentage unit increase in pregnancy per AI was associated with every unit increase in BCS (Ambrose et al., 199 9). Supplementation of cows with SY improved milk yield that was likely associated with improved health and metabolic efficiency (greater T3:T4 molar ratio) during the first month of lactation However, no differences in group DMI were observed. An additio nal increase in milk yield occurred in the fifth to eighth month of lactation in association with a lower milk S CC In the later stages of lactation, SY was beneficial to mammary gland health. No differences in

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139 clinical mastitis were detected that would ha ve accounted for greater milk production. Somatic cell count was highest during the first month of lactation and declined to the third month in both experimental diets. Thereafter S CC remained lower for SY cows but increased for cows in the SS group at 6 t o 8 months of tested milk. A greater incidence of udder infections occurs during dry off and first month of lactation with a decline in incidences with advancing of lactation ( Erskine et al., 1988 ). Although innate and humoral responses were enhanced in th e early postpartum for cows i n the SY diet (Chapter 5) S CC in milk were not less in this group in the first month of lactation. B ulk tank S CC decreased significantly as Se concentration in plasma increased (Weiss et al., 1990) and as plasma glutathione peroxidase activity, a seleno enzyme, increased ( Erskine e t al., 1987). Jukola and other s (1996) follow 551 dairy cows for a period of one year and observed that an increase in whole blood Se concentration was associated with a decrease in mammary infectio ns caused by Staphylococcus aureus, Actinomyces pyo genes and Corynebacterium (Jukola et al., 1996). R eduction in milk yield and greater milk S CC were observed in cows with low (0.03 g/mL) compared with adequate (0.132 g/mL) concentrations of Se in whole blood when the mammary gland was challenged with E scherichia coli (Erskine et al., 1989). The level of S CC in milk is an important indicator of subclinical mastitis and is correlated inversely with milk yield ( Philpot, 1984 ). Generally, replacing inorganic Se with SY did not influence yie lds of milk and milk components (Heard et al., 2007; Juniper et al., 2006; Weiss and Hogan, 2005). Differences in the percentage of milk fat and protein were not observed between the diets. However, because milk yields were greater for cows supplemented with SY the total output of 3.5% FCM, fat and protein were greater in the SY diet. In a concurrent Californian study (Rutigliano et al., 2008 ) in which experimental design was replicated exac tly as the present study, background Se concentration (mg/kg of DM) in the

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140 diets were in the range of 0.20 and 0.31 in the pre and postpartum periods compared with concentrations below 0.10 of Se in the present study. Overall Se concentrations in the pre partum diets differed substantially between Florida (0.44 [ SS ] and 0.49 [ SY ] mg/kg of DM) and California (0. 54 [sodium selenite] and 0. 66 [ SY ] mg/kg of DM) studies. Differences in Se concentrations (mg/kg of DM) for the postpartum diets were even greater; that is, Florida with 0.36 for SS and 0.36 for SY and California with 0. 53 for SS and 0. 64 for SY Furthermore, in most feedstuffs, Se is present in the organic form (Weiss, 2003), which resembles those of SY Indeed, in the Californian study, feeding SY c ompared to SS during the last 25 days of gestation had no impact on plasma concentrations of Se (> 0.10 g/ mL ). However, yield of 3.5% FCM, milk fat, and milk fat content during the first 90 days of lactation were increased in cows fed Se yeast with no effects detected on yields of milk, true protein, concentration of true protein, and S CC Also, effects of source of Se were not observed for incidence of puerperal metritis, subclinical endometritis, and rates of ovarian cyclicity and pregnan cy. R utigliano and others (2008 ) observed that concentrations of Se in the basal diets were in the range of 0.20 and 0.31 mg/kg of DM in the pre and postpartum periods, respectively. Analyses of the composite feed samples for both experiments identified clear differences in dietary Se concentrations. Based upon the greater overall Se concentrations in the California experiment, it was not surprising that clinical and production responses did not differ between experimental diets In contrast, Florida was essent ially a Se deficient environment in which most of dietary Se was derived via the two supplements. Under these conditions, SY fed in the ration beginning in the pre partum period elevated plasma Se concentrations, improved uterine health and milk yield ass ociated with lower SSC and increased second service pregnancy per AI during summer. In conclusion, health and

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141 production responses of dairy cows are benefited by SY supplementation when Se status of cows and feedstuff compositing diets are inadequate.

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142 Ta ble 4 1. Ingredients and chemical composition of pooled experimental diets 1 during the pre and postpartum periods for groups supplemented with sodium selenite and selenium yeast Composition % of Dry m atter (DM) Ingredients Pre partum Postpartum Corn s ilage 29.4 0 28.9 0 Alfalfa h ay 12.6 0 11.8 0 Coastal b ermuda h ay 3 .3 0 Wet b rewer s grain 5.6 0 5.7 0 Soybean m eal 7.0 0 9. 70 Soy bean h ulls 10. 30 3.7 0 Corn h ominy 1 1 .10 19.6 0 Whole c ottonseed 7.6 8.0 Corn d g rain 7. 5 8.0 Mineral and vitamin mix 2 5.6 4.6 Chemical composition NE L 3 Mcal/Kg of DM 1.44 1.54 CP, % of DM 18.37 17.82 ADF, % of DM 29.65 24.22 NDF, % of DM 44.89 37.95 Lignin, % of DM 4.27 3.25 Ether extract, % of DM 5.05 4.70 Ash, % of DM 9.86 9.55 Ca, % of DM 1.61 1.19 P, % of DM 0.40 0.45 Mg, % of DM 0.32 0.32 K, % of DM 1.45 1.62 S, % of DM 0.24 0.35 Na, % of DM 0.11 0.39 Cl, % of DM 0.71 0.58 Mn, mg/Kg of DM 87.0 108.0 Zn, mg/Kg of DM 113.0 131.0 Cu, mg/Kg of DM 24.0 23.0 Fe, mg/Kg of DM 430.0 443.0 Se, mg/Kg of DM 0.44 0.36 1 Ingredients and chemical composition of the diets are similar for SS and SY groups, therefore values are pooled. 2 Se lenium was s upplemented at same concentration in the pre partum ( 0.445 mg/Kg of DM) and postpartum ( 0.3 mg/Kg of DM) diets with sodium selenite or selenium yeast ( Sel P lex, Alltech Inc, Nicholasville, KY). Pre partum mineral mix contained 20.5% CP; 1.0% fat; 1.5% fibe r; 3% ADF; 20.5% Ca; 8 mg/kg of Se; 114,000 IU/Kg of vitamin A; 63.5 mg/Kg of monensin. Postpartum mineral mix contained 30.0 % CP; 14% fat; 2.0% fiber; 2.0% ADF; 9.95% Ca; 0.75% P; 2.4% NaCl; 6.7% Na; 6.63 mg/kg of Se; 68,000 IU/Kg of vitamin A. 3 Calculat ed using NE L values published by NRC (2001).

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143 Table 4 day during the first 10 days postpartum, adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance Variable % (n) AOR 95% C.I P value Primiparous SS 1 39.9 (41/104) Ref Ref SY 2 41.6 (42/101) 1.36 0.6 3.2 NS Multiparous SS 25.4 (46/181) Ref Ref SY 13.3 (25/188) 0.46 0.5 0.9 < 0.05 BCS 3 at parturition 3.5 3.75 24.8 (67/270) Ref Ref 3.25 27.2 (70/257) 1.97 1.1 3.6 < 0.05 2.75 3.0 37.2 (16/43) 3.52 1.3 9.9 < 0.01 Cervical d ischarge at day 10 postpartum Clear 17.0 (27/159) Ref Ref Mucopurulent 24.9 (43/173) 1.49 0.8 2.7 NS Purulent 50.8 (33/55) 5.25 2.5 11.1 < 0.01 1 Sodium selenite (SS); 2 Selenium yeast (SY; Sel Plex Alltech Inc, Nicholasville, KY). Selenium was s upplemented at same concentration in the pre partum ( 0.445 mg/Kg of DM) and postpartum ( 0.3 mg/Kg of DM) periods. 3 Body Condition Score. Ref. = Reference; NS = non significant

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144 Table 4 3. Frequency distribution of cows among cervical discharge categories diagnosed at days 5 and 10 postpartum Variable (n) Clear or f lecks % (n) Mucopurulent % (n) Purulent % (n) P value Diet 1 SS (437) 35.0 (153) 47.8 (209) 17.2 (75) Ref SY (460) 47.2 (217) 43.5 (200) 9.4 (43) < 0.01 Day postpartum D5 (476) 42.3 (201) 47.3 (225) 10.5 (50) Ref D10 (421) 40.1 (169) 43.7 (184) 16.2 (68) < 0.05 Parity Primi parous (306) 29.4 ( 306 ) 51.6 (158) 19.0 (58) Ref Multi parous (591) 47.4 ( 591 ) 42.3 (251) 10.2 (60) < 0.01 Calving assistance 2 No (213) 54.0 (115) 35.2 (75) 10.8 (23) Ref Minor and major (681) 37.4 (255) 48.8 (332) 13.8 (94) < 0.05 RFM 3 No (834) 43.8 (365) 46.8 (390) 9.4 (79) Ref Yes (62) 8.1 (5) 30.6 (19) 61.3 (38) < 0.01 1 Sodium selenite (SS), selenium yeast (SY; Sel Plex Alltech Inc, Nicholasville, KY). Selenium was s upplemented at same concentration in the pre partum ( 0.445 mg/Kg of DM) and postpartum ( 0.3 mg/Kg of DM) periods. Frequency distribution of cows among cervical discharges for diets is presented as pooled from days 5 and 10 postpartum. 2 Calving assistance: No = no assistance; Minor = use of minor force; Major = use of a jack with minor or major force. 3 Retained fetal membranes; Diet by parity: non significant. Ref. = Reference.

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145 Table 4 4. Frequency distribution of cows with subclinical endometritis obtained by uterine flushing at day 37 postpartum adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance Variable % (n) AOR 95% C.I. P value Diet 1 SS 31.5 (85/270) Ref. Ref SY 31.0 (85/274) 0.80 0.5 1.3 NS Parity Primi parous 29.6 ( 59/199 ) Ref. Ref Multi parous 32.2 ( 111/345 ) 1.20 0.7 2.0 NS RFM 2 No 29.1 (143/492) Ref. Ref. Yes 54.0 (27/50) 3.60 0.8 15.9 = 0.09 Cervical discharge (D5) Clean 27.8 (31/183) Ref. Ref. Mucopurulent 33.5 (63/188) 1.30 0.8 2.3 NS Purulent 52.8 (19/36) 3.90 1.1 13.6 < 0.05 Cervical discharge (D10) Clean 23.0 (36/156) Ref. Ref. Mucopurulent 36.2 (63/174) 1.60 0.9 2.9 < 0.10 Purulent 33.8 (21/64) 1.40 0.6 3.4 NS Flush score Clean 20.5 (90/438) Ref. Ref. Flecks 66.7 (22/33) 12.50 3.9 40.0 < 0.01 Mucopurulent 70.6 (24/34) 6.80 2.6 17.6 < 0.01 Purulent 87.2 (34/39) 14.40 4.5 46.0 < 0.01 1 Sodium selenite (SS), selenium yeast (SY; Sel Plex Alltech Inc, Nicholasville, KY). Selenium was s upplemented at same concentration in the pre partum ( 0.445 mg/Kg of DM) and postpartum ( 0.3 mg/Kg of DM) periods. 2 Retained fetal membranes. Diet by parity: non significant. Ref. = Reference; NS = non significant

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146 Table 4 5. Proportion of cyclic cows, adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance Variable Cycli c 2 % (n) AOR 95% C.I. P value Diets 1 SS 82.3 (214/260) Ref. Ref. SY 82.3 (205/249) 1.0 0.6 1.6 NS Pre synchronization protocol Presynch 85.6 (220/257) Ref. Ref. CIDR 79.0 (199/252) 0.72 0.4 0.8 NS Parity Multiparous 84.5 (274/324) Ref. Ref. Primiparous 78.4 (145/185) 0.53 0.3 0.9 < 0.01 Calving assistance no assistance 91.0 (102/112) Ref. Ref. minor assistance 81.8 (131/160) 0.43 0.2 0.9 < 0.05 mild assistance 79.7 (114/143) 0.46 0.2 0.9 < 0.05 BCS 3 at 37 dpp 89.5 (197/220) Ref. Ref. = 2.75 81.8 (131/160) 0.48 0.3 0.8 < 0.01 < 2.75 67.5 (77/114) 0.20 0.1 0.4 < 0.01 1 Sodium selenite (SS), selenium yeast (SY; Sel Plex Alltech Inc, Nicholasville, KY). Selenium was s upplemented at same concentration in the pre partum ( 0.445 mg/Kg of DM) and postpartum ( 0.3 mg/Kg of DM) periods. 2 Determined based upon plasma concentrations of progesterone. 3 Body Condition Score. Ref. = Reference; NS = non significant.

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147 Table 4 6 First and second services p regnanc ies per AI at days 27 to 30 and 55 after insemination and first service pregnancy loss for cows fed diet supplements of sodium selenite (SS) and selenium yeast (SY) and differentially pre synchronized ( Presynch and CIDR ) Variables SS % ( n ) SY % ( n ) P value First s ervice D27 to D 30 23.6 (62 /262 ) 2 4.9 (62 /249 ) NS D55 14.9 (39 /262 ) 1 4.9 (37 /248 ) NS Loss 1 37 .1 (23 /62 ) 39.3 (24 /61 ) NS Second s ervice D55 11.3 (24 /211 ) 17.1 (34 /199 ) < 0.0 5 Variable Presynch % (N) CIDR % (N) P value First s ervice D27 to D 30 24.8 (64 /258 ) 23.7 (60 /253 ) NS D55 15.5 (40 /257 ) 14.2 (36 /253 ) NS Loss 3 6.5 (23 /63 ) 40 .0 (24 /36 ) NS Second s ervice D55 12.9 (27 /209 ) 15.4 (31 /201 ) NS 1 Pregnancy losses from 27 and 30 to 55 days after insemination. NS = non significant

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148 Table 4 7 First service p regnancy per AI at 27 to 30 days after insemination diagnosed by per rectum ultrasonography Variables Pregnancy % (n) AOR 1 95% CI 1 P value 1 BCS at enrollment 3.75 4.25 17.6 (21/119) Ref Ref 3.5 22.7 (47/207) 1.57 0.8 3.0 NS 3.25 28.3 (34/120) 2.5 0 1.2 5.0 NS 2.75 3.0 32.8 (21/64) 3.65 1.6 8.3 < 0.01 BCS at 37 dpp 3 3.75 27.7 (61/220) Ref Ref 2.75 22.4 (36/161) 0.62 0.3 1.1 < 0.10 2 2.5 20.0 (23/115) 0.53 0.3 0.9 < 0.01 Ovulated (P 4 ) 2 Yes 28.3 (111/392) Ref Ref No 3.6 (3/83) 0.1 0 0.03 0.3 < 0.01 1 Adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance (NS = non significant). 2 Cows were considered to have ovulated a synchronized follicle if plasma concentration of progesterone (P 4 ) was below 1 ng/mL at artificial insemination and greater than 1 ng/ mL at 7 days after artificial insemination

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149 Table 4 8. Least squares means and pooled standard errors (S.E.) for milk parameters obtained monthly during the first 8 months postpartum for cows in the sodium selenite (SS) and Se yeast (SY) diet s Diets Parameters 1 SS (n = 262) SY (n = 249) S.E. P value Milk (Kg/day) 36.4 37.2 0.31 < 0.05 3.5% FCM 2 (K g/ d) 35.3 36.2 0.18 < 0.05 Milk fat % 3.37 3.39 0.017 NS g/day 1227 1265 5.0 < 0.05 Milk true protein % 2.88 2.89 0.005 NS g/day 1040 1065 4.50 < 0.05 S CC 3 (x1000) 286.6 283.2 32.3 NS S CS 4 2.60 2.56 0. 04 NS S e lenium was s upplemented at same concentration in the pre partum ( 0.445 mg/Kg of DM) and postpartum ( 0.3 mg/Kg of DM) periods for SS and SY diets 1 All parameters were obtained from the Dairy Herd Improvement Association. 2 Fat corrected milk (milk yield [Kg] x ( 0.4255 + (16.425 x {fat [ % ] /100 } ) ; 3 Somatic cell count. 4 Somatic cell score ( SCS = Log 10 [ somatic cell count /12,500 ] /Log 10 [2])

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150 Figure 4 1 Least squares means ( S.E.) hourly v aginal temperatures for cows in the sodium selenite ( n = 11) and selenium yeast ( --, n = 12) diets and recorded every 15 min for a period of 5 days per cow across three replications from 12 August to 27 August Cows were housed in separate pens in single free stall barn. Diets (non significant), hour (P < 0.01). 38,6 38,8 39 39,2 39,4 39,6 39,8 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Temperature ( o C) Hour

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151 Figure 4 primiparous and multiparous cows fed diets containing sodium selenite ( ) or selenium yeast ( --) in the pre partum (n = 18/diet; 0.445 mg of selenium/Kg of dry matter) and postpartum (n = 20/diet; 0.30 mg of selenium/Kg of dry matter) periods. Diet (P < 0.01), parity and diet by parity (non significant). Pr e partum values non significant. 0,05 0,06 0,07 0,08 0,09 0,1 0,11 25 0 7 14 21 37 Plasma selenium (g/mL) Days relative to parturition

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152 Figure 4 3. Leas t squares means ( S.E) plasma triiodothironine (T3) to t hyroxine (T4) molar ratios (x10 2 ) for primiparous (A) and multiparous (B) cows fed diets containing sodium selenite ( ) or selenium yeast ( --) in the pre partum (n = 18/diet; 0.445 mg Se/Kg of dry matter) and postpartum (n = 20/diet; 0.30 mg Se/Kg of dry matter) periods. Parity (P < 0.05), diet at day 0 (parturition) in A (P = 0.10) and B (P < 0.05). 2 3 4 5 6 25 0 7 14 21 37 T3:T4 molar ratio (x10 2 ) Days relative to parturition A 2 3 4 5 6 25 0 7 14 21 37 T3:T4 molar ratio (x10 2 ) Days relative to parturition B

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153 Figure 4 4. Least squares means ( S.E.) milk yields for cows fed diets containing sodium selenite ( ; n = 262 ) or selenium yeast ( --; n = 249 ) in the pre partum (25 days pre partum, 0.445 mg Se/Kg of dry matter) and postpartum (0.30 mg Se/Kg of dry matte r) periods. Diet (P < 0.05), month (P < 0.01) and diet by month (P < 0.05) effects. 30 32 34 36 38 40 1 2 3 4 5 6 7 8 Milk (Kg) Months postpartum

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154 Figure 4 5. Least squares means ( S.E.) milk somatic cell score ( SCS = Log 10 [ somatic cell count /12,500 ] /Log 10 [2]) for cows fed diets containing sodium selenite ( ; n = 262) or selenium yeast ( --; n = 249) in the pre partum (25 days pre partum, 0.445 mg Se/Kg of dry matter) and postpartum (0.30 mg Se/Kg of dry matter) periods. Diet (non significant), month (P < 0.01) and diet by month (P < 0.05) effects. 1,5 2 2,5 3 3,5 1 2 3 4 5 6 7 8 SCS Months postpartum

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155 CHA PTER 5 EFFECT OF SOURCE OF SUPPLEMENTAL SELENIU M ON NEUTROPHIL ACTI VITY AND HUMORAL IMMUNE R ESPONSES IN THE PERI PARTUM PERIOD OF DAIRY COWS IN FLORIDA Introduction Selenium (Se) is an important component of antioxidant enzymes. Selenium in the form of selenocysteine is an essential component of a family of antioxidant enzymes such as glutathione The most common fo rms of Se consumed by ruminants are selenomethionine ( SeMet ), selenocystein (SeCys), selenite and selenate Feedstuffs and selenized yeast ( Se yeast ) provide Se mostly as Se containing amino acids, whereas inorganic supplements provide selenite and selenat e ( Whanger, 2002). In a review by Weiss (2003) 18 % and 90% increase s in whole blood and milk Se concentrations were reported when Se yeast was fed compared to selenite in ten studies using beef or dairy cattle. Low absorption of Se in ruminants is due to ruminal selenite reduction to insoluble compounds such as elemental Se or selenides. Neutrophils are phagocytes that can engulf and kill many microbes. Each phagocytic event results in the formation of a phagosome into which reactive oxygen species (ROS) and hydrolytic enzymes are secreted. The consumption of oxygen during the generation of ROS has been termed the oxidative burst (Paape et al., 2003 ) and involves the activat ion of the enzyme NADPH oxidase Also, opsonized bacteria or zymosan, aggrega ted IgG, and protein kinase C activators such as phorbol myristate acetate (P MA) can activate NADPH oxidase (Czuprynski and Hamilton, 1985; Leino and Paape, 1993, 1996; Rambeaud et al., 2006). The NADPH oxidase facilitates the shuttling of electrons from c ytosolic NADPH to oxygen present in the phagosome or in the proximal extracellular environment to produce large quantities of superoxide anion (O 2 ) (Weiss, 1989) Superoxide anion is the most proximally

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156 generated ROS by NADPH oxidase and forms as a result of one molecule of oxygen acting as an acceptor for a single donated electron (Weiss, 1989) Superoxide is dismutate d spontaneously or through catalysis via an enzyme known as superoxide dismutase to form hydrogen peroxide (H 2 O 2 ) which is then converted to hypochlorous acid (HOCl ) by the enzyme myeloperoxidase (Weiss, 1989). The generation of ROS can be harmful to the neutrophil and surrounding tissues and is denominated as oxidative stress Glutathione peroxidase, a selenoprotein, and catalase, an iron containing enzyme, are important enzymes to reduce the excessive amounts of ROS produced by cells and tissues (Paape et al., 2003) Particularly, g lutathione peroxidase catalyzes the reduction of hydroperoxides, including hydrogen peroxides, by reduced glu tathione and functions to protect the cell from oxidative damage. One mole of purified glutathione peroxidase contains four gram atoms of Se (Rotruck et al., 1973). Most of all of the g lutathione peroxidase enzymes are tetramers of four identical subunits. Each subunit contains a selenocysteine in the active site which participates directly in the two electron reduction of the peroxide substrate (Ceballos Picot et al., 1992; Forstrom et al., 1978). Peripheral neutrophil function is suppressed during the per iparturient period (Kehrli et al., 1989; Cai et al., 1994). Reduced chemotaxis and bacterial killing ability of neutrophils were associated with retained fetal membranes (Gunnink, 1984; Kimura et al., 2002), puerperal metritis (Cai et al., 1994), subclinic al endometritis (Hammon et al., 2006) and mastitis (Heyneman et al., 1990; Vandeputte Van Messom et al., 1993). Mechanisms of immunosuppression at parturition are related to the release of glucocorticoids (Preisler et al., 2000), increasing concentrations of plasma NEFA and BHBA (Hammon et al., 2006) and hypocalcemia (Kimura et al., 2006).

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157 Neutrophil bactericidal activity (phagocytosis and killing) has been reported to be enhanced in cows supplemented with Se (i.e., sodium selenite) compared with c ows not s upplemented ( Boyne and Arthur, 1979 ; Grasso et al., 1990 ; Hogan et al., 1990). Moreover, Se often giving in conjuction with vitamin E supplementation (i.e., intra muscular injections ), reduced the incidence of ret ained fetal membranes (Archiga et al., 1994 ; Trinder et al., 1973), metritis (Harrison et al., 1986) and mastitis ( Smith et al., 1984 ; Jukola et al., 1996 ) Also, b ulk tank somatic cell count ( Erskine e t al., 1887) and clinical mastitis ( Weiss et al., 1990 ) decreased as plasma concentration of Se and gluthatione peroxidase activity increased ( Erskine e t al., 1987). More recently, the effec ts of feeding Se yeast or selenate diets, 0.3 mg of Se/Kg of the dry matter, to non lactating and early lactation dairy cows increased the serum Se concentrati ons at parturition and at day 28 posptartum but neutrophil phagocytic and bactericidal activit ies were not altered at day 28 postpartum (Weiss and Hogan, 2005) which is a time beyond the periparturient immunossupressed period To our knowledge, there is no information about the effects of organic Se on innate (neutrophil function) and adaptive humoral (serum immunoglobulin) immunity in peri parturient dairy cows. The objectives were to compare the effect of dietary organic Se ( S e yeast, Sel Plex Alltech, Inc, Nicholasville, KY) with inorganic sodium selenite when supplemented during pre and postpartum periods on responses such as: a) colostrum quality; b) n eutrophil phagocytic and oxidative burst activity and c) s erum anti oval bumin antibody The hypotheses were that feeding an organic source of Se prior to parturition and during the puerperium of dairy cows increases the absorption and retention of Se that enhances Se

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158 containing enzymes activity (reduces oxidative stress) resul ting in a greater n eutrophil phagocytic and oxidative burst activity and serum anti ovalbumin antibody responses. Material and M ethods Animals Experimental Design and Feeding The present study was part of a large field trial conducted at a n orth Florida dairy farm comprised of 3, 0 00 Holstein cows that were milked thrice daily A sub sample of cows was used to carry out the objectives. Therefore information regarding animals, facilities, diets and chemical composition of diets was described in Chapter 4 Briefly, cows were allocated randomly into two experimental diets begun at approximately 25 days prior to the expected date of parturition and continued until 8 0 days postpartum ( dpp ) C ontrol diet was supplemented with an inorganic source of Se ( sodium selen ite; SS) and treatment diet supplemented with an organic source of Se (Se yeast ; SY; Sel Plex ; Alltech Inc., Nicholasville KY ) which is comprised of approximately 60% selenomethionine and 40% other selenoaminoacids according to the supplier C ows were fed a total mixed ration to meet or exceed the requirements of the NRC (2001) for lactating Holstein cows weighing 650 kg and producing > 40 kg/d of milk cont aining 3.5% fat. The pre partum diets were formulated to provide supplemental Se at a concentration of 0.445 mg/Kg of dry matter according to the recommendation of the farm veterinarian and 0.3 mg/Kg of dry matter in the postpartum diets. Primiparous and multiparous cows of both experimental diets were housed together during the pre (enrol lment to parturition) and postpartum (0 to 30 dpp) periods. Pre and postpartum cows were housed in open dry lots of the same size that was separated solely by the feed bunk area (oriented face to face) and longitudinal fence. Pens were equipped with a sha de structure, fans, and waterers of the same size, orientation and location. Fans were equipped with high pressure nozzles and both fans and nozzles were activated once ambient temperature reached

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159 23 o C Pre and postpartum pens housed an average of 85 and 114 cows at any time during the study respectively. Health Monitoring T rained farm employees monitored p re partum cows for signs of parturition daily in a 24 hour period. After parturition cows were moved to a separate free stall barn for first milkin g and recording of volume and quality of colostrums Colostrum quality (globulin concentration) was classified into low ( ) moderate ( 40 to 80 mg/ml ) and high ( ) quality based upon specific gravity with the use of a colostrometer (Fleen or and Stott, 1980). C alving assistance categories were recorded routinely Only animals with calving assistance 1 (no assistance), 2 (minor assistance) and 3 (mild assistance) were continued in the experiment. Cows that had dystocia requiring veterinary a ssistanc e (i.e., caesarian section, fetotomy ) were excluded from the experiment Cows that had not shed their fetal membranes within 12 hours based upon visual inspection of the vulva and perineum were considered to have a retained fetal membrane (RFM). Ad ditionally, cows were evaluated for cervical discharge on days 5 and 10 postpartum using a disposable foil lined cardboard vaginal speculum (Milburn Distributors, Ocala, FL ) Appearance of cervical discharge was categorized into clear mucus without flecks, clear mucus with flecks lochia, muc o purulent (50% clear mucus and approximately 50% of pus) and purulent (> 50% pus to brown and foul smell). Body C ondition S core (BCS) Body condition of all cows w as scored at enrollment ( i.e., approximately 25 pre partum) parturition and 37 dpp Cows were scored for body condition o n a scal e of 1 (emaciated) to 5 (obese) with 0.25 unit increments as described by Edmonson et al (1989).

