AN INVESTIGATION OF BOAR SPERM MOTILITY USING A NOVEL COMPUTERIZED ANALYSIS SYSTEM CLARE HOLT Thesis submitted to the University of London for the degree of Doctor of Philosophy InstituteZoology Department of Biology Regent's Park University College London London NW1 4RY University of London Gower Street London WG1E 6BT ProQuest Number: 10046139 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10046139 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT A novel computer-assisted sperm analysis (CASA) system was validated and used to investigate the value of sperm motility in predicting the fertility of semen samples. Appropriate Hobson Sperm Tracker set-up parameters were derived for the analysis of boar spermatozoa and techniques developed to facilitate CASA analysis of this species. The accuracy and precision of the system were evaluated and minimum sperm number and minimum velocity criteria were developed to ensure the quality of the analyses performed. Relationships between the CASA measurements were investigated and ejaculates compared before and after storage. Cluster analysis of the track data was performed to investigate whether sub-populations of sperm could be distinguished. Using this methodology, hyperactivated boar spermatozoa could be identified from the quantitative analysis of motility data. The motility characteristics of semen samples during incubation at 39*C were compared to data from two artificial insemination trials. Each employed a controlled sperm dose (1.5 x 10® spermatozoa) and standardized semen preparation and insemination protocols. The results revealed a significant relationship between sperm motility measurements and the fertility data (maximum correlation with average litter size P=0.007; maximum correlation with non-return rate P=0.00002). Overall, sperm velocity over a two hour incubation period proved to be the most informative motility characteristic. The quality of sperm motility is altered on interaction with the zona pellucida and objective analysis of this response might be useful for fertility estimation. Zonae ghosts were collected and solubilized; the addition of this preparation to boar sperm samples severely impaired motility and prevented analysis using the Hobson Sperm Tracker. Further experiments indicated that this was a palcium- dependent effect. This research supports the hypothesis that sperm movement characteristics are indicative of fertility. ACKNOWLEDGEMENTS I would like to acknowledge John Rymer and JSR Healthbred Limited, the Meat and Livestock Commission and the Institute of Zoology for supporting this studentship. In particular I wish to express my gratitude to Hugh Reed from JSR Healthbred for his help over this period. I would like to thank all the people at the Institute of Zoology who have helped me during my time there. Special mention must go to my supervisor Dr Bill Holt who has shown patience, kindness and wisdom throughout my time as a student. His advice and insight have been invaluable and I am grateful to him for the support he has given as both tutor and friend. I am indebted to Professor Harry Moore and Dr Alison Moore for sharing their thoughts on this research with me and for helping me understand molecular biology. I am grateful also to Professor Jeremey Hyams from the Department of Cell Biology, UCL, for acting as my external supervisor. There are a number of individuals to whom I am indebted and my thanks go the following: Bob Curnock and all at the Thorpe Willoughby Pig Breeding Centre for their assistance and hospitality on my visits; Dr John Aitken and Margaret Patterson from the Medical Research Council Reproductive Biology Unit, Edinburgh, for the gift of antisera to the porcine zona pellucida; Alireza Fazeli and Professor Ben Colenbrander from the Department of Herd Health and Reproduction, Utrecht University, for their help with the hemi-zona assay; Dr Bart Gadella and his wife Marjon for their kindness during my stay in Utrecht; Terry Dennett from the Institute of Zoology for providing slides and photographs for this thesis; Dr Paul Watson for his advice with all aspects of my research. I am very grateful to Geoff Hobson and Heather for their aid as I wandered through the minefields of engineering and computer systems. The unflagging enthusiasm they displayed whilst nurturing the Hobson Sperm Tracker was always encouraging and I wish them every success in this venture. I wish to thank all those who have made my time at the Institute a happy one, especially Dave C, Pippa, and Stella. Two people in particular have shared this experience with me - my thanks go to Viv Marshall and Linda Penfold, for providing encouragement, sympathy and humour whenever these were needed. Special thanks go to all my 'non-scientists' friends who have witnessed the ups and downs over the last few months and shown endless support throughout, (and especially to Ros for her unique blend of gin and sympathy!). I am also grateful to my family for the encouragement and joy that they continually provide. My warmest thanks are reserved for Rob who has been at my side throughout the last two years. His patience, generosity of spirit and sense of humour have helped me to this goal, and kept me smiling along the way. To my father TABLE OF CONTENTS ABSTRACT .................................................................................................. 2 ACKNOWLEDGEMENTS........................................................................................ 3 TABLE OF CONTENTS........................................................................................... 6 LIST OF TABLES .................................................................................................. 12 LIST OF FIGURES ................................................................................................ 13 LIST OF ABBREVIATIONS ................................................................................. 15 CHAPTER 1. - INTRODUCTION AND LITERATURE R EVIEW .................... 16 1.0 INTRODUCTION......................................................................................... 17 1.1 MAMMALIAN SPERMATOZOA - STRUCTURE AND FUNCTION . 18 1.1.1 Introduction...................................................................................... 18 1.1.2 The sperm flagellum ....................................................................... 18 1.1.3 Generation of sperm motility ........................................................ 21 1.1.4 Biochemical control of sperm motility.......................................... 22 1.1.5 Physical effects on sperm m otility............................................... 23 1.1.6 Hyperactivation .............................................................................. 24 1.2. THE PASSAGE OF SPERM IN THE FEMALE TRACT........................... 28 1.2.1 Insemination ................................................................................... 28 1.2.2 Sperm reservoir formation ........................................................... 28 1.2.3 Fertilization ...................................................................................... 29 1.2.4 Thé zona pellucida.......................................................................... 30 1.2.5 Penetration of the zona pellucida ................. 31 1.3 COMPUTER ASSISTED SEMEN ANALYSIS........................................ 31 1.3.1 The principles of CASA ................................ 32 1.3.2 Motility parameters recorded during C A S A ................................ 34 1.3.3 Advantages and disadvantages of C A S A ................................... 36 1.3.4 Clinical significance of CASA ...................................................... 37 1.3.5 CASA of domestic sp ecies ........................................................... 38 1.3.6 CASA of boar sperm ..................................................................... 39 1.4 PIG ARTIFICIAL INSEMINATION............................................ 40 1.4.1 Introduction...................................................................................... 40 1.4.2 The economics of AI ..................................................................... 40 1.4.3 Semen assessment techniques in pig AI ................................... 41 CHAPTER 2 - VALIDATION OF THE HOBSON SPERM TRACKER FOR USE WITH BOAR SPERM.................................................................... 43 2.0 INTRODUCTION......................................................................................... 44 2.0.1 CASA software and hardware ...................................................... 44 2.0.2 The Hobson Sperm T ra c k e r ........................................................ 45 2.1 SAMPLE PREPARATION........................................................................... 46 2.1.1 Introduction ................................................................................... 46 2.1.2 Materials and methods .................................................................