Associations between Canine Male Reproductive Parameters and Serum Vitamin D and Prolactin Concentrations by Adria Julianne Kukk A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Veterinary Science in Population Medicine Guelph, Ontario, Canada ¤ Adria Julianne Kukk, December, 2011 ABSTRACT ASSOCIATIONS BETWEEN CANINE MALE REPRODUCTIVE PARAMETERS AND SERUM VITAMIN D AND PROLACTIN CONCENTRATIONS Adria Julianne Kukk Advisor: University of Guelph, 2011 Professor C.J. Gartley Maintaining reproductive health and diagnosing and treating conditions of infertility in stud dogs is important in canine theriogenology. However, there is still a great deal to be learned about reproductive physiology and factors that affect reproductive organs and semen quality in dogs. This thesis is an investigation of two factors in the male dog; serum 25-hydroxy Vitamin D (25OHVD) and prolactin (PRL) concentrations, and their possible associations with benign prostatic hyperplasia (BPH), prostate volume and/or sperm morphology and motility characteristics. 28 (Vitamin D Study) and 29 (28 plus one for the Prolactin study) client dogs of various breeds from the Ontario Veterinary College and Graham Animal Hospital in Southwestern Ontario, Canada were enrolled in the study from March to December 2009. Of these dogs 22 were successfully collected for semen. BPH was diagnosed using prostate volume measured by ultrasound, as well as clinical signs including blood in the ejaculate. Semen analysis was performed using manual microscopic techniques for morphology and computer assisted sperm analysis equipment for motility. In the vitamin D study, no associations were found between BPH and serum 25OHVD concentrations. In contrast, several sperm motility (motility, progressive motility, beat cross frequency (BCF), distance average path (DAP), curvilinear distance (DCL), linear distance (DSL), average path velocity (VAP), curvilinear velocity (VCL) and straight line velocity (VSL), amplitude lateral head displacement (ALH) and average orientation change (AOC)) and morphology characteristics (percentage normal sperm, head defects and detached heads) had desirable outcomes with 25OHVD concentrations between 120-180 nmol/l. Using bivariable analysis, positive associations were observed with 25OHVD and some semen quality characteristics from 4 to 8 years of age (motility, progressive motility, BCF, DCL, VCL, ALH, AOC) and at transformed prostate volumes smaller than or equal to 4.5 (motility, progressive motility, DCL, VCL, and normal morphology) while negative associations of these semen parameters were found at ages greater than 8 years and transformed prostate volumes greater than or equal to 5.5. Head defects were negatively associated with 25OHVD. Vitamin D may have an impact on spermatogenesis and normal sperm physiology that warrants further research. The prolactin study showed no statistically significant associations between serum PRL and BPH and serum PRL and sperm motility characteristics. However, two sperm morphology characteristics (percentage proximal droplets and percentage midpiece defects) had significant negative associations with PRL concentrations. Age interaction with PRL was also a factor in the percentage of midpiece defects with desirable outcomes associated at 4 years of age compared with older ages. Overall, undesirable outcomes occurred at PRL concentrations less than 2.5 ng/ml. In conclusion, both 25OHVD and PRL may have important roles in spermatogenesis and normal sperm physiology in the dog. ACKNOWLEDGEMENTS The past few years have been an exciting and important part of my life. I would not have been able to complete this great undertaking without the support and knowledge of a great many people. First I would like to thank my advisor Dr. Cathy Gartley for her wealth of knowledge in the field of theriogenology, for her mentorship, her kindness and lastly, for her sense of humour to make me laugh even on the greyest of days. Also, I would like to thank members of my advisory committee: Drs. Tracey Chenier, Andria Jones, Stephanie Nykamp and Heather Chalmers for their patience and expertise in guiding me through my research and for their critical advice in pushing me to create a body of work I can be proud of. For his statistical expertise, I am grateful to William Sears whose help was invaluable in the analysis of my data. For their friendship, support and encouragement during my D.V.Sc. programme I would like to thank Drs. Rasa Levstein and Cyril Stephen, our veterinary technicians Jim Rahn and Karen DiCaro, as well as my fellow graduate students Drs. Mariana Diel De Amorim and Leslie Gonzalez. I would also like to thank Dr. Walter Johnson for his encouragement and for sharing his knowledge