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160 Neutrophil P hagocytic and O xidative B urst A ctivity Neutrophil phagocytic and oxid ative burst activities were assessed by means of a modified dual color flow cytometry assay (Smits et al., 1997). In a preliminary study (Silvestre et al., 2005) bovine whole blood from non lactating and non pregnant cows was used for assay o ptimization of reagents, bacterial concentration and incubation times. Blood samples for analysis of neutrophil function were collected at 25 days pre partum (8 primiparous and 10 multiparous cows per diet) and at parturition, 7, 14, 21 and 37 dpp (10 primiparous and 10 multiparous cows per diet). Isolates of Echerichia coli ( E. coli ) were obtained from a cow with mastitis and grown in triptic soy broth for 18 hours at 37 o C. T he E. coli concentration was determined by colony counts from serial dilutions of the broth cul ture. The E. coli were heat killed at 56 o C for 60 minutes, washed, re suspended in sterile PBS to original volume, and added to an equal volume of p ropidium i odide ( PI, Sigma Aldrich, Saint Louis, MO) L in sterile PBS. After mixing by continuous rotation for 60 minutes at 23 o C, the PI labeled bacteria were re suspended in sterile PBS to a concentration of 10 6 cell/ L and stored at 4 o C in a light proof container. Dihydrorhodamine 123 (DHR, Sigma Aldrich, Saint Louis, MO) was dissolved in dimethyl sulfoxide to a stock concentration of 500 o C in a light proof container. Dihydrorhodamine 123 is a membrane permeable compound t hat fluoresces upon oxidation by hydrogen peroxide (Walrand et al. 2003). Phorbol 12 miristate 13 acetate (PMA, Sigma Aldrich, Saint Louis, MO) was dissolved in ethanol and stored at 10 o C. B lood samples were collected by puncture of coccygeal vessels into evacuated 10 mL s pray d ried s odium h eparin tubes ( Vacutainer, Becton Dickinson, Franklin Lakes, NJ ). Samples were maintained at room temperature and transported to the laboratory for analysis within 4 hours of collection. Upon arrival in the laboratory, samples were kept in a rotation

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161 station to avoid blood clotting. A 25 L aliquot was removed from each blood sample for determination of total leukocyte concentration in a hemocytometer after hemolysis of erytrocytes (Unopette, Becton Dickinson & Co, Franklin Lakes, NJ). A differential count of neutrophils (i.e., perce nt neutrophils among total leukocyte) was obtained by smearing 10 L of whole blood onto glass slide, stained (Protocol Hema 3, Fisher Diagnostics, Middletown, VA) and examine d at magnification of 40x with oil immersion using the battlement method (Schalm et al., 1975 ). Number of neutrophils per volume of whole blood was calculated based upon total leukocytes multiplied by percent of neutrophils. A 100 L aliquot of blood was pipetted into each of three 5 mL polystyrene round bottom tubes (12 x 75 mm). A 10 L of a 50 M DHR solution (100 L of DHR stock [500 M] and 900 L of PBS) was added to all tubes. Tubes were incubated at 37 o C for 10 minutes on a rotation platform to allow the loading of DHR into the neutrophils. After incubation, tube one was used a s a negative control. In tube two (positive control) a 10 L of PMA (activates neutrophil oxidative burst) at a concentration of 2 g/mL was added. In tube three a volume of the E coli suspension solution was added accordingly to the number of neutrophils to achieve a 40:1 bacteria to neutrophil ratio per 100 L of whole blood. All tubes were further incubated at 37 o C for 30 minutes on a rotation platform. After incubation tubes were placed immediately into ice to stop neutrophil phagocytosis and oxidati ve burst activity. Tubes were then processed for flow cytometry using reagents for red blood cell lysis (88% formic acid), white blood cell buffer and cell fixative (1% paraformaldehyde). An automated lysing system (Q Prep Epics immunology workstation, Cou lter Corp, Miami, Fl) was used to add reagents followed by further addition of 500 L of distilled water for completion of hemolysis and 10 L of 0.4% trypan blue solution for quenching extracellular oxidized DHR.

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162 The tubes underwent flow cytometry (FACSor t, Becton Dickinson Immunocytometry Systems, San Jose, CA) utilizing a 488 nm argon ion laser for excitation and 15 mW of power. Density cytograms were generated by linear amplification of the signals in the forward scatter and side scatter channels. Samp les were analyzed within 2 hour after addition of fixatives. Neutrophils were selectively gated from acquisition of 10,000 cells/sample based on their sizes and complexity in the density cytogram (Jain et al., 1991). Data was processed and further analyzed by computer software (CellQuest, version 3.3, Becton Dickinson Immunocytometry Systems, San Jose, CA). Parameters analyzed from the fluorescent cytograms included the percentage of neutrophils that phagocytized bacteria and the percentage of neutrophils w ith a phagocytosis induced oxidative burst. Also, histogram analysis for mean fluorescence intensity (MFI) of green (DHR oxidation) and red (PI labeled bacteria) wave lengths were used as an estimation of the total gated neutrophil mean oxidative burst int ensity (indicator of mean intensity of ROS produced per neutrophil) and mean phagocytic activity (indicator of mean number of bacteria per neutrophil), respectively. Serum A nti O valbumin A ntibody Humoral immunity was evaluated based upon antibody response following ovalbumin challenge for cows in SS (20 primiparous and 26 multiparous) and SY (14 primiparous and 24 multiparous) diets. Briefly, animals were injected (5 mL, s.c.) with 1 mg of ovalbumin (Type VII, Sigma Chemical Co.) dissolved in an E c oli Bac terin J 5 s train vaccine (Upjohn Co., off day) and 22 (initiation of experimental diets) days before the expected day of parturition. At parturition (0 dpp), animals were injected (2 mL, s.c.) fo r the third immunization with 2 mg of ovalbumin diluted in

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163 commercial adjuvant (Quil A, Accurate Chemical, Westbury, N Y). Ovalbumin was chosen as an inert test antigen to which the animals had not likely been exposed previously. B lood samples were collected by puncture of coccygeal vessels into 10 mL non heparinized plain tubes ( Vacutainer, Becton Dickinson, Franklin Lakes, NJ ) at 60 and 22 days before expected day of parturition and at parturition, 21 and 42 dpp. Serum was harvested after blood sa mples were allowed time for coagulation and further centrifuged at 2,000 x g for 20 minutes. Serum was stored at 20 C and used to monitor total a nti ovalbumin antibody, measured by an Enzyme Linked ImmunoSorbent Assay (ELISA) as described previously (Ma llard et al., 1997) Briefly, flat bottom 96 well polystyrene plates ( Thermo Electron Co. Waltham, MA ) we re coated (100 L/well) with a solution of ovalbumin dissolved in carbon ate bicarbonate coating buffer ( pH = 9.6) for final concentration of 1.4 mg/mL Plates were incubated at 4 C for 48 hours washed (200 L/well) with P BS and 0.05% Tween 20 solution (pH = 7.4). Plates were blocked (200 L/well) with a PBS 3% Tween 20 bovine serum albumin (Sigma Chemical, St. Louis, MO ) solution and incubated at room temperature for 1 h our Plates were washed (200 L/well) and diluted (l/50 and l/200) sera samples and controls were added in duplicate (100 L/well) using a quadrant system (Wright, 1987). Positive and negative control sera were obtained from pools of se ra known to have high (21 days after a third ovalbumin immunization) and low (non immunized animals) concentrations of anti ovalbumim antibody, respectively. Sequential s era samples from each animal were analyzed in the same plate and plates contained balanced numbers of animals from each diet. Plates were incubated at room temperature for 2 h ours and washed (200 L/well) with previously described buffer solution Subsequently alkaline phosphatase conjugate rabbit anti bovine IgG whole molecule (Sigma Chemical, St. Louis, MO ) was dissolved in wash buffer (1/10) added to the plates (100 L/well)

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164 and incubated for 2 hours at room temperature After incubation, plate were washed (200 L/well) once more and P n itrophenyl p hosphate d isodium ( Sigma Chemical, St. Louis, MO ) was added to the plates (80 L/well) and incubated at room temperature for 30 min Plates were examine on an automatic ELISA plate reader ( MRX Revelation ; Dynex Technologies Inc. Chantilly, VA ) and the optical density (OD) was recorded at 405 and 6 50 nm. Prior to the initiation of experimental serum sample assays, sera positive controls for anti ovalbumin antibody were analyzed at 1/50 and 1/200 dilutions in order to calculate positive controls mean and standard deviation (sd). Furthermore, sera positive controls results from plates containing experimental samples were continuously added with previous results to calculate accumulated mean and sd. Plates with positive control mean above or below 1.5 sd of total accumulated positive controls w ere repeated. Inter assay coefficient of variation for positive control samples was 9%. Plates were also repeated when the coefficient of variation of positive control samples were above 20% within a plate. A correction factor was calculated for each plat e by dividing the mean from the accumulated positive control results with the total mean of the positive controls from each plate. Experimental sample results were obtained from the product of the sum of the average of each duplicated sample dilution by th e correction factor of each plate. Statistical Analyses The generalized linear model (G ENMOD ) procedure of SAS was used to analyze categorical responses such as frequency distribution of body BCS categories (i.e., at enrolment, parturition and 37 dpp), cal ving assistance (no assistance minor assistance and major assistance), colostrum quality (low, medium and high) and cervical discharge (no, clear or lochia, mucupulent and purulent). A multinomial distribution of response and a logit link function were us ed. The mathematical model included diet parity and the interaction. Additionally, for cervical discharge scores a Generalized Estimating Equations (GEE) with an

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165 independent covariance structure, and multinomial distribution of response and a logit link function were used. The mathematical model included diet day (i.e., days 5 and 10 postpartum), parity, calving assistance categories and higher order interactions. Cow was used as a repeated subject Continuous responses such as neutrophil function (i.e., percent phagocytosis and/or oxidative burst, MFI phagocytosis and oxidative burst ) and sera concentration of anti ovalbumin antibody were analyzed using the mixed model procedure of SAS (Littell et al., 1996). The covariance structure that provided the best relative goodness of fit based upon penalty criteria (Bayesian criterion) was used. The statistical models consisted of diet, parity, day relative to parturition and diet by day interaction. Cow, the random variable was considered as the experimental unit nested within diet and parity Differences among diet means of the diet by day interaction were tested using the slice option of the mixed procedure. Results Co lostrum Q uality Colostrum volume and quality was assessed in the total population of cows enrolled in the large field trial (SS [n = 227] and SY [n = 233]). Colostrum volume was measured by number of jars (1,890 mL) filled with colostrum. Colostrum volume did not differ for SS and SY diets (3.0 1.6 jars). Frequency distribution of cows among quality colostrum categories of low (5.7 and 6.9%), medium (33.5 and 39.1%) and high (60.8 and 54.0%) did not differ between SS and SY diets, respectively. Neutrophil P hagocyt ic and Oxidative Burst Activity A total of 36 animal s (primiparous [n=16] and multiparous [n=20]) were sampled before initiation of diets. During the postpartum period, cows (SS [n=8] and SY [n=10]) that received antibiotic or anti inflammatory treatment were discontinued with sampling and not used in the

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166 an alysis. Therefore, primiparous (SS [n=10] and SY [n=10]) and multiparous (SS [n=10] and SY [n=10]) cows with complete sampling in the postpartum period were analyzed. Length of pre partum feeding did not differ between SS (24.2 1.6 days) and SY (27.4 1 .6 days) cows and did not affect neutrophil function responses. Proportion of cows among body condition score at the day of enrollment (3.0 to 3.25 [n = 12]; 3.5 to 3.75 [n = 28]) and parturition (3.0 to 3.25 [n = 15]; 3.5 [n = 25]) did not differ between SS and SY diets. Also, frequency distribution of cows among calving assistance categories (no assistance, minor assistance and major assistance) was not different for cows in SS (4, 13, 3 cows) and SY (5, 9, 6 cows) diets, respectively. A total of 2, 3, 8, 7 cows in the SS and 2, 4, 13, 1 cows in the SY diets were diagnosed with either no, clear or lochia, mucupulent and purulent cervical discharge at 10 dpp and were not treated with antibiotics or anti inflammatory drugs. L) of whole blood was not affected by diet and parity or the interaction, but number of neutrophils decreased (P < 0.01) from pre partum and parturition samples to postpartum samples (i.e., 7 to 35 dpp; Figure 5 1). Neutrophil phagocytic and oxidative burs t activity increased (P < 0.01) as the number of neutrophils increased per 5 2). Neutrophil phagocytic and oxidative burst activities measured before initiation of diets w ere not affected by diet, parity or diet by parity interaction. During the post partum sampling period an interaction of diet by parity by day was detected (P < 0.05, Figure 5 3 A and B). Neutrophil phagocytic and oxidative burst activities were greater (P < 0.01) for SY compared with SS in primiparous cows at 7, 14 and 37 dpp (Figure 5 3A), and in multiparous cows at parturition (Figure 5 3 B).

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167 Mean fluorescence intensity of the of the green wave length, an indicator of intensity of ROS produced per neutro phil, was greater for primiparous cows of in the SY diet at 7 (P < 0.01) and 14 (P < 0.01) dpp and in multiparous cows at 7 (P < 0.01) dpp when samples were stimulated with E. coli (Figure 5 4A and B). The MFI of the red wave length, an indicator of number of bacteria phagocytized per neutrophil, was greater (P < 0.05) for multiparous cows of the SY diet at 7 dpp when samples were stimulated with E. coli (Figure 5 4B). A capture index (percent of neutrophil with phagocytic and oxidative burst activity x [MF I for the red light/100]) was calculated (Rainard et al., 2000). Capture index was consistently greater (P < 0.01) for cows in SY (17.84 2.4) compared with SS (9.43 2.4) diets during the postpartum (parturition to 37 dpp). Serum A nti O valbumin A ntibody Serum anti ovalbumin antibody concentrations (OD) was analyzed for 34 primiparous (SS [n = 20 ] and SY [n = 1 4 ]) and 50 multiparous (SS [n = 26 ] and SY [n = 24 ]) cows The period from the first (dry off) to the second (initiation of diets) ovalbumin immunization (17.8 and 14.0 days; S.E. = 6.2) and from the second to the third immunization (parturition; 21.0 and 24.2 days; S.E. = 6.0) were not different for cows in SS and SY diets, respectively. Cows were equally distributed among b ody condition score s at the day of initiation of experimental diets ( 2.75 to 3.25 [n = 30 ]; 3. 5 to 3. 75 [n = 54 ] ). However, cows were unequally (P < 0.01) distributed among BCS categories at partur ition between SS ( 3.0 to 3.5 [n = 32 ]; 3. 5 to 3. 75 [n = 14 ] and SY ( 3.0 to 3.5 [n = 16 ]; 3. 5 to 3. 75 [n = 22 ] ) diets. Frequency distribution of cows among calving assistance categories (no assistance minor assistance and major assistance ) were not differe nt between SS (24, 18, 4 cows) and SY (21, 15, 2 cows) diets, respectively Serum anti ovalbumin antibody was affected (P < 0.01) by the interaction of diet by parity by day. Serum a nti ovalbumin antibody increased (P < 0.01) from undetectable values in

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168 sa mples before initiation of immunization scheme (0.18 0.01 OD) to increasingly higher values after first and second immunizations, and 21 days after third immunizations that was followed by a decrease (P < 0.01) until day 42 after third immunization (i.e ., 42 dpp). Serum a nti ovalbumin antibody did not differ between diets in primiparous cows before initiation of immunization scheme or after immunization days (Figure 5 6A). However, in multiparous cows, serum a nti ovalbumin antibody was greater at parturition (P < 0.07), 21 and 42 dpp (P < 0.01) for cows fed SY compared with those fed SS diets (Fig ure 5 6B). Discussion Supplementation of organic Se during the peri parturient period of dairy cows increased plasma concentrations of Se ( C hapter 4 ) in a ssociation with improved whole blood neutrophil bactericidal activity and serum humoral response to an inert antigen. Immune responses were manifested differently between primiparous and multiparous cows supplemented with Se yeast. In the pr esent study so urce of Se did not affect colostrum total immunoglobulins, using a colostrometer for measurement of total immunoglobulin. Colostrum s from beef cows supplemented with adequate quantities of Se had greater concentrations of IgG and IgM compared with cows fed inadequate quantities of Se (Awadeh et al., 1998) Also, greater colostrum volume represented more IgG produced when beef cows were supplemented with Se (Lacetera et al., 1996). Colostrum from beef cows had a higher IgG concentration when cows were supple mented with sodium selenite compared with unsupplemented and deficient counterparts ( Swecker et al., 1995). A positive correlation between concentration of IgG in serum and colostrum of beef cows was described (Awadeh et al., 1998) In the present experime nt the greater anti ovalbumin response in cows supplemented with Se yeast alluded to a greater specific immunoglobulin secretion in the colostrum However, colostrums were not collected for analysis of specific immunoglobulins. Additionally, in a review by Weiss (2003)

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169 90% increase s in milk Se concentrations were reported when Se yeast was fed compared to selenite in ten studies using beef or dairy cattle. Statistical differences were detected for concentration of Se in the majority of the studies. The cle ar impact of Se yeast supplementation on milk Se content is a result of the large amounts of selenomethionine incorporation for the secretion of milk protein, mainly casein. The increased Se contents in colostrum and milk can benefit offspring. Calves born and nursed by cows fed Se yeast had higher blood concentrations of Se at birth and higher glutathione peroxidase activity than cows fed inorganic Se (Weiss and Hogan, 2005). Furthermore, calves at weaning had a greater macrophage phagocytic activity when nursed by cows fed Se yeast (Gunter et al., 2003). Therefore, feeding Se yeast during the last 60 days of gestation may have beneficial effects on calf health by improving the Se status of the calf through milk that was not tested in the present study. The greater n eutrophil activity in cows supplemented with Se yeast was associated with adequate plasma concentrations of Se ( 0.087 g/ml ), contrary to cows in the sodium selenite diet which were in a marginally deficient Se status ( 0.0 69 g/ml ; C hapter 4 ). The greater proportion of neutrophil s with phagocytic and oxidative burst activity indicated that during the period of incubation with E. coli more neutrophils were active and/or neutrophils remained alive for longer time because of a better capacity to el iminate or reduce the damaging intracellular effects of ROS. Low er efficiency of anti oxidant enzymes, as observed in the cows supplemented with sodium selenite at parturition (i.e., lower T3:T4 molar ratio), could damage and impair microbicidal and metabo lic function and thus cell survivability. Mammalian selenoproteins catalyze biochemical reactions associated with antioxidant defense systems, thyroid hormone metabolism and redox control of cell reactions (Arthur, 1999) M acrophage bactericidal activity i s disturbed if there is reduced glutathione peroxidase activity during an oxidative burst, but

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170 enzyme activity in these cells can be reestablished by the addition of Se to an in vitro ex vivo model (Ebert Dmig et al., 1999). Moreover, i n vitro supplementa tion with Se enhanced the production of chemotaxins by cow macrophages, improved neutrophil chemotactic random migration and production of superoxide following stimulation with PMA (Ndiweni and Finch, 1995 and 1996). Supplementation of sodium selenite to b rain derived neural progenitor cell in culture reduced cell death after induction of H 2 O 2 release ( Yeo and Kang 2007). To test if neutrophils were active for longer periods of time, because of their ability to reduce more efficiently ROS, time dependent studies in which cells are incubated for incremental per iods of time need to be addressed. Research indicate s that Se de ficiency in cattle (Boyne & Arthur, 1979; Gyang et al., 1989; Grasso et al 1990; Hogan et al 1990) and pigs ( Wuryastuti et al., 1993 ) reduces neutrophil microbicidal activity Reduced neutrophil killing activity in Se deficient steers (Boyne & Arthur, 19 79) and rat peritoneal macrophages (Serfass and Ganther, 1976) was associated with absence of neutrophil glutathione peroxidase activity. The g reater proportion of neutrophil s with bactericidal activity could be due to a greater random migration. R andom mi gration and phagocytic activity of PMN from goats fed a Se deficient diet was depressed severely and incubation of these cells with Se resulted in a marked functional enhancement (Aziz et al., 1984). Selenium deficiency in goats decreased the production o f leukotrine B 4 by neutrophils and leukotrine B 4 mediated chemotaxis compared with Se adequate animals (Aziz and Klesius, 1986a). Moreover, supernatants derived from neutrophils isolated from goats fed the Se deficient diet had a significantly decreased capacity to stimulate neutrophil chemotaxis and chemiluminescence ( Aziz and Klesius, 1986b). Moreover, i nflux of neutrophils into mammary quarters infused with E. coli was more rapid in Se

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171 supplemented cows result ing in more rapid elimination of the bacter ia and less severe infections (Erskine et al., 1989). The nutritional importance of Se is attributable to its function as a component of the active site of the enzyme glutathione peroxidase. This enzyme is one of several mammalian scavenging systems which protect cells and membranes from damage by injurious oxygen radicals. The substrates for glutathione peroxidase are H 2 O 2 generated during biological oxidation processes of phagocytes and lipid peroxides formed during free radical chain reactions. A ctivated neutrophils from patients with congenital glutathione reductase deficiency have normal oxygen consumption and H 2 O 2 production during the first 5 to 10 minutes of zimozan ingestion; thereafter oxygen is no longer consumed and H 2 O 2 is not produced (Roos et al., 1979). Indeed prior incubation of neutrophils in an H 2 O 2 generating system results in a decreased ability of the cells to produce O 2 that affected to a greater degree glutathione peroxidase deficient mice This decrease in respiratory burst activity was due to a decrease in membrane associated NADPH oxidase activity resulting in impairment of neutrophil functions (i.e phagocytosis, degranulation of ROS contents) that are sensitive to H 2 O 2 intoxication ( Baker and Cohen, 1983 ). In the present study, i ncreased cellular damage of neutrophils in cows with marginally deficient plasma concentrations of Se could explain the l ower oxidative burst activity, measured by the degree of DHR oxidation in the postpartum period for primiparous cows and at parturitio n for multiparous cows The inability of the Se inadequate neutrophils to reduce ROS (i.e., H 2 O 2 ) may prevent the continuous production of ROS upon bacteria stimulation leading to cell stagnation and death. Generation of H 2 O 2 in macrophages from Se deficie nt mice was reduced in response to p horbol myristic acetate This was possibly due to increased cellular damage since glutathione peroxidase activity was lower (Parnham et al., 1983) which can compromise

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172 neutrophil half life and antimicrobial cell functio n Grasso et al. (1990) reported that v iability of neutrophil was reduced by incubation with S. aureus in Se deficient compared to Se adequate cows. Also, lipid peroxidation of bovine arterial endothelial cells is increased when cells are incubated in Se d eficient media resulting in reduced survivability of cells upon and stimulus to induce arachidonic acid peroxidation in a non enzymatic fashion (Hara et al., 2001). The effect of the source of Se on the oxidation of DHR per neutrophil was not accompanied b y an increased in bacterial phagocytosis per neutrophil, except at day 7 postpartum for multiparous cows. In vitro supplementation of Se to macrophages increased the release of TNF 1 and IL 6 cytokines upon lipopolysaccharide stimulation that can ser ve as a stimulus for phagocytosis (Safir et al., 2003). Cytokine production per neutrophil was not tested in the present study. The phagocytic activity per neutrophil was not enhanced for most of the postpartum period, but because a greater proportion of c ells were active the microbicidal activity was higher in Se yeast supplemented animals as expressed by the capture index. In the present experiment an enhancement in antibody response to a n inert antigen (i.e., ovalbumin) was evident only for multiparous cows being fed Se yeast. Therefore, Se yeast supplementation increased Se status of cows from moderate Se deficiency to adequate plasma concentrations resulting in beneficial effect s on the humoral immune response. Indeed, supplementation of Se to correct a Se deficiency has improved the a nti body response to sheep red blood cells in mice (Spallholz et al., 1973), ponies (Knight and Tyznik, 1990) and chickens ( Larsen et al., 1997; Marsh et al., 1981). Also, Se supplementation in sheep increased whole blood concentrations of Se in association with a greater antibody titer against a Chlamydia psittaci vaccine (Giadinis et al., 200). Feed s upplementa tion of Se in calves increased antibod ies to hen egg lysozyme an inert antigen (Swecker et al., 1989) and sheep red blood cells

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173 (Nicholson et al., 1993). Also, s timulation of lymphocyte proliferation with either c oncanavalin A, phytohemagglutinin, pokeweed mitogen or streptolysin stimulation was suppress ed completely in Se deficient dogs (Sheffy & Schultz, 1978) I t ha s been suggested that Se has a modifying role in the interactions of macrophages and lymphocytes (Afzal et al., 1984). The macrophage is an antigen presenting cell that links the innate with the adaptive immune system. If the beneficial results observed in neutrophils were extended to the macrophages, then greater antigen (i.e ovalbumin) ingestion and presentation could possibly benefit initiation of a humoral response. Peripheral blood lymphocytes isolated from Se deficient cows exhibited a reduced pr oliferation response to c oncavalin A, a lymphocyte mitogen in a time dependent manner (Cao et al., 1992) Lymphocytes isolated from Se deficient and Se adequate cows had similar proliferation rates for 36 hours of incubation, which was followed by a continuing increase in the Se adequate cows versus a decrease in proliferation for Se deficient cows through 96 hours of incubation (Cao et al., 1992) Moreover, i n vitro supplementation with Se enhanced cow lymphocyte prolifer ation in response to concavalin A (Ndiweni and Finch, 1995 and 1996) Additionally, lymphocytes from Se deficient mice had lower proli ferati on rates, upon concavalin A stimulation, that was associated with reduced expression of surface IL 2 receptors S elenium supplementation enhanced the number of high affinity IL 2 receptors that accelerated clonal expansion (Kiremidjian Schumacher et al., 1 992; Roy et al, 1992). Antigen binding to the T lymphocyte receptor s timulates the secretion of IL 2 and the expression of IL 2 receptors. The IL 2/IL 2 receptor inter action then stimulates growth, differentiation and survival of antigen selected cytotoxic T lymphocytes via a ctivation of the expression of specific genes. In contrast cows supplemented with high levels of dietary sodium selenite (i.e., 12 ppm) in the diets had reduced in vitro lymphocyte proliferation and production

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174 of antibodies ( Yaeger et al., 1998). L ow doses of Se (i.e., 0.5 to 2.0 M) added as sodium selenite or selenomethionine to human lymphocytes in vitro did not alter the secretion of antibodies, but at higher levels ( i.e., 5.0 to 10.0 M) an inhibitory effect was detected for sodium selenate compared with a progressive increase in immunoglobulin production observed after exposure to selenomethionine (Borella et al., 1996). Indeed, human lymphocytes had greater chromosomal aberrations (i.e., chromatid breaks, gaps and fragments) when incubated with sodium selenite compared with selonomethionin e at high concentrations ( i.e., 8.0 10 5 M ) (Khalil et al., 1989). T he mechanism s of toxicity and mutagenicity of selenite are believed to result from interaction s with thiol groups possibly lead ing to the destruction of macromolecules (Khalil et al., 1989) Thus, supplementation with organic Se is less detrimental when toxicity becomes an issue. Additionally, t hioredoxin reductase, a seleno enzyme, has been identified as a lymphocyte growth f actor and may be involved in the cross talk of macrophages and lymphocytes ( Ebert Dmig et al., 1999). Moreover, lymphocytes upon antigen presentation undergo clonal expansion for antibody production. Nuclear factor k B (NFkB) is a heterodimer nuclear tran scription factor involved in the gene transcription related to cell proliferation and differentiation. Glutathione when oxidized by H 2 O 2 is converted into glutathione disulfide which at high concentrations can convert NFkB heterodimer into a reversible oxi dized form with little or no DNA binding activity (Droge et al., 1994). Therefore, lymphocytes containing gluthathione peroxidase of greater activity can maintain a lower glutathione disulfide:glutathione ratio that is beneficial for clonal expansion. Peri pheral neutrophil function is suppressed during the periparturient period (Kehrli et al., 1989; Cai et al., 1994) which is associated with increased incidence of uterine and mammary gland disorders in the postpartum period of dairy cows (Gunnink, 1984; Kim ura et al., 2002; Cai

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175 et al., 1994; Hammon et al., 2006; Heyneman et al., 1990; Vandeputte Van Messom et al., 1993). Based upon reports in the literature and results of the present study t he beneficial effects of inorganic or organic Se supplementation wer e expressed when animals were Se deficient. In the present study, based upon plasma Se concentrations, cows were considered to be in a Se deficient status when the maximum concentration of Se was supplied in the form of sodium selenite as opposed to an ade quate status when fed Se yeast as a source of selenomethionine. In a concurrent Californian study (Rutigliano et al., 2008 ) in which experimental design was replicated as the present study feeding Se yeast compared to sodium selenite during the last 25 da ys of gestation of cows had no impact on plasma concentrations of Se (> 0.10 g/ml), total IgG concentration in colostrum, neutrophil phagocytic and oxidative burst activity and antibody response to ovalbumin immunization. Rutigliano et al. ( 2008 ) observed that concentrations of Se were in the range of 0. 60 and 0. 74 mg/kg of DM of pre and postpartum diets. Furthermore, in most feedstuffs, Se is present in organic forms (Weiss, 2003), which resembles those of Se yeast. In the present study, concentrations w ere 0.44 and 0.36 mg/Kg after Se supplements were added to pre and postpartum diets, respectively. The site differences suggest that innate and humoral immune responses of dairy cows fed different sources of Se are benefitted when fed Se yeast when Se sta tus of cows and feedstuffs are inadequate. Concentration of supplemental Se above 0.3 ppm is not permitted. A general recommendation can be made from the present studies. Sources of Se that are better absorbed and retained, such as Se yeast, are beneficial to high producing dairy cows in a marginally deficient Se status. Furthermore, recommended feeding levels of supplemental Se should be revised for dairy cows that are deficient in this mineral, and with the current legal level of Se feeding (i.e., 0.3 ppm ) an organic source of Se (e.g., selenomethionine) would be most effective.

