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Spermatozoal Quality of Cryopreserved Canine Semen Using Different Extenders and Chloroquine Phosphate

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Spermatozoal Quality of Cryopreserved Canine Semen Using Different Extenders and Chloroquine Phosphate SPERMATOZOAL QUALITY OF CRYOPRESERVED CANINE SEMEN USING DIFFERENT EXTENDERS AND CHLOROQUINE PHOSPHATE ____________________________________________ JOVI RICHARD OTITE MATRIC No. 63116 SPERMATOZOAL QUALITY OF CRYOPRESERVED CANINE SEMEN USING DIFFERENT EXTENDERS AND CHLOROQUINE PHOSPHATE BY Jovi Richard OTITE B.sc Animal Science (Ibadan), M.sc Animal Science (Ibadan) MATRIC No. 63116 A THESIS SUBMITTED TO THE DEPARTMENT OF ANIMAL SCIENCE FACULTY OF AGRICULTURE AND FORESTRY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF THE UNIVERSITY OF IBADAN. FEBRUARY, 2012. i CERTIFICATION I certify that this work was carried out by Mr. Jovi Richard OTITE of the Department of Animal Science, University of Ibadan, Ibadan, Nigeria under my supervision. ................................................ SUPERVISOR Prof. D.O. ADEJUMO Professor of Animal Reproductive Physiology Department of Animal Science University of Ibadan Ibadan. ii DEDICATION This work is dedicated to my parents Prof. and Dr. Mrs. Onigu Otite, my wife Yohi and son Aman. iii ACKNOWLEDGMENT My sincere gratitude goes to my supervisor, Prof. D.O. Adejumo for his good advice, guidance and supervision of this project. I owe a lot of appreciation to my wife Yohi Mersha Otite, my parents Prof. and Dr. Mrs Otite as well as other members of my family for all their support throughout the span of this work. I wish to express my gratitude to Prof. G.N. Egbunike, Prof. A.O. Akinsoyinu, Prof. A.D. Ologhobo, Dr. O.A. Sokunbi, Dr. A.O. Ladokun, Dr. Dele Ojo, Miss Temitope Lawal, Dr. O.A. Ogunwole, Dr. O.J. Babayemi, Mr. O. Alaba, Dr. A.B. Omojola, Dr. O.A. Adebiyi, Mr. O.O. Aiyepeku and Mr. O.H. Osaiywvu for their assistance in making this work a success. I also wish to express my sincere gratitude to Prof. O.O. Tewe, Dr. A.E. Salako, Dr. O.A. Abu, Mr. Akin Adamu, Dr. Kilani, Mr. Zhattu Bani and Mr. Daniel Udofia. iv TABLE OF CONTENTS TITLE PAGE i CERTIFICATION ii DEDICATION iii ACKNOWLEDGEMENT iv LIST OF TABLES xv LIST OF FIGURES xvi ABSTRACT xviii CHAPTER ONE 1.0 GENERAL INTRODUCTION 1 1.1 HISTORY OF THE DOG 1 1.2 ANCESTRY OF THE DOG 4 1.3 IMPORTANCE OF THE DOG 7 1.4 THE NIGERIAN DOG TRADE INDUSTRY 10 1.5 OBJECTIVE OF STUDY 13 1.6 JUSTIFICATION 14 CHAPTER TWO 2.0 LITERATURE REVIEW 15 2.1 REPRODUCTION IN THE DOG 15 2.1.1.1 HORMONES OF REPRODUCTION 15 v 2.1.1.2 Releasing Hormones 15 2.1.1.3 Gonadotropin Releasing Hormone (GNRH) 16 2.1.1.4 Follicular stimulating hormone (FSH) 16 2.1.1.5 Luteinizing hormone (LH) 17 2.1.1.6 Inhibin 17 2.1.1.7 Androgens 18 2.1.1.8 Estrogens 18 2.1.1.9 Prolactin 18 2.1.1.10 Oxytocin 19 2.1.1.11 The placental gonadotropins 19 2.1.1.12 Prostaglandins 19 2.1.2 The reproductive cycle of the dog: 20 2.1.2.1 Characteristics of dog semen 20 2.1.2.2 First Fraction 21 2.1.2.3 Second Fraction 21 2.1.2.4 Third Fraction 21 2.1.2.5 Concentration 22 2.1.2.6 Mass Activity 23 2.1.2.7 Progressive motility 23 2.1.2.8 Morphology 24 2.1.2.9 Structure of Canine Spermatozoa 24 vi 2.1.2.10 The head 24 2.1.2.11 The neck 24 2.1.2.12 The tail 25 2.1.2.13 Seminal plasma 25 2.1.2.14 Survival time of Canine spermatozoa 26 2.1.3 Factors affecting semen quality 26 2.1.3.1 Age 26 2.1.3.2 Breed 26 2.1.3.3 Nutrition 27 2.1.3.4 Excessive weight 28 2.1.3.5 Scrotal Thickness 28 2.1.3.6 Testis size/diameter 29 2.1.3.7 Frequency of collection 29 2.1.3.8 Temperature 29 2.1.3.9 Light 30 2.1.3.10 pH 30 2.1.3.11 Time of collection 31 2.1.3.12 Libido 31 2.1.3.13 Starvation 31 2.1.3.14 Health 31 2.1.3.15 Unilateral Cryptorchidism 32 vii 2.1.4 Semen collection in the dog 32 2.1.4.1 Massage Technique 33 2.1.4.2 Artificial Vagina 34 2.1.4.3 Electro ejaculator 36 2.1.4.4 Balloon Artificial Vagina 36 2.1.5 Semen Preservation and extension 37 2.1.5.1 Buffers 38 2.1.5.1.1 Organic Buffers 38 2.1.5.1.2 Inorganic Buffers 38 2.1.5.2 Citrates 38 2.1.5.3 Glucose and Fructose 39 2.1.5.4 Additives 39 2.1.5.4.1 Antibiotics 39 2.1.5.4.2 Egg yolk 39 2.1.5.4.3 Coconut Water 40 2.1.5.4.4 Zobo (Hibiscus sabdarifa) 40 2.1.5.4.5 Skim milk 42 2.1.5.4.6 Orvus ES Paste (OEP) 42 2.1.6 Cryopreservation 42 2.1.7 Fertilization and implantation in the dog 44 2.1.8 Artificial insemination in the dog 45 viii 2.1.9 Techniques of Artificial Insemination 47 2.1.9.1 Non surgical insemination 47 2.1.9.2 Trans-cervical insemination 47 2.1.9.3 Trans-cervical insemination with the Norwegian Catheter 48 2.1.9.4 Surgical insemination 51 2.2.9.5 Intratubal Insemination 51 2.1.10 Future concerns of artificial insemination using thawed frozen semen 51 (i) Conception Rate 52 (ii) The safety of the insemination process on the health of the bitch 52 (iii) Sex Ratio 52 2.1.11 Optimum time for insemination 53 (i) Cytology 53 (ii) Day Count 53 (iii) Male check 54 (iv) Electrical resistance test 54 2.1.11.1 Hormonal Assay 54 CHAPTER THREE PRELIMINARY STUDY OF THE EFFECTS OF EXTENDERS ON SEMEN QUALITY 3.0 INTRODUCTION 57 3.1 MATERIALS AND METHODS 58 ix 3.2 Experimental dogs 58 3.3 Collection of semen 58 3.4 Semen evaluation 58 3.4.1 Volume 58 3.4.2 Concentration 60 3.4.3 Mass activity 60 3.4.4 Percentage progressive motility 60 3.4.5 Morphology 61 3.4.6 pH 61 3.5 Semen Extenders 61 3.6 Semen Extension 61 3.7 Semen preservation 62 3.8 Addition of Penicillin and Streptomycin 62 3.9 Statistical analysis 62 3.9 RESULTS 63 3.10 Semen preservation at room temperature 64 3.11 Semen preservation at refrigeration temperature 70 3.12 DISCUSSION 77 3.13 SEMEN PRESERVATION AT ROOM TEMPERATURE 77 3.13.1 Progressive motility 77 3.13.2 Mass activity 78 x 3.13.3 Survival time 79 3.13.4 Percentage abnormalities 79 3.14 SEMEN PRESERVATION AT REFRIGERATION TEMPERATURE 80 3.14.1 Motility 80 3.14.2 Mass activity 81 3.14.3 Survival time 81 3.14.4 Percentage abnormalities 81 3.15 CONCLUSION 82 CHAPTER FOUR CRYOPRESERVATION OF CANINE SEMEN 83 4.1 INTRODUCTION 83 4.2 MATERIALS AND METHODS 84 4.2.1 Experimental dogs 84 4.2.2 Collection of semen 84 4.2.3 Semen evaluation 84 4.2.3.1 Acrosome integrity 84 4.2.3.2 DNA Integrity 85 4.2.4 Semen Extenders 85 4.2.5 Semen Extension 85 4.2.6 Addition of Penicillin and Streptomycin 85 4.2.7 Cryopreservation 85 xi 4.2.8 Thawing 86 4.2.9 Post preservation analysis 86 4.2.10 Statistical analysis 86 4.3 RESULTS 87 4.4 DISCUSSION 100 4.4.1 Progressive motility 100 4.4.2 Mass activity 101 4.4.3 Survival time 102 4.4.4 Percentage abnormality 103 4.4.5 Acrosome integrity 104 4.4.6 DNA integrity 105 4.5 CONCLUSION 105 CHAPTER FIVE THE EFFECTS OF CHLOROQUINE PHOSPHATE ON CRYOPRESERVED CANINE SPERMATOZOA 107 5.1 INTRODUCTION 107 5.2 MATERIALS AND METHODS 109 5.2.1 Experimental dogs 109 5.2.2 Semen Collection 109 5.2.3 Semen evaluation 109 5.2.4 Semen Extenders 109 xii 5.2.5 Semen Extension 109 5.2.6 Cryopreservation 110 5.2.7 Thawing 110 5.2.6 Post preservation analysis 110 5.2.7 Addition of Chloroquine Phosphate 110 5.2.8 Addition of Penicillin and Streptomycin 110 5.2.9 Statistical analysis 110 5.3 RESULTS 111 5.4 DISCUSSION 123 5.4.1 Progressive motility 123 5.4.2 Mass activity 124 5.4.3 Post thaw survival time 126 5.4.4 Percentage abnormality 127 5.5 CONCLUSION 127 CHAPTER SIX SUMMARY, CONCLUSION AND RECOMMENDATION 129 6.1 CONCLUSION 129 6.2 RECOMMENDATION 130 REFERRENCES 131 APPENDIX 150 COMPOSITIONS OF THE DIFFERENT SEMEN EXTENDERS 150 xiii PREPARATION OF BUFFERS 152 PREPARATION OF EXTENDERS 152 PREPARATION OF ADDITIVES 153 xiv LIST OF TABLES Table 1: Dog breeds showing places, dates of origin and probable ancestors 3 Table 2: Nutritional value of Dog meat (per 100g) 10 Table 2.1: The nutritional content of Zobo (Hibiscus sabdarifa) 41 Table 2.2: Boiling Point (BP) of various cryogenic gases 43 Table 3.1: Progressive motility (%) of canine semen stored at room temperature (28oC) 65 Table 3.2: Mass Activity (%) of canine semen stored at room temperature (28oC) 67 Table 3.3: Progressive Motility (%) of canine semen stored at refrigeration temperature (4oC) 71 Table 3.4: Mass Activity (%) of canine semen stored at room temperature (4oC) 73 Table 3.5: Summary of results obtained for survival time and percentage abnormalities of canine spermatozoa preserved at room temperature (28oC) and refrigeration temperature (4oC) 76 Table 4.1: Motility (%) of spermatozoa in four different extenders 90 Table 4.2: Mass Activity (%) of spermatozoa in four different extenders 92 Table 4.3: Summary of results 94 Table 4.4: Effects of preservation temperature on percentage abnormality of canine spermatozoa 95 Table 5.1: The effects of Chloroquine Phosphate on the Progressive Motility of post thawed spermatozoa in 4 different extenders 114 Table 5.2: The influence of Chloroquine phosphate on the progressive motility of spermatozoa 115 Table 5.3: The effects of Chloroquine Phosphate on the Mass Activity of post thawed spermatozoa in 4 different extenders 117 Table 5.4: The influence of Chloroquine phosphate on the mass activity of spermatozoa 118 Table 5.5: Influence of Chloroquine phosphate on the percentage abnormality and post thaw survival time of spermatozoa 122 xv LIST OF FIGURES Fig.
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