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UNIVERSITY of CALGARY a Comparison Of UNIVERSITY OF CALGARY A Comparison of Low - Versus High - Fertility Holstein Bulls For Identification of Fertility Markers by Habib Allah Shojaei Saadi A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF MEDICAL SCIENCE DEPARTMENT OF MEDICAL SCIENCES CALGARY, ALBERTA DECEMBER, 2011 © Habib Allah Shojaei Saadi 2011 Library and Archives Bibliothdque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'fedition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre r6ttrence ISBN: 978-0-494-83424-4 Our file Notre inference ISBN: 978-0-494-83424-4 NOTICE: AVIS: The author has granted a non­ L'auteur a accords une licence non exclusive exclusive license allowing Library and permettant d la Bibliothdque et Archives Archives Canada to reproduce, Canada de reproduce, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lntemet, prdter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, d des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, 6lectronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propri6t6 du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette thdse. Ni thesis. Neither the thesis nor la thdse ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent dtre imprimis ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conform6ment & la loi canadienne sur la Privacy Act some supporting forms protection de la vie priv6e, quelques may have been removed from this formulaires secondares ont 6t6 enlev6s de thesis. cette th&se. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada UNIVERSITY OF CALGARY FACULTY OF GRADUATE STUDIES The undersigned certify that they have read, and recommend to the Faculty of Graduate Studies for acceptance, a thesis entitled "A comparison of low- versus high-fertility Holstein bulls for identification of fertility markers" submitted by Habib Allah Shojaei Saadi in partial fulfilment of the requirements of the degree of Master of Medical Sciences. Supervisor, Dr. Jacob C Thundathil. Department of Medical Sciences Dr. Frans van der Hoorn. Department of Biochemistry and Molecular Biology Dr. John Kastelic. Department of Medical Sciences Dr. Leluo Guan. Department of Medical Science Dr. Robert McCorkell. Faculty of Veterinary Medicine Date ii Abstract The objectives were to compare low versus high-fertile bulls for testicular physical characteristics, sperm characteristics, expression patterns of testicular genes, and sperm protein profiles for identification of fertility markers in Holstein bulls used for artificial insemination (AI). Ten Holstein bulls (4-5 y old) were classified as either low- fertility (LF) or high-fertility (HF; n=5 each), based on adjusted 56-d non-return rates (NRR; range 48.0 to 74.0%). Testicular physical characteristics were not significantly different between the two groups. Several indices of sperm motion (based on computer- assisted sperm analysis) at post-thaw and post-swim up were correlated with NRR. Thirty-one protein spots differed between total protein extracted from LF and HF sperm. However, ASB5 was the only common gene differentially expressed between LF and HF bulls (P < 0.05). In conclusion, fertility of mature Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but evaluation of sperm characteristics and genomic and proteomic approaches have considerable promise to predict fertility potential in Holstein bulls. iii Acknowledgements Foremost, I would like to express my sincere gratitude to my supervisor Dr. Jacob Thundathil for his patience, motivation, enthusiasm, and assistance. His guidance helped me throughout the research and writing of this thesis. This is a great opportunity to appreciate Dr. Frans van der Hoorn for his support, encouragement and valuable advice in scientific discussions. I gratefully thank Dr. John Kastelic for all of his constructive criticism during the period of this study and time commitment for critically reading this thesis and my manuscripts. My sincere thanks to Dr. Leluo Guan for sharing her expertise on microarray analysis and providing me a great opportunity to work in her laboratory at the University of Alberta. I take the opportunity to acknowledge Natural Sciences and Engineering Research Council of Canada (NSERC), Agriculture and Food Council, Alberta Livestock Industry Development Fund, Semex Alliance, L'Alliance Boviteq (LAB) inc. and Westgen and University of Calgary for financial assistance and support for this study. I also thank the University