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176 Figure 5 fed diets containing sodium selenite ( ) or selenium yeast ( --) in the pre partum (n = 18/diet; 0.445 mg Se/Kg of dry matter) and post partum (n = 20/diet; 0.30 mg Se/Kg of dry matter) periods. Diet, parity and diet by parity (non significant, day (P < 0.01) 500 1000 1500 2000 2500 3000 25 0 7 14 21 37 Neutrophils (/L) Days relative to parturition

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177 Figure 5 2. Least squares means ( S.E.) percentage of neutrophils with phagocytotic and oxidative burst activity upon exposure to heat killed E s cherichia coli in whole blood collected for all experimental cows at pre partum (n = 36) and post partum period s (n 7177 neutrophils/L). 20 30 40 50 60 1 2 3 4 % Neutrophil Quartiles of neutrophil per L of whole blood

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178 Figure 5 3. Least squares means ( S.E.) percentage of neutrophils with phagocytotic and oxidative burst activity upon exposure to heat killed E s c herichia coli in whole blood for primiparous (A) and multiparous (B) cows fed diets containing sodium selenite ( ) or selenium yeast ( --) in the pre partum (n = 18/diet; 0.445 mg Se/Kg of dry matter) and post partum (n = 20/diet; 0.30 mg Se/Kg of dry matt er) periods. Diet, parity and day interaction (P < 0.05). Diet (P < 0.01) at 7, 14 and 37 dpp in A and at parturition (P < 0.01) in B. 15 25 35 45 55 65 25 0 7 14 21 37 % Neutrophil Days relative to parturition A 15 25 35 45 55 65 25 0 7 14 21 37 % Neutrophil Days relative to parturition B

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179 Fi gure 5 4. Least squares means ( S.E.) neutrophil mean fluorescence intensity (MFI) of the green ( indicator of in tensity of ROS produced/neutrophil ; lines) and red (indicator of number of bacteria/neutrophil; bars) wave lengths of fluorescence following exposure to heat killed E s cherichia coli in whole blood for primiparous (A) and multiparous (B) cows fed diets containing sodium selenite (SS) or selenium yeast (SY) in the pre partum (n = 18/diet; 0.445 mg Se/Kg of dry matter) and post partum (n = 20/diet; 0.30 mg Se/Kg of dry matter) periods For lines, diet (P = 0.06), day (P < 0.05), parity (non significant) and higher order interactions (non significant). Diet by day (overall: non significant; except day 7 and 14 in A [P < 0.05] and day 7 in B [P < 0.05]). For bars, diet (P < 0.10), day (non significant), parity (non significant) and higher order interaction (non significant). Diet by day (overall: non significant; except day 7 in B [P < 0.05]). 0 100 200 300 400 25 0 7 14 21 37 MFI Days relative to parturition A SS SY SS SY 0 100 200 300 400 25 0 7 14 21 37 MFI Days relative to parturition B SS SY

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180 Figure 5 5. Least squares means ( S.E.) optical density for serum anti ovalbumin antibody in primiparous (n = 35; A) and multiparous (n = 49; B) cows fed diets containing sodium selenite ( ) or selenium yeast ( --) in the pre partum (0.445 mg Se/Kg of dry matter) and postpartum (0.30 mg Se/Kg of dry matter) periods. Values at initiation of immuni zation (38.5 9 days pre partum) were undetectable (0.18 0.01 OD). Diet (non significant), day (P < 0.01) and diet by day (non significant) in A. Diet at parturition (P < 0.07), 21 and 42 days postpartum (P < 0.01) in B. 0,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 24 0 21 42 Optical density (OD) Days relative to parturition A 0,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 21 0 21 42 Optical density (OD) Days relative to parturition B

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181 CHAPTER 6 EFFECT OF D IFFERENTIAL SUPPLEMENTATION OF FATTY ACIDS DURING T HE PERIPARTUM AND BREED ING PERIOD ON UTERIN E AND METABOLIC STAT US ES REPRODUCTION AND LAC TATION IN HIGH PRODU CTION DAIRY COWS Introduction The transition from the non lactating, pregnant state to lactation is a critical phase of the life cycle of the dairy cow The transition period, typically considered from the last 3 weeks pre partum until 3 weeks postpartum, is marked by a declin e in dry matter intake ( DMI ) as the cow approaches parturition and by an increase in nutrient demands with the onset of lactation E nergy intake in early lactation usually is less than what is required for maintenance and milk production Thus a negative energy status prevails when lactation is initiate d (Staples et al., 1990) Also, innate immunossupression during the transition period is characterized by reduction in neutrophil chemotaxis (Kimura et al., 2003), expression of adhesion molecules (i.e., L selectin) (Weber et al., 2001) and generation of reactive oxygen spe cies (Kimura et al., 2002; Hammon et al., 2002) that increase the risk of uterine diseases (Kimura et al., 2002; Hammon et al., 2002). In the early postpartum period, a very intense secretion of uterine PGF 2 occurs as evidenced by dramatic increases in co ncentration s of plasma 13, 14 dihydro, 15 keto PGF 2 metabolite ( PGFM ) (Guilbault et al., 1984 ) Prostaglandins are derived from the membrane phospholipid stores of arachidonic acid (C20:4 n 6 ), which are synthesized from the essential dietary fatty acid li noleic acid (C18:2 n 6 ). Also, lipoxygenases, acting on arachidonic acid, generate leukotriene B 4 which is a potent neutrophil chemo attractant (Heidel et al., 1989). Seals et al (2002) reported that concentrations of PGFM in jugular plasma were elevated during the first 6 days postpartum for cows that did not develop metritis Indeed, PG F 2 has tonic effects on the uterus during the estrous cycle (Eiler et al., 1984) or postpartum period (Gajewski

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182 et al., 1999). Moreover, in response to S taphylococcus au reus ex vivo bovine neutrophils had increased chemotaxis response and phagocytic activity in the presence of arachidonic acid metabolites such as PGF and LTB 4 (Hoedemaker et al., 1992). Elmes et al (2004) fed pre partum pregnant ewes a linoleic acid enriched diet that increas ed the proportion of arachidonic acid in the endometrium and allantochorion and increased secretion of PGF and PGE 2 in cultured placental explants Also, supplementation of pre partum cows with linoleic acid enriched calcium sa lts of fatty acids at 2% of the diet dry matter resulted in greater concentrations of plasma PGFM in the postpartum compared with unsupplemented cows (Cullens 2005). In studies with variable designs and sample sizes, pregnancy rates were improved for postpartum lactating dairy cows supplemented with fish meal ( Bruckental et al., 1989; Armstrong et al., 1990; Carrol et al ., 1994 and Burke e t al ., 1997 ). Also, cows fed flaxseed linolenic acid (C18:3n 3), had either increased first service pregnancy per artificial insemination (Petit et al., 2001 ), no effect (Ambrose et al., 2006; Fuentes et al., 2008) or reduced pregnancy loss from 30 to 50 days of pregnancy (Petit and Twagiramungu, 2006). Fatty acids of the n 3 family are thought to reduce uterine pulsatile secretion of PGF 2 that can possibly delay luteolysis (Mattos et al., 2004). S uppression of luteolytic PGF 2 secretion and maintenance of the CL are obligatory steps for establishment of pregnancy in cows (Thatcher et al., 1994) The objectives were to d etermine the optimum fatty acid supplementation program during the transition (linoleic acid enriched supplement) and breeding (eicosapentaenoic acid [C20:5n 3; EPA] and docosahexanoic acid [C22:6n 3; DHA] enriched supplement) per iods on: a) fatty acid profiles of cotyledon and caruncle tissues; b) metabolic status and uterine health during the

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183 early postpartum period; c) p regnancy per artificial insemination after two postpartum inseminations ; d ) p regnancy losses after th e first and second postpartum inseminations and e) lactation performance. The hypotheses were that feeding a supplement enriched in calcium salts of linoleic acid (safflower oil) prior to parturition and during the first 30 days postpartum (dpp) would increase the percentages of linoleic and arachidonic acids in caruncular tissue and thereby enhance synthesis of pro inflammatory eicosanoids, alter neutrophil function and improve uterine health. Furthermore, feeding calcium salts of EPA and DHA enriched supplement ( fish oil), beginning at 30 dpp until the completion of two inseminations, would increase pregnancy per artificial insemination and reduce pregnancy losses. An optimal fatty acid supplementation program will lead to physiological responses associated with i mprovements in cow health as well as reproductive and lactation performance. Material and Methods Animals Experimental Design and Feeding The experiment was conducted at a n orth Florida dairy farm comprised of 3,500 Holstein cows that were milked thrice d aily Cows were housed in free stall barns during pre and postpartum stages of the study. C ows (n = 1,582) were allocated randomly into two experimental transition diets begun at approximately 30 days prior to the expected date of parturition and continue d until 30 dpp. Primiparous and multiparous cows were blocked when assigned to diets. After 30 dpp cows within each transition diet were blocked by parity (i.e., primiparous and multiparous) and allocated randomly to the experimental breeding diets that w ere fed until 160 dpp. Experimental transition (Tables 6 1 and 6 2 ) and breeding (Table 6 2) diets differed only i n the source of supplemental fatty acid.

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184 Transition diets (i.e., pre partum to 30 dpp) consisted of calcium salts (CS) of palm oil ( PO 1.5% of DM; Ener G II, Virtus Nutrition, Corcoran CA, USA) or CS of safflower oil (SO; 1.5% of DM, Prequel 21, Virtus Nutrition, Corcoran CA, USA). Breeding diets consisted of CS of palm ( PO; 1.5% of DM; Ener G II) or CS of fish oil ( FO ; 1.5% of DM, StrataG Vir tus Nutrition, Corcoran CA, USA) The refore, the combinat ional experimental diets fed during the transition and breeding periods were PO PO, PO FO, SO PO and SO FO respectively. Diets were formulated to meet or exceed NRC (2001) nutrient requirements for net energy of lactation (NE L ), crude protein (CP), fiber, mineral and vitamins (Tables 6 1 and 6 2 ) and fed to obtain an intake of 200 and 400 g/d of CS for pre and postpartum cows, respectively Diets were fed as a total mixed ration twice daily target ing 5% orts. Group DM intake s w ere calculated daily based on the total feed offered minus refus ed throughout the study. Primiparous and multiparous cows of both experimental transition diets were housed together during the pre (enrollment to parturition) and postpartum (0 to 30 dpp) periods. Pre and postpartum cows were housed in common free stall pens that were located within separated barns for each period. After 30 dpp primiparous and multiparous cows were located in separ ate barns that housed pens for the two experimental breeding diets. Cows that had been fed for less than 14 days in the pre partum period or had a dystocia requiring veterinary assistance (i.e., cesarian section, fetotomy) and gestation lengths less than 2 65 days were excluded from the experiment. T he free stall pens within the barns used during the transition and breeding periods were identical in design, size, location, orientation and were equipped with rows of fans; one row above the feed bunk and the o thers above the stalls. Moreover, at the time in which approximately one half of the total target population of cows calved, the prepartum and

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185 postpartum cows with their respective transition diets interchanged pens within the pre and postpartum free stal l barns. Also, at the time in which one half of the total target population of cows reached approximately 90 dpp, multiparous and primiparous cows with their respective breeding diets interchanged pens within their free stall barns. T emperature ( o C) and re lative humidity (%) data were obtained from the Florida Automated Weather Network ( http://fawn.ifas.ufl.edu/scripts/reportrequest.asp ) f r om November 2006 to August 200 7 The weather station is located in Alachua, FL, approximately 40 miles from the experim ental location. Average daily temperature was converted from Celsius to Fahrenheit using the equation o F = ( o C x 9/5) + 32 The temperature humidity index (THI) was calculated as: THI = td (0. 55 0.55 RH/100) (td 58), where td is the dry bulb temperat ure in F and RH is relative humidity expressed as a percentage (NOAA, 1976) Seasons were defined Placentome C ollection A sub sample of PO (n = 11) and SO (n= 12) cows was used for collection of cot y ledonary caruncular tissue. Only cows fed the pre partum diet for more than 20 days were included. Cotyledonary caruncle units (placentome) were extracted manually through the vagina using a shoulder length sleeve. The perineal area was w ashed with p ovidone iodine s crub (0.75% titratable iodine and 1% povidone solution, Agripharm, Memphis, TN, USA) and dried before collection. Collection was within 7 hours after parturition (average 3 hours) and before placental expulsion. None of the cows developed a retain ed placenta After collection, caruncles and cotyledons were separated manually, wash ed with water put into respective plastic bags each plunged into liquid nitrogen and stored at 80 o C. Tissues were then freeze dried for 72 hours and gr ound in an aluminum blender for determination of fatty acid composition.

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186 Fatty acids in freeze dried tissue samples were converted to methyl esters in 0.5 M sodium methoxide in methanol followed by a second methylation in acetyl chloride:methanol (1:10, vol/vol) to prevent epimerization and isomerization of conjugated acids (Kramer et al., 1997 ) Methyl esters were separated by GLC (HP 5890, Agilent Technologies, Palo Alto, CA) on a 100 m x 0.25 mm x 0.2 m film thickness SP2560 capillary column with spli t (80:1) injectors. The temperature program used for the tissue fatty acids was 140C initially (held for 4 min), raised 13.0C/min to 160C (held for 44.0 min) then raised 4C/min to 220C and held for 23.47 min. Peak s were identified by comparison of ret ention times to reagent grade fatty acid standards. Vaginosc opy All cows were evaluated once between 8 to 10 dpp for cervical discharge using a disposable foil lined cardboard vaginal speculum (Milburn Distributors, Ocala, FL). Appearance of discharge was categorized into clear mucus without flecks, clear mucus with flecks, lochia, mucopurulent (50% clear mucus and approximately 50% of pus) and purulent (> 50% pus to brown and foul smell). Estrous S ynchronization and R e synchronization P rotocols Cows, begin ning at 43 dpp, were enrolled once week ly into a Presynch protocol with two injections of PGF ( 25 mg dinoprost tromethamine i.m., Lutalyse ; Sterile Solution; Pfizer Animal Health, New York, NY ) injected 14 days apart. The Ovsynch protocol was initiated 14 days after the second injection of PGF Or the Presynch with a GnRH injection (100 g; gonadorelin diacetate tetrahydrate, i.m., Cystorelin Merial Ltd., Athens, GA ) followed 7 days later by an injection of PGF and a final i njection of GnRH 56 hours later. Timed artificial insemination (TAI) for first service was performed 16 hours after the second GnRH injection of the Ovsynch protocol.

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187 All cows received a controlled internal drug releasing (CIDR, EAZI BREED; Pfizer Animal H ealth, New York, NY ) containing 1.38 g of progesterone at 18 days after the first TAI followed 7 days later by removal of the CIDR device an injection of 100 g of GnRH. At 32 days after first TAI, cows were examined ultrasonographically per rectum to iden tify presence of an embryo and an embryonic heart beat Non pregnant cows were injected with 25 mg of PGF and then injected with 100 g of GnRH 56 hours later. A TAI was performed 16 hours after the last GnRH for the second service. Cows were examined ul trasonographically per rectum 32 days after second service. All cows diagnosed pregnant after first and second services were re examined ultrasonographically at 60 days after insemination to determined pregnancy losses. Blood S amples and P rogesterone A ssay Blood samples were collected from all cows before the second injection of PGF of the Presynch and at the time of the first GnRH of the Ovsynch protocol for first postpartum AI. Blood samples were collected by puncture of coccygeal vessels into evacuated tubes containing K2 EDTA (Vacutainer, BD, Franklin Lakes, NJ, USA). Samples were placed immediately into an ice bath until centrifugation for 20 minutes at 2619 x g. After centrifugation, plasma was harvested and stored frozen at 20 C until assayed. Wit h this set of blood samples, cows were considered to have initiated estrous cycles if plasma concentration of progesterone (P4) was greater than 1 ng/ml in at least one of the two samples (cyclic) or non cyclic or anovulatory if both samples were below 1 n g/mL. Concentrations of plasma P4 were determined by radioimmunoassay using a Coat A Count Kit (DPC Diagnostic Products Incorporation, CA, USA) designed for the quantitative measurement of P4 in plasma. Duplicated reference samples for plasma P4 were low Intraassay coefficients of variation of low, medium and high samples were 4.1%, 4.7% and 6.0%

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188 respectively. Interassay coefficients of variation were 6.1%, 7.2% and 10.4%, respectively. S ensitivity of the assay was 0.1 ng/mL. Body C ondition S core Cows were evaluated for body condition score ( BCS ) at the day of enrollment, at parturition, 43 dpp, and at the first TAI Scores were given by two veterinarian s based on a 1 (thin) to 5 (obese) scale using a quarter point system (Edmonson et al., 1989). Changes in BCS were obtained by subtracting : BCS at parturition from BCS at enrollment, BCS at 43 dpp from BCS at parturition, and BCS at TAI from BCS at 4 3 dpp. The BCS gain or loss was used as an indicator of energy status. Milk Y iel d and Feed Samples Milk weights were recorded once a month for all cows. First measurements occurred at different times after parturition for the first monthly sample depending upon the day of parturition in relation to the day of the monthly milk test for the herd. Because cows were assigned to the treatments randomly on a weekly basis, the day of the first postpartum measurement was balan ced between treatments. The single measurement of milk production for each month was considered as the average for the month. Data from the first 5 months of lactation were used. Total mixed rations were sampled weekly at the time of feeding from respectiv e feed bunks and immediately frozen in plastic bags. Diets were composited (i.e., 4 samples/month), freeze dried, further composited into a 3 month sample and analyzed by wet chemistry methods for chemical composition (Dairy One, Ithaca, NY ) (Tables 6 1 an d 6 2). Total lipid concentration and fatty acid profile of fat supplements and diets were determined using the extraction and methylation procedures of Kramer et al. (1997). An internal standard of C19:0 (0.5 mg) was added in order to calculate total fat concentration. The fatty acid methyl esters were

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189 determined using a Varian CP 3800 gas chromatograph (Varian Inc., Palo Alto, CA) equipped with an auto sampler (Varian CP 8400), a flame ionization detector, and a Varian capillary column (CP Sil 88, 100 m x 0.25 mm x 0.2 m). The carrier gas was He, the split ratio was 10:1, and the injector and detector temperatures were maintained at 230C and 250C, respectively. The sample (1 L) was injected with an auto sampler. The oven temperature was set initially a t 120C for 1 min, increased by 5C/min up to 190C, held at 190C for 30 min, increased again by 2C/min up to 220C, and held at 220C for 40 min. The fatty acid peaks of samples were identified and concentrations calculated based up on the retention time and peak area of known standards. PGFM and B lood M etabolites A sub sample of cows (PO [n = 15] and SO [n = 17]) was used for blood sampling during the early postpartum period. Blood samples were collected and processed as described above for progesterone measurements. Samples were collected once daily from parturition to 10 dpp for measurement of plasma PGFM concentrations as an index of uterine PGF secretion. Plasma samples were analyzed for concentrations of PGFM using a modification of the radioimmuno assay procedure described by Mattos et al ( 2004 ). The PGFM standard solutions w ere made by serial dilutions in a buffer of a stock solution (1 g/m L in 10% ethanol and 90% PBS buffer) of authentic PGFM (Sigma, S t. Louis, MO). Standards (100 L ) were run i n duplicates at the following concentrations: 31.2, 62.5, 125, 250, 500, 1000, 2000, 4000, and 8000 pg/m L The standard curve included 100 L of prostaglandin free plasma, which was obtained from a cycling non lactating beef cow pre treated twice with an i .m. injection of 1 g of flunixin meglumine (Banamine, Intervet/ Schering Plough Animal Health Kenilworth, NJ ), a prostaglandin endoperoxide synthase ( PGHS ) inhibitor. Samples of prostaglandin free plasma had undetectable concentrations (< 0.0 1 n g/m L ) of PGFM when assayed utilizing standard curves

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190 in 0.05 M PBS buffer. The PBS buffer contained 2.3 g/ L NaH 2 PO 4 H 2 O, 4.76 g/ L Na 2 HPO 4 7H 2 O, 1 g/ L sodium azide, and 8.41 g/ L NaCl. The buffer pH was adjusted to 7.5 with NaOH. Activity and volume of radioactive ly labeled PGFM ( Amersham Pharmacia Biotech, Piscataway, NJ ) used were 10,000 cpm and 100 L respectively. For unknown samples, final assay volume was 400 L ; it comprised 100 L of sample, 100 L of rabbit antiserum to PGFM ( 1:10,000; Meyer et al., 1995) 100 L of buffer, and 100 L of labeled PGFM. After a 12 h incubation at 4C, free PGFM was separated using 750 L of a solution of charcoal coated dextran (1.25% dextran [Sigma, St. Louis, MO], 12.5% activated charcoal [Sigma, St. Louis, MO] in a PBS bu ffer). After centrifugation for 20 min at 3565 g the supernatant was transferred to scintillation vials and mixed with 4.5 m L of scintillation fluid (Scintiverse II, Fisher Scientific, Pittsburgh, PA). Activity was measured using a liquid scintillation counter (model LS6000 IC, Beckman, Fullerton, CA USA ). Inter and intraassay coefficients of variation for one reference sample (1 432.1 pg/mL) were 3.65% and 4.15%, respectively. Inter and intraassay coefficients of variation for a second reference samp le (3 292.7 pg/mL) were 4.38 and 6.65%, respectively. Sensitivity of the assay was 31.2 pg/mL. The same sub sample of cows were sampled for blood thrice weekly between 10 and 35 dpp to analyze p lasma concentrations of non esterified fatty acids ( NEFA ) (NEF A HR 2 kit; Wako Diagnostics, Richmond, VA) as modified by Johnson and Peters ( hydroxy butyric acid ( BHBA ) ( Wako Diagnostics, Richmond, VA) A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to determine thrice weekl y concentrations of plasma urea nitrogen following a modification of Coulombe and Favreau (1963) as described in Bran + Luebbe Industrial Method #339 01 and plasma glucose following

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191 a modification of Gochman and Schmitz (1972) as described in Bran + Luebbe Industrial Method #339 19. Statistical Analyses Milk yield, proportion of cyclic cows, pregnancy per AI, pregnancy losses and c umulative proportion of pregnant cows after two services were analyzed using pre determined statistical contrasts to t est the effects of the transition diets ( PO PO + PO FO vs. SO PO + SO FO ), breeding diets ( PO PO + SO PO vs. PO FO + SO FO ) and the interaction of transition and breeding diets ( PO PO + SO FO vs. PO FO + SO PO ). Proportion of cyclic cows, pregnancy per AI pregnan cy losses and cumulative proportion of pregnancy cows after two services were analyzed by logistic regression (SAS Institute Inc; Version 9.1). The logistic regression stepwise selection procedure was used for all independent variables and possible intera ctions with pre determined significance levels set for a variable to be entered with a P P Mathematical model for cyclic responses included diet, parity, calving assistance (no assistance, minor assistance, maj or assistance), cervical discharge score, 5 month accumulated milk weight, BCS, changes in BCS and all higher order interactions. Mathematical model for pregnancy responses included diet, parity, calving assistance, cervical discharge score at vaginoscopy, cyclic status (cyclic or non cyclic), inseminator, sire, season ( i.e., THI < 7 2 2 ), accumulated milk weight, BCS, changes in BCS and all higher order interactions. Accumulated milk weight and BCS data were categorized into quartiles using the Univar iate procedure of SAS. After identifying variables that were significant, the model was reduced and analyzed using the generalized linear model (G ENMOD ) of SAS with a binomial distribution and a logit link function to obtain odds ratios, confidence interva ls and probability values.

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192 Milk yield data for the first 5 months were analyzed using the repeated measures analyses of the mixed model of SAS. Covariance structures tested included compound symmetry, autoregressive 1, and unstructured. Autoregressive 1 had the best relative goodness of fit based upon penalty criteria (Bayesian criterion). The statistical model consisted of diet, parity, month postpartum, BCS (i.e., at enrollment, parturition, 43 dpp and at TAI) and higher order interact ions. Cow, the random variable was considered as the experimental unit nested within diet and parity Milk yield also w as analyzed using the test of homogeneity of polynomial regression of the general linear models (GLM) procedure of SAS. This approach de termines whether individual curves for each treatment provide a better fit for the data than a single pooled curve (Guilbault et al., 1985). The GENMOD procedure of SAS was used to analyze cervical discharge scores with a multinomial distribution of respon se and a logit link function. The mathematical model included diet (i.e., PO and SO), parity, diet by parity interaction, calving assistance (i.e., no assistance, minor assistance, major assistance), retained fetal membranes (i.e., yes or no) and BCS (enro llment and parturition ). Binomially distributed health data (i.e., mastitis, ketosis, lameness, and displacement of abomasum) obtained from farm records were analyzed in GENMOD procedure of SAS as w ith a binomial distribution and a logit link function Pla sma concentrations of PGFM and blood metabolites (i.e., BHBA, NEFA, plasma urea nitrogen and glucose) were analyzed using repeated measures responses of the mixed model procedure of SAS. Data were tested for normal distribution of the residuals by the PROC UNIVARIATE procedure of SAS. Residuals were considered to be normally distributed when the Shapiro Wilk statistic was equal to or greater than 0.90, and log transformed if required. For each dependent variable, the autoregressive 1 covariance structure wa s selected because it had

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193 the best relative goodness of fit based upon penalty criteria (Bayesian criterion). The mathematical model contained diet (PO and SO), day and diet by day interaction. The random variable was cow nested within treatment. Group DMI was analyzed using the repeated measures analyses of the mixed model of SAS. Covariance structures were tested in order to determine the best relative goodness of fit. A weekly DMI average was used in a mathematical model consisting of diet, parity, perio d (i.e., before or after cows and their diets were interchanged between pens within the free stall barns), experimental weeks nested in period and higher order interactions. R esults Animals, Diets, DMI, BCS, Vaginoscopy A total of 1,582 cows (PO [n = 800] and SO [n = 782]) were enrolled and calved during the period of November 2006 to February 2007. A total of 202 cows (PO [14.8%; 119/800] and SO [10.6%; 83/782]) were excluded at parturition because the length of pre partum feeding was less than 14 days. Therefore 1,380 cows (primiparous [n = 460] and multiparous [n = 920]) remained in the experiment. Length of pre partum feeding was shorter (P < 0.05) for primiparous (26.9 0.47 days) than multiparous (28.25 0.38 days) cows and did not differ between t ransition diets (27.5 0.47 days). Of the 1 380 parturitions 1 092 cows had breeding records for calculation of gestation length. Gestation lengths were longer (P < 0.01) for primiparous (288.3 1.5 days) compared with multiparous (280.7 1.0 days) cows but it did not differ between transition diets (284.5 1.28 days). Parturitions throughout the experimental period occurred at the same monthly rate for the two transition diets. Of the 1,380 experimental cows, 297 cows (PO PO [19%; 65/343], PO FO [18.6 %; 66/355], SO PO [24.5%; 83/339], and SO FO [24.5%; 84/343]) were excluded before first service pregnancy diagnosis and the frequency was greater

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194 2 = 6.63) for SO than PO transition diet. Exclusion was because cows were moved inadvertently out of experimental lots, or for health, culling and death. Chemical compositions of pre and postpartum transition diets (i.e., PO and SO; Table 6 1 and 6 2) and breeding diets (Table 6 2) were similar. Evaluation of the fatty acid compositions for the fat s upplements (Table 6 3) indicated a descending n 6: n 3 ratio of fatty acids for the SO (49.7), PO (29.4) and FO (< 1) supplements, respectively. The greater n 6: n 3 ratio in the SO supplement was because of the high quantity of linoleic acid in this suppl ement (63.6%). The low n 6: n 3 ratio in the FO supplement was because of the greater concentrations of EPA (5.4%) and DHA (5.3%) compared with the other fatty acid supplements. Fatty acid compositions of the total mixed rations (Table 6 4) were characteri zed by greater proportions of linoleic acid and EPA DHA in diets supplemented with SO and FO, respectively. Mean group DMI was greater (P < 0.01) in the SO (14.4 and 19.0 Kg/d) compared with the PO (13.5 and 17.6 Kg/d) diets at pre and postpartum stages, respectively. During the breeding period DMI was greater (P < 0.01) in the PO (23.8 Kg/d) compared with the FO (22.2 Kg/d) diet. Frequency distribution of cows among different BCS quartiles for all stages by diets is summarized in Table 6 5. Frequency dist ribution of cows among different BCS quartiles did not differ at the time of initiation of transition diets (BCS close up) and was not affected by transition diets at parturition and at 43 dpp (Table 6 5). Furthermore, proportion of cows that lost, maintai ned or gained BCS did not differ between the transition diets. A greater (P < 0.01) proportion of FO cows were in the high BCS quartile category (i.e., 3.2 to 3.75 BCS) compared with PO breeding diets. Indeed, a greater proportion of cows gained BCS from 4 3 dpp to TAI in the FO (11% [lost 0.75 to 0.25]; 47% [maintained]; 42% [gained 0.25 to 1.0] compared with PO (16% [lost 0.75 to 0.25]; 47% [maintained]; 37% [gained 0.25 to 1.0] diets. The increased

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195 proportion of cows that gained BCS in the FO diet (i.e., 5%) was related to a decrease in the proportion of cows that lost BCS (4.4%; Table 6 5). Also, a relationship between milk yield and BCS was detected. There was a lower (P < 0.01) proportion of cows in the lower BCS category that produced less than 43 kg/d of milk compared with those that produced more that 43 kg/d at both 43 dpp (2.0 to 2.75 [57.5% (n = 320) vs. 75.3% (n = 444)]; 3.0 to 3.5 [42.5% (n = 236) vs. 24.7% (n = 146)]) and at TAI (2.25 to 2.75 [46.3% (n = 236) vs. 63.0% (n = 354)], 3.0 to 3.75 [5 7.7% (n = 274) vs. 37.0% (n = 208)], respectively. Frequency distribution of cows among calving assistance categories was not affected by diets, BCS at enrollment and parturition. A greater (P < 0.01) proportion of primiparous (n = 344) cows had minor assi stance (18.9% vs. 10%) to major assistance (i.e., use of a calf jack; 22.7% vs. 5.7%) compared with multiparous (n = 759) cows, respectively. Frequency distribution of cows among cervical discharge categories examined at 8 dpp (Table 6 6) was not affected by transition diets; however, primiparous cows, calving assistance and retained fetal membrane were variables associated with greater (P < 0.01) proportion of purulent cervical discharge. Fatty A cid P rofile s in C ot i ledon and C aruncle Tissues Fatty acid pro files in cotyledon and caruncle tissues for a sub sample of SO (n = 12) and PO (n = 11) cows are summarized in Table 6 7. The sub sample of cows was fed during the pre partum period for 33 9 days. Total fatty acid content was l ower (P < 0.01) in fetal co tyledonary tissue than maternal caruncular tissue (4.5 < 8.6 g/100g tissue; Table 6 8). The predominant fatty acid in the cotyledon and caruncle was oleic acid (C18:1n 9; 24.5%) and stearic acid (C18:0; 20.8%), respectively. Saturated fatty acid content wa s less (P < 0.01) in cotyledon compared with the caruncle, and a greater (P < 0.01) content of unsaturated fatty acid was in the cotyledonary tissue (Table 6 8). Cotyledon had a less (P < 0.01) content of myristic acid (C14:0) and stearic

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196 acid, and a great er (P < 0.01) content of oleic acid, C18:1 cis 11, CLA t ran s 9, t rans 11 and DHA. Moreover, the cotyledonary content of linoleic acid was less (P < 0.01) compared to the caruncle. Consequently, the n 6: n 3 ratio was less in the cotyledon (Table 6 8). Caru ncular content of linoleic acid tended to be greater (P < 0.10) in SO compared to PO diets (Table 6 7). Additionally, palmitic acid (16:0) was less (P < 0.01) in cows supplemented with SO compared with PO (Table 6 7). First Service Pregnancy per AI Transition, breeding and interaction of diets did not affect the proportion of cyclic cows (Table 6 9). However, proportion of cyclic cows was less (P < 0.01) in primiparous than multiparous cows, cows with a purulent cervical discharge and those with BCS equal to or lower than 2.75 (Table 6 9). Transition, breeding and interaction of diets did not affect pregnancy per AI at 32 and 60 days after TAI (Table 6 10). Pregnancy loss from day 32 to day 60 after TAI (10.3% [39/380]) was not affected by any variabl es other than breeding diets (Table 6 1 0 ). Cows in the FO (6.3% [12/190]) diet had less (P < 0.05) pregnancy losses compared with cows in the PO (13.6% [27/198]) diet (Table 6 1 0 ). First service pregnancy per AI examined at 32 days after TAI was not differ ent between primiparous and multiparous cows. However, it was less (P < 0.01) in non cycling than cyclic cows (Table 6 11). Pregnancy per AI was less (P < 0.01) for cows diagnosed by vaginoscopy at 8 dpp with flecks cervical discharge compared to cows with clear cervical discharge (Table 6 11). Pregnancy per AI was numerically less for cows diagnosed with mucopurulent and purulent cervical discharge than cows with clear cervical discharge (Table 6 11). Pregnancy per AI was less (P < 0.01) for cows with BCS at TAI equal to or less than 3.0 compared with BCS > 3.0 (Table 6 11). Pregnancy at 60 days after TAI was affected by cyclic status and cervical discharge scores in a manner similar to day 32 pregnancy per AI (Table 6 11).

