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Cryopreservation of Equine Semen with a Mechanical Control Rate Freezer

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Cryopreservation of Equine Semen with a Mechanical Control Rate Freezer CRYOPRESERVATION OF EQUINE SEMEN WITH A MECHANICAL CONTROL RATE FREEZER By Sossi Marianne Iacovides, B.S. A THESIS IN ANIMAL SCIENCE Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE ApProved Dr. Samuel Prien CHairPerson Dr. Samuel Jackson Dr. Leslie Thompson Dr. Mark Sheridan Dean of the Graduate School December, 2014 Copyright 2014, Sossi Marianne Iacovides, B.S. Texas TecH University, Sossi M. Iacovides, December 2014 ACKNOWLEDGMENTS ‘It’s hard to soar with eagles, if you won’t jump off the cliff.’ At the beginning of my graduate school process, I had the choice to start somewhere fresh and new. While it may have been the right move for some, it was not for me; I knew that I wasn’t finished with here. The Department of Animal & Food Sciences has become home, from the abundance of moral, ethical and personal support; it has been a blessing and privilege to spend the past 4 years here. Dr. Glenn Blodgett, you believed in my research, and that means the world to me. Without the access to the 6666 Ranch, and your indisPensible personnel, this project would have never gained flight. Mallory Canaday, the true “Swimmie Specialist,” you were an indispensable resource for me and I have learned more from you then I could have ever imagined. The lab personnel: my countless questions never went unanswered. I hope that you’ll walk the hallways and hear my snaughles forever more. The minions, my fellow graduate students, from the countless nights of studying, to the late night research – I could not have hoped for a better group to have survived these last two years with! Dr. Lindsey Penrose, where do I begin? You have been a foundation in my life since I began this journey. Your never-ending words of encouragement; the ideas we bounced back and forth; having been your chief minion in training…. You managed to keep this hot-blooded Greek girl off the edge! Can I finally have a (small) crown, Turtle Queen?! ii Texas TecH University, Sossi M. Iacovides, December 2014 Dr. Sam Jackson, starting with undergrad transfer orientation day, to sheep production and throughout my graduate career, you have always been available for help and encouragement. Thanks General. Dr. Leslie Thompson: I knew you would be a great asset after working with you for the IDEAL camps. Your crucial guidance in my exploration of your Alma Mater in Florida was invaluable, and I cannot thank you enough. The Captain, Dr. Sam Prien: I don’t think it’s possible to sum up what you mean to me in either a single word or even a few run on sentences. You have been so much more than just my mentor, confidant, and rock throughout my undergraduate and graduate careers. From presenting my research at different venues, to the times I have been at my wits end, I could not have made it this far without your endless understanding and support. I know my neon colors tend to get too bright and the shiny things too, but you put up with the true dark cutter and even managed to get a few songs out of it! You’re a phenomenal person, teacher and friend. Both you and your promise, “You will always have a home here,” will forever have a place in my mind and heart. Girls, I don’t know how we all survived the past few years, but we definitely kept each other sane. The ranch, if it wasn’t for the push you gave me years ago, I never would have made it this far following livestock as my passion. Mom, Mougs (and Chimmie) the late night phones calls; the crying over failed trials; the rejoicing when I finally finished. While I may sound like I’m speaking Chinese, you know the blood, sweat and tears that I poured into this. Thanks for iii Texas TecH University, Sossi M. Iacovides, December 2014 putting up with my OCD, dark cutting ways and standing by my decisions to keep at it. A self-proclaimed perpetual student, without all of your help, I could not have lasted this long. But we did it! So the question is… Are y’all ready for the next 4 years?! “You know what you gotta do when life gets you down? Just keep swimming, just keep swimming, just keep swimming, swimming, swimming!” - Dory, Finding Nemo, Disney® (add a snaughle or two for good measure!) iv Texas TecH University, Sossi M. Iacovides, December 2014 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. ii LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES ........................................................................................................ vii ABSTRACT ............................................................................................................... viii CHAPTERS I. LITERATURE REVIEW .................................................................................................. 