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Comparison of Epididymal and Ejaculated Sperm Collected from the Same Holstein Bulls Michael A Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2012 Comparison of epididymal and ejaculated sperm collected from the same Holstein bulls Michael A. Stout Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Animal Sciences Commons Recommended Citation Stout, Michael A., "Comparison of epididymal and ejaculated sperm collected from the same Holstein bulls" (2012). LSU Doctoral Dissertations. 1160. https://digitalcommons.lsu.edu/gradschool_dissertations/1160 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. COMPARISON OF EPIDIDYMAL AND EJACULATED SPERM COLLECTED FROM THE SAME HOLSTEIN BULLS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical Collage in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The School of Animal Sciences by Michael A. Stout B.S., University of Louisiana Lafayette, 2004 M.S., Louisiana State University, 2006 August 2012 ACKNOWLEDGMENTS I would like to thank my major professor and advisor, Dr. Robert A. Godke, Boyd Professor for giving me the great opportunity to join the Reproductive Physiology Program. I am deeply honored to have had the privilege to work with a scientist of your stature and prestige. Thank you for guiding me through this journey and encouraging me to perform at the highest level. Thank you for your unending support, patience, guidance, encouragement and friendship that you have bestowed upon me, for this I am grateful. To Dr. Kenneth Bondioli, I would like to extend special thanks for always lending his expert knowledge of reproductive physiology and laboratory practice. Thank you for always taking the time to listen to my ideas and giving advice and perspectives to help my research. It has been a great honor to have worked, watched and learned from you. Thank you to Dr. Glen Gentry, for freely giving of your advice and encouragement to help me along this journey. Thank you for always taking the time to discuss my research and suggesting topics that I had not taken into account and were important for my research. It has been a privilege to have worked with you. To Dr. Bruce Eilts, thank you for your patience, guidance and support through the completion of this dissertation. Thank you for your participation in our laboratory research meetings, and for making yourself available to discuss my research. I would like to thank Dr. John Battista, for his guidance and patience through the completion of this dissertation. Thank you to Dr. Ramachandra Devireddy, for your collaboration and assistance with the differential scanning calorimeter and determining the optimal cooling rate, I consider it an honor to have worked with you. To Dr. Gary Hay, thank you for believing in me and for your financial assistance for without this, I would not have been able to attain this lifelong goal. Thank you to Dr. Charles Hutchison, for your effort in locating quality bulls for use in my research. To Dr. John Lynn, thank you for your guidance in determining fluorescent probes for use in my experiments, it has been a privilege to have worked with you. ii Thank you to Dr. Laura Gentry and Dr. Jerome La Peyre, for your assistance and for allowing me to utilize your laboratories to conduct my testosterone and cholesterol assays. Thank you to Dr. Bruce Olcott, for performing all surgical procedures on our research animals. Mr. James Chenevert, thank you for always being willing to participate in my research, for allowing me to utilize your resources and making Genex such an open and enabling environment to conduct research. I owe you a debt of gratitude for your encouragement, advice and utmost your friendship. I have learned so much from your expertise, thank you. Mr. Adam Foster and Mr. Ulysses Wright, thank you for your assistance with handling bulls and taking samples during those early mornings and late afternoons. Ms. Sally Turner, thank you for always being there when I needed you and for always having a warm, welcoming and open heart. My fellow graduate students, past and present, thank you for all of your assistance during this research. I know that I cannot adequately describe the level of support that each of you have provided. Special thanks to Jesse Saenz, for your friendship and always being there when I need assistance. To my wife Christine and son Everett, thank you for your support and standing with me through this journey. To my parents, Linda and William Stout, thank you for your unending support and guidance and for teaching me to always give my best, without you, none of this would have been possible. I would like to give a special thank you to my mom, who has been there from day one and has never given up on me, without you, I would have never made it this far. Mrs. Pam Cheek and Mrs. Bridget Strother, thank you for stepping up to the bat for me when I needed it. I would not be here without your help and guidance. I would also like to thank the late Dr. Lora Lana Goodeaux, for the foundation from which my interest in assisted reproduction originated. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii LIST OF TABLES vii LIST OF FIGURE viii ABBREVIATIONS USED IN DISSERTATION xiii ABSTRACT xiv CHAPTER I INTRODUCTION 1 CHAPTER II LITERATURE REVIEW 5 Function of the Testis 5 Hormone Production 5 Sperm Production 8 Function of the Epididymis 9 Maturation 10 Transport and Storage 10 Protection 11 Cholesterol regulation 12 Collection of sperm 13 Ejaculated 13 Epididymal 13 Ejaculated and Epididymal Sperm Collection 14 Seminal Plasma 15 Bovine Seminal Plasma Proteins 17 Seminal Plasma Lipid Concentration 18 Decapacitation Factors 18 Cryopreservation of Sperm 18 Cholesterol 20 Cryopreservation of Epididymal Sperm 23 Capacitation of Sperm 28 Cryopreservation Induced Capacitation 33 Epididymal Sperm in Assisted Reproductive Techniques 34 Artificial Insemination (AI) 34 In Vitro Fertilization (IVF) 36 Factors Associated with Reproduction 43 Factors Associated with Embryonic Development 43 Factors Associated with Sperm Function 46 Preimplantation Factor 48 CHAPTER III MEMBRANE PERMEABILITY DURING CRYOPRESERVATION OF BOVINE EJACULATED AND EPIDIDYMAL SPERM FROM THE SAME HOLSTEIN BULLS 50 Introduction 50 Materials and Methods 51 Experimental Design 51 Experimental Procedure 52 Statistical Analysis 57 Results 59 iv Discussion 66 CHAPTER IV INFLUENCE OF CASTRATION-INDUCED TESTOSTERONE DEFICIENCY ON CIRCULATING PLASMA CHOLESTEROL LEVELS IN HOLSTEIN BULLS 71 Introduction 71 Materials and Methods 72 Experimental Design 72 Experimental Procedure 73 Statistical Analysis 74 Results 80 Discussion 80 CHAPTER V CAPACITATION AND ACROSOME REACTION OF EJACULATED AND EPIDIDYMAL SPERM POST-THAW AND THE EFFECTS OF HEPARIN DURING IN VITRO CULTURE 85 Introduction 85 Materials and Methods 87 Experimental Design 87 Experimental Procedure 88 Statistical Analysis 93 Results 93 Discussion 113 CHAPTER VI EFFECTS OF PREIMPLANTATION FACTOR (PIF) ON BOVINE CRYOPRESERVED SPERM 117 Introduction 117 Materials and Methods 119 Experimental Design 119 Experimental Procedure 120 Statistical Analysis 123 Results 123 Discussion 137 CHAPTER VII THE EFFECTS OF EJACULATED AND EPIDIDYMAL SPERM FROM THE SAME HOLSTEIN BULLS ON IN VITRO FERTILIZATION RATES AND SUBSEQUENT EMBRYO DEVELOPMENT 142 Introduction 142 Materials and Methods 144 Experimental Design 144 Experimental Procedure 145 Statistical Analysis 148 Results 148 Discussion 149 CHAPTER VIII SUMMARY AND CONCLUSION 161 LITERATURE CITED 165 APPENDIX I 193 APPENDIX II 194 v APPENDIX III 195 APPENDIX IV 196 APPENDIX V 197 APPENDIX VI 198 APPENDIX VII 199 APPENDIX VIII 201 VITA 203 vi LIST OF TABLES 3.1 Bull conformation values collected from mature Holstein bulls 60 3.2 Ejaculated and epididymal sperm collection values from the same mature Holstein bulls 61 3.3 Best fit water transport parameters for the volumetric shrinkage response of ejaculated Holstein sperm without CPA and with glycerol, at a cooling response of 20⁰C/minute 62 3.4 Best fit water transport parameters for the volumetric shrinkage response of ejaculated and epididymal Holstein sperm without CPA and with glycerol, at a cooling response of 20⁰C/minute 63 4.1 Mean plasma testosterone and cholesterol concentration before and after bilateral castration of mature Holstein bulls 75 5.1 Viability, acrosomal integrity and capacitation of cryopreserved ejaculated and epididymal sperm collected from the same four Holstein bulls exposed to varying concentration of heparin during 6 hours of in vitro culture 95 6.1 The effects of preimplantation factor (PIF) and heparin on cryopreserved ejaculated bovine sperm during in vitro culture 124 6.2 Effect of adding preimplantation factor (PIF) and or heparin (Hep) during bovine in vitro fertilization, on embryo cleavage and development 125 7.1 Fertilization rate following IVF with ejaculated or epididymal sperm from the same Holstein bulls with or without heparin 151 7.2 Cleavage and embryo development following IVF with ejaculated or epididymal sperm with or without heparin 153 vii LIST OF FIGURES 3.1 Epididymal sperm collection. (A) An isolated epididymis, the vas deferen is cannulated with the tip of a modified syringe. (B) Epididymal sperm being collected by retrograde flush of the cauda epididymis 58 3.2 Comparative volumetric shrinkage response of ejaculated bovine sperm from the four bulls used in this study, shown as open circles (bull A & B) and open squares (bull C & D). DSC technique was used to obtain the volumetric response, at a cooling rate of 20⁰C/minute. Water transport data for bovine sperm with no CPAs (Figure, A) and pre-loaded with glycerol (Figure, B) are depicted.
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