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197 Second Service Pregnancy per AI Second service pregnancies per AI for all diets are summarized in Table 6 1 2 A smaller proportion of cows were inseminated for the second service during the cool season (31.9% [193/604]) compared with the warm season (68% [411/604]). Pregnancies per AI ra tes examined at 32 and 60 days after second TAI were less (P < 0.01) for cows inseminated during the warmer season (Table 6 1 3 ). Breeding diet altered (P < 0.05) pregnancy per AI and a tendency (P < 0.10) for an interaction was detected between transition and breeding diets (Table 6 13) The increase in day 32 pregnancy per AI cause d by FO was greater in cows fed the SO transition diet, whereas there was no increase in pregnancy per AI in cows fed the PO breeding diet regardless of transition diet (Table 6 13) Likewise, a similar transition by breeding diet interaction (P < 0.05) was detected for the 60 day pregnancy per AI response (Table 6 13) An interaction of transition and breeding diets by season (P < 0.05) was detected. The interaction of transition and breeding diets by season was associated with a differential effect of FO and PO breeding diets fed during the warm season compared to the cool season for days 32 (Figure 6 1B) and 60 (Figure 6 2B) of pregnancy Fish oil fed during the warm season had a stimulatory effect on pregnancy per AI if fed following SO in the transition period, whereas PO fed in the breeding period had no beneficial effect, perhaps even a negative effect, on pregnancy per AI following SO fed in the transition period. During the cool season, breeding diet differences were minimal regarding whether PO or SO were fed during the transition period for days 32 (Figure 6 1B) and 60 (Figure 6 2B) of pregnancy Pregnancy loss from day 32 to day 60 after TAI (6.5% [12/185]) for the second service was not affected by any variables including transition and breeding diets or interaction of diets (Table 6 12) Second service pregnancy per AI at days 32 and 60 after insemination was not affected by parity, but was less (P < 0.05) in cows that l ost BCS from day 43 postpartum to the day of first insemination service (Table 6 14)

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198 Accumulated Proportion Pregnant Accumulated proportion pregnant and pregnancy loss comprise the total pregnancies examined following first and second services. Analyses were conducted only for contrast of diets, because other variables associated with pregnancies were examined separately for first and second services. Accumulated proportion pregnant at day 32 after TAI was not affected by transition or breeding diets, but tended to be significant (P = 0.10) for the interaction of diets (Table 6 15 ). Accumulated proportion pregnant at 60 d ays after TAI was not affected by transition diets, but it was greater (P < 0.01) for cows supplemented with FO compared with PO during the breeding period. A tendency (P = 0.07) was observed for interaction between transition and breeding diets (Table 6 1 5 and 6 16). Accumulated pregnancy losses were not affected by transition or interaction of transition and breeding diets, but it was less (P < 0.01) in cows supplemented with FO compared with PO (Table 6 15 ). Milk Yield Average milk yield for combinations of transition and breeding diets were PO PO (41.1 0.6 Kg/day; n = 295), PO FO (41.3 0.7 Kg/day; n = 280), SO PO (41.7 0.6 Kg/day; n = 302) and SO FO (42.1 0.7 Kg/day; n = 289). Average milk yield was affected (P = 0.02) by transition diets such th at cows supplemented with SO (41.9 0.4 Kg/day; n = 591) during the transition period had a greater average milk yield for the 5 months postpartum compared with cow fed PO (41.2 0.4 Kg/day; n = 575). Average milk yield was not affected by breeding diet or the interaction between transition and breeding diets. Analysis of lactation curves detected an interaction of diet and months (P < 0.01) with a greater increase in milk production from the first month postpartum to the remaining months in the SO supple mented cows compared with cows fed PO in the transition period (Figure 6 3A). A breeding diet by month interaction also was detected because cows fed FO had greater milk yield

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199 in the second and third months of lactation than cows fed PO, followed by a grea ter decline during the fourth and fifth months compared with PO supplemented cows (Figure 6 3B). These differences were detected by homogeneity of regression analyses (Wilcox et al., 1990) that indicated a better fit when four cubic curves (i.e., four diet ary treatments) were generated as opposed to a single pooled lactation curve. Further analyses indicated that the transition diets (SO vs. PO) and breeding diet (FO vs. PO) curves were not parallel. Transition diets curves (Figure 6 3A) for cows supplement 25.00625 + 15.193 x 3.88351 x 2 + 0.31132012 x 3 ) or SO 23.7949 + 15.815x 3 .5985 x 2 + 0.242324 x 3 ) were not parallel (Figure 6 3A; P < 0.01). Similarly, breeding diets curves represented by cows supplemented with 25.7589 + 12.7894 x 2.75097 x 2 + 0.18201599 x 3 22.9287 + 18.4347 x 4.823854 x 2 + 0.38279733 x 3 ) differed (Figure 6 3B; P < 0.01). Multiparous cows (46.0 0.4 Kg/day; n = 744) produced more milk (P < 0.01) than primiparous cows (37.0 0.5 Kg/ day; n = 422) over the first 5 months of lactation. Average milk yield across all diets and cows peaked between months 3 and 4 of lactation (P < 0.01). PGFM and B lood M etabolites Blood sampling during the postpartum period was terminated in 10 cows of each diet because of treatments with antibiotics and anti inflammatory drugs. Therefore, 15 PO and 17 SO cows were used for analyses of concentration of PGFM and blood metabolite in plasma. The subset of cows selected for blood sampling were fed the SO diet for a longer (P < 0.01) period of time (34.8 1.4 days) compared to PO (30.8 1.3 days) cows in the pre partum period. Proportion of cows in BCS categories at the day of enrollment did not differ between diets. All cows had a normal parturition (i.e., no assistance), e xcept for one cow requiring minor assistance. A total of 10, 2, 1, 2 cows in the PO and 9, 5, 0, 3 cows in the SO diets were

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200 diagnosed with either clear or normal lochia, flecks, mucupulent or purulent cervical discharge at 8 dpp and were not treated with antibiotics or anti inflammatory. Plasma concentrations of PGFM was not affected by transition diets (Figure 6 4) except for days 4 and 7 postpartum, in which a greater (P < 0.01) concentration was detected for the SO (2 809 310 and 2 667 314 pg/mL) c ompared with PO (2 081 325 and 1 443 325 pg/mL) diets, respectively. Mean plasma concentration of NEFA (316 38 and 372 37 mEq/L), BHBA (5.5 0.4 and 5.3 0.4 mg/dL), plasma urea nitrogen (21.9 5.5 and 19.3 5.4 mg/dL) and glucose (55.9 5.4 a nd 58.6 5.3 mg/dL) were not affected by PO or SO transition diets, respectively. However, NEFA and BHBA increased (P < 0.01) whereas glucose and plasma urea nitrogen decreased after parturition (P < 0.01). D iscussion Supplementation of CS of fatty acids enriched with linoleic acid during the transition period resulted in an occasional increases in plasma concentrations of PGFM, did not improve uterine health at 8 10 postpartum, but increased milk yield and DMI that may have been caused by a better overall cow health. After 30 days postpartum, supplementation of CS of fatty acid enriched with fatty acids of the n 3 family increased second service pregnancy per AI and the accumulated proportion of pregnant cows after the first two postpartum AI, particularly when cows were fed SO in the transition period. The combination of n 6 fatty acids during the transition period followed by n 3 fatty acids during the breeding period represented the best combination of fatty acids for dairy cows in the current study. The CS of palm oil supplement was comprised of small concentrations of n 6 and n 3 fatty acids allowing for a suitable comparison with the CS of safflower oil, which contained high concentration of linoleic acid, and CS of fish oil which was rich in n 3 fatty acids. Indeed the

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201 fatty acid profile of the total mixed rations reflected an approximate 15% increase in linoleic acid in the SO compared with PO transition diet. Also, the presence of EPA and DHA in the CS of fish oil resulted in approximately a 2% incre ase in these fatty acids in the total mixed ration fed in the breeding period. The greater proportion of linoleic acid in the SO supplement during the pre partum period resulted in a greater incorporation of this fatty acid into maternal caruncular tissue s. The absence of detectable concentrations of arachidonic acid in the caruncular tissue agrees with Mattos et al (2004) and may reflect a continual turnover of arachidonic acid for the extensive synthesis and secretion of PGF characteristic of the immed iate postpartum period of dairy cows (Guilbault et al., 1984). However, the greater content of linoleic acid in the caruncle of SO supplemented cows may reflect a greater availability of linoleic acid as a precursor of arachidonic acid and possibly greater synthesis of eicosanoids. Clearly, endometrium of lactating dairy cows is enriched in arachidonic acid on day 17 of the estrous cycle (approximately 15.3 g/100 g of fatty acids; Bilby et al. 2006) when PGF secretion is much less than the peri parturient period. Neutrophil responses (i.e., bactericidal activity and presence of adhesion molecules) were greater in cows supplemented with SO during the early postpartum (4 and 7 dpp, C hapter 7 ). These responses are known to be stimulated by arachidonic acid pr oducts such as PGF ( Badwey et al., 1981; Badwey et al., 1994; Hardy et al., 1991; Poulos et al., 1991) and leukotriene B 4 (Jannace et al., 1992). Significant increases in plasma PGFM concentrations associated with SO supplementation occurred on days 4 an d 7 postpartum but no overall sustained difference was detected. Feeding sources of n 6 fatty acids during the transition period have increased PGFM concentrations in the first 10 dpp (Cullens et al., 2006) in cows and at the first day postpartum in primip arous cows (Juchem et al., 2008). On the other hand, supplementation of dairy cows with

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202 CS of fish oil has the potential to increase n 3 fatty acids in caruncular (Mattos et al., 2004) and endometrial tissues (Bilby et al., 2007), and attenuate inflammator y responses. This study is the first to report that dairy cows supplemented with calcium salts of SO that is enriched in linoleic acid can increase linoleic acid and the n 6: n 3 ratio in caruncular tissue. Therefore, daily feeding of calcium salts of fatty acids can strategically impact tissue fatty acid content. Caruncular total fatty acid content was almost 2 fold greater than in the cotyledon The greater and lesser content of oleic acids and linoleic acid, respectively, in the cotyledon compared with the caruncular tissue is in agreement with Elmes et al (2004) who found much lower concentrations of linoleic acid in the fetal placenta of sheep, represented by the allantochor ion compared with the caruncule However oleic acid was not repo rted. Feeding SO supplement slightly increased the li noleic acid content and the n 6: n 3 ratio in the cotyledon In the cotyledonary placenta of the cow the exchange between maternal and fetal circulation occurs in the placentomes where maternal caruncula r tissues and fetal allantochorion interdigitate Therefore, placenta fatty acid composition is predisposed to changes accordingly to maternal nutrition. Importantly, changes in the n 6: n 3 ratio of fatty acids in the diet of cows fed SO or PO did not cha nge gestation lengths either in primiparous or multiparous cows. This suggests that altering the fatty composition of the diet fed in late gestation does not alter gestation length of dairy cows. The desaturation ratio calculated based upon the ratio of C 18:1 n 9/C18:0 was greater for the cotyledon (1.80) than the caruncle (0.76), regardless of diet. The differences between these tissues may indicate a depressed activity of the enzyme delta 9 desaturase in the caruncle tissue that maintained a greater satu ration of fatty acids in this tissue compared with the cotyledon. The delta 9 desaturase also converts trans 11 C18:1 to c is 9 t rans 11 CLA. T he ratio c is 9 t rans 11

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203 CLA : t rans 11 C18:1 was 0.26 for cotyledon on palm oil and 0.14 for caruncle on both diets which agrees that the desaturase was inhibited. The c alculat ion is not possible for cotyledon on safflower oil because the concentration of c is 9 t rans 11 CLA was 0 g/100 g fatty acid In this experiment BCS did not differ between diets until 43 dpp but a greater proportion of cows gained BCS from 43 dpp to first insemination day (i.e., approximately 80 dpp) in cows supplemented with FO compared with PO This increase in BCS occurred during the period in which FO cows were producing more milk (i.e., months 2 and 3 of lactation). Group DMI was lower for cows in the FO compared with PO diets. An association of DMI with greater proportion of cows gaining BCS in the FO diet is not possible because it is not known the DMI for each cow at specific months p ostpartum. However, taking in account the postpartum period in which FO diet was fed, equal milk yields, greater proportion of cows gaining BCS at lower DMI may represent a better metabolic efficiency in cows fed FO compared with PO. Metaboli tes measured in plasma in a sub sample of cows during the postpartum period were not affected by SO or PO transition diets. Metabolic responses are influenced mainly by energy balance which is represented by the difference in energy consume d minus ener gy expended for maintenance and lactation. The difference becomes negative during the transition period, starting before parturition and reach ing a maximum nadir in the first week after parturition (Grummer, 1995). The degree of negative energy balance in the postpartum p eriod depends on body condition score at parturition the degree in reduction of intake, and quality of the diet (Garnsworthy and Topps, 1982). The frequency distribution of cows among the body condition scores was the same between transition diets for the sub sample of cows and for all experimental cows. Moreover, the energy content of the transition diets was the same. The dry matter intake was greater in the SO supplemented cows that may have contributed to subsequent

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204 increase in milk yield. Mattos et al (2004) found greater plasma concentrations of glucose and lower of BHBA in cows fed olive oil compared with those fed fish oil These differences may be attributable to a lower DMI in the fish oil fed group. The proportions of cows with clear, flecks, mu copurulent or purulent discharge were not different between diets. Junchem et al (2008) reported that feeding of CS of l inoleic and trans o ctadecenoi c acids during the transition period reduced the incidence of puerperal metritis. Indeed, c ows with greater PGFM concentrations during the first 5 dpp had lower risk of metritis (Seals et al., 2002; Silvestre et al., 2005). Greater proportions of primiparous cows had minor to major occurrences of assisted calving that resulted in greater incidences of mu copurulent or purulent cervical discharges Stress related with manipulation of cows and ascendant contamination of the uterus can contribute to this relationship. This result emphasizes the importance of managing heifers to be bred at an optimum body size and choices of sire related to calving ease. Cows diagnosed with cervical discharges containing flecks had the lowest first service pregnancy per AI. In this group of cows, only 7% were treated with antibiotics compared with 38% for cows with mucopurulent and purulent discharges during the early postpartum period. Cows diagnosed with mucopurulent and purulent discharges had greater pregnancy per AI than cows with flecks. Possible antibiotic treatments may have ameliorated uterine bacterial clearance that r esulted in a partial improvement of fertility. Alternatively, cows with discharges of flecks may have developed minor uterine contamination that was not sufficient to induce an immune response resulting in a chronic uterine bacterial contamination. Inciden ce of subclinical endometritis was not evaluated in this study, and the relationships between antibiotic treatment and cervical discharge score in the early postpartum period warrants further investigation.

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205 Proportion of cows cycling was not affected by tr ansition, breeding or the interaction of diets. Cows with purulent discharge in the early postpartum had a greater frequency of anestrous. Also, proportion of cows cycling was less when BCS was less than 3.0 at 43 dpp. The association of uterine bacterial infection on day 7 postpartum was associated with delayed first postpartum ovulation (Williams et al., 2007; Sheldon et al., 2002) and a negative association between BCS and the incidence of anovulatory/anestrous cows has been reported (Santos et al., 2004 ; Moreira et al., 2001). The energy status of dairy cows affects resumption of ovarian cycles, and BCS is an indicator of body reserves, mostly body fat, in dairy cows (Ferguson et al., 1994). In the present study the response to feeding FO differed betwee n the first and second postpartum AI. Pregnancy per AI increased with feeding FO in the second postpartum AI, whereas maintenance of pregnancy was improved by FO in the first postpartum AI. Taken together, feeding FO improved the accumulated proportion of pregnant cows following the first two postpartum inseminations. Differences in the nature of reproductive benefits regarding timing could have resulted from duration of fatty acid supplementation that was greater when second service occurred (90 days) comp ared with first service (50 days). However, the 50 day period allowed for FO priming was sufficient to change the profile of n 3 fatty acids in the uterus at 17 days of cycle (Bilby et al., 2006). Feeding of fish meal improved the proportion of pregnancy cows in several studies ( Armstrong et al., 1990; Carrol et al., 1994; Burke et al., 1997). Also, Petit and Twagiramungu (2002) observed that embryonic mortality from day 30 to 50 post insemination tended to be reduced in cows that received linseed (i.e., n 3 enriched) compared with cows that received CS of palm oil.

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206 Up to 40% of total embryonic losses are estimated to occur between d 8 and 17 of pregnancy (Thatcher et al., 1994). This high proportion of losses precedes or occurs concurrently with the perio d when the embryo inhibits uterine PGF 2 secretion. Thus some loss may occur because non viable or undersized embryos are unable to inhibit secretion of PGF 2 T he ability of n 3 fatty acids supplements to delay luteolysis has been proposed as a potential method of improving cattle fertility by giving the conceptus longer to develop before the onset of luteolysis (Mattos et al., 2004). Therefore strategies to partially inhibit secretion of PGF 2 may increase pregnancy rates and embryonic survival. Mattos and colleagues (2000) suggested that fish meal could reduce uterine secretion of PGF 2 that delayed completion of functional luteolysis. Indeed, supplementation of n 3 PUFA in the form of fish oil increased fatty acid concentrations of EPA and DHA in carun cular (Mattos et al., 2002) and endometrial (Bilby et al., 2006) tissues. The greater availability of these fatty acids in the uterus can competitively inhibit the oxygenation of arachidonic acid by cyclooxygenase and therefore reduce the synthesis of PGF 2 (Mattos et al.,2003; Mattos et al., 2004) leading to increased synthesis of prostanoids of the 3 series at the expense of the prostanoids of the 2 series. Prostanoids of the 3 series are less bioactive (Needleman et al., 1979), and there appears to be no evidence for their role in ruminant luteolysis. Prostaglandin F 3 has only 25% affinity for the ovine luteal receptor compared with PGF 2 (Balapure et al., 1989). The greater pregnancy per AI at 30 and 60 days after the second service for FO fed cows w as detected during the warm season of the year and when FO supplemented cows were fed SO during the transition period prior to FO feeding (i.e., transition and breeding diet by season interaction). One possibility contributing to the interaction with seaso n was that fewer cows received their second service during the cool season (n = 193) compared with the warm season (n

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207 = 411) of the year. In parallel, most early embryonic mortality associated with the warm season occurs between d 6 and 14 of pregnancy (Ry an et al., 1993) or within 7 days after insemination when experimental application of a heat stress caused rectal temperature to rise to 41.1C (Putney et al. 1988). Indeed, the effects of FO on fertility in the second service were observed by day 32 after TAI, with no changes in maintenance of pregnancy between 32 and 60 d of gestation. Greater pregnancy rates at second service and less pregnancy losses at first service in cows supplemented with FO were also associated with less TNF ophils were stimulated with LPS in a sub sample of cows around the time of insemination ( C hapter 7 ). An immunological suppression or tolerance is present during pregnancy; the fetus and fetal component of the placental unit is an allograft that needs to av oid rejection by the maternal unit for the duration of pregnancy (Siiteri and Stites, 1982). Maintenance of an immunologically favorable, immunosuppressive environment in the uterus is needed for embryo survival (Raghupathy, 2001; Hunt et al., 2005). An im mune response to a foreign body starts with the induction of an inflammatory response that is amplified by cytokines produced by cells (i.e., epithelial cells, macrophages and later neutrophils) in the vicinity of the foreign body. Since cytokine productio n of neutrophils is less in FO fed cows, perhaps there is an overall lower inflammatory response of the cows that could enhance tolerance to the embryo/fetal placental units. Indeed, the localized released of INF by the trophectoderm and the production o f progesterone by the corpus luteum creates an anti inflammatory envi ronment contributing to immune tolerance. For example, progesterone, through the actions of uterine serpins can reduce lymphocyte proliferation (Tekin et al., 2005) and progesterone itse lf can reduce the production of reactive oxygen species by macrophages (Siiteri and Stites, 1982). Additionally, it is currently

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208 believed that for the continuous normal development of pregnancy, production of pro inflammatory cytokines (i.e., IL 2, TNF nd IFN should be suppressed, whereas production of anti inflammatory cytokines (i.e., IL 4, IL 6 and IL 10) is enhanced (Wegmann et al., 1993). Immune cells, especially the macrophages, are a major source of cytokines and growth factors and they contribute to the maintenance of the adequate balance between pro and anti inflammatory cytokines at the placental bed Inappropriate activation or accumulation of endometrial macrophages is associated with pregnancy loss (Haddad et al., 1997) and reduced trophoblast invasiveness (Reister et al., 2001). The inhibitory actions of progesterone and anti inflammatory cytokines are believed to represent an important mechanism by which the allogenic conceptus escapes graft rejection by the maternal immune system. The greatest accumulated proportion of pregnant cows following the 2 postpartum inseminations was observed in cows with a sequential supplementation consisting of SO during the transition period followed by FO fed in the breeding period. Once parturition occurs the immune competence of the uterus must be restored to successfully reject remaining placental tissues and to pre vent ascendant bacterial growth. The pro inflammatory state induced by feeding the SO supplement (i.e., enriched with linoleic acid) t o enhance numerous aspects of neutrophil physiology and a greater acute phase tissues and improving both general and uterine health. Cows supplemented with SO compared with PO during the transition period produced more milk during the 5 month period of lac tation. This was caused partially by the greater DMI during the transition period for SO fed cows. Chouinard et al (1998) reported that milk yield increased linearly as degree of unsaturation of the dominant FA in the CS of fatty acid increased such that s linolenic acid) had the highest

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209 milk yields and lower milk fat content (Chouinard et al., 1998; Chouinard et al., 1999), as did cottonseed (Bertrand et al., 1998) which is high in linoleic acid. Howev er feeding a 2.2% PO supplement postpartum (i.e., high saturated fat) also improved milk yield compared with no fat supplementation ( Garcia Bojalil et al., 1998). Additionally, a greater state of immune competence in cows supplemented with SO may have enha nced health status that contributed to a greater milk yield without affecting the metabolic status of these animals compared with PO supplemented cows. There was no increase in the overall milk yield during the entire period of FO feeding. An interaction of breeding diet by month was represented by greater milk yield in months 2 and 3 of lactation in cows fed FO, but a decline in months 4 and 5 of lactation. Stage of lactation is one variable suggested to be related to the variation in milk response to fat ty acids supplementation (Abu Ghazaleh et al., 2001). During later stages of lactation when DMI is highest and thus greater ingestion of FO supplement possible negative effects of FO on fiber digestion may reduce DMI. In the present study, differential ch anges in DMI between diets during the curse of lactation are not possible because it is not known the DMI for each cow at specific months postpartum. No effects on milk yield by adding FO to diets have been reported (Spain et al., 1995; Abu Ghazaleh et al. 2001; Mattos et al., 2002), whereas others have reported increase in milk yield in which all were at early stages of lactation (Keady et al., 2000; Bilby et al., 2006; Moussavi et al., 2007). In conclusion, strategic feeding of fatty acids can impact ti ssue fatty acid composition. Feeding supplements enriched with linoleic acid during the transition period improved milk yield. Following the transition period, feeding CS enriched in n 3 fatty acids during the breeding period response ( C hapter 7 ) may play an important role in remodeling of the endometrium as

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210 demonstrated by the reduced pregnancy loss after first service and increased pregnancy per AI after the second service that was augment when n 6 fatty acids were previously fed in the transition period. Therefore, we propose a strategy of fatty acid supplementation comprised of feeding n 6 fatty acids during the transition period followed by n 3 fatty acids in the breeding period to maximize dairy cow production and reproduction.

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211 Table 6 1 Ingredient and chemical composition of diets fed to pre partum cows Ingredient % of Dry m atter (DM) Corn silage 26.6 6.6 13.3 3 6 15 3.3 3 9.5 7.86 4.16 1.5 Ryegrass silage Sorgum silage Alfalfa hay Wheat hay Cotton seed hulls Brewers grain wet Soybean meal Soy Plus 1 Corn grain ground Mineral mix Fat supplement PO SO Chemical Composition NE L Mcal/Kg of DM 1.43 1.45 CP, % of DM 13.2 13.8 Ether extract, % DM 3.7 3.7 NDF, % of DM 49.5 52.8 ADF, % of DM 33.2 34.7 Ca, % of DM 0.89 0.87 P, % of DM 0.33 0.32 K, % of DM 1.31 1.32 Mg, % of DM 0.29 0.29 Na, % of DM 0.2 0.2 Cl, % of DM 1.21 1.45 S, % of DM 0.19 0.19 Mn, mg/Kg of DM 79 68 Cu, mg/Kg of DM 24 21 Zn, mg/Kg of DM 85 80 Fe, mg/Kg of DM 283 260 Mo, mg/Kg of DM 0.8 0.9 1 West Central Soy, Ralston, IA. PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA).

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212 Table 6 2 Ingredient and chemical composition of diets fed to lactating cows Postpartum 1 Breeding 2 Ingredient, % of DM 29 14.5 4.9 2.23 3.27 5.45 5.45 4.9 1.8 23.36 3.45 1.5 29.1 14.54 4.9 2.23 3.27 5.45 3.45 4.9 1.81 23.36 3.45 1.5 Corn silage Ryegrass silage Cotton seed hulls Liquid blend 3 Cotton seed whole Brewers grain wet Soybean meal Soy Plus 4 Soybean hulls Corn grain ground Promix Fat supplement PO SO PO FO Chemical Composition NE L Mcal/Kg of DM 1.54 1.60 1.67 1.63 CP, % of DM 15.7 16.0 16.8 16.3 Ether extract, % DM 4.3 4.5 5.1 4.6 NDF, % of DM 38.6 38.8 34.2 34.8 ADF, % of DM 25.0 23.0 22.2 21.9 Ca, % of DM 0.78 0.75 0.81 0.88 P, % of DM 0.38 0.39 0.39 0.39 K, % of DM 1.37 1.40 1.20 1.20 Mg, % of DM 0.25 0.25 0.24 0.25 Na, % of DM 0.23 0.22 0.25 0.27 Cl, % of DM 0.57 0.40 0.51 0.55 S, % of DM 0.22 0.22 0.24 0.24 Mn, mg/Kg of DM 69 66 66 70 Cu, mg/Kg of DM 16 16 17 19 Zn, mg/Kg of DM 70 71 76 81 Fe, mg/Kg of DM 179 211 214 181 Mo, mg/Kg of DM 0.9 0.9 1.1 1 1 Diet fed from par turition to 30 days postpartum; 2 Diet fed from 30 to 160 days postpartum. 3 Blend of molasses, glycerin and corn steep 4 West Central Soy, Ralston, IA. PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA).

