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Identification of Sperm Parameters and Gene Variants Influencing Boar Fertility

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Identification of Sperm Parameters and Gene Variants Influencing Boar Fertility Philosophiae Doctor (PhD), Thesis 2018:37 (PhD), Doctor Philosophiae Norwegian University of Life Sciences Faculty of Biosciences Philosophiae Doctor (PhD) Thesis 2018:37 Nina Hårdnes Tremoen Nina Hårdnes Identification of sperm parameters and gene variants influencing boar fertility Identifisering av sædparametere og gen- varianter som påvirker fertilitet hos råne Nina Hårdnes Tremoen Identification of sperm parameters and gene variants influencing boar fertility Identifisering av sædparametere og gen-varianter som påvirker fertilitet hos råne Philosophiae Doctor (PhD) Thesis Nina Hårdnes Tremoen Norwegian University of Life Sciences Faculty of Biosciences Department of Animal and Aquacultural Sciences Ås 2018 Thesis number 2018:37 ISSN 1894-6402 ISBN 978-82-575-1509-6 Acknowledgements This study was carried out at Department of Biotechnology, Inland Norway University of Applied Sciences and partly at the Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences. The work was funded by the Norwegian Pig Breeder’s Association (Norsvin) and the Research Council of Norway (Grant number 207568). First, I would like to express my gratitude to my three supervisors for giving me this opportunity, and for your support and guidance throughout these years. Frøydis Deinboll Myromslien, I would never have managed this without you! Your encouragement and advises have been priceless, and our discussions, both professional and personal, have meant so much for me. Dag Inge Våge, your expertise and patience have given me the opportunity of getting new knowledge and to see the bigger picture, and Eli Grindflek, your knowledge and enthusiasm have inspired me. I would also like to thank the members of the project group, Ann Helen Gaustad, Maren von Son and Ina Ranberg-Andersen. Although you have not been official supervisors, you have spent precious time giving me advice and guidance, and I really appreciate all of you. I wish to thank all my colleagues at INN and Biohus for welcoming me and for giving me such a great working environment during this period. I would especially thank Kirsten Frydenlund, Else-Berit Stenseth and Teklu T. Zeremichael for technical support in the laboratory. Also, a huge thanks for fellow PhD students, Fride Berg Standerholen and Relindis Njah, for discussions, sharing experiences and frustrations. Finally I would like to thank my family and friends for standing by me in these years, and for listening to all the strange things I do at work. Especially my husband, Anders, and our three beautiful children, Amalie, Teodor and Olava, - you are my world! Table of contents Abstract ................................................................................................................................ 3 Sammendrag ........................................................................................................................ 5 List of abbreviations ............................................................................................................ 7 List of papers........................................................................................................................ 8 1. Introduction ...................................................................................................................... 9 1.1 Reproduction physiology in pigs ............................................................................. 10 1.1.1 Boar reproductive system ................................................................................. 10 1.1.2 Spermatogenesis and the maturation of the sperm cells ................................... 11 1.1.3 The spermatozoa ............................................................................................... 13 1.1.4 Sow reproductive system .................................................................................. 16 1.2 Sperm transport in the female reproductive organ and fertilization ........................ 18 1.2.1 The sperm reservoir and capacitation process .................................................. 19 1.2.2 Fertilization ....................................................................................................... 20 1.3 Evaluation of fertility ............................................................................................... 21 1.4 Evaluation of semen quality..................................................................................... 22 1.4.1 Evaluation of sperm motility parameters .......................................................... 23 1.4.2 Evaluation of ATP levels in semen ................................................................... 24 1.4.3 Flow cytometric evaluation of sperm DNA integrity ....................................... 25 1.5 Fertility genomics .................................................................................................... 26 1.5.1 Candidate genes ................................................................................................ 27 1.5.2 Identifying gene variants affecting fertility ...................................................... 29 1.6 Pig breeding in Norway ........................................................................................... 29 1.6.1 Norwegian Landrace ......................................................................................... 30 1.6.2 Norwegian Duroc .............................................................................................. 31 2. Objectives ...................................................................................................................... 32 3. Results: summary of individual papers .......................................................................... 33 Paper I: ........................................................................................................................... 33 Paper II: .......................................................................................................................... 34 Paper III: ........................................................................................................................ 35 Paper IV: ........................................................................................................................ 36 4. Discussion ...................................................................................................................... 37 4.1 Semen quality parameters and boar fertility ............................................................ 37 4.1.1 Sperm motility parameters ................................................................................ 37 4.1.2 Sperm DNA integrity ........................................................................................ 40 4.2 Gene variants and boar fertility................................................................................ 41 4.2.1 Assessment of underlying genetics ................................................................... 41 4.3 Breed differences ..................................................................................................... 44 4.4 Storage capacity ....................................................................................................... 46 5. Concluding remarks ....................................................................................................... 47 7. References ...................................................................................................................... 48 Abstract Boar fertility has a major impact on overall pig reproductive efficiency, and good semen quality is essential for successful fertilization and proper embryo development. Thus finding semen characteristics that can predict fertility traits like pregnancy rate and total number of piglets born (TNB) is of great importance. However, the results of pregnancy rate and TNB are not available until the boars have been used in production for a period, and determination of phenotypes with effects on fertility at an earlier stage in the boars’ lives would be beneficial for estimating breeding values. To find methods to evaluate the boar’s sperm quality in relation to TNB and thus the boar’s fertility is of great importance also for the semen produced for sale. Identifying gene variants affecting these traits is of equal importance. We found significant breed differences in the motility characters comparing ejaculates from Norwegian Landrace (NL) and Norwegian Duroc (ND). The percentage of hyperactivated sperm cells increased significantly upon storage in NL. In ND a larger portion of sperm cells with a hyperactive swimming pattern were detected at day 0, and the size of this population decreased upon storage. A significant decrease in the ATP level (p<0.0001) was also found in both breeds during storage. The motility characters linearity and wobble showed an effect on TNB in NL, at the day of collection and after storage, respectively. For ND, the percentage of motile cells, curvilinear velocity and amplitude of lateral lateral head displacement at the day of collection and linearity after storage showed an effect on TNB. A significant negative effect on TNB was found for boars with contrasting DNA fragmentation index (DFI). This might explain some of the variation in TNB caused by the sperm quality of the individual boars, although the effect was moderate. Transcriptome profiling by RNA sequencing (RNAseq) of testis tissue from NL and ND boars showed that 308 and 374 genes displayed significant different expression between
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