NUTRACEUTICAL AND HORMONAL REGULATION OF IMMUNITY, UTERINE HEALTH, FERTILITY, AND MILK PRODUCTION OF POSTPARTUM DAIRY COWS By FLÁVIO 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 1 © Flávio Teixeira Silvestre 2 To all dairy cows and producers of the world 3 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 opportunity to be exposed to Dr. Thatcher‘s work through scientific publications. The experience of reading Dr. Thatcher‘s work magnified my interest in reproductive physiology of the dairy cow and in science. The importance of his work has inspired many students like me to pursue experimental science and the discovery of the unknown. Dr. Thatcher provided me with all the tools necessary for my accomplishments during these seven fruitful years of my graduate career and he will be forever a friend. I would like to extend my appreciation to Dr. José E. P. Santos who accepted to be on my committee, even while he was not part of our department. Dr. Santos 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 career since the master‘s years; 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 postpartum 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 data handling; Marie-Joelle is the most altruistic person I have encountered at the University of Florida. She spent countless hours helping me, even when it was not part of her responsibilities. Marie-Joelle‘s teachings were of great importance to me during my graduate career 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 closely involved in my PhD program and his 4 friendship will not be forgotten. I am very grateful to Dr. Cynda Crawford, Dr. Tom Jenkins and Evanne Thies for their collaboration with sample analyses. I also would like to thank Dr. Max Paape for welcoming me during a productive week at the USDA-Beltsville and providing 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 working with me in the farms or enduring late hours in the laboratory. These 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 handling, data acquisition and sample analyses. 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, John Karanja, Saide, Angel, Mario, José and Raul) and Alliance Dairies (Nilo, Amadeo, June, Kátia, Junia, Antônio, Eraldo, Melvin, Fabrício, 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 the 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 Joann M. Fischer (student 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. Aydin Guzeloglu, Dr. Jeremy Block, Dr. Dean Jousan, Cristina Caldari, Lílian Oliveira, Davi Araújo, 5 Fabio Lima and Bárbara Loureiro. I also extend my appreciation to all friends in the Tanglewood football group (Evandro, Carlos and Cristovan, Valério, Cláudio, Andrés, Pato, Pepe, Federico, Alejandro, Sérgio and Sebastian brothers, Marcelo, Geraldo, Pablo, Thakadu, Bernard and everyone else who came to play) for the evenings of great entertaiment. Special thanks are extended to members of my family (Pai, mãe, Gustavo/Roberta/Arthur and João 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/Sérgio/Camila, Léo, Henrique, Ricardo, Claudine, Ronaldinho, André and Dulce; Tios: Ronald†, Luizinho†, José Roberto; Tias: Júlia, Lígia and Lucília) I appreciate their support and friendship. Special appreciation also is extended to Vó Cinira† and Tereza for their endless support and positive thinking of me. Last but not least, I extend my most sincere recognition and appreciation to Maria Juliana Silvestre de Sá. Her never-ending unconditional support brought me where I am today. Without Juliana nothing in the past seven years could have been accomplished. To Juliana I give all my heartfelt thanks. 6 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 Transport 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 Acute Phase Response and Proteins .......................................................................................61 Haptoglobin .....................................................................................................................61 Fibrinogen ........................................................................................................................62 Ceruloplasmin ..................................................................................................................63 The Neutrophil ........................................................................................................................64 Cytokines .........................................................................................................................67 Adhesion Molecules ........................................................................................................68