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213 Table 6 3. Fatty a cid profile s of f at s upplements Fat Supplement 1 Fatty a cid PO SO FO ----g/100 g of FA ----C14:0 1.2 0 1.0 4.5 0 C16:0 46.6 0 12.9 0 38.7 0 C16:1 0.0 0.13 0.0 C17:0 0.1 0 0.1 0 0.3 0 C18:0 4.3 0 4.2 0 4.6 0 C18:1, cis 9 38.2 0 16.7 0 30.8 0 C18:2 n 6 9.3 0 63.6 0 8.0 C18:3 n 3 0.3 0 0.2 0 1.0 0 C20:4 n 6 0.0 0.0 0.4 0 C20:5 n 3 0.0 0.6 0 5.4 0 C22:5 n 3 0.0 0.1 0 0.9 0 C22:6 n 3 0.0 0.4 0 5.3 0 n 6/n 3 31.0 48.92 0.6 3 1 Fat supplement: PO (Palm oil; EnerGII; S.O.= Prequel 21 ; F.O. = StrataG. All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA).

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214 Table 6 4. Fatty acid profile s of diets Pre partum 1 Postpartum 2 Breeding 3 Fatty acid PO S O P O S O P O F O ----g/100 g of FA ----C14:0 0.69 0.65 0.66 0.51 0.67 1.5 0 C16:0 28.11 19.61 27.34 16.55 27.76 24.52 C18:0 3.54 3.30 3.51 3.24 3.47 3.61 C18:1, cis 9 23.59 18.32 23.50 17.76 23.52 21.62 C18:2 n 6 37.91 50.28 37.24 53.04 37.82 40.74 C18:3 n 3 6.16 7.84 7.76 8.90 6.75 5.86 C20:5 n 3 0.0 0.0 0.0 0.0 0.0 1.03 C22:5 n 3 0.0 0.0 0.0 0.0 0.0 0.22 C22:6 n 3 0.0 0.0 0.0 0.0 0.0 0.92 n 6 / n 3 6.15 6.41 4.79 5.95 5.60 5.07 1 Diet fed for at least 14 before parturition; 2 Diet fed from parturition to 30 days postpartum; 3 Diet fed from 30 to 160 days postpartum. PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA).

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215 Table 6 5. Frequency distribution of cows among body condition scores (BCS) quartiles at close up, parturition, 43 days postpartum (dpp) and at timed artificial insemination (TAI) for palm oil (PO), safflower oil (SO) and fish oil (FO) diets BCS close up 1 BCS parturition 2 BCS 43 dpp 3 BCS TAI 4 BCS Quartiles PO % (n) SO % (n) PO % (n) SO % (n) PO % (n) SO % (n) PO a % (n) FO b % (n) 1 42.3 (284) 43.7 (300) 21.9 (144) 25.9 (176) 27.6 (169) 29.4 (181) 19.4 (110) 15.0 (83) 2 23.7 (159) 25.3 (174) 39.3 (259) 34.4 (234) 39.6 (242) 36.8 (227) 38.8 (220) 37.5 (208) 3 19.5 (131) 18.9 (130) 29.2 (192) 26.7 (181) 24.8 (152) 27.1 (167) 29.1 (165) 29.8 (165) 4 14.5 (98) 12.1 (83) 9.6 (63) 13.0 (49) 8.0 (49) 6.7 (41) 12.7 (72) 17.7 (98) Total 672 687 658 640 612 616 567 554 Median 3.25 3.25 3.0 3.0 2.75 2.75 2.75 2.75 1 BCS quartiles: 1 = 2.25 to 3.0; 2 = 3.25; 3 = 3.5; 4 = 3.75 to 4.0. 2 BCS quartiles: 1 = 2.25 to 2.75; 2 = 3.0; 3 = 3.25; 4 = 3.5 to 4.0. 3 BCS quartiles : 1 = 2.0 to 2.5; 2 = 2.75; 3 = 3.0; 4 = 3.25 to 3.5 4 BCS quartiles: 1 = 2.25 to 2.5; 2 = 2.75; 3 = 3.0; 4 = 3.25 to 3.75. ab Diet (P < 0.01); Parity (P < 0.01) at all BCS stages; Diet by parity (non significant) at all BCS stages. PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA).

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216 Table 6 6 Frequency distribution of cows among cervical discharge categories diagnosed between days 8 to 10 postpartum Variable Clear or Flecks Mucopurulent Purulent P value Diet 1 PO (554) 57.6 (319) 14.4 (80) 28.0 (155) Ref. SO (562) 59.6 (335) 10.0 (56) 30.4 (171) NS Parity Multi parous (768) 65.8 ( 505 ) 12.6 (97) 21.6 (166) Ref. Prim iparous (348) 42.8 ( 149 ) 11.2 (39) 46 (160) < 0.01 Calving assistance 2 No (841) 65.3 (549) 10.7 (90) 24 (202) Ref. Minor (141) 46.1 (65) 14.2 (20) 39.7 (56) < 0.01 Major (121) 27.3 (33) 21.5 (26) 51.2 (62) < 0.01 RFM 3 No (892) 63.7 (568) 11.8 (105) 24.5 (219) Ref. Yes (91) 9.9 (9) 11.0 (10) 79.1 (72) < 0.01 1 PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). 2 Calving assistance: No = no assistance; Minor = use of minor force; Major = use of a jack with minor or major force. 3 Retained fetal membranes.

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217 Table 6 7. Least square means and pooled SE of the cotyledon and caruncle fatty acid profiles (% of the total fatty acid; g/100 g of fatty acids) collected at the time of parturition for cows supplemented with palm oil or safflower oil Palm oil Safflower oil Fatty acid C ot yledon C aru ncle C ot yledon Caruncle SE Diet Tissue Int. C14:0 0.71 0.93 0.66 0.85 0.04 NS ** NS C15:0 0.59 0.44 0.61 0.41 0.02 NS ** NS C16:0 17.04 16.66 16.21 14.89 0.35 ** ** NS C16:1 0.69 0.64 0.92 0.73 0.07 NS NS C17:0 1.08 1.10 1.09 1.08 0.03 NS NS NS C18:0 13.60 21.10 13.66 20.64 0.33 NS ** NS C18:1 t 11 0.19 0.60 0.29 0.84 0.04 ** ** NS C18:1 t 12 0.72 0.66 0.69 0.58 0.05 NS NS NS C18:1 c 9 24.58 15.8 24.5 15.93 0.54 NS ** NS C18:1 c 11 3.98 2.7 4.24 2.84 0.12 ** NS C18:2 n 6 5.10 9.8 5.53 11.06 0.46 ** NS CLA 1 c 9, t 11 0.05 0.08 0.0 0.13 0.01 NS ** ** CLA t 9, t 11 1.04 0.51 0.75 0.45 0.07 ** ** NS C18:3 n 3 0.20 0.30 0.0 0.29 0.02 ** ** ** C20:0 0.67 0.64 0.34 0.73 0.05 ** ** C20:5 n 3 0.09 0.10 0.10 0.15 0.02 NS NS NS C22:0 5.66 5.51 5.59 5.13 0.29 NS NS NS C22:1 4.07 8.20 3.87 7.13 0.31 ** NS C22:4 n 6 0.80 1.06 0.83 0.99 0.04 NS ** NS C22:5 n 3 2.33 1.42 2.07 1.30 0.07 ** ** NS C22:6 n 3 2.14 0.50 1.91 0.51 0.09 NS ** NS C24:0 0.56 0.70 0.62 0.67 0.04 NS NS NS Others 13.8 10.4 15.4 12.5 0.33 ** ** NS Diet fed from 3 3 days prepartum to 30 days postpartum. Palm oil (EnerGII) and Safflower oil ( Prequel 21 ) All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA) 1 CLA = Conjugated linoleic acid. P P P significant

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218 Table 6 8 Least squares means and pooled SE for total fatty acids (g/100 g of freeze dried tissue) and different fatty acid percentages (% of the total fatty acid; g/100 g of fatty acids) in the cotyledon and caruncle tissues collected at the time of parturition f or cows supplemented with palm oil or safflower oil Palm oil Safflower oil Fatty acid Cotyledon Caruncle Cotyledon Caruncle SE Diet Tissue Int Total 4.3 8.6 4.7 8.7 0.26 NS ** NS SFA 39.9 46.8 38.8 44.5 0.40 ** ** NS UNSFA 46.5 42.6 45.7 43.0 0.60 NS ** NS MUSFA 34.7 28.9 34.5 28.1 0.57 NS ** NS PUFA 11.8 13.8 11.2 14.9 0.50 NS ** NS n 6/n 3 1.3 5.4 1.6 6.3 0.04 ** NS Diet fed from 33 days pre partum to 30 days postpartum. Palm oil (EnerGII) and Safflower oil ( Prequel 21 ) All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). SFA = Saturated fatty acids, UNSFA = unsaturated fatty acids, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids. n6/ n3 = (C18:2 + C22:4 )/(C18:3 + C20:5 + C22:6). P P P ; NS = non significant

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219 Table 6 9. Proportion of cyclic cows, adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance Variable Cycli c 1 % (n) AOR 95% C.I. P value Transition diets PO PO + PO FO 81.8 (464/567) Ref. Ref. SO PO + SO FO 78.2 (430/550) 0.76 0.54 1.1 NS Breeding diets PO PO + SO PO 81.7 (460/563) Ref. Ref. PO FO + SO FO 78.3 (434/554) 0.78 0.56 1.12 NS Interaction of diets PO FO + SO PO 79.3 (446/562) Ref. Ref. PO PO + SO FO 80.7 (448/555) 1.14 0.81 1.61 NS Parity Multi parous 85.2 (628/737) Ref. Ref. Prim iparous 70 (266/380) 0.37 0.26 0.54 < 0.01 Cer vical d ischarge Clear 86.5 (327/378) Ref. Ref. Flecks 83 (156/188) 0.87 0.53 1.44 NS Mucopurulent 78.5 (84/107) 0.65 0.37 1.14 NS Purulent 71.8 (189/263) 0.56 0.37 0.86 < 0.01 BCS 2 at 43 dpp 3.0 3.75 85.4 (327/383) Ref. Ref. 2.75 77.9 (335/430) 0.60 0.40 0.92 < 0.01 2.0 2.5 76.7 (231/301) 0.48 0.30 0.76 < 0.01 1 Determined based upon plasma concentrations of progesterone collected at 60 and 74 days postpartum. 2 Body condition score. PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). NS = non significant

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220 Table 6 10 First service pregnancy per AI at 32 and 60 days after insemination and pregnancy loss for experimental diets Diets Di et c ontrasts 1 ( P value ) PO PO S O P O P O FO SO FO C1 C2 C3 D 32 % (n) 38.7 (107/276) 35.8 (96/268) 39.1 (103/263) 35.8 (89/248) NS NS NS D 60 % (n) 33.7 (92/273) 29.7 (79/266) 37.0 (97/262) 32.8 (81/247) NS NS NS Loss % (n) 11.5 (12/104) 15.9 (15/94) 4.9 (5/102) 7.9 (7/88) NS < 0.05 NS 1 C ontrast are C1 (transition diets [PO PO + PO FO vs. SO PO + SO FO ]); C2 (breeding diets [ PO PO + SO PO vs. PO FO + SO FO ]); and C3 (interaction of diets [ PO PO + SO FO vs. PO FO + SO PO ]). PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA).

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221 Table 6 11. First service pregnancy per AI, adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance for the risk of pregnancy for cows inseminated at 81 days postpartum (dpp) and evaluated at 32 and 60 days after insemination Variables D 32 pregnancy % (n) AOR 95% C.I. P value Parity Multiparous 38.8 (260/670) Ref. Ref. Primiparous 35.0 (135/385) 0.89 0.64 1.22 NS Cycling 1 Yes 39.5 ( 322/816 ) Ref. Ref. No 30.5 ( 62/203 ) 0. 72 0. 49 1.06 < 0.10 Cervical d ischarge Clear 44.4 (153/345) Ref. Ref. Flecks 28.2 (49/174) 0.49 0.33 0.73 < 0.01 Mucopurulent 36.3 (37/102) 0.73 0.46 1.17 NS Purulent 38.1 (93/244) 0.90 0.62 1.28 NS BCS 2 at TAI (81 dpp ) 3. 25 3.75 46.3 (76/164) Ref. Ref. 3.0 35.1 (110/313) 0.58 0.37 0.89 < 0.01 2.75 35.9 (142/395) 0.59 0.39 0.90 < 0.01 2.0 2.5 37.8 (65/172) 0.62 0.38 1.02 = 0. 10 Variables D 60 pregnancy % (n) AOR 95% C.I. P value Parity Multiparous 34.5 (229/664) Ref. Ref. Primiparous 31.2 (120/384) 0.91 0.66 1.27 NS Cycling Yes 35.4 (287/810) Ref. Ref. No 25.7 (52/202) 0.64 0.43 0.94 < 0.0 5 Cervical d ischarge Clear 39.5 (134/339) Ref. Ref. Flecks 25.8 (45/174) 0.54 0.36 0.81 < 0.01 Mucopurulent 32.7 (33/101) 0.75 0.47 1.22 NS Purulent 34.0 (83/244) 0.87 0.61 1.24 NS 1 Determined based upon plasma concentrations of progesterone collected at 60 and 74 days postpartum. 2 Body Condition Score. NS = non significant

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222 Table 6 12 Second service pregnancy rates at 32 and 60 days of pregnancy and pregnancy loss for experimental diets Diets Diets Contrast 1 (P value) PO PO S O P O P O FO SO FO C1 C2 C3 D 32 % (n) 27.7 (43/155) 26.7 (41/154) 30.3 (44/154) 43.3 (65/150) NS < 0.05 = 0.10 D 60 % (n) 21.0 (38/152) 22.5 (34/151) 27.3 (39/143) 41.3 (62/150) NS < 0.01 < 0.05 Loss % (n) 5.0 (2/40) 10.0 (4/38) 7.1 (3/42) 4.6 (3/65) NS NS NS 1 C ontrast are C1 (transition diets [PO PO + PO FO vs. SO PO + SO FO ]); C2 (breeding diets [ PO PO + SO PO vs. PO FO + SO FO ]); and C3 (interaction of diets [ PO PO + SO FO vs. PO FO + SO PO ]). PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Cor coran, CA, USA).

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223 Table 6 13. Second service pregnancy per AI, adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance for the risk of pregnancy for cows inseminated at 126 days postpartum (dpp) and evaluated at 32 and 60 days after insemination Variables D 32 pregnancy % (n) AOR 95% C.I. P value Transition diets PO PO + PO FO 29 (87/300) Ref. Ref. SO PO + SO FO 34.8 (106/304) 1.13 0.75 1.72 NS Breeding diets PO PO + SO+PO 27.2 (84/309) Ref. Ref. PO FO + SO +FO 36.9 (109/295) 1.41 1.0 2.0 < 0.05 Interaction of diets PO FO + SO PO 28.4 (85/299) Ref. Ref. PO PO + SO FO 35.4 (108/305) 1.82 1.43 2.85 < 0.1 0 Season THI < 72 38.3 (74/193) Ref. Ref. THI > 72 28.9 (119/411) 0.40 0.23 0.69 < 0.01 Variables D 60 pregnancy % (n) AOR 95% C.I. P value Transition diets PO PO + PO FO 26.0 (77/295) Ref. Ref. SO PO + SO FO 31.9 (96/301) 1.2 0.78 1.84 NS Breeding diets PO PO + SO+PO 23.8 (72/303) Ref. Ref. PO FO + SO +FO 34.5 (101/293) 2.22 1.21 4.16 < 0.01 Interaction of diets 1 PO FO + SO P O 24.8 (73/294) Ref. Ref. PO P O + SO FO 33.1 (100/302) 1.94 1.21 3.10 < 0.0 5 Season THI < 72 35.2 (68/193) Ref. Ref. THI > 72 26.0 (105/403) 0.42 0.23 0.74 < 0.01 PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). Interaction of transition and breeding diets by season was significant (P < 0.01). Pregnancy losses from day 32 to 60 after insemination were not affected by diets or other variables.

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224 Table 6 14. Second service pregnancy per AI, adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance for the risk of pregnancy for cows inseminated at 126 days postpartum (dpp) and evaluated at 32 and 60 days after insemination Variables D 32 pregnancy % (n) AOR 95% C.I. P value Parity Multiparous 33.2 (123/370) Ref. Ref. Primiparous 29.9 (70/234) 0.86 0.59 1.25 NS BCS 1 difference Gained 0.25 0.75 34.3 (79/230) Ref. Ref. Maintained 32.3 (90/279) 0.90 0.62 1.32 NS Lost 0.25 0.75 21.8 (17/78) 0.50 0.27 0.94 < 0.05 Variables D 60 pregnancy % (n) AOR 95% C.I. P value Parity Multiparous 29.4 (107/364) Ref. Ref. Primiparous 28.45 (66/232) 0.95 0.65 1.40 NS BCS difference Gained 0.25 0.75 31.0 (70/226) Ref. Ref. Maintained 30.0 (82/273) 0.93 0.62 1.38 NS Lost 0.25 0.75 18.2 (14/77) 0.46 0.24 0.91 < 0.05 1 Body condition score resulted by the difference of BCS at TAI and 43 dpp. NS = non significant. Pregnancy losses from day 32 to 60 after insemination were not affected by diets or other variables.

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225 Table 6 1 5 Accumulated proportion pregnant and pregnancy losses after the first and second services, adjusted odds ratio (AOR), 95% confidence interval (C.I.) and levels of significance for the risk of pregnancy for cows inseminated at 81 and 126 days postpartum (dpp) and evaluated at 32 and 60 days after insemination Variables D 32 pregnancy % (n) AOR 95% C.I. P value Transition diets PO PO + PO FO 54.2 (299/552) Ref. Ref. SO PO + SO FO 54.8 (292/532) 1.04 0.81 1.33 NS Breeding diets PO PO + SO+PO 52.5 (290/552) Ref. Ref. PO FO + SO +FO 56.8 (301/532) 1.19 0.93 1.51 NS Interaction of diets PO FO + SO PO 52.2 (285/546) Ref. Ref. PO PO + SO FO 56.8 (306/538) 1.21 0.95 1.54 = 0.10 Variables D 60 p regnancy % (n) AOR 95% C.I. P value Transition diets PO PO + PO FO 49.3 (268/543) Ref. Ref. SO PO + SO FO 48.8 (257/526) 1.01 0.79 1.29 NS Breeding diets PO PO + SO+PO 45.5 (246/541) Ref. Ref. PO FO + SO +FO 52.8 (279/528) 1.35 1.06 1.72 < 0.01 Interaction of diets PO FO + SO PO 46.5 (250/538) Ref. Ref. PO PO + SO FO 51.8 (275/531) 1.24 0.97 1.58 = 0.07 Variables Pregnancy l oss % (n) AOR 95% C.I. P value Transition diets PO PO + PO FO 7.6 (22/290) Ref. Ref. SO PO + SO FO 10.1 (29/286) 1.36 0.75 2.56 NS Breeding diets PO PO + SO+PO 11.8 (33/279) Ref. Ref. PO FO + SO +FO 6.0 (18/297) 0.48 0.26 0.88 < 0.01 Interaction of diets PO FO + SO P O 9.7 (27/277) Ref. Ref. PO P O + SO FO 8.0 (24/299) 0.86 0.47 1.59 NS PO (Palm oil; EnerGII); SO (Safflower oil; Prequel 21 ); (Fish oil; StrataG). All fats were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA).

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226 Figure 6 1. Interaction of transition and breeding diets by season (P < 0.05) for second service pregnancy per AI examined at 32 days after timed artificial insemination (TAI). Transition diets (axis: PO [palm oil] and SO [safflower oil]) and breeding diets (legend: PO [palm oil, ] and FO [fish oil, --]). In A cows were inseminated during the cool season (THI < 72), in B cows were inseminated during the warm season 15 25 35 45 55 PO SO % A PO FO 15 25 35 45 55 PO SO % B PO FO

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227 Figure 6 2. Intera ction of transition and breeding diets by season interaction (P < 0.05) for second service pregnancy per AI examined at 60 days after timed artificial insemination (TAI). Transition diets (axis: PO [palm oil] and SO [safflower oil]) and breeding diets (leg end: PO [palm oil, ] and FO [fish oil, --]). In A cows were inseminated during the cool season (THI < 72), in B cows were inseminated during 15 25 35 45 55 PO SO % A PO FO 15 25 35 45 55 PO SO % B PO FO

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228 Figure 6 3. Milk yield regression curves for transition and breeding diets we re not parallel based on tests for homogeneity of regression (transition diets: palm oil [ ] and safflower oil [ --] in A, P < 0.01); breeding diets breeding diets: palm oil [ ] and fish oil [ --] in B, P < 0.01). 34 36 38 40 42 44 46 1 2 3 4 5 Kg Month of lactation A 34 36 38 40 42 44 46 1 2 3 4 5 Kg Month of lactation B

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229 Figure 6 4 Least squares means ( S.E.) for plasma concentrations of 15 keto 13,14 dihydro prostaglandin F (PGFM) for the first 14 days postpartum in a subsample of cows fed calcium salts of palm oil ( ; n = 15) or safflower oil ( --; n = 17) during the pre partum period (at least 20 days) to 35 days postpartum. Means at 4 and 7 dpp are greater (P < 0.05) in SO than P.O. diets. 100 1100 2100 3100 4100 5100 0 1 2 3 4 5 6 7 8 9 10 12 14 pg/ml Days postpartum

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230 CHAPTER 7 EFFECT OF DIFFERENTIAL SUPPLEM ENTATION OF FATTY AC IDS DURING THE PERIPARTUM AND BREED ING PERIOD ON INNATE IMMUNITY: NEUTROPHIL FUNCTION AND ACUTE P HASE PROTEIN Introduction The transition period, typically considered from the time 3 weeks pre partum until 3 weeks postpartum, is marked by declining dry matter intake ( DMI ) as the cow approaches parturition and n egative nutrient balance when lactation begins (Grummer, 1995). The constraints imposed by deficient feed intakes, coupled with other stressors associated with parturition and adjustments to lactation contribute to the high incidence of health disorders dur ing the transition period ( Grhn and Rajala Schultz, 2000 ) Neutrophils are part of the innate immune system acting upon antigens in a non specific manner as the first line of defense against pathogens. T he migration of neutrophils from the vasculature is regulated by at least three distinct steps. The first step involves capture and rolling of the neutrophils with adhesion mediated by selectins. After this initial step, the neutrophil must be activated by chemoattractants, and the third step is firm adhesi on or arrest of the cells to the endothelium by activated integrins. An essential feature is that these are sequential steps and not parallel events I nhibition of any one of these steps causes complete, rather than partial, inhibition of neutrophil migrat ion (Paape et al., 2003) Neutrophils are phagocytes that can internalize and kill many microbes Each phagocytic event results in the formation of a phagosome into which reactive oxygen species (ROS) and hydrolytic enzymes are secreted. The consumption of oxygen during the generation of ROS has been termed the oxidative burst (Paape et al., 2003) Concurrently, the release of inflammatory cytokines (i.e., TNF he site of infection and also stimulate the release of acute phase proteins from the liver in a coordinated systemic innate

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231 response (Petersen et al., 2004). The acute phase response is considered to be a dynamic process involving systemic and metabolic changes that provid es an early nonspecific defense mechanism against insult before specific immunity is achieved Stress, when it occurs, is an undesirable aspect of livestock pr oduction as it results in immune suppression and increase s the likel ihood of an infection. Mechanisms of immune suppression at parturition are related to the release of cortisol (Preisler et al., 2000) in association with hypocalcemia (Kimura et al., 1999) and elevated BHBA ( Hoeben et al ., 1997) and NE FAs (Hammon et al., 2 006) during this time Innate immune suppression during the transition period is characterized by reductions in neutrophil chemotaxis (Kimura et al., 2003), reduction in expression of adhesion molecules (i.e., L selectin) ( Kimura et al., 1999; Weber et al. 2001) and decreased generation of ROS (Kimura et al., 2002; Hammon et al., 2002). Collectively this increases the risk of uterine diseases (Kimura et al., 2002; Hammon et al., 2002). Prostaglandins are derived from the membrane phospholipid stores of ara chidonic acid (C20:4 n 6 ), which are synthesized from dietary linoleic acid (C18:2 n 6 ), an essential fatty acid. During the early postpartum period a very intense secretion of uterine PGF 2 occurs measured in plasma by its metabolite ( 13, 14 dihydro, 15 keto PGF 2 [ PGFM ] ) (Guilbault et al., 1984 ) Cows without cases of metritis had greater plasma concentrations of PGFM in the early postpartum compared with cows with metritis (Seals et al., 2002). Uterine PGF 2 has tonic effects in the uterus (Gajewski et al., 1999) and promotes neutrophil chemotaxis and phagocytosis ( Hoedemaker et al., 1992). Also, lypoxygenases can generate the 4 series of leukotrienes from arachidonic acid, a neutrophil chemoattractant. The o bjectives were to investigate effects of a linoleic acid enriched supplement (calcium salts of safflower oil) fed during the transition period of cows on: a) blood neutrophil surface

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232 expression of L 2 integrin; b) phagocytic and oxidative burst activit ies of blood neutrophils; c) neut rophil cytokine production (i.e., TNF response (i.e., plasma concentrations of haptoglobin, fibrinogen and ceruloplasmin). Another objective was to investigate the effects of an n 3 fatty acid enriched supplement (i.e., calc ium salts of fish oil) during the breeding period on neutrophil cytokine production. The hypotheses were that feeding calcium salts (CS) of a linoleic acid enriched supplement before parturition and during the first 30 days postpartum (dpp) would increase the percentages of linoleic and arachidonic acids in the fatty acid profiles of uterine caruncular tissue and neutrophils. This was expected to enhance synthesis of pro inflammatory eicosanoids and neutrophil function, and induce a greater acute phase resp onse. Furthermore, feeding n 3 fatty acid enriched supplement (fish oil), beginning at 30 d pp would increase the percentages of eicosapentaenoic acid (C20:5n 3; EPA) and docosahexanoic acid (C22:6n 3; DHA) in neutrophils that may attenuate neutrophil cytok ine production sensitivity to antigen. Material and Methods Animals E xperimental D esign and F eeding The present study was part of a large field trial, involving 1 582 cows, conducted at a n orth Florida dairy farm comprised of 3,500 Holstein cows that were milked thrice daily A sub sample of cows was used to carry out these objectives. Therefore information regarding animals, facilities, chemical composition of diets and fatty acids content was described in C hapter 6. Briefly, cows were allocated randomly into two experimental transition diets begun at approximately 30 days before the expected date of parturition and continued until 3 5 dpp. After 3 5 dpp cows within each transition diet were randomly allocated into the experimental breeding diets that were f ed until 160 dpp. Experimental transition and breeding diets differed only i n the source of supplemental fatty acid.