of bovine theriogenology. A special thank-you to Sally, Linda, Julie and Karla in the Population Medicine office, and Mary Elliot for helping me with the bureaucratic aspects of graduate studies and for making my life that much easier. To Dr. Carol Graham, all the staff at Graham Animal Hospital, and to all our clients who agreed to enroll their dogs in this study, I extend a warm v appreciation for their participation. Without them there wouldn’t have been any data to analyse. I wish to also thank the Pet Trust for providing the funding to make this study possible. Lastly, I would like to thank two very special people in my life, my mother Eva and my husband Anthony (Toncsi) for the much needed emotional support during my graduate programme. Their patience and unending belief in me gave me the strength I needed to achieve my goals. vi DECLARATION OF WORK PERFORMED I declare that with the exception of the items listed below, all work presented in this thesis was performed by me. The Animal Health Laboratory, University of Guelph, performed urinalysis and culture and sensitivities for urine, prostatic fluid and semen. The Endocrinology Division of the Diagnostic Center of Population and Animal Health, Michigan State University, performed the radioimmunoassay for Vitamin D analysis. The New Animal Science Laboratory, Washington State University, performed the enzyme-linked immunosorbent Assay for Prolactin analysis. vii TABLE OF CONTENTS CHAPTER ONE ………………………………………………………………… 1 INTRODUCTION, RESEARCH OBJECTIVES AND LITERATURE REVIEW …………………………………………................................... 1 1. INTRODUCTION …………………………………………………….. 1 2. RESEARCH OBJECTIVES..………………………………………… 3 3. LITERATURE REVIEW.……………………………………………… 4 3.1 PHYSIOLOGY OF CANINE SPERMATOGENESIS AND METHODS OF SEMEN ANALYSIS………………………… 4 3.1.1 Reproductive Physiology of the Testes and the Role of Hormones and Local Factors In Male Reproduction….. 4 3.1.2 Spermatogenesis ………………………………………. 13 3.1.3 Factors Affecting Semen Quality.…………………… 16 3.1.4 Methods of Semen Evaluation……………………… 17 3.2 THE CANINE PROSTATE GLAND ……………………….. 31 3.2.1 Anatomy and Physiology of the Prostate Gland ……... 31 3.2.2 Pathophysiology of Benign Prostatic Hyperplasia…….. 33 3.2.3 Conventional Treatment of Benign Prostatic Hyperplasia ……………………………………………….. 39 3.2.4 Ultrasonography of the Prostate.……………………….. 42 3.3 VITAMIN D AND ITS ROLE IN MALE REPRODUCTION.. 44 3.3.1 Vitamin D Metabolism ………………………………… 45 3.3.2 Vitamin D and the Prostate …………………………… 49 3.3.3 Role of Vitamin D in Sperm Production ……………… 54 3.4 PROLACTIN AND ITS ROLE IN MALE REPRODUCTION.. 56 3.4.1 Prolactin and Ovarian Physiology.……………………. 56 3.4.2 Prolactin and Male Physiology.………………………. 58 3.4.2.1 Prolactin and the Prostate.…………………………. 58 3.4.2.2 Prolactin, Male Fertility and Semen Quality ……… 60 viii References ………………………………………………………… 67 CHAPTER TWO …………………………………………………………… 81 VITAMIN D, BENIGN PROSTATIC HYPERPLASIA, PROSTATE VOLUME AND SEMEN PARAMETERS IN THE DOG………… 81 Abstract.……………………………………………………………... 81 1. Introduction……………………………………………………….. 82 2. Materials and Methods.………………………………………….. 84 3. Results.……………………………………………………………. 92 4. Discussion and Conclusions.…………………………………… 98 References ………………………………………………………….. 143 CHAPTER THREE.…………………………………………………………… 147 PROLACTIN, BENIGN PROSTATIC HYPERPLASIA, PROSTATE VOLUME AND SEMEN PARAMETERS IN THE DOG.. 147 Abstract.………………………………………………………………... 147 1. Introduction …………………………………………………………. 148 2. Materials and Methods ……………………………………………. 151 3. Results ……………………………………………………………… 155 4. Discussion and Conclusions ……………………………………… 157 References.…………………………………………………………….. 178 CHAPTER FOUR …………………………………………………………….. 180 SUMMARY AND CONCLUSIONS ………………………………… 180 References……………………………………………………………. 184 APPENDICES ……………………………………………………………….. 185 ix LIST OF TABLES Table 1.1 Selected Physical and Chemical Analyses of Prostatic Fluid in Dogs with Healthy Prostates.…………………………………………… 33 Table 1.2 Genes influenced by Vitamin D receptor ligands and their effects 48 Table 1.3 Prolactin values in Dogs (ELISA) ……………………………………65 Table 2.1 Technical Parameters for SpermVision™ CASA …………………111 Table 2.2 Classification scheme for determination of the presence of BPH by ultrasonography in 29 dogs, March-December 2009, Ontario, Canada ………………………………………………………….112 Table 2.3 Characteristics of the study population of 28 dogs, in Ontario, Canada, March-December 2009.……………………………………….113 Table 2.4 Characteristics of the study population of 22 dogs from whom semen was collected in Ontario, Canada March-December 2009…………...114 Table 2.5 Breed, Vitamin D concentration, age and fertility data on 22 dogs from whom semen was collected in Ontario, Canada, 2009…………115 Table 2.6 Mean serum Vitamin