1 HISTORY OF ARTIFICIAL INSEMINATION ...................................................................... 1 HISTORY OF CRYOPRESERVATION ............................................................................. 4 MULTI SPECIES INDUSTRY TRANSFORMATION ............................................................. 7 GENERAL CONCEPTS OF EQUINE SEMEN .................................................................. 11 TYPES OF EXTENDERS ........................................................................................... 19 CRYOPROTECTANT CLASSES ................................................................................... 25 FREEZING TECHNIQUES ........................................................................................ 34 STALLION SPERM PHYSIOLOGY & CRYOPRESERVATION ................................................ 37 THAWING: ISSUES & IMPROVEMENTS TO POST-THAW QUALITY .................................... 40 GENERAL DISCUSSION OF SPERM LOSS .................................................................... 42 FUTURE OF SEMEN CRYOPRESERVATION .................................................................. 48 II. CRYOPRESERVATION OF EQUINE SEMEN USING A MECHANICAL CONTROL RATE FREEZER ..... 50 INTRODUCTION ................................................................................................... 50 MATERIALS & METHODS ...................................................................................... 52 SEMEN COLLECTION & PREPARATION ...................................................................... 52 SEMEN FREEZING ................................................................................................ 53 SEMEN THAWING ................................................................................................ 54 MORPHOLOGY & ACROSOMES ............................................................................... 57 STATISTICAL ANALYSIS .......................................................................................... 58 RESULTS ............................................................................................................ 58 DISCUSSION ....................................................................................................... 81 III. CONCLUSION ....................................................................................................... 83 LITERATURE CITED ..................................................................................................... 86 APPENDIX ................................................................................................................ 96 v Texas TecH University, Sossi M. Iacovides, December 2014 LIST OF TABLES 1. COMPARISON OF INTERMEDIATE SPEED PARAMETERS BY TREATMENT & TIME ..................... 64 2. COMPARISON OF AVERAGE MOVEMENT CLASSIFICATIONS BY TREATMENT & TIME ............... 66 3. QUANTITATIVE COMPARISON OF TREATMENTS ............................................................. 80 4. PERCENT RETENTION OF CONCENTRATION, MOTILITY & RAPID CELLS ................................. 96 5. PERCENT RETENTION OF MORPHOLOGICAL VALUES ....................................................... 97 6. PERCENT RETENTION OF ACROSOME VALUES ................................................................ 97 vi Texas TecH University, Sossi M. Iacovides, December 2014 LIST OF FIGURES 1. COMPARISON OF CONCENTRATIONS BY TREATMENT & TIME ........................................... 60 2. COMPARISON OF MOTILITIES BY TREATMENT & TIME .................................................... 61 3. COMPARISON OF RAPID CELL PROGRESSION BY TREATMENT & TIME ................................. 62 4. COMPARISON OF NORMAL MORPHOLOGY BY TREATMENT & TIME .................................... 68 5. COMPARISON OF ABNORMAL HEAD MORPHOLOGY BY TREATMENT & TIME ........................ 70 6. COMPARISON OF ABNORMAL MIDPIECE MORPHOLOGY BY TREATMENT & TIME ................... 71 7. COMPARISON OF OTHER ABNORMAL TAIL MORPHOLOGY BY TREATMENT & TIME ................ 73 8. COMPARISON OF CURLED ABNORMAL TAIL MORPHOLOGY BY TREATMENT & TIME ............... 74 9. COMPARISON OF INTACT ACROSOMES BY TREATMENT & TIME ........................................ 76 10. COMPARISON OF PARTIALLY ACROSOMES BY TREATMENT & TIME .................................. 77 11. COMPARISON OF NON-INTACT ACROSOMES BY TREATMENT & TIME .............................. 78 vii Texas TecH University, Sossi M. Iacovides, December 2014 ABSTRACT With the evolving use of assisted reproductive technologies (ART) in the equine industry, there is a need for a cost effective, field method of preparing stallion semen for cryopreservation.
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