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233 Transition diets (i.e., pre partum to 35 dpp) consisted of CS of palm oil ( PO 1.5% of DM; Ener G II, Virtus Nutrition, Corcoran CA USA) or CS of safflower oil (SO; 1.5% of DM, Prequel 21, Virtus Nutrition, Corcoran CA, USA). Breeding diets consisted of CS of palm ( PO; 1.5% of DM; Ener G II) or CS of fish oil ( FO ; 1.5% of DM, StrataG Virtus Nutrition, Corcoran, CA, USA) To test the effects of fish oil, only cows treated with palm oil in the transition period were used. Therefore, transition diets were tested for PO versus SO and breeding diets were tested for PO versus FO. Diets were formulated to meet or exceed NRC (2001) nutrient requirement s for net energy of lactation (NE L ), crude protein (CP), fiber, mineral and vitamins and fed to obtain intake s of 200 and 400 g/d of CS for pre and postpartum cows, respectively Diets were fed as a total mixed ration twice daily targeting 5% orts. Body C ondition S core and V aginoscopy Cows were evaluated for body condition score ( BCS ) at the day of enrollment, parturition, 43 dpp, and at the first insemination (i.e., approximately 87 dpp) Scores were given by two veterinarian s based on a 1 (thin) to 5 (o bese) scale using a quarter point system (Edmonson et al., 1989). All cows were evaluated once between 8 to 10 dpp for cervical discharge using a disposable foil lined cardboard vaginal speculum (Milburn Distributors, Ocala, FL ) Appearance of discharge wa s categorized into clear mucus without flecks, clear mucus with flecks lochia, mucopurulent (50% clear mucus and approximately 50% of pus) and purulent (> 50% pus with a brown and foul smell). Acute P hase P roteins Blood samples were collected thrice weekly from parturition to 35 dpp for analys e s of plasma concentrations of haptoglobin, fibrinogen and ceruloplasmin. Blood samples were

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234 collected by puncture of coccygeal vessels into evacuated tubes containing K2 EDTA (Vacutainer, BD, Franklin Lakes, NJ USA). Samples were placed immediately into an ice bath until centrifugation for 20 minutes at 2 619 x g. After centrifugation, plasma was harvested and stored frozen at 20 C until assayed. Plasma fibrinogen was determined from a standard curve generate d using a human reference (Sigma Diagnostics). Plasma haptoglobin concentrations were determined by measuring haptoglobin/hemoglobin complexing by the estimation of differences in peroxidase activity as described previously (Makimura and Suzuki, 1982). Cer uloplasmin oxidase activity was measured using colorimetric procedures described by Demetriou et al. (1974). Neutrophil Phagocytic and Oxidative Burst A ctivity Neutrophil phagocytic and oxidative burst activities were assessed by means of a modified dual color flow cytometry assay (Smits et al., 1997). In a preliminary study (Silvestre et al., 2005) bovine whole blood from non lactating and non pregnant cows was used for assay o ptimization of reagents, bacterial concentration and incubation times. Blood samples were collected at approximately 30 days pre partum, within 7 hours after parturition, and at days 4 and 7 postpartum by puncture of coccygeal vessels into evacuated tubes containing 10 mL s pray d ried s odium h eparin tubes ( Vacutainer, Becton Dickinson, Franklin Lakes, NJ ). Samples were maintained at room temperature and transported to the laboratory for analysis within 4 hours of collection. Upon arrival in the laboratory, samples were kept in a rotation station to avoid blood clotting. A 25 L aliquot was removed from each blood sample for determination of total leukocyte concentration in a hemocytometer after hemolysis of erytrocytes (Unopette, Becton Dicki nson & Co, Franklin Lakes, NJ). A differential count of neutrophils (i.e., percent neutrophils among total leukocyte) was obtained by smearing 10 L of

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235 whole blood onto glass slide, stained (Protocol Hema 3, Fisher Diagnostics, Middletown, VA) and examine d at magnification of 40x with oil immersion using the battlement method (Schalm et al., 1975 ). Number of neutrophils per volume of whole blood was calculated based upon total leukocytes times percent of neutrophils. A 100 L aliquot of blood was pipetted i nto each of four 5 mL polystyrene round bottom tubes (12 x 75 mm). A 10 L of a 5 M d ihydrorhodamine 123 (DHR, Sigma Aldrich, Saint Louis, MO) solution (100 L of DHR stock [500 M] and 900 L of PBS) was added to all tubes. Tubes were incubated at 37 o C for 10 min on a rotation platform to allow the loading of DHR into the neutrophils. After incubation, tube one was used as a negative control. In tube two (positive control) 10 L of Phorbol 12 miristate 13 acetate ( PMA; activates PM burst Si gma Aldrich, Saint Louis, MO) at a concentration of 2 g/mL was added. In tube three ( E scherichia coli ) and four ( Sthaphylococcu s aureus ) a volume of bacterial isolated suspension solution was added accordingly to the number of neutrophils to achieve a 40: 1 bacteria to neutrophil ratio. Bacteria (10 6 bacteria/L) were previously heat killed ( 18 hours at 37 o C ) and labeled with Propidium Iodide (PI; Sigma Aldrich, Saint Louis, MO). All tubes were further incubated at 37 o C for 30 min on a rotation platform. Af ter incubation tubes were placed immediately into ice to stop phagocytosis and oxidative burst activity. Tubes were then processed for flow cytometry using reagents for red blood cell lysis (formic acid at 88%), white blood cell buffer and cell fixative of 1% paraformaldehyde An automated lysing system (Q Prep Epics immunology work station, Coulter Corp, Miami, FL ) was used to add reagents followed by further addition of 500 L of distilled water for completion of hemolysis and 10 L of 0.4% trypan blue sol ution for quenching extracellular oxidized DHR.

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236 Samples were subjected to flow cytometry (FACSort, Becton Dickinson Immunocytometry Systems, San Jose, CA) utilizing a 488 nm argon ion laser for excitation and 15 mW of power. Density cytograms were generate d by linear amplification of the signals in the forward scatter and side scatter channels. Samples were analyzed within 2 hour s after addition of fixatives. Neutrophils were selectively gated from acquisition of 10,000 cells/sample based on their sizes an d complexity in the density cytogram (Jain et al., 1991). Data w ere processed and further analyzed by computer software (CellQuest, version 3.3, Becton Dickinson Immunocytometry Systems, San Jose, CA). Parameters analyzed from the fluorescent cytograms inc luded the percentage of neutrophils that phagocytized bacteria and the percentage of neutrophils with a phagocytosis induced oxidative burst. Also, histogram analysis for mean fluorescence intensity (MFI) of green (DHR oxidation) and red (PI labeled bacter ia) wave lengths were used as an estimation of the total gated neutrophil mean oxidative burst intensity (indicator of mean intensity of ROS produced per neutrophil) and mean phagocytic activity (indicator of mean number of bacteria per neutrophil), respec tively. Neutrophil and M ononuclear B lood C ells A dhesion M olecules Abundances of adhesion molecules on neutrophil and mononuclear cells surfaces were analyzed using the same sub sample of cows and collection dates used for neutrophil activities. Monoclonal mouse antibodies against bovine L selectin (CD62L, clone CC32, IgG 1 isotype, Serotec) and canine 2 integrin (CD18, clone CA1.4E9, IgG1 isotype, Serotec) that cross reacts with bovine CD18 were used. An isotype mouse control antibody (clone MCA 928, IgG1 isotype, Serotec) was used to correct for non specific binding of C D62L and CD18 antibodies to the cells.

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237 B lood samples were collected by puncture of coccygeal vessels into evacuated 10 mL s pray d ried s odium h eparin tubes. Samples were maintained in room temperature and transported to the laboratory for analysis within 4 hours of collection. A total of 10 6 neutrophils in a 100 L suspension are needed for the immunostaining procedure. Therefore 4 mL of whole blood (approximately 3 x 10 6 neutrophils/mL) was added drop wise into a red blood cell lysing solution of NH 4 Cl (8.9 9 g NH 4 C l, 1 g KHCO 3 and 0.037 g EDTA in 1 L of distilled H 2 O) and left at room temperature for 10 min. Samples were centrifuged at 1,600 rpm for 15 min, supernatant decanted, and the cell pellet re suspended in 10 mL of the NH 4 Cl solution. Samples were left at room temperature for 10 min and centrifuge at 1,600 rpm for 10 min. Supernatant was decanted and cells re suspend in 10 mL of FACS buffer (2% of fetal bovine serum and 0.1% of sodium azide in PBS) and centrifuged at 1,600 rp m for 10 min. Supernatant was discarded and cells re suspended in 1 mL of FACS buffer at an approximate concentration of 10 7 neutrophils/mL or 10 6 neutrophils/100 L. Cells were kept in an ice for the antibody staining procedure. The cell suspension (100 L) was added into three separate 5 mL tubes for immunostaining of each antibody. Antibodies (12 L) were added to each individual tube (1:10 dilution of CD62L, CD18 and control antibody in FACS buffer) and refrigerated for 30 min. Two mL of FACS buffer was pipetted into tubes and centrifuged at 1,000 rpm for 5 min, supernatants decanted, each tube received 5 L of Phycoerythrin conjugated goat anti mouse IgG (STAR81PE, Serotec) and then refrigerated for another 30 min. Cells were washed with FACS buffer (2 mL) and centrifuged at 1,000 rpm for 5 min. Supernatants were decanted and 0.5 mL of the FACS fixative solution (2% of fetal bovine serum and 0.1% of sodium azide in 0.5% formalin) was pipetted into each tube to re suspend the cell pellet.

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238 A flow cytometer (FACScan; Becton Dickinson, San Jose, Ca) was used to acquire and analyze the neutrophil CD62L and CD18 data. Data from 10,000 events per sample were acquired and analyzed using CellQuest software (Becton Dickinson, San Jose, CA). The neutrophil and monon uclear cell populations were gated out separately based upon their forward and side scatter characteristics on dot plots (Jain et al., 1991). Percent of neutrophils and mononuclear cells positive for CD62L and CD18 were obtained based upon gated cells and used to further calculate number of positive cells per volume of blood using hemocytomer cell count results. Also, the geometric mean fluorescent intensity of the labeling kit, an indicator of the number of receptors on the surface of cells, was obtained i n the histogram for the gated cell populations. Neutrophil I solation, C ulture and F atty A cids A nalysis To measure neutrophil fatty acid profiles and c ytokine production, sampl es were collected at enrollment 35 dpp (PO vs. SO) and 2 days after PGF inject ion of the Ovsynch ( C hapter 6 ) at approximately 85 dpp (PO vs. FO). B lood samples (150 mL) were collected by puncture of coccygeal vessels into evacuated 10 mL s pray d ried s odium h eparin tubes and n eutrophils were isolated using the procedure described by Sohn et al ( 2006 ) Briefly, blood was c entrifuged at 500 x g for 5 min, at 4 o C Plasma, buffy coat and 1/ 3 of the red blood cells were removed. The remaining red blood cells and white blood cells (~10 mL) were s uspended d rop wise into a double volume (20 mL ) of cold 0.2% NaCl solution and gently mixed for 5 min to induce lysis of red blood cells Immediately, half the original volume of a cold 3.7% NaCl solution equivalent to half of the original volume of red and white blood cells (5 mL ) was added to restore isotonicity. The suspension was centrifuged at 500 x g for 15 min at 4 o C, supernatant was discarded and cell pellet re suspended with 5 mL of PBS. Another round of lysis was performed and cell pellet was washed twice with 20 mL of PBS solution for final re suspension

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239 in 2 mL of RPMI 1640 (Bio Whittaker, Walkersville, MD, USA). Cell viability (> 98%) was examined in a hemocytometer, and neutrophil purity determined on a glass microscopic slide stained (Protocol Hema 3, Fisher Diagnostics, Middletown VA) for differential cell counts (i.e., > 85% neutrophils present). Neutrophils were adjusted to a concentration of 5 x 10 6 cells/mL in RPMI 1640 and added to a 96 well flat bottom micro for each cytokine assay of interest. Duplicate wells were stimulated or not with 20 L of LPS stock solution ( 1 mg/ mL; Escherichia coli 0111:B4 L3024 purified by ion exchange chromatography; Sigma Chemical Co., Saint Louis, MO, USA ). Volume per well was adjusted to 200 L with RPMI 1640 and plates incubated at 37 o C, in a 5% CO 2 incubator for 18 h ours After incubation plates were centrifuged, supernatants collected and frozen at 20 o C for analysis of TNF 1. When samples yielded excess of isolated neutrophils (> 10 8 neu trophils), cells were centrifuged at 500 x g for 5 min, supernatant discarded, cells re suspended in 5 mL of PBS, and stored in 80 o C to further determine fatty acid profile. Enzyme L inked I mmuno s orbent A ssay s ( ELISA) TNF For measurement of TNF in supernatants of neutrophil incubations, a TNF kit was used T h e kit contai ned c oating and biotinylated d etection a ntibodies, r ecombinant s tandard s, st reptavidin H orseradish Peroxidase (SR HRP) TMB s ubstrate s olution and a s top s olution (Bovine TNF Screening Set Thermo Scientific Inc ., Rockford, IL) All samples of each animal were analyzed in the same plate that was balanced for numbers of animals for PO and SO or PO and FO diets. Lyophilized TNF Dulbecco's Phosphate Buffered Saline ( DPB

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240 buffer (0.2 M). One hundred microliters were added to a 96 well microtiter plate sealed and wrapped in aluminum foil for incubation overnight at 4 o C. After incubation coating antibody was aspirated by vacuum and plate washed three times with 300 L of wash solution (150 mL Tween 20 and 300 mL of DBPS, pH = 7.4). Blocking o each well. Plates were sealed and wrapped in aluminum foil, and incubated for 1 hour at room temperature. Blocking buffer was aspirated and plates washed with 200 L of wash solution. Standards of TNF n DBPS, pH = 7.4), and 100 L of standards and experimental samples were added to respective wells and incubated for 1 h our at room temperature. Plates were washed three times with 200 L of wash solution. Reconstituted TNF was diluted in reagent diluent (1:100) and added to each well for 1 h our incubation at room temperature and washed three times with 200 L of wash solution. The SR HRP reagent was diluted in reagent diluents (1:400), 100 L added to each well, and incubat ed for 30 min at room temperature in the dark. Plates were washed three times with 200 L of wash solution, and TMB substrate solution (100 L) added to each well for 2 min in the dark. Reaction was stopped (100 L of 2M sulfuric acid) and absorbance read at 450 nm. Internal standards diluted in RPMI 1640 were added to each plate to calculate a correction coefficient for experimental samples. IL Measurement of IL in f lat bottom 96 well plates (Nunc Maxisorp C) and using wash solution (Tris buffered saline, 0.05% Tween 20, pH=8.0,) and TMB tetramethylbenzidine ) substrate obtained from Bethyl Laboratories ( ELISA Starter Kit Montgomery, Texas, USA). Coating (MCA1658), detecti on (AHP423 ) and HRP conjugated (STAR 54) antibodies were obtained from Serotec ( Raleigh, NC, USA ). Standard recombinant

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241 bovine IL Rockford, IL ). All samples of each animal were analyzed in the same plate that was balanced for numbers of animals for PO a nd SO or PO and FO diets. Plates were coated overnight at 4 o C with 5 mL of mouse anti bovine IL washed three times (300 L), and blocked (100 L/well, 2% fish skin gelatin [45% solution, Sigma Aldrich] in wash solution ) for 1 h our at room temperatur e. Blocking buffer was aspirated and plates washed. Standards of IL experimental samples (100 L) were added to wells and incubated for 2 h ours at room temperature. Plates were washed three times. Bo vine IL solution (1:500), was added (100 L) to each well for 1 h our incubation at room temperature and washed three times. Anti IL conjugated antibody diluted in wash solution containing 0.2% fish gelatin (1:5 00) was added to each well (100 L) and incubated for 30 min at room temperature in the dark. Plates were washed three times and TMB substrate solution (100 L) added to each well for 5 min in the dark. Reaction was stopped (100 L of 2M sulfuric acid) and absorbance read at 450 nm. Internal standards diluted in RPMI 1640 were added to each plate to calculate a correction coefficient for experimental samples. Statistical A nalyses Plasma concentrations of acute phase proteins (i.e., haptoglobin, fibrinogen and ceruloplasmin), neutrophil activity (i.e. percent phagocytosis and/or oxidative burst, MFI phagocytosis or oxidative burst) and expression of neutrophil adhesion molecules (i.e., CD62L and CD18 ) were analyzed using repeated measures responses of the m ixed model procedure of SAS (SAS Institute Inc; Version 9.1). Data were tested for normal distribution of the residuals by the PROC UNIVARIATE procedure of SAS. Residuals were considered to be normally distributed when the Shapiro Wilk statistic was equal or greater than 0. 1 0 and log transformed if

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242 not normally distributed For each dependent variable, the covariance structure that had the best relative goodness of fit based upon penalty criteria (Bayesian criterion) was used. The mathematical model contain ed diet, day and diet by day interaction with cow (random variable) nested within treatment. For responses that can be affected by inflammation (i.e., acute phase proteins, neutrophil activity and adhesion molecule expression) and because vaginoscopy score was a significant variable, vaginoscopy score was retained in the statistical model for estimating potential diet effects. Furthermore, cows with purulent and mucopurulent discharge also were excluded from the data set for additional analyses to avoid con founding. Analyses of neutrophil cytokines (i.e., TNF least squares using the general linear model (GLM) procedure of SAS. Cytokines concentrations were compared within LPS non stimulated, LPS stimulated and increa sed mass (i.e., LPS stimulated minus LPS non stimulated). Mathematical model consisted of diet. R esults Acute P hase P roteins A total of 32 multiparous cows (PO [n = 15] and SO [n = 17]) were used for analysis of acute phase proteins. Ten cows in each transition diet were excluded because sampling was terminated during the postpartum period after antibiotic and/or anti inflammatory treatments. Cows in the SO were fed experiment al diets for a longer period (34.8 1.4 days) compared with PO (30.8 1.3 d ays) before parturition. Frequency distribution of cows among BCS at the day of enrollment (2.5 to 2.75 [n = 16]; 3.0 to 3.25 [n = 14] and 3.75 [n=2]) and at parturition (2.5 to 2.75 [n = 8]; 3.0 to 3.25 [n = 20] and 3.5 to 3.75 [n=4]) did not differ betwe en diets. All cows had a normal parturition (no assistance), except for one cow that had minor assistance. A total of 10, 2, 1, 2 cows in the PO and 9, 5, 0, 3 cows in the SO diets were diagnosed with either clear or

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243 lochia, flecks, mucupulent or purulent cervical discharge at 8 dpp, respectively and were not treated with antibiotics or anti inflammatory agents. Mean plasma concentrations of haptoglobin (Figure 7 1) and fibrinogen (Figure 7 2) were greater (P < 0.05) for cows fed SO compared with PO transit ion diets. Cows diagnosed with cervical discharge classified as clear, flecks or lochia (0.01 0.002 AU and 205.3 12 mg/dl) had lower (P < 0.01) mean concentrations of haptoglobin and fibrinogen, respectively, compared with cows diagnosed with mucopurul ent or purulent discharges (0.04 0.005 AU and 248.8 12 mg/dl). An additional analysis was conducted only with cows that had cervical discharges clear, flecks or lochia (n = 26) and mean plasma concentrations of haptoglobin and fibrinogen was greater (P < 0.01) for cows fed SO (0.02 0.002 AU, 221.7 11 mg/dl) compared with PO (0.01 0.002 AU, 172.5 12 mg/dl) transition diets, respectively. Plasma concentrations of ceruloplasmin were not different between PO (8.0 0.6 mg/dl) or SO (8.2 0.6 mg/dl ) transition diets. There was an interaction (P < 0.05, Figure 7 3) between days postpartum and cervical discharge score on concentrations of ceruloplasmin in plasma. Cows diagnosed with clear, flecks or lochia discharge had lower ceruloplasmin concentrati ons after 6 dpp compared with cows with mucopurulent or purulent discharges. Basal concentrations of ceruloplasmin were elevated and sustained in cows with an abnormal cervical discharge. Neutrophil P hagocytic and O xidative B urst P hagocytic and o xidative b urst of neutrophils were analyzed in whole blood collected from 47 multiparous cows at 32 days pre partum (n= 18), at parturition (2.8 1.8 hours after delivery), and 4 and 7 dpp. Frequency distribution of cows among BCS categories at 32 days pre partum w as not different between diets (median = 3.25) and at parturition (median = 3.0). At parturition all cows had normal or minor assistance for delivery of calves. Frequency distribution of cows among cervical discharge scores examined at 8 dpp was not differ ent between diets

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244 (clear or flecks [n = 3 0 ] and mucopurulent or purulent [n = 1 4 ]). Body condition scores, calving assistance categories, cervical discharge categories and their interaction with diets were not associated with neutrophil responses and were removed from the statistical analyses. Hemocytometer analyses and differential staining of white blood cells allowed for quantification of total white blood cells per volume of blood and the percentage of neutrophils and monocytes to calculate the number o f each cell population per volume of blood. Total number of white blood cells was not affected by transition diets, but was slightly less (P = 0.09; Figure 7 4) after parturition compared with pre partum values. Total number of mononuclear cells in blood was not affect by transition diets, day or interaction of diet by day (Figure 7 4). However, number of neutrophils in blood increased from 32 days pre partum to t he time of parturition and then underwent an appreciable decrease (P < 0.01; Figure 7 4) at 4 and 7 dpp. The number of neutrophils was less (P < 0.01) for cows supplemented with SO and the decrease in total blood neutrophils tended (diet by day; P < 0.08) to be greater for cows supplemented with SO at 4 and 7 dpp (Figure 7 5). Percentage of neutrophils in whole blood with phagocytic and oxidative burst activit ies was not affected by transition diets when challenged with either E. coli (Figure 7 6A) or S. au reus (Figure 7 6B). The lowest activity was observed at the time of parturition, but within 4 days postpartum values returned to pre partum levels (Figure 7 6A). Neutrophil activity was greater (P < 0.01) for samples challenged with E. coli compared with S aureus (Figure 7 7) and increased (P < 0.01) as the number of neutrophils increased in whole blood of samples collected during the postpatum period (Figure 7 7). Mean fluorescence intensity of the red light, an indicator of number of bacteria phagocytize d per neutrophil, was greater (P < 0.05) for cows in the SO at 4 dpp when samples

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245 were stimulated with either E. coli (Figure 7 8A) or S. aureus (Figure 7 8B). Moreover, MFI of the green wave length, an indicator of intensity of ROS produced per neutrophil was greater for cows in the SO at days 4 (P < 0.05) and 7 (P = 0.08) postpartum when samples were stimulated with E. coli (Figure 7 8A) and on day 4 (P < 0.05) postpartum when stimulated with S. aureus (Figure 7 8B). Neutrophil A dhesion M olecules: CD62L and CD18 A dhesion molecules associated with n eutrophil and mononuclear cells were analyzed using the same samples for neutrophil function assays. Percent mononuclear cells positive for CD62L ( Table 7 1 ) and CD18 (Table 7 1 ) were low (P < 0.01) at parturit ion and increased at 4 and 7 dpp with the responses being greater (P < 0.05) for SO supplemented cows. Percentages of mononuclear cells positive for CD62L (56.8% and 48.8 %; S E = 3.1) and CD18 (57.2% and 48.9%; S E = 2.3) were greater (P < 0.01) for cow s diagnosed with mucopurulent or purulent (n = 14) cervical discharges compared with discharges clear or with flecks (n = 30), respectively. When analys es of percent of mononuclear cells that were positive for CD62L and CD18 were conducted in cows with onl y clean or flecks cervical discharges similar diet by day interactions were detected as for the total population of cows sampled. B ecause the percentage of mononuclear cells positive for CD62L and CD18 are obtained from a fixed total number of 10 000 cells in the flow cytometer, the increase in the monocyte population percentage after parturition is a result of the decrease in the neutrophil population percentage observed after parturition. Indeed, the actual number of monocytes per volume of blood does not change in the peri partum (Figure 7 4). Therefore, the percentage of monocytes positive for CD62L and CD18 were re analyzed using the percentage of neutrophils as a covariate. Results indicated that the percent of monocytes positive for CD62L and CD18 inc reased (P < 0.01) after parturition (Table 7 1) and w ere greater (P < 0.05) for cows in the SO

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246 (50.7% and 46.5%, S.E. = 3.0) diets at 4 dpp compared to cows in the PO (44.6% and 39.3%, S.E. = 2.4) diets respectively Number of mononuclear cells positive f or CD62L and CD18 per volume of blood increased after (P < 0.05) parturition that was the same for both transition diets (Table 7 1) However, quantities of adhesion molecules per mononuclear cell expressed by the MFI of CD62L (550.0 and 492.5; S E = 37) and CD18 (154.6 and 126.3; S E = 35) were not affected by PO and SO diets, respectively, day cervical discharge score and respective interactions Percent ages of neutrophils positive for CD62L and CD18 were highest (P < 0.01) in samples collected at parturition and then decreased (P < 0.01) at 4 and 7 dpp ( Table 7 2 ). Furthermore, the decrease was greater (P < 0.05) for SO cows compared with PO supplemented cows ( Table 7 2 ). Percent age of neutrophils positive for CD62L ( 28.2% and 37.1 %; S E = 1.9) and CD18 (26.3% and 37.2%; S.E. = 1.9) were less (P < 0.01) for cows diagnosed with mucopurulent or purulent (n = 14) cervical discharges compared with discharges classified as clear or flecks (n = 30), respectively. Analy s es for percent age of neutrophils positive for CD62L and CD18 only in cows with clear or flecks cervical discharges resulted in similar diet by day interactions observed for the total population of animals sampled. Number of neutrophils positive for CD62L and CD18 per volume of blood decreased ( P < 0.01 ) after parturition ( Table 7 2 ) to lesser (P < 0.08) numbers for SO cows at 4 and 7 dpp compared with PO supplemented cows (Table 7 2) Neutrophil MFI for CD62L increased (P < 0.05) after parturition and was grea ter (P < 0.05) at 4 and 7 dpp for cows supplemented with SO compared with PO diets ( Table 7 2 ). Neutrophil MFI for CD62L (1109.2 and 757.6; S E = 61) was greater (P < 0.01) for cows diagnosed with mucopurulent or purulent (n = 14) cervical discharge compa red with clear or

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247 flecks (n = 30), respectively. Analy s es for neutrophil CD62L MFI only in cows with clear or flecks cervical discharges (n = 30) resulted in similar diet by day interaction observed previously for the total population of animals. Neutrophi l MFI of CD18 was not affected by diet, day cervical discharge score and respective interactions ( Table 7 2 ). Neutrophil C ytokine s Mean concentration of TNF at 30 dpp was greater (P < 0.05) for cows supplemented during the transition period with SO compared with PO and also when cells were stimulated with LPS (Figure 7 9A). Because the concentration of TNF was constitutively (no LPS) greater (P < 0.05) in SO oil supernatants, the mas s increase did not differ between diets. In contrast at 80 dpp, 50 days after initiation of breeding diets (i.e., PO and FO), the mean concentration of TNF (Figure 7 9B). However, at this time TNF duction in response to LPS was attenuated (P < 0.05) in cows fed FO compared with th ose fed PO. Consequently, the mass increase in response to LPS was l ess (P < 0.01) in the FO compared with PO supplemented cows. When cows were supplemented during the tran sition period with SO, mean concentration of IL1 at 30 dpp was greater (P < 0.01) when cells were stimulated with LPS (Figure 7 10A) and a greater mass increase of IL1 0.01, Figure 7 10A). At 8 0 dpp, concentrations of IL1 when neutrophils were either stimulated or not (Figure 7 10B). Neutrophil Fa tty A cid C omposition Analysis of f atty acid profiles in neutrophils collected from pre partum cows (n = 14befo re initiation of experimental diets, at 30 dpp (PO [n = 13] vs. SO [n = 13], and at 80 dpp (PO [n = 15] vs. FO [n = 14]) are summarized in Table 7 3. All cows used at 80 dpp were fed PO from 3 0 days pre partum to 30 dpp. Palmitic acid (C16:0), behenic acid (C22:0) and erucic acid (C22:1n

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248 9) were greater (P < 0.01) in cows of fed PO compared with SO (Table 7 3). Vaccenic acids ( C18:1 t 11 and C18:1 t 12 ) were le ss (P < 0.01) with conjugated linoleic acid ( CLA t 9, t 11 ) and linolenic acid (C18:3n 3) slightly greater (P < 0.10) in neutrophils of PO compared with SO cows (Table 7 3). Linoleic acid, although numerically, was not significantly greater (P = 0.19) in cows fed SO compared with PO supplements (Table 7 3). The predominant fatty acids in the neutrophils at 30 dpp were linoleic, stearic (C18:0), palmitic, oleic (18:1n 9) and erucic acids which comprised approximately 72% of all fatty acids (Table 7 3). Moreover, the ratio n 6: n 3 fatty acids tended (P = 0.07) to be greater for cows fed SO compared with PO diets (Table 7 4). N eutrophils collected from cows fed FO had less (P < 0.01) stearic, oleic, behenic, erucic and adrenic (C22:4n 6) fatty acids compared with PO fed cows at 80 dpp (Table 7 3). However, C18:1 tr ans isomers ( C18:1 t 1 0, C18:1 t 11 and C18:1 t 12 ), EPA ( C20:5n 3 ), DPA ( C22:5n 3 ) and DHA ( C22:6n 3 ) were all greater (P < 0.01) in cows fed FO compared with PO (Table 7 3). Consequently, the ratio of n 6:n3 fatty acid s were less (P < 0.01) in cows fed F O compared with PO (Table 7 4). The predominant fatty acids in the neutrophils at 80 dpp were linoleic, stearic, palmitic, oleic and erucic acids which comprised approximately 62% of all fatty acids (Table 7 3). Discussion Supplementation of CS enriched with linoleic acid (i.e., safflower oil) during the transition period induced a pro inflammatory state in cows that was illustrated by the increased neutrophil expression of adhesion molecules, production of cytokines, enhanced bactericidal activi ty, and increased circulating acute phase proteins. After 30 dpp, supplementation of CS enriched with n 3 family of fatty acids (i.e., fish oil) induced an anti inflammatory state that was illustrated by the attenuation of neutrophil cytokine production.