of Calgary, faculty of Veterinary Medicine for the UCVM graduate student entrance award and Faculty of Medicine, Department of Medical Sciences, for travel awards. Special thanks and gratitude to Dr. Esmail Behboodi for his advice, comments, insight and inspiration throughout my work and life. I also acknowledge Mr. Doug Nickel for his help and technical support. Thanks to the past and present members of Thundathil lab, who provided a very friendly lab environment and during the period of this project: Drs. Ajitkumar Menon, Sulochana Krishna Kumar, Gayatheri Rajamanickam and Ms. Alysha Dance iv Many thanks to Dr. Amin Rezvanfar, Dr. Sadegh Karimi, Dr. Adel Pezeshki, Dr. Sepideh Abbasi & Dr. Mustafa Gozlan, Yasha and Majid for their moral support. Last but not the least, I thank Mahboubeh, Mona & Hamed being supportive and caring siblings. v Dedication This thesis is dedicated to my parents who have supported me all the way since the beginning of my studies and offered me unconditional love and support. Also, this thesis is dedicated to my wife who has been a great source of motivation and inspiration. vi Table of Contents Approval Page ii Abstract iii Acknowledgements i v Dedication vi Table of Contents vii List of Tables ix List of Figures and Dlustrations x CHAPTER ONE: INTRODUCTION 1 1.1 Background 1 1.2 Relevant literature review 1 1.2.1 Testicular physical characteristics 1 1.2.2 Sperm motion characteristics 4 1.2.3 Sperm plasma membrane integrity 8 1.2.4 Sperm selection through sodium hyaluronate swim-up medium 10 1.2.5 Transcriptomics 11 1.2.6 Proteomics 13 1.3 Objectives and hypothesis 18 CHAPTER TWO: MATERIALS AND METHODS 19 2.1 Testicular physical characteristics 19 2.1.1 Selection of bulls 19 2.1.2 Evaluation of testicular physical characteristics 20 2.2 Evaluation of sperm characteristics 21 2.2.1 Evaluation of sperm motion characteristics 21 2.2.2 Evaluation of sperm plasma membrane viability 22 2.2.3 Evaluation of sperm characteristics after swim-up 23 2.2.3.1 Swim-up procedure 23 2.2.3.2 Sperm motion characteristics 24 2.2.3.3 Determination of sperm concentration 24 2.2.3.4 Evaluation of sperm viability 25 2.3 Transcriptome analysis 25 2.3.1 Microarray hybridization and data analysis 25 2.3.1.1 Preparation of slides 27 2.3.1.2 Reverse transcription 27 2.3.2 Bioinformatics 29 2.3.3 Q-PCR analysis 30 2.3.4 Evaluation of the ASB5 protein expression in testis 31 2.4 Proteomics 32 2.4.1 Semen samples 32 2.4.2 Two-dimensional gel electrophoresis 32 2.4.2.1 Extraction and quantification of total proteins 32 2.4.2.2 Rehydration of IPG strips 33 2.4.2.3 Isoelectric focusing 33 2.4.2.4 Second dimension 34 vii 2.4.2.5 Staining, imaging and gel analysis 34 2.4.2.6 Determination of the pi and molecular mass of DE proteins 36 2.5 Statistical analyses 36 CHAPTER THREE: RESULTS 38 3.1.1 Testicular physical characteristics 38 3.1.2 Testicular echotexture 38 3.1.3 Scrotal thermography 38 3.2 Sperm characteristics 39 3.2.1 Sperm motion characteristics 39 3.2.2 Sperm plasma membrane viability 39 3.2.3 Sperm concentration 40 3.3 Transcriptome analysis using microarray 48 3.3.1 Bioinformatics: 56 3.3.2 ASB5 protein expression in testes: 67 3.4 Proteomics 68 3.4.1 Sperm protein profile 68 CHAPTER FOUR: DISCUSSION 73 REFERENCES 87 viii List of Tables Table 1. Fertility data for low- versus high-fertility Holstein bulls used in the present study 20 Table 2. Motion characteristics (mean ± SD) of frozen-thawed sperm from high- versus low-fertility Holstein bulls 41 Table 3. Mean (± SD) sperm motion characteristics after swim-up in high- versus low-fertility Holstein bulls 42 Table 4. Correlation coefficients between sperm motility end points and non-return rate (NRR) in Holstein bulls 43 Table 5. Viability of frozen-thawed sperm and its correlation with NRR in high- versus low-fertility Holstein bulls 44 Table 6. The number of differentially expressed genes with 2-fold and higher changes between high- and low-fertility bulls 48 Table 7. Two genes differentially expressed (DE) in common between all low- and high-fertility bulls 55 Table 8. Three experiments calculating ACt. for ASB5 for all HF and LF bulls 60 Table 9. Mean (± SD) ACt values for ASB5 in HF and LF bulls based on 3 replicates....61 Table 10. Mean(± SD) fold change for ASB5 gene expression in HF vs. LF bulls 61 Table 11. Differentially expressed sperm proteins between high- versus low-fertility Holstein bulls 70 ix List of Figures and Illustrations Figure 1.
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