FUNCTION AND GENE EXPRESSION OF CIRCULATING NEUTROPHILS IN DAIRY COWS: IMPACT OF MICRONUTRIENT SUPPLEMENTATION A Dissertation Presented to The Faculty of the Graduate School At The University of Missouri In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy By Xavier S. Revelo Dr. Matthew R. Waldron, Dissertation Supervisor July, 2012 ©Copyright by Xavier S. Revelo 2012 All rights reserved The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled: FUNCTION AND GENE EXPRESSION OF CIRCULATING NEUTROPHILS IN DAIRY COWS: IMPACT OF MICRONUTRIENT SUPPLEMENTATION Presented by Xavier Revelo A candidate for the degree of Doctor of Philosophy in Animal Sciences And hereby certify that, in their opinion, it is worthy of acceptance. Dissertation Examination Committee: ________________________ Advisor Matthew R. Waldron, Ph.D. ________________________ Kevin L. Fritsche, Ph.D. ________________________ Matthew C. Lucy, Ph.D. ________________________ James W. Perfield, Ph.D. ACKNOWLEDGEMENTS First of all, I would like to thank my family for their patience, love and inseparable support even after all the years that I have been away from home. Thanks to my mother Sonia for dedicating her life to the guidance, inspiration, and nurturance of our family. I have been blessed with a loving and devoted family who gave me the motivation I needed to pursue my professional goals. I would also like to offer my sincerest gratitude to the friends I made while pursuing my doctorate degree at the University of Missouri. Special thanks to Daniel Mathew, Brad Scharf, Ky Pohler, Eric Coate, and Emma Jinks for sharing their time and friendship. I have great memories of my time in Missouri and will never forget all the trouble we produced during the prime years of our lives. I would also like to express my appreciation to all the people involved in the research presented in this dissertation. In this regard, I would like to thank former and present members of the Waldron lab who contributed to my research. In particular, I would like to acknowledge Ann Kenny and Nicole Barkley for their hard work during the execution of the animal studies. In a similar way, my most cordial gratitude to Nathan Bivens and Wade Davis for their assistance with the execution and analysis of the RNA- sequencing experiments. I would also like to thank all the faculty and staff at the Division of Animal Science for their academic and technical support during my tenure at the University of Missouri. I would like to thank the members of my graduate program defense committee Kevin Fristche, Matthew Lucy, and James Perfield for their help and input in the realization of this dissertation. Not only they have contributed to my career as teachers ii and mentors, but also cared about me as a person throughout my graduate program at Mizzou. Finally, special thanks to my advisor Matthew R. Waldron for the opportunity to conduct research in his laboratory and for all the guidance received throughout my graduate program. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ ii TABLE OF CONTENTS ............................................................................................... iv LIST OF FIGURES ...................................................................................................... vii LIST OF TABLES ..........................................................................................................x ABSTRACT ................................................................................................................. xii CHAPTER ONE: REVIEW OF LITERATURE ..............................................................1 INTRODUCTION ..................................................................................................1 MAMMARY GLAND IMMUNOBIOLOGY .........................................................2 IMMUNE DYSFUNCTION DURING THE PERIPARTURIENT PERIOD ......... 16 NUTRITIONAL APPROACHES TO IMPROVE IMMUNITY ............................ 33 CONCLUSION .................................................................................................... 40 CHAPTER TWO: IN VITRO EFFECTS OF ESCHERICHIA COLI LIPOPOLYSACCHARIDE ON THE FUNCTION AND GENE EXPRESSION OF NEUTROPHILS ISOLATED FROM THE BLOOD OF DAIRY COWS ...................... 43 ABSTRACT ......................................................................................................... 43 INTRODUCTION ................................................................................................ 45 MATERIALS AND METHODS .......................................................................... 48 RESULTS ............................................................................................................ 63 DISCUSSION ...................................................................................................... 85 CONCLUSIONS .................................................................................................. 93 CHAPTER THREE: EFFECT OF A B-VITAMIN AND FUNGAL DIETARY SUPPLEMENT ON THE FUNCTION OF NEUTROPHILS HARVESTED FROM PERIPARTURIENT DAIRY COWS ............................................................................ 95 ABSTRACT ......................................................................................................... 95 INTRODUCTION ................................................................................................ 97 MATERIALS AND METHODS .......................................................................... 99 RESULTS .......................................................................................................... 111 iv DISCUSSION .................................................................................................... 120 CONCLUSIONS ................................................................................................ 125 CHAPTER FOUR: EFFECT OF A B-VITAMIN AND FUNGAL DIETARY SUPPLEMENT ON THE GLOBAL GENE EXPRESSION PROFILE OF NEUTROPHILS HARVESTED FROM PERIPARTURIENT DAIRY COWS............ 127 ABSTRACT ....................................................................................................... 127 INTRODUCTION .............................................................................................. 129 MATERIALS AND METHODS ........................................................................ 132 RESULTS .......................................................................................................... 143 DISCUSSION .................................................................................................... 158 CONCLUSIONS ................................................................................................ 163 CHAPTER FIVE: EFFECT OF TRACE MINERAL SUPPLEMENTATION SOURCE ON THE FUNCTION OF NEUTROPHILS COLLECTED FROM PERIPARTURIENT DAIRY COWS ............................................................................................................ 181 ABSTRACT ....................................................................................................... 181 INTRODUCTION .............................................................................................. 183 MATERIALS AND METHODS ........................................................................ 186 RESULTS .......................................................................................................... 200 DISCUSSION .................................................................................................... 217 CONCLUSIONS ................................................................................................ 225 CHAPTER SIX: CHAPTER SIX: EFFECT OF TRACE MINERAL SUPPLEMENTATION SOURCE ON THE GLOBAL GENE EXPRESSION PROFILE OF NEUTROPHILS COLLECTED FROM PERIPARTURIENT DAIRY COWS ...... 227 ABSTRACT ....................................................................................................... 227 INTRODUCTION .............................................................................................. 230 MATERIALS AND METHODS ........................................................................ 233 RESULTS .......................................................................................................... 245 DISCUSSION .................................................................................................... 266 CONCLUSIONS ................................................................................................ 272 v CHAPTER SEVEN: INTREGRATED DISCUSSION FROM THE STUDY OF NUTRITIONAL TACTICS TO IMPROVE INNATE IMMUNITY IN DAIRY COWS .................................................................................................................................... 304 BIBLIOGRAPHY ....................................................................................................... 314 VITA ........................................................................................................................... 339 vi LIST OF FIGURES Figure 2.1. Effect of Escherichia coli lipopolysaccharide (LPS) on an index of the intracellular reactive oxygen species (ROS) production by bovine neutrophils (PMNL). .................................................................................. 65 Figure 2.2. Effect of Escherichia coli lipopolysaccharide (LPS) on an index of the total reactive oxygen species (ROS) production by bovine neutrophils (PMNL).69 Figure 2.3.