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249 C alcium salts of palm oil used in this experiment as a fat supplement control was comprised of a minimal concentration of n 6 and n 3 fatty acids allowing for a suitable comparison with the CS of safflower oil that contained a high concentration of linoleic acid. Fatty acid profile of the total mixed ration showed an approximate 15% increase in linoleic acid for the SO diet ( C hapter 6 ). Also, the CS of fish oil increased EPA and DHA to approximately 2% of the total fatty acids in the total mixed ration (C hap ter 6 ). In the present study feeding cows with a linoleic acid enriched supplement numerically increased the proportion of this fatty acid in the total fatty acid profile of neutrophils and arachidonic acid, a product of linoleic acid was not detected. The se fatty acids are present in the phospholipid fractions of cells, which comprise mainly cell membranes. Because fatty acid profiles were conducted after total fatty extraction of neutrophils, neutral fatty acids, largely comp rised of saturated fat ty acids and present in cell cytoplasm, may have undermined the detection of certain polyunsaturated fatty acids Arachidonic acid comprised over 20% of the phospholipid fraction of fatty acids approximately three times its proportion in the neutral lipid fractio n in lymphocytes (Calder et al., 1994). Moreover, feeding the SO supplement increased the proportion of linoleic acid in caruncular tissue of parturient cows ( C hapter 6 ). The PO supplementation fed during the transition period increased the proportion of s ome saturated fat ty acids (i.e., palmitic and behenic acids), but the proportion of total saturated fatty acids did not differ from SO fed cows. Nonetheless, the ratio of n 6:n 3 fatty acids tended to be greater in cows supplemented with SO compared with P O. The feeding of FO for approximately 50 day period increased the proportion of the n 3 family of fatty acids (i.e., EPA, DHA and DPA) in neutrophils resulting in major reduction in proportion of monounsaturat ed fatty acids and in the ratio of n 6:n 3 fat ty acid s Indeed, CS of

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250 FO supplemented to lactating dairy cows increased the proportion of EPA and DHA in endometrium, liver, muscle, mammary gland, milk (Bilby et al., 2006) and caruncle (Mattos et al., 2004). Importantly, neutrophil fatty acid composition is predisposed to changes accordingly to maternal nutrition. The incubation with either linoleic acid, arachidonic acid, EPA or DHA, resulted in an increase of these fatty acids in the phospholipid fraction of lymphocytes (Calder et al., 1994). Also, when healthy adult human s were supplemented with arachidonic acid and fish oil for a period of 12 weeks, the arachidonic acid, EPA and DHA fatty acid concentrations in the phospholipid fraction of peripheral blood mononuclear cells were increased by 15%, 220%, and 16%, respectively (Thies et al., 2001). The high variability of PGFM in postpartum dairy cows precluded the detection of consistent differences of this metabolite between transition diets although spurious increases were evident in cows fed SO (Chapter 6). The uterus is the main source of PGF synthesis during the early postpartum period in the cow (Guilbault et al., 1984). Metabolism of arachadonic acid to products such as PGF can activate neutrophil ba ctericidal activity, and leukotriene B 4 that has chemoattractant properties towards neutrophils ( Hoedemaker et al., 1992 ). Neutrophil migration and bactericidal activity are pivotal for uterine health postpartum. The acute phase response provid es an early nonspecific defense mechanism against insult before specific immunity is achieved (Peterson et al., 2004). Haptoglobin is a hemoglobin scavenger that prevent s the loss of iron by formation of very stable complexes with free hemoglobin in the blood This ph enomenon functions as a b acteriostatic environment by restricting availability of iron necessary for bacterial growth and p revents pro oxidant activity of hemoglobin ( Eaton et al., 1982 ). Fibrinogen is the precursor of fibrin a protein involved in

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251 hemostasis. Hemos tatic and inflammatory systems are activated similarly and are highly integrated. Fibrin can control neutrophil activation in vitro through TL (i.e., toll like) receptors and M 2 integrin receptor to induce i ntracellular calcium mobilizat ion p hosphor y lation events NF kB activation (i.e., expr ession of cytokines, COX 2), and both c ell adhesion and migration ( Flick et al., 2004 ) Therefore, the enhanced acute phase response in cows supplemented with SO can represent an important mechanism to prevent systemic pathogen growth and septicemia. The acute phase response is induced by pro inflammatory cytokines (i.e., TNF 1) produced by epithelial cells, macrophages and later neutrophils present at the sites of inflammation (Petersen et al., 2004). Cytokines act as messengers between the local site of injury and the hepatocytes to synthesiz e acute phase proteins ( Petersen et al., 2004) Cows with mucopurulent or purulent cervical discharge, characteristic of bacterial contamination and inflam mation, had greater concentrations of haptoglobin, fibrinogen and ceruloplasmin. Indeed, Sheldon et al (2001) indicated that the severity of bacterial contamination in the uterine lumen, as determined by the total bacterial growth score, was associated wit h greater concentrations of 1 acid glycoprotein, haptoglobin and ceruloplasmin. The possible confounding effects of uterine contamination on dietary treatment effects on the acute phase response was eliminated by analyzing only co ws with cervical discharge scores classified as clear, fleckes and lochia. Also no other infection (e.g., mastitis) was diagnosed in this group of cows allowing them to be a suitable platform for comparison of transition dietary effects. The greater concen trations of haptoglobin and fibrinogen in cows supplemented with SO are possibly a result of the greater cytokine production as determined in the neutrophil culture. Although cytokine production was only investigated in supernatant of cultured neutrophils and not in the circulating blood, it is expected that other cells at sites of inflammation would be more

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252 prone to synthesize cytokines upon a pathogen challenge in SO fed cows. Such responses would increase the overall acute phase response. Also, fats rich in n 6 polyunsaturated fatyy acids can increase liver responsiveness to cytokines by altering the composition of membrane phospholipids. As a consequence of alterations in phospholipid composition, membrane fluidity may change, altering binding of cytokin es to receptors and G protein activity (Grimble, 199 8 ). Tappia et al (1997) reported that sensitivity of murine macrophages to TNF IL 1 production was increased when animals were supplemented with corn oil, a rich source of linoleic acid (18:2 n 6). Decreased DMI prior to parturition is well documented and associated with mobilization of lipids, which are released as NEFA from adipose tissue ( Grummer et al., 2004). Increase NEFA in blood generally enhances hepatic ketogenesis as a rep onse to dispose excess of fatty acids reaching the liver. Hoeben et al (1997) reported that exposure of neutrophils to elevated levels of BHBA reduced PMN respiratory burst and concluded that BHBA may, in part, be responsible for the higher susceptibility to local and systemic infections during the postpartum period Moreover, cows diagnosed with puerperal metritis and endometritis had greater concentration of NEFA and BHBA, associated with lower neutrophil activity, during the peripartum period (Hammon et al., 2006). Metabolic parameters (BHBA, NEFA, plasma urea nitrogen and glucose) measured during the postpartum period were not affected by SO or PO transition diets (C hapter 6 ), and, therefore it is unlikely that differences in innate immune responses be tween transition diets were caused by altered metabolic status. The number of white blood cells in blood decreased slightly after parturition particularly because of neutropenia at 4 and 7 dpp The number of mononuclear cells did not change during the tra nsition period. The neutrophilia at the time of parturition observed in this study is

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253 physiological and a result s from cortisol down regulation of CD62L at this time (Weber e t al., 2001). At 4 and 7 dpp, the neutropenia was greater in SO supplemented cows possibly because the quantity of CD62L on neutrophils was greater at these days. Therefore a greater proportion of these cells were likely marginalizing to the vascular endothelium. Rolling of neutrophils on the vascular endothelium is mediated by CD62L an d is an essential event that precedes neutrophil exit from blood to possible sites of infection (Janeway, 2005). Indeed, cows detected with endometritis at 4 weeks postpartum had greater blood neutrophil numbers beginning at one week pre partum (Kim et al. 2005). This may be a result of less adhesion molecules on the cell surface. Differential quantities of CD18 were not detected between transition diets as opposed to differential response in CD62L. Detection of dietary or day effects on CD18 on circulatin g neutrophils may be difficult because this adhesion molecule, when up regulated, interacts with vascular endothelium with a high affinity that results in anchoring of the neutrophil. Consequently, CD18 quantities on the surface of circulating pool of neut rophils, represented by the mean fluorescence intensity, would not be detected. Also, the proportion of neutrophils identified as CD62L or CD18 positive cells were not different between transition diets because they are expressed, at some degree, in more t han 98% of the neutrophils. The proportion of CD62L+ neutrophils decreases at parturition (Meglia et al., 2001). In fact, these changes may contribute to increased susceptibility of infections in the puerperium. In mononuclear cells the quantities of CD62L and CD18 per cell were not affected by diets or day, but were less compared with neutrophils. The proportion of mononuclear cells positive for CD62L and CD18 was greater after parturition in both transition diets, but greater in the SO

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254 supplemented cows p ossibly because of pro inflammatory mediators released in this period that was greater for the SO group of cows. Neutrophil bactericidal activity, represented by its phagocytic and killing activity (i.e., ability to generate ROS), is essential during the period after parturition. Impairment of activity in cows was associated with increased incidence of retained fetal membranes (Kimura et al., 2002) and metritis (Hammon et al., 2002) in cows. Also, phagocytic and bactericidal activities are reduced in cows with endometritis (Zerbe at al., 1996, Kim et al., 2005). Neutrophils from cows supplemented with SO had greater phagocytic activity per cell towards E. coli (4 dpp) and S. aureus (4 dpp) concurrently with greater generation of ROS (4 and 7 dpp). The grea ter production of cytokines, such as TNF neutrophil activity. Although cervical discharge score was not different between diets at approximately 8 dpp ( C hapter 6 ), possible uterine health could have been improved at later stages postpartum. The absence of a direct measurement of uterine health at later stages in this study prevents further speculation other than second service pregnancy rate was improved in cows fed SO during the transition period pr eceding the period o f feeding the FO breeding diet. Immune cells detect bacterial components such as endotoxins and lipopolissacharide via toll like receptors, which activate downstream signaling to stimulate the release of cytokines such as TNF 1 (Beutler et al., 2003). These cytokines provide a positive feed back loop to further increase immune cell mobilization and are potent stimulants of oxidant molecule production. In particular, nitric oxide, hydrogen peroxide, and superoxide radi cals are pr oduced by phagocytes (Farante et al., 1988). Also sensitivity of murine macrophages to TNF to produce IL 1 was increased when animals were supplemented with corn oil that is rich in 18:2 n 6 fatty acid (Tappia et al., 1997). Additionally, the gr eater concentration of fibrinogen in the

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255 blood samples of cows supplemented with SO may have benefitted the phagocytic and oxidative burst activity of the neutrophil since whole blood was used without the need of cell isolation. Indeed i n vitro studies ( R ubel et al., 2002, Shi et al., 2005 ) have shown that fibrinogen can profoundly alter leukocyte function, leading to changes in cell migration, phagocytosis, NF B mediated transcription, producti on of chemokines and cytokines and degranulation Changes in t he fatty acid profile of the neutrophils, favoring a greater ratio of n 6:n 3 fatty acids could have led to a greater production of inflammatory mediators such as prostaglandins. Lymphocyte stimulation with concavalin A, a mitogen, resulted in the decreas ed in the linoleic for nuclear translocation (Camandola et al., 1996), thus increased TNF IL by monocy tes (Baldie et al., 1993, Sinha et al 1991). Indeed, neutrophil activity increased positively with increasing numbers in the blood. More neutrophils may result in a greater production of eicosanoids and cytokines in the confines of the in vitro system. The greater productions of these media tors can stimulate in an autocrine manner the activity of neutrophils. Moreover, the lesser number of neutrophils per volume of blood in the SO diet was sufficient to not affect neutrophil activity although number of bacteria engulfed per neutrophil was in creased in this group of cows. Suppression of TNF and numerical decrease of IL production in cows supplemented with FO is possibly due to an increased proportion of the n 3 family of fatty acids (i.e., EPA and DHA) with a concurrent decrease in n 6 family of fatty acids within the neutrophils. The n 3 fatty acids, especially EPA and DHA, can compete for intracellular enzymatic pathways to generate prostaglandins of the 3 series and leukotrienes B 5 both having anti inflammatory properties (Mattos et al, 2000). Indeed EPA and DHA inhibited production of IL

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256 TNF Also, Caughey et al. (1996) demonstrated that a diet enriched with flaxseed followed by FO inhibited IL 1 and TNF p roduction by monocytes that was negatively correlated with the EPA content in the fatty acid profile of these cells. Supplementation of f ish meal, an enriched source of the n 3 fatty acids, improved pregnancy rates in few studies ( Armstrong et al., 1990; C arrol et al., 1994; Burke et al., 1997). Also, Petit and Twagiramungu (2002) observed that embryonic mortality from day 30 to 50 post insemination tended to be reduced in cows that received linseed compared with cows that received CS of palm oil, indicat iv e that linseed improved embryonic survival after embryonic attachment to the uterus that occurs gradually beginning at approximate 17 to 25 days of pregnancy. In addition to the possible suppression of omega 3 fatty acids on PGF 2 secretion (Mattos et al., 2003; Mattos et al., 2004) and possible attenuation of luteolysis during the time of pregnancy recognition, g reater pregnancy rates and lesser pregnancy losses in cows supplemented with FO were observed ( C hapter 6 ). The possibility of progesterone be ing a confounding factor for the immune suppression of neutrophils was excluded, because all animals were sampled 2 to 3 days after the last PGF injection of the Ovsynch. An immunological suppression or tolerance is need ed during pregnancy; the allograft em bryo/fetus placental unit (Siiteri and Stites 1982) An immune response to a foreign body starts with the induction of an inflammatory response that is amplified by cytokines produced by cell s (i.e., epithelial cells, macrophages and later neutrophils) in the vicinity of the foreign body. Since cytokine production of neutrophils was less responsive in FO primed cows, we hypothesize that an overall lower inflammatory response

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257 within the materna l unit placental unit. Indeed, the localized released of INF tau by the trophectoderm and the production of progesterone by the corpus luteum creates an anti inflammatory environment need ed for immun e tolerance in pregnancy Progesterone, through the actions of uterine serpins can, for example, reduce lymphocyte proliferation ( Tekin et al., 2005) and progesterone itself can reduce the production of ROS by macrophages (Siiteri and Stites, 1982). These inhibitory actions are believed to represent an important mechanism by which the allogeneic conceptus escapes graft rejection by the maternal immune system. M aintenance of an immunologically favorable, immunosuppressive environment in the uterus is need ed for embryo survival (Raghupathy, 2001; Hunt et al., 2005). I nappropriate activation or accumulation of endometrial macrophages is associated with pregnancy loss (Haddad et al., 1997) and reduced trophoblast invasiveness (Reister et al., 2001). During preg nancy, ewes undergo an accumulation of macrophages in the endometrium, which was limited to the stromal compartment, with lymphocytes present in the epithelial compartment T hese two types of cells can presumably communicate through cytokines secretion (Te kin and Hansen, 2004). Regulation of macrophages and lymphocytes by the placenta may be complex as there is evidence that signals from the conceptus may modulate macrophage function to provide immunoprotection to the conceptus. In conclusion, CS of safflow er oil, a fat supplement rich in linoleic acid can improve innate immunity (i.e., neutrophil function and acute phase response) during the transition period. Cows supplemented with safflower oil presented an overall pro inflammatory state (i.e., an immune alert state) suitable for coping with the stressful and highly contaminated postpartum period. Calcium salts of fish oil fed during the breeding period exert an anti inflammatory

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258 environment that may attenuate immune responses in early pregnancy and upon environmental challenges (i.e., heat stress) that may benefit embryonic survival.

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259 Table 7 1 Least square s means ( S.E .) percent of mononuclear cells percent mononuclear cells adjusted by percent of neutrophils and log number of mononuclear cells positive per L of blood for L 2 Integrin (CD18) in whole blood during the transition period for cows supplemented with palm oil ( PO; n= 23) or safflower oil ( SO; n = 24) Days r elative to p arturition P value Diet 32 0 4 7 S.E. Diet Day Diet x d ay CD62L + % Mononuclear cells PO 34.5 29.0 51.1 52.7 3.1 0.05 0.01 NS SO 32.9 31.6 64.0 61.4 3.0 % Mononuclear cells adjusted PO 33.9 37.4 44.6 46.2 2.6 0.08 0.01 NS SO 33.3 40.0 50.7 48.6 2.6 Log e cells/L of blood PO 3.4 3.4 3.5 3.6 0.09 NS 0.05 NS SO 3.3 3.4 3.6 3.6 0.09 CD18 + % Mononuclear cells PO 20.0 26.1 45.2 46.4 3.2 0.05 0.01 NS SO 23.7 27.9 58.3 55.8 2.8 % Mononuclear cells adjusted PO 18.4 33.5 39.3 40.4 2.8 NS 0.01 NS SO 24.0 35.1 46.5 42.8 2.8 Log e cells/L of blood PO 3.1 3.3 3.4 3.5 0.09 NS 0.06 NS SO 3.2 3.3 3.5 3.5 0.09 Diet s fed from 3 2 days pre partum to 30 dpp Palm oil (EnerGII) and s afflower oil ( Prequel 21) fat supplements were manufactured as calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). NS = non significant.

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260 Table 7 2 Least square s means ( S.E ) percent of neutrophils log number of neutrophils per L of blood positive and mean fluorescence intensity (MFI) for L Selectin 2 Integrin (CD18) in whole blood during the transition period for cows sup plemented with palm oil (PO; n= 23) or safflower oil ( SO; n = 24 ) Days r elative to p arturition P value Diet 32 0 4 7 S.E. Diet Day Diet x d ay CD62L + % Neutrophils PO 33.8 49.5 28.9 28.5 3.1 0.05 0.01 0.01 SO 36.2 49.8 18.7 20.4 2.9 Log e neutrophils/L of blood PO 3.4 3.6 3.2 3.3 0.07 0.05 0.01 NS SO 3.3 3.6 3.0 3.0 0.07 MFI PO 551.6 744.0 862.5 892.8 95.8 0.05 NS 0.01 SO 619.0 761.5 1205.3 1134.2 96.2 CD18 + % Neutrophils PO 33.8 49.1 28.1 28.3 2.7 0.05 0.01 0.05 SO 38.3 48.4 18.3 17.9 2.6 Log e neutrophils/L of blood PO 3.4 3.6 3.2 3.3 0.07 0.05 0.01 NS SO 3.3 3.5 3.0 3.0 0.07 MFI PO 189.6 206.2 153.7 215.4 31.8 NS NS NS SO 164.0 203.0 179.1 210.7 31.9 Diet s fed from 3 2 days pre partum to 30 dpp Palm oil (EnerGII) and s afflower oil ( Prequel 21) fat supplements were manufactured as of calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). NS = non significant.

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261 Table 7 3 Least squares means and pooled SE of the neutrophil fatty acid profiles ( g/100 g of fatty acids) collected at th e time of initiation of diets ( 30 pre partum, pooled, n = 14 ), 30 days postpartum (dpp) for cows supplemented with palm oil (PO n = 13 ) or safflower oil (SO n =13 ) and at 80 days postpartum for cows supplemented with palm oil (PO n = 15 ) or fish oil (FO n = 14 ) 30 dpp 1 80 dpp 2 P value Fatty a cid 30 days p re partum PO SO SE PO FO SE 30 dpp 80 dpp C12:0 4.72 3.13 4.49 1.57 4.69 5.49 1.6 7 NS NS C14:0 0.12 0.18 0.29 0.08 0.20 0.36 0.0 7 NS NS C15:0 0.35 0.15 0.13 0.05 0.15 0.36 0.0 6 C16:0 10.90 12.3 0 10.6 0 0.24 11. 63 11.6 0 0. 31 ** NS C16:1 0.47 0.33 0.51 0.10 0.27 0.34 0.0 8 NS NS C17:0 0.78 0.87 1.04 0.19 0.91 0.78 0.1 4 NS NS C18:0 17.40 19.9 19.2 0 0.42 20.7 0 18.1 0 0. 38 NS ** C18:1 t 10 0.21 0.01 0.06 0.27 0.06 1.58 0.2 0 NS ** C18:1 t 11 0.39 0.45 0.96 0.13 0.21 0.74 0. 08 ** ** C18:1 t 12 0.11 0.15 0.47 0.07 0.17 0.72 0.07 ** ** C18:1 12.20 11.14 10. 60 0.51 9.86 8.0 3 0. 48 NS ** C18:2 n 6 18.10 20.61 23.23 1.37 21.63 22.21 1. 28 NS NS CLA 3 c 9, t 11 0.90 1.69 0.85 0.35 2.18 1. 44 0. 53 NS C18:3 n 3 1.25 1.43 1.02 0.15 0.48 0.74 0.1 2 NS C20:0 0.37 0.28 0.59 0.17 0.29 0.38 0. 08 NS NS C20:5 n 3 0.58 0.52 0.36 0.09 0.3 0 1.5 0 0.1 3 NS ** C22:0 3.40 3.47 2. 60 0.24 3.82 2.11 0.19 ** ** C22:1 10.40 9.48 7.9 0 0.51 9.56 6.48 0.4 4 ** ** C22:4 n 6 3.58 1.67 1.7 5 0.20 2.16 0.81 0.2 0 NS ** C22:5 n 3 3.43 2.4 0 2.13 0.22 2.33 3.48 0. 32 NS ** C22:6 n 3 0.96 0.11 0.20 0.05 0.11 1.65 0. 11 NS ** C24:0 0.20 0.04 0.06 0.03 0.05 0.05 0.03 NS NS Others 9.18 9.44 10.9 6 1.25 8.24 11.05 0.80 NS ** 1 Diet s fed from 3 0 days pre partum to 30 dpp 2 Diets fed from 30 dpp to 80 dpp. All cows used at 80 dpp were fed PO from 3 0 days pre partum to 30 dpp. Palm oil (EnerGII) s afflower oil ( Prequel 21) and fish oil (StrataG) fat supplements were manufactured as of calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). 3 CLA = Conjugated linoleic acid. P P P 0.01; NS = non significant.

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262 Table 7 4 Least squares means and pooled SE for total fatty acids (g/100 g of freeze dried tissue) and different fatty acid percentages (% of the total fatty acid; g/100 g of fatty acids) in neutrophils collected at the time of initiation of diets (30 pre partum, pooled, n = 14), 30 days postpartum (dpp) for cows supplemented with palm oil (PO, n = 13) or s afflower oil (SO, n =13) and at 80 days postpartum for cows supplemented with palm oil (PO, n = 15) or fish oil (FO, n = 14) 30 dpp 1 80 dpp 2 P value Fatty acid 30 days p re partum PO SO SE PO FO SE 30 dpp 80 dpp Total 2.42 3.85 4.46 0.41 3.43 3.51 0.37 NS NS SFA 38.3 40.4 39.1 1.60 42.3 39.3 1.36 NS NS UNSFA 52.7 50.1 50.0 1.40 49.4 49.6 1.20 NS NS MUSFA 23.8 21.6 20.49 0.88 20.2 17.9 0.75 NS 0.05 PUFA 28.8 28.5 29.5 1.42 29.2 31.7 1.17 NS NS Trans FA 0.7 0.63 1.51 0.17 0.46 3.05 0.29 0.01 0.01 n 6 21.7 22.29 24.95 1.36 23.8 23.08 1.29 NS NS n 3 4.9 3.05 2.07 0.29 2.75 6.64 0.5 NS NS n 6/n 3 5.9 7.23 9.16 0.73 8.48 3.75 0.58 0.07 0.01 1 Diet s fed from 3 0 days pre partum to 30 dpp 2 Diets fed from 30 dpp to 80 dpp. All cows used at 80 dpp were fed PO from 3 0 days pre partum to 30 dpp. Palm oil (EnerGII) and Safflower oil ( Prequel 21) and fish oil (StrataG) fat supplements were manufactured as of calcium salts by Virtus Nutrition, LLC (Corcoran, CA, USA). SFA = Saturated fatty acids, UNSFA = unsaturated fatty acids, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids. n6 = C18:2 + C22:4. n3 = C18:3 + C20:5 + C22:6 NS = non significant.

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263 Figure 7 1 Least squares means ( S.E.) for plasma concentration of haptoglobin, adjusted for cervical discharge score, for cows fed calcium salts of palm oil ([ ]; n =15) or safflower oil ([ --]; n = 17) during the pre partum period (at least 20 days) to 35 days postpartum. Cervical discharge score was examined at 8 days postpartum. Transition diet (P < 0.05) days postpartum (P < 0.01) and cervical discharge score (P < 0.01) effects. 0,01 0,02 0,03 0,04 0,05 0,06 0 3 6 9 12 17 21 26 30 35 Arbitrary units Days postpartum

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264 Figure 7 2 Least squares means ( S.E.) for plasma concentration of fibrinogen, adjusted for cervical discharge score, for cows fed cal cium salts of palm oil ([ ]; n =15) or safflower oil ([ --]; n = 17) during the pre partum period (at least 20 days) to 35 days postpartum. Cervical discharge score was examined at 8 days postpartum. Transition diet (P < 0.05) days postpartum (P < 0.01) an d cervical disc harge score (P < 0.01) effects. 150 200 250 300 0 3 6 9 12 17 21 26 30 35 mg/dL Days postpartum

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265 Figure 7 3 Least squares means ( S.E.) for plasma concentration of ceruloplasmin for cows diagnosed with cervical discharge of clear, flacks or lochia ([ ]; n = 26) and mucopurulent or purulent ([ --]; n = 6). Cervical discharge score was examined at 8 days postpartum. Days postpartum by cervical discharge score interaction (P < 0.05) effect. 5 6 7 8 9 10 11 0 3 6 9 12 17 21 26 30 35 mg/dL Days postpartum

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266 Fi gure 7 4. Least squares means ( S.E.) number of white blood cells ( ), neutrophils ( ) and mononuclear presented as pooled and log transformed. White blood cells (diet and diet by day: non significant; day: P = 0.09); mononuclear (diet, day and diet by day: non significant) and neutrophils (diet: P < 0.05; day: P < 0.01; diet by day: non significant). 2,7 2,9 3,1 3,3 3,5 3,7 3,9 4,1 32 0 4 7 Log n of cells /L of blood Days relative to parturition

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267 Fi gure 7 5. Least squares means ( S.E.) n umber of neutrophils (log transformed) in whole blood from cows supplemented with palm oil ([ ]; n = 23) or safflower oil ([ --]; n = 24) during th e transition period. Diet (P < 0.01), day (P < 0.01) and diet by day (non significant, except day 4 and 7 postpartum, P = 0.08). 2,7 3 3,3 3,6 3,9 32 0 4 7 Log n of cells /L of blood Days relative to parturition

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268 Figure 7 6. Least squares means ( S.E.) percentage of neutrophils with phagocytic and oxidative burst activity in whole blood stimulated with E. coli (A) or S. aureus (B) from cows supplemented with palm oil ([ ]; n = 23 ) or safflower oil ([ --]; n= 24) during the transition period. In B pre partum value is missing. Diet (non significant), day (P < 0.1) and diet by day (n on significant). 15 25 35 45 55 65 32 0 4 7 % Days relative to parturition A 15 25 35 45 55 65 0 4 7 % Days relative to parturition B

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2 69 Fi gure 7 [1,412 to 2,290], 3 [2,290 to 3,637] and 4 [> 3,637]) on the least square means ( S.E.) percentage of neutrophils with phagocytic and oxidative burst activity when stimulated with E. coli S. aureus days postpartum). Bacteria (P < 0.01), number of neutrophils per L (quartile, P < 0.01), bact eria by quartile (non significant). 0 20 40 60 80 1 2 3 4 % N of neutrophils/ l (quartile)

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270 Fi gure 7 8. Least squares means ( S.E.) of neutrophil mean fluorescence intensity (MFI) of the red (indicator of number of bacteria phagocytised per neutrophil; bars) and green ( indicator of intensity of reactive oxygen species produced per neutrophil ; lines) wave lengths in neutrophils from whole blood stimulated with E. coli (A) or S. aureus (B) from cows supplemented with palm oil (PO; n = 23) or safflower oil (SO; n = 24) during the transition period. Bars in A and B: Diet at day 4 for A and B [P < 0.05]). Lines in A and B: Diet at day 4 for A and B [P < 0.05] and day 7 for A [P = 0.08]). 0 200 400 600 800 1000 32 0 4 7 MFI Days relative to parturition A PO SO PO SO 0 100 200 300 400 0 4 7 MFI Days relative to parturition B P.O. S.O. PO SO

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271 Fi gure 7 9. Least squares means ( S.E.) for TNF neutrophils cultured for 18 hours in RPMI 1640 stimulated or not with lypopolysaccharide (LPS) incubated at 37 o C and 5% CO 2 at 30 days postpartum (dpp) for cows supplemented with the transition period (A) or at 80 dpp for cows supplemented with mass inc rease (diet: non significant). In B no LPS (die t: non significant), LPS and mass increase (diet: P < 0.01). 0 25 50 75 100 125 150 no LPS LPS Mass Increased pg/mL A 0 25 50 75 100 125 150 no LPS LPS Mass Increased pg/mL B

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272 Fi gure 7 10. Least square s means ( S.E ) IL neutrophils cultured for 18 hours in RPMI 1640 stimulated or not with lypopolysaccharide (LPS) incubated at 37 o C and 5% CO 2 at 30 days postpartum (dpp) for cows supplemented with the transition period (A) or at 80 dpp for cows supplemented with p or fish oil (n = 14 ; significant), LPS and mass increase (diet: P < 0.01). In B no LPS, LPS and mass increase (diet: non significant). 0 0,5 1 1,5 2 no LPS LPS Mass Increased ng/mL A 0 0,5 1 1,5 2 no LPS LPS Mass Increased ng/mL B

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273 CHAPTER 8 GENERAL DISCUSSION A ND CONCLUSIONS Development of protocols for s ynchronizatio n of ovulation have been optimized to a point where pregnancy rates on commercial dairies have been improved, and it has become evident that further advances will be dependent on improving specific biological windows that currently are associated with sub fertility. Su b fertility in high producing dairy cows is a global problem and is characterized by multifactorial components that will necessitate a multidisciplinary approach to improve fertility of the lactating dairy cow Preparation for a new pregnancy starts even before parturition with dietary feeding of adequate quantities of energy, protein and minerals Also, management of proper body condition and health of the cow housing and delivery of the calf can prevent disorders associated with parturition such as retained fetal membranes metritis and metabolic disorders known to reduce subsequent fertility in lactating dairy cows Appropriate physical involution of the uterus, normal placental and endometrial sloughing, proper uterine bacterial clearance due to the the innate immune system, and recrudescence of the endometrium are important steps towards optimal fertility. Additionally, during early postpartum, recrudescence of follicular development and ultimately ovulation should happen at a time in whic h uterine bacterial clearance has been resolved. Early ovulation postpartum when the uterus is contaminated can result in prolonged luteal phases due to pyometra that prolongs calving to first insemination interval. Also, extreme uterine contamination can prolong calving to first ovulation preventing reoccurrence of estrous cycles that are important towards optimal fertility (Thatcher and Wilcox, 1972). Delayed first ovulation is also a result of metabolic imbalances resulting f rom onset of lactation and su b optimum energy intake during early lactation. At this time energy intake is less

