CRYOPRESERVATION OF CROSSBRED-GOAT (ALPINE X BEETAL) SEMEN IN DIFFERENT SEASONS

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1 CRYOPRESERVATION OF CROSSBRED-GOAT (ALPINE X BEETAL) SEMEN IN DIFFERENT SEASONS THESIS SUBMITTED TO THE NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN LIVESTOCK PRODUCTION MANAGEMENT BY NARWADE BABAN MADHAVRAO LIVESTOCK PRODUCTION MANAGEMENT NATIONAL DAIRY RESEARCH INSTITUTE (I.C.A.R.) KARNAL (HARYANA), INDIA 2013 Regn. No

2 Dedicated to My beloved mother and late father who have encouraged me to fulfill my dreams and aspirations

3 CRYOPRESERVATION OF CROSSBRED-GOAT (ALPINE X BEETAL) SEMEN IN DIFFERENT SEASONS By NARWADE BABAN MADHAVRAO THESIS SUBMITTED TO THE NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY) KARNAL (HARYANA) IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN LIVESTOCK PRODUCTION MANAGEMENT Approved By (EXTERNAL EXAMINER) (T. K. MOHANTY) MAJOR ADVISOR & CHAIRMAN (GUIDE) Members, Advisory Committee 1. Dr. V. S. RAINA Ex. Principal Scientist 2. Dr. S.K. ATREJA Principal Scientist, ABC Division 3. Dr. A.K. Gupta Principal Scientist, DCB Division 4. Dr. D. MALAKAR Principal Scientist, ABTC On leave 5. Dr. A. K. Mohanty Principal Scientist, ABTC

4 LIVESTICK PRODUCTION MANAGEMENT NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY) KARNAL (HARYANA), INDIA Dr. T. K. MOHANTY, Ph.D. Principal Scientist (Animal Reproduction) Artificial Breeding Research Centre CERTIFICATE This is to certify that the thesis entitled CRYOPRESERVATION OF CROSSBRED-GOAT (ALPINE X BEETAL) SEMEN IN DIFFERENT SEASONS submitted by Mr. Narwade Baban Madhavrao towards the partial fulfilment of the requirement for the award of the degree of Doctor of Philosophy in Livestock Production Management of the National Dairy Research Institute (Deemed University), Karnal (Haryana), India, is a bonafide research work carried out by him under my supervision and guidance and no part of the thesis has been submitted for any other degree or diploma. Dated: 31 st July, 2013 (Tushar Kumar Mohanty) Major Advisor & Chairman (Guide)

5 ACKNOWLEDGEMENT I would like to take this opportunity to express my deep sense of gratitude to my guide and major advisor, Dr. T. K. Mohanty principal scientist, Livestock Production Management section, National Dairy Research Institute, Karnal for his able guidance, valuable suggestions, sustained encouragement and constructive criticism during the entire course of the present research work. It is a matter of great pleasure and pride to get an unique opportunity of working with an able, affectionate and benevolent scientist. I wish to express my deep reverence to Dr. A.K. Srivastva, Director, NDRI, Karnal, for providing the necessary facilities throughout the tenure of this work. I express my gratitude to this institute for keeping my financial worries at bay in the form of N.DR.I fellowship. I sincerely acknowledge the valuable suggestions, erudite guidance and all possible cooperation extended by Dr. V. S. Raina Principal Scientist Ex. I/C Head LPM, Dr. Shiv Prasad, Head, L.P.M.in several phases of this investigation. I would like to convey my regards and sincere thanks to the members of my Advisory Committee Dr. S. K. Atreja, Principal Scientist, Animal Biochemistry, Dr. A. K. Gupta, Principal Scientist, DCB, Dr. D. Malakar, Principal Scientist, Animal Biotechnology and Dr. A. K. Mohanty, Principal Scientist Animal Biotechnology for their concrete suggestions, worthy criticism and univocal help in shaping and execution of the study. I express my sincere thanks to all faculty members and staff of Artificial Breeding Research Center for their co-operation and support. Especially, I would like to acknowledge Dr. Bhagat, Senior Scientist, LPM Section, Dr. J.K. Pundir, Mr. Dharampal, Dr. Amarpal, Mr. Nihal Singh, Mr. