EFFECTS OF GROWING PROGRAMS AND β- ADRENERGIC AGONISTS ON GENE EXPRESSION IN ADIPOSE TISSUE DEPOTS IN FINISHING BEEF STEERS By DANIEL RAY STEIN Bachelor of Science in Agriculture Ecology Northwestern Oklahoma State University Alva, Oklahoma 1978 Master of Science in Animal Science Oklahoma State University Stillwater, Oklahoma 2004 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY July, 2009 EFFECTS OF GROWING PROGRAMS AND β- ADRENERGIC AGONISTS ON GENE EXPRESSION IN ADIPOSE TISSUE DEPOTS IN FINISHING BEEF STEERS Dissertation Approved: Dr. Clint R. Krehbiel Dissertation Adviser Dr. Udaya E. DeSilva Dr. Patricia J. Ayoubi Dr. J. Brad Morgan Dr. Rodney D. Geisert Dr. A. Gordon Emslie Dean of the Graduate College ii Many doors had to be opened at the right time for this achievement to occur at this stage in my life. At times when I thought all doors were closed, one would open and a new direction taken. I thank the Lord, Jesus Christ for his unconditional love and for watching over me, opening (and closing) those doors, and directing my life in a path to better serve him. In all your ways acknowledge him and he shall direct your paths (Proverbs 3:6). iii ACKNOWLEDGMENTS I would first like to express my sincere appreciation to Dr. Rodney Geisert for giving me the opportunity to pursue my Ph.D. in Animal Science at Oklahoma State University. Not often does a person have the opportunity and good fortune to attain a dream or goal they have set in life, let alone the opportunity and possibility to attain a second. My first goal, to be successful in the registered seedstock business – made my resolve for my second goal, to attain a Ph.D. degree, teach at the university level, and make a difference in the lives of young people, permissible. Dr. Geisert, your understanding, support, guidance, and mentorship are truly appreciated. The teaching opportunity given me after you accepted the position of Department Head at the University of Missouri awarded me a great number of opportunities and recognitions never imagined. I would also like to express my deepest gratitude to Dr. Clint Krehbiel and Dr. Udaya DeSilva for not only serving on my Ph.D. graduate committee, but also for agreeing to serve as my Ph.D. committee co-advisors. Your insight and guidance in the research and teaching arena and your support through my Ph.D. program were very much appreciated. I would also like to thank Dr. Patricia Ayoubi and Dr. Brad Morgan for serving on my Ph.D. graduate committee. Dr. Ayoubi, I truly appreciate the time you took to answer the many questions that came up while performing the microarrays and iv completing the data analysis; your expertise, support and friendship through this endeavor was greatly appreciated. Dr. Morgan, a big “Thanks” for your support, encouragement, and perspective of things along the way. I would like to acknowledge and truly thank my mother, Rosevelyn, for her encouragement, support, and prayers through these past seven years. It has been a struggle at times, but a sincere “Thank You” for believing in my goals and my ability to attain them. To the rest of my family and in-laws, I truly appreciated your prayers, support, and encouragement. I would also like to acknowledge my many friends, the entire OSU Animal Science Family including; all faculty (past and present), fellow graduate students, all support personel, and my undergraduate students who have assisted, encouraged, and supported not only me, but Jana, through our time at OSU. I also need to express my gratitude and appreciation to Morgan Ashworth for his friendship and encouragement over the years. Above all, I would like to thank my wife, Jana, for her never-ending love and support. It has been a long seven years since we began this journey; I could have never accomplished this undertaking without your love, patience, friendship and understanding. I truly appreciate the sacrifices that you have made for our future. You have constantly helped me keep a perspective on what is important in life and for that I am truly indebted. All my love. v Dedications I would like to dedicate this manuscript to my father, Leroy, who did not live long enough to see me finish my Ph.D. degree. It is not often a son has the opportunity and privilege to work side by side with his father for nearly thirty years – for that I am truly blessed. Dad, your wisdom, character and spirit gave me strength when needed. I'm Just a Farmer, Plain and Simple I'm just a farmer, plain and simple. Not of royal birth, but rather a worker of the earth. I know not of riches, but rather of patches on my britches I'm just a farmer, plain and simple. I know of drought and rain, of pleasure and pain. I know the good, the bad, the happy and the sad. I'm a man of emotions. A man who loves this land and the beauty of its sand. I'm just a farmer, plain and simple. I know the spring's fresh flow and autumn's golden glow. Of a new born calf's hesitation and an eagle's destination. I'm just a farmer, plain and simple. I know of tall pines and long waiting lines. I know the warmth of campfires and the agony of flat tires. I'm just a farmer, plain and simple. I'm a man who loves his job And the life that I live. I'm just a farmer, plain and simple I'm a reaper of harvest. I'm the sower of seeds and I'm the tender of stock. I'm just a farmer, plain and simple. I know of planting wheat and baling hay and animals going astray. I live in a complex world, but my faith guides me. I'm just a farmer, plain and simple. I am a man who works with God. I cannot succeed without his help. For you see, I'm just a farmer, plain and simple. vi TABLE OF CONTENTS Chapter Page Chapter I. INTRODUCTION……………………………………………………………………..….1 RESEARCH OBJECTIVES……………………………………………………………....4 Chapter II. REVIEW OF LITERATURE…………………………………………………………......5 Introduction……………………………………………………………………………5 Adipose Tissue……………………………………………………………………….10 Adipose Development………………………………………………………………..15 Adipose Related Genes……………………………………………………………....21 Fatty Acid Binding Protein 4……………………………………………………..21 Stearoyl-CoA Desaturase…………………………………………………………24 CD36……………………………………………………………………………...28 Fatty Acid Synthase...…………………………………………………………….31 Zilpaterol Hydrochloride…………………………………………………………….40 Adrenergic Receptors………………………………………………………………...43 β -Adrenergic Agonists……………………………………………………………….47 Effect of Zilpaterol Hydrochloride on Feed and Carcass Performance……………...55 Chapter III. EFFECTS OF DIFFERENT WINTER GROWING PROGRAMS ON GENE EXPRESSION IN DIFFERENT ADIPOSE TISSUE DEPOTS IN BEEF STEERS AT THE END OF THE GROWING PHASE……………………………..58 Abstract………………………………………………………………………………58 Introduction…………………………………………………………………………..59 Methods and Materials……………………………………………………………….61 Results………………………………………………………………………………..73 Discussion……………………………………………………………………………79 vii Chapter Page Chapter IV. EFFECTS OF DIFFERENT WINTER GROWING PROGRAMS ON GENE EXPRESSION IN DIFFERENT ADIPOSE TISSUE DEPOTS IN BEEF STEERS AT THE END OF THE FINISHING PHASE….………………………………………116 Abstract……………………………………………………………………………..116 Introduction…………………………………………………………………………118 Methods and Materials……………………………………………………………...119 Results………………………………………………………………………………132 Discussion…………………………………………………………………………..138 Chapter V. EFFECTS OF THE β− ADRENERGIC AGONIST, ZILPATEROL HYDROCHLORIDE, ON GENE EXPRESSION IN MUSCLE AND ADIPOSE TISSUE DEPOTS IN BEEF STEERS …………………………...…….......................174 Abstract…………………………………………………………………………….174 Introduction………………………………………………………………………...176 Methods and Materials……………………………………………………………..180 Results……………………………………………………………………………...189 Discussion………………………………………………………………………….193 ABBREVIATIONS………...…………………………………………………………..220 LITERATURE CITED………………………………………………………………...221 APPENDICES…………………………………………………………………………258 viii LIST OF TABLES Table Page 3.1. Sequences, accession number, and melting temperatures of the bovine specific primers for the selected target genes of interest. Accession number is from the National Center for Biotechnology Information (NCBI) available at http://www.ncbi.nlm.nih.gov/ ........................................................95 3.2. The number of differentially expressed up- and down-regulated genes in each of the functional categories in Initial and Wheat Pasture, Silage-fed, and Program-fed diets of the Growing Phase………………………………....96 3.3. Comparison of mRNA expression (i.m. relative to s.c.) detected in the cDNA microarray analysis to mRNA expression detected in RT-PCR. Fold change values represent i.m. adipose tissue expression relative to s.c. adipose tissue ………………………………………………………………...97 3.4. Principal Networks generated for Initial, Wheat Pasture, Silage-fed, and Program-fed diets of Growing Phase using Inginuity Pathway Analysis. Focus genes were overlaid into a globular molecular network deveolped from information contained in the IPA Knowledge Base. Networks of these focus genes were then algorithmically generated based on their connectivity…………………………………………………………….……102 3.5. Networks were generated for Initial using Inginuity Pathway Analysis. Genes differentially expressed only in Initial are highlighted in yellow. Genes highlighted in bold are focus genes. Genes in regular print were used to connect to other genes in the network………………………………103 3.6. Networks were generated for the Wheat Pasture diet of the Growing Phase using Inginuity Pathway Analysis. Genes differentially expressed only in Wheat Pasture diet of Growing Phase are