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274 than what is required for maintenance and milk production. Thus a negative energy status prevails (Staples et al., 1990) and is characterized by high circulating GH and low concentrations of IGF 1 and insulin. Therefore, metabolism is tied to reproductive events through a hormonal link that involves the digestive (i.e., rumen, small intestine, liver) adipose hypothalamic pituitary ovarian uterine axis (es). Moreover, minera l deficiencies (i.e., calcium and selenium) and mobilization of body reserves from fats to accommodate the requirements of energy for lactation results in increasing concentrations of NEFA and BHBA. These dynamic responses are associated with depression of neutrophil activity during the peri partum period. Another area for improvement is embryonic survival, which has an direct impact on conception rates. Early embryonic loss in lactating dairy cows has been estimated to be as high as 40% between days 7 and 19 after insemination which is likely related to aberrations in oocyte quality, embryo development and encompasses the time in which the conceptus must secrete sufficient amounts of interferon secretion and sustain the corpus luteum to maintain the pregnancy. Further, if embryo implantation is successful, an immunological suppression or tolerance is needed during pregnancy; the embryo/fetal placental ration of pregnancy (Siiteri and Stites, 1982). In a previous study (Silvestre et al., 2008), we determined that continuous treatment of postpartum dairy cows with a GnRH agonist (Deslorelin, 5 mg) accelerated the rate of uterine involution and reduced the proportion of cows with a purulent cervical discharge postpartum; however the prolonged anestrous caused by desensitization of the pituitary after termination of treatment had a major negative impact on responsiveness of cows to programs for synchronizati on of ovulation. Therefore, we further pursued this concept of continuous treatment

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275 with Deslorelin but with the administration of smaller doses, and use of a degradable form of the implant to make it practical for producers since it eliminates the need fo r removal. The objectives were to improve rate of uterine involution, reduce occurrence of a purulent discharge, and not in occur a prolonged anestrous at the time when ovulations are synchronized for breeding (i.e., TAI). In Chapter 3 increasing doses of a degradable Deslorelin implant (i.e., 2.1 and 4.2 mg) were administered to suppress ovarian follicular activity while enhancing uterine and cervical involution to reduce the occurrence of uterine infection in dairy cows after parturition Both doses of t he GnRH a gonist treatment s caused desensitization of the pituitary leading to suppress ed follicular growth that was restricted to follicles of 2 3 mm in diameter Cows treated with both doses of Deslorelin had faster rates in reductions of diameters of bot h uterine horns. Also, the reductions in diameter of the uterine horns were accompanied by reductions in the width of the endometrial and myometrial layers compositing the uterine horns that were not different among cows treated or not with Deslorelin. How ever, diameter of the uterine lumen was reduced in a greater rate for cows treated with both doses of Deslorelin. Additionally, uterine ton e measured by per rectal palpation, was increased in the Deslorelin treated cows. Therefore, the reduction in sizes of the uterine horns was likely greater because of enhanced uterine contractility of the longitudinal and circular muscle fibers compositing the myometrial layers of the uterine horns. Mechanisms involved in greater uterine contractilityand accelerated red uction in uterine horn sizes could be explained by the lack of progesterone, known to induce relaxation of the uterus, or increased PGF to cause uterine contractility. However, both progesterone and PGFM concentrations concentrations were comparable for the control and experimental groups during the period from parturition until the time in which uterine sizes were no longer changing. Therefore, a greater rate in reduction of uterine sizes are possibl y due to

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276 direct affects of the GnRH agonist on the ute rus an d increased tonicity due to decreased secretion of pituitary gonadotrophin s (FSH and LH). B inding of FSH and LH to its receptors is associated with a direct activation of adenyl cyclase to produce cAMP and activation of signaling pathways that increa se expression of COX 2 and production of PGE 2 in the myometrium and cervix (Shemesh et al., 2001). Both cAMP and PGE 2 induce relaxation of these tissues. A sequence of events was proposed to occur during involution of the uterus: reduction in size, loss of tissue, and tissue repair (Marion et al., 1968). Additionally, Silvestre et al (2008) proposed that resolution of uterine infection must occur before first ovulation to reduce the risk of prolonged luteal phases and thus pyometra. Although the frequency distribution of cows among cervical discharge categories did not differ across treatments, greater rates of reduction in size of the uterine lumens could benefit uterine clearance of bacterial and necrotic tissues. Mattos et al. (2001) reported that insert ion of a degradable Deslorelin implant at day 7 postpartum induced follicular suppression for a period no longer than 40 days which permitted similar rates of synchronization of ovulation compared with non treated animals. In Chapter 3, one of the Deslorel in treated group of cows received a similar dose of Deslorelin as used by Mattos et al. (2001). H owever the anestrous rate w as greater than expected. Insertion of the GnRH agonist implant at the time of parturition may account for the difference as compar e with insertion of the Deslorelin implant 7 days postpartum (Mattos et al., 2001) The positive effects of a Deslorelin treatment on uterine involution can potentially have a great impact in the dairy industry to reduce cost associated with antibiotic treatment, milk withhold, labor and culling rates due to reproductive failure. Moreover, reduction of uterine infections in the postpartum period can reduce calving to conception interval, services per conception, and increase first service conception rat es However, t he benefits of the GnRH agonist on the overall physical

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277 involution of the reproductive tract early postpartum will not be of practical use until better drug delivery systems are develop ed to permit the utilization of this therapy in a shorter window of time (i.e., first 15 to 18 days postpartum) in which termination of treatment is practical for herd health personnel Furthermore, treatment must permit an earlier recrudescence of follicular activity leading to a proper synchronization of ovula tion at the time set for insemination. A nutraceutical is defined as a product isolated or purified from feeds that is demonstrated to have a physiological benefit or provide protection against chronic disease s Selenium (Se) supplementation has long been associated with reduction s in the incidence s of retained fetal membranes, clinical mastitis, calf mortality and milk somatic cell counts (SCC). Selenium is an essential component for the redox activity of Se enzymes required for biological functions includ ing antioxidant defense and thyroid hormone production. Therefore, adequate dietary intakes of Se are essential. The soil and forages produced in the Southeast of the United States are consider ed deficient in Se. Supplementation of this mineral in diets of ten utilize an inorganic form of the mineral (i.e., sodium selenite) and is limited to a maximum of 0.3 ppm in the dry matter of rations due to regulat i o ns set forth by the Food and Drug Administration. However oral supplementation of ruminants with inorga nic forms of Se results in low absorption caused by reduction of selenite to insoluble elemental Se and selenide compounds in the rumen and high e xcret ion mainly via the urine but also feces and exhalation. In Chapter s 4 and 5, effects of supplemental orga nic Se (Se yeast containing selenomethionine) were compared with sodium selenite on fertility uterine health, milk yield and peri parturient immune responses during the summer heat stress period. Treatments initiate before parturition and continue until a minimum of 81 days postpartum. Supplementation of Se

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278 yeast elevated plasma concentrations of Se from marginally deficient ( 0.069 g/ml) to an adequate ( 0.087 g/ml ) concentration Besides the eff ect of supplemental Se source on plasma concentration of selenium is the fact that oral administration of inorganic Se could not provide sufficient selenium to dairy cows, even when fed maximum concentrations in the diet In Chapter 4, health monitoring of postpartum cows conclusively demonstrated the beneficial effects of the Se yeast to reduce occurrences of fever and purulent cervical discharge. These findings were supported by results from Chapter 5 in which neutrophil function was enhanced in cows supp lemented with Se yeast. The overall benefit in cow health led to a greater milk yield i n the first month of lactation. Factors associated with Se yeast supplementation that increased neutrophil activity likely are caused by increased redox activity of sele enzymes (i.e., glutathione peroxidase) on hydrogen peroxide produced during the respiratory oxidative burst. Hydrogen peroxide is used by the neutrophil as a microbicidal coumpound; however, its generation and accumulation are toxic to the cell. Thus t he greater proportion of neutrophil s with phagocytic and oxidative burst activity indicated that neutrophils remained alive for longer time because of a better capacity to eliminate the damaging intracellular effects of reactive oxygen species Moreover, all cells in the organism produce, to a certain degree, free radicals from cell respiration that are toxic to the cells and surrounding tissues. The decreased SSC at later stages of lactation in cows supplemented with Se yeast likely is related to a reduction in mammary epithelial cell death and therefore less shedding of mammary epithelial cells. Indeed, mammary epithelial cells supplemented with Se methionine in primary culture had greater survivability associated with increased glutathione peroxidase activit y (Miranda et al., 2007). R estoration of a favorable uterine env ironment to support a pregnancy, particularly after a late embryonic loss, is possibly associated with a greater

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279 second service pregnancy per insemination in Se yeast fed cows. Additionnaly, m ammalian embryos are particularly sensitive to heat shock C ulture of embryos at elevated temperatures causes retardation in development, malformations and embryonic death caused by the generation of superoxide and hydrogen peroxide wich act s on peroxidation of lipid membranes, DNA damage and enzyme inactivation. Antioxidants such as glutathione and enzymes involved in removal of superoxide and hydrogen peroxide play an important role in intracellular thermoprotection Indeed, thermoprotection of bovine morulae embryos from heat shock can be achieved in vitro by administration of glutathione (Ealy et al., 1992) The bovine caruncular endometrium is a major source of PGF during the early postpartum period with secretion increasing from day of pa rturition to approximately 4 days postpartum. This increase is followed by a progressive decline to basal concentrations at day 14 postpartum (Guilbault et al., 1984), when sloughing of caruncular structures is completed. Cows with greater PGF metabolite (PGFM) in plasma, which is related to the degree of PGF secretion from the uterus, are at lower risk of metritis (Seals et al., 2002). Prostaglandins are derived from the membrane phospholipid stores of arachidonic acid in which arachidonic acid is synt hesized from linoleic acid an essential dietary fatty acid. The PGF activates neutrophil bactericidal activity (Hoedemaker et al., 1992) and has important functions on uterine contractility (i.e., physical involution of the uterus). Also, lipoxygenases, acting on arachidonic acid, generate leukotriene B4 which is a potent neutrophil chemo attractant (Heidel et al., 1989). I n Chapter 6 and 7 feeding calcium salts (CS) of linoleic acid (safflower oil ; SO ) prior to parturition and during the first 30 days p ostpartum numerically increase d the proportion of linoleic acid in the caruncular tissue and significantly increased the n 6/n 3 fatty acid ratio compared with the palm oil (PO) supplemented diets. The absence of detectable portions of

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280 a rachidonic acid in the caruncular tissue indicates a possible rapid turnover of this fatty acid immediately after parturition for production of PGF Additionally, supplementation with CS of SO and fish oil (FO) increased and decreased the n 6/n 3 fatty acid ratios in neutr ophils, respectively. Bilby et al. (2006) reported that other tissues such as endometrium, muscle, liver and the mammary gland are subject to changes in their fatty acid profile after feeding CS of FO. Collectively, daily feeding of CS of selective fatty a cids is a practical approach in which tissue fatty acid composition can be manipulated accordingly to the stage of the life cycle of the cow. In Chapter 6, differences in plasma concentrations of PGFM were sporadically greater for cows fed the SO compared with PO on certain days. Mean concentration of PGFM was numerically greater in the SO fed cows; however variability among cows and days prevented the detection of an overall statistical difference between diets. Increase in sampling frequency, perhaps twic e daily samples, could have improved statistical power to detect differences between diets. Additionally, in Chapter 7, other markers of inflammation (i.e., acute phase response) and responses associated with inflammation (i.e., neutrophil adhesion molecules, neutrophil phagocytic activity and cytokine production) were greater in cows fed SO compared with PO I t is worthwhile to mention that studies designed to measure dietary effects on inflammatory and immune responses in the postpartum cow must us e clinically similar groups of healthy cows avoid disorders that involve inflammation (i.e., mastitis and metritis) and treatment interventions with anti inflammatory or antibiotic s This will prevent confounding with dietary treatments. As it was mention ed in Chapter 7, cows with mucopurulent and purulent cervical discharge had the greatest responses in acute phase proteins and quantities of adhesion molecules o n the surface of neutrophils. However, significant differences were apparent after deletion of these cows from the

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281 analysis. Also, in Chapter 3, antibiotic treatments acutely reduce d plasma concentration of PGFM, possibly by eliminating bacteria, the triggering factor of inflammation. The acute phase response of the liver is induced by pro inflammat ory cytokines (i.e., TNF 1) produced by epithelial cells, macrophages and later neutrophils present at the sites of inflammation (Petersen et al., 2004). The acute phase response is often used as a marker of stress; therefore often confused with an u nwanted response. However, the acute phase response provides an early non specific defense mechanism against insult before specific immunity is achieved (Peterson et al., 2004). Blood haptoglobin binds free hemoglobin restricting the availability of iron n ecessary for bacterial growth in the blood and prevents pro oxidant activity of hemoglobin on tissues ( Eaton et al., 1982 ). Fibrinogen is the precursor of fibrin a protein that activates neutrophil toll like receptors and M 2 integrin receptor to induce i ntracellular calcium mobilization, phosphorylation events, NF kB activation (i.e., expr ession of cytokines, COX 2), and both c ell adhesion and migration ( Flick et al., 2004 ). It was demonstrated that supplementation of SO during the peri partum increased t he acute phase response (i.e., haptoglobin an d fibrinogen) associated with increased number of bacteria phagocytised per neutophil, quantities of adhesion molecules on the surface of neutrophils ( i.e ., L selectins) and neutrophil cytokine production ( i.e., TN F 1). W hen measured in whole blood these cellular responses are likely to be correlated because an increased cytokine production, although measured in one type of cell (i.e., neutrophil) is likely to occur in other types of cells such as epithe lial and vascular endothelial cell s Therefore, greater neutrophil phagocytic activity and expression of L Selectin could be induced by greater circulating inflammatory markers. Also peroxisome proliferator activated receptors (PPARs) are a family of nucle ar receptors activated by selected long chain fatty acids that upon ligand binding, heterodimerize with the retinoid

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282 receptor RXR and interact with specific PPAR response elements in the promoter region of genes associated with cytokine production and neut rophil activity. Also, changes in the n 6 fatty acid composition of neutrophils can increase e icosanoid production leading to greater cytokine production upon challenge. Indeed, eicosanoids (Camandola et al., 1996) that increas es TNF IL in monocytes (Baldie et al., 1993, Sinha et al., 1991). Collectively, feeding a linoleic fatty acid enriched diet beginning in the close up ration pre a state involves a lower threshold for initiation of an inflamm atory response and increased sensitivity of cells upon stimuli. Inflammation is the first step for initiation of an immune response. Increased milk yield in cows supplemented with SO during the transition period was more likely caused by the increased dry matter intake in this group of cows Additionally, a greater state of immune competence in cows supplemented with SO may have enhanced health status that contributed to a greater milk yield without affecting the ir metabolic status compared with PO suppleme nted cows. In Chapter 6, cows fed FO diets had increased pregnancy per insemination at day 32 after insemination and less embryonic losses from days 32 to 60 of pregnancy. Causes for pre implantation embryo loss may involve extra uteri inflammatory mediado rs (i.e., TNF IL nitric oxide and PGF ) that can affect embryonic development by acting either on the oocyte or on the deleveloping embryo (Hansen et al., 2004). Also, f ollowing the transition period, feeding a FO enriched diet may help to reduce t he pulsatile secretion of PGF from the endometrium which, if not suppressed, results in corpus luteum regression and embryonic death A h igh

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283 proportion of losses (~40%) precedes or occurs concurrently with the period when the embryo inhibits uterine PGF 2 secretion. Embryonic loss es may occur because non viable or undersized embryos secrete sub optimum quantities of interferon to inhibit secretion of PGF 2 Feeding n 3 fatty acids in the form of fish oil increased concentrations of EPA and DHA fatty acids in caruncular (Mattos et al., 2002) and endometrial (Bilby et al., 2006) tissues. The greater availability of these fatty acids in the uterus can competitively inhibit the oxygenation of arachidonic acid by cyclooxygenase and therefore reduce t he synthesis of PGF (Mattos et al., 2003; Mattos et al., 2004). Moreover, once implantation has occurred, an immunological suppression of the uterine environment must take place f or tolerance of the placental /embryo allograft. In Chapter 7, neutrophil se nsitivity to a challenge for production of TNF (numerically) was diminished in cows fed the FO supplements. This result could indicate a higher threshold for stimulation of an inflammation/immune response in tissues that may benefit avoidance o f placental/embryo units by the uterus. The emigration of neutrophils from the vasculature occurs in sequential distinct steps. The first step involves a capture and rolling adhesion mediated by the selectins. After this initial step, the leukocyte must b e activated by chemo attractants, and the third step involves firm adhesion or arrest of the cells to the endothelium by the activated integrins. Once anchored to the endothelial cell, the neutophil can migrate from the circulation into the infected tissue by diapedisis.Phagocytosis of bacteriais then stimulated by pro inflammatory cytokines released by surrounding cells (i.e., paracrine action) and the neutrophil itself (i.e., autrocrine action). Phagocytosis results in generation of reactive oxygen specie s (oxidative burst) for bacterial killing that must undergo reduction by a complex of enzymes to prevent cellular damage (oxidative damage). Feeding diets enriched in linoleic acid and selenium yeast may improve all

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284 of the local inflammatory steps describe d above in the peri parturient dairy cow. An essential feature in these steps is that they do not occur in parallel but rather in a sequential manner, thus impairment of one step can be detrimental for the entire process. These dietary strategies have pro found implications for the dairy industry, because prevention of diseases and improvements in heard health can be achieved without the need of individual cattle treatments and manipulations. Also, daily feeding provides continual delivery of these compound s to all animals at specific physiological stages as required. In Chapters 4 and 6, large numbers of animals were used in both studies allowing multi variable statistical analyses and detection of important associated biological responses related to anima l health and well being, milk production and fertility. Several associations were common between studies even thought experimentation was carried out on two different dairy operations ed with multiparous cows. Primiparous cows had increased risk of calf delivery problems which increased the need of human assistance. Stress related to first parturition, mechanical injury of reproductive tracts, and possible ascend ing contamination caused by human manipulation place primiparous cows to increased risk of severe uterine contamination, systemic illness (i.e., fever), subclinical endometritis and anovulation. Therefore, it is paramount that management of heifers must be addressed carefully. Ve terinarians and producers should work together to implement management strategies to target proper weigh and sizes for heifer insemination along with the choices of proper sires for these heifers. This also includes reproductive technology such as use of s exed semen and ultrasonography for identification of twin pregnancies etc. Additionally, maternity personal should be trained to identify problems and handle differently the first parturition cow.

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285 Regardless of parity, constant interrelationships of a stre ssful parturition, uterine disorders (retained fetal membranes and metritis, with or without fever), and severe loss of body condition after onset of lactation with subsequent reductions in ovarian cyclicity and first insemination fertility are indicative that sub optimal fertility of modern dairy cows is a multifactorial problem. Solutions to this require a multidisciplinary approach (i.e., veterinary medicine, nutrition, physiology and immunology). In conclusion, hormonal and nutritional programs can benefit the performance of lactating dairy cows in the periparturient and breeding periods of their life cycle. Continuuous treatment with a GnRH agonist improves physical involution of the postpartum uterus but additional research is needed to counteract prolonged periods of acyclicity. Feeding Se yeast enhanced neutrophil function that was associated with a reduction in mucopurulent and purulent discharges of the postpartum uterus. Feeding Se yeast improved selenium status and increased the capacity of ne utrophils to remove toxic reactive oxygen species. Sequential feeding of diets enriched in linoleic acid followed by diets enriched in EPA and DHA during the periparturent and breeding periods, respectively altered immune responses that benefited overall c ow performance and fertility. The threshold for triggering an immune response (i.e., creating a pro inflammatory state that can respond greatly upon challenge) upon feeding fatty acid precursors of pro inflammatory eicosanoids can benefit the postpartum i mmunity (innate immunity and secretion of acute phase proteins) of dairy cows. This would set the stage for greater milk production. Following a health transition period, continual supplementation Se yeast and initiation of FO feeding can improve milk prod uction and fertility of the dairy cow. Such feeding strategies warrant economic analyses to evaluate cost benefit.

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286 LIST OF REFERENCES AbuGhazaleh, A. A., D. J. Schingoethe, A. R. Hippen, K. F. Kalscheur, and L. A. Whitlock. 2002. Fatty acid prof iles of milk and rumen digesta from cows fed fish oil, extruded soybeans, or their blend. J. Dairy Sci. 85:2266 2276. Aldred, A. R., A. Grimes, G. Schreiber, and J. F. Mercer. 1987. Rat ceruloplasmin. Molecular cloning and gene expression in liver, choro id plexus, yolk sac, placenta, and testis. J. Biol. Chem. 262:2875 2878. Allison R D ., and R. A. Laven 2000. Effect of vitamin E supplementation on the health and fertility of dairy cows: a review. Vet Rec 147 : 703 708 Allen, M. S. 2000. Effects of diet on short term regulation of feed intake by lactating dairy cattle. J. Dairy Sci. 83:1598 1624. Allen, W. M., R. Bradley, S. Berrett, W. H. Parr, K. Swannack, C. R. Barton, and A. Macphee. 1975. Degenerative myopathy with myoglobinuria in yearling cattle. Br. Vet. J. 131:292 308. Alsemgeest, S. P., H. C. Kalsbeek, T. Wensing, J. P. Koeman, A. M. van Ederen, and E. Gruys. 1994. Concentrations of serum amyloid A (SAA) and haptoglobin (HP) as parameters of inflammatory diseases in cattle. Vet. Q. 16 : 21 23 Ambrose D J. M. Drost, R. L. Monson, J. J. Rutledge, J. J. Leibfiied Rutledge, M. J. Thatcher, T. Kassa, M. Binelli, P. J. Hansen, P. J. Chenoweth and W. W. Thatcher. 1999. Efficacy of timed embryo transfer with fresh and frozen in vitro produ ced embryos to increase pregnancy rates in heat stressed dairy cattle. J Dairy Sci 82:2369 2376. Ambrose, D. J., J. P. Kastelic, R. Corbett, P. A. Pitney, H. V. Petit, J. A. Small, and P. Zalkovic. 2006a. Lower pregnancy losses in lactating dairy cows fed a diet enriched in alpha linolenic acid. J Dairy Sci. 89 : 3066 3074 Ambrose, D. J., C. T. Estill, M. G. Colazo, J. P. Kastelic, and R. Corbett. 2006b. Conception rates and pregnancy losses in dairy cows fed a diet supplemented with rolled flaxseed. Proc 7th International Ruminant Reproduction Symposium, Wellington, New Zealand. abstract 50. Andersen, L.E., Y L. Wu, S J. Tsai and M .C. Wiltbank 2001. Prostaglandin F receptor in the c orpus luteum: recent information on the gene, messenger ribonu cleic acid, and protein. Biol Reprod 64:1041 1047. Archiga, C.F., O. Ortz, and P. J. Hansen. 1994. Effect of prepartum injection of vitamin E and selenium on postpartum reproductive function of dairy cattle. Theriogenology 41 : 1251 1258.

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287 Armstrong, J. D., E. A. Goodall, F. J. Gordon, D. A. Rice, and W. J. McCaughey. 1990. The effects of levels of concentrate offered and inclusion of maize gluten or fish meal in the concentrate on reproductive performance and blood parameter of dairy cows. Anim. Prod 50:1 10. Arthur, J. R., P. C. Morrice, and G. J. Beckett. 1988. Thyroid hormone concentrations in selenium deficient and selenium sufficient cattle. Res. Vet. Sci. 45:122. Arthur, J.R. 1999. Functional indicators of iodine and selenium status. Proc N utr Soc. 58:507 12. Arthur J R ., and G. J. Beckett. 1999. Thyroid function. Br. Med. Bull. 55 : 658 668. Awadeh, F. T., R. L. Kincaid, and K. A. Johnson. 1998. Effect of level and source of dietary selenium on concentrations of thyroid hormones and immunoglobulins in beef cows and calves. J. Anim. Sci. 76:1204 1215. Aziz, E.S., P. H. Klesius and J. C. Frandsen. 1984. Effects of selenium on polymorphonuclear leukocyte function in goats. Am J Vet Res 45 : 1715 1718 Aziz E S., and P. H. Klesius 1986a. Effect of selenium deficiency on caprine polymorphonuclear leukocyte production of leukotriene B4 and its neutrophil chemotactic activity.Am J Vet Res. 47 : 426 428. Aziz E S ., and P. H. Klesius. 1986b. Depressed neutrophil chemotactic stimuli in supernatants of ionophore treated polymorphonuclear leukocytes from selenium deficient goats. Am. J. Vet. Res. 4 7 : 148 151. Badwey, J.A., J. M. Robinson, M. J. Karnovsky, and M. L. Karnovsky. 1981. Superoxide production by an unusual aldehyde oxidase in guinea pig granulocytes. Characterization and cytochemical localization. J Biol Chem. 256 : 3479 3486 Badway, J. A., J. T. Curnutte, J. M. Robinson, C. B. Berde, M. J. Karnovsky, and M. L. Karnovsky. 1984. Effects of free FA on release of superoxide and on change of shape by human neutrophils. Reversibility by albumin. J. Biol Chem. 259: 7870 7877. Baker, S. S., and H. J. Cohen. 1983 Altered oxidative metabolism in selenium deficient rat granulocytes. J. Immunol. 130 : 2856 2860. Baker, S S ., and H. J. Cohen 1984. Increased sensitivity to H 2 O 2 in glutathione peroxidase deficient rat granulocytes. J Nutr. 114 : 2003 200 9 Balapure, A. K., C. E. Rexroad, Jr., K. Kawada, D. S. Watt, and T. A. Fitz. 1989. Structural requirements for prostaglandin analog interaction with the ovine corpus luteum prostaglandin F 2 receptor. Implications for development of a photoaffinity probe. Biochem. Pharmacol. 38:2375 2381.

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289 Bilby, T. R ., T. Jenkins C. R. Staples and W. W. Thatcher 2006. Pregnancy, bovine somatotropin, and dietary n 3 Fatty acids in lactating dairy cows: III. Fatty acid distribution. J Dairy Sci. 89:3386 3399. Bonnamy, P. J., A. Benhaim, and P. Leymarie. 1990. Estrous cycle related changes of high affinity luteinizing hormone/human chorionic gonadotropin binding sites in the rat uterus. Endocrinology. 126 : 1264 1269 Borella P A. Bargellini and C. I. Medici 1996. Chemical form of se lenium greatly affects metal uptake and responses by cultured human lymphocytes. Biol Trace Elem Res. 51:43 54 Boyne, R., and J. R. Arthur. 1979. Alterations of neutrophil function in selenium deficient cattle. J Comp Pathol. 89 : 151 158 Broekmans, F.J. 1996. GnRH agonists and uterine leiomyomas. Hum. Reprod. 11:3 25. Bruckental, I., D. Dori, M. Kaim, H. Lehrer, and Y. Folman. 1989. Effects of source and level of protein on milk yield and reproductive performance of high producing primiparous and multiparous dairy cows. Anim. Prod. 48:319 329. Buergelt, C.D., D. Sisk, P. J. Chenoweth, J. Gamboa, and R. Nagus. 1996. Nutritional myodegeneration associated with dorsal scapular displacement in beef heifers. J Comp Pathol. 114 : 445 450 Burke, J. M ., C. R. Staples, C. A. Risco, R. L. De La Sota, and W. W. Thatcher. 1997. Effect of ruminant grade menhaden fish meal on reproductive performance of lactating dairy cows. J. Dairy Sci. 80:3386 3398. Cai, T. Q., L. A. Weston, L. A. Lund, B. Brodie, D. J. McKenna, and W. C. Wager. 1994. Association between neutrophil functions and periparturient disorders in cows. Am. J. Vet. Res. 55: 934 943. Calder, P. C., and S. Kew. 2002. The immune system: a target for functional foods. Br. J. Nutr. 2:165 177. Calder, P. C., C. Yaqoob, D. J. Harvey, A. Watts, and E. A. Newsholme. 1994. Incorporation of FA by concanavalin A stimulated lymphocytes and the effect on fatty acid composition and membrane fluidity. Biochem J. 1300:509 518. Calder, P. C ., J. A. Bond D. J. Harvey S. Gordon and E. A. Newsholme 1990. Uptake and incorporation of saturated and unsaturated FA into macrophage lipids and their effect upon macrophage adhesion and phagocytosis. Biochem. J. 269:807 814

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BIOGRAPHICAL SKETCH Flv io Teixeira Silvestre was born in 1973, to Paulo Augusto Alves Silvestre and Maria Angela Teixeira Silvestre in Campinas, So Paulo, Brazil. He is the second son of four. In 1997, he received his degree in veterinary medicine from the Universidade Federal de Minas Gerais, located in Belo Horizonte, Minas Gerais. After his degree, he worked as a practitioner for a dairy cooperative located in Esprito Santo State during and soon after he join ed the University of 2 years as a trai ning student at North Florida Holsteins Dairy Farm. In the summer of 2001 he with emphasis in reproductive biology at the University of Florida under supervision of Dr. William W. Thatcher. In the spring of 2004 Flvio was awar ded the Animal Molecular and Cellular Biology Fellowship to pursue his doctoral degree with Dr. William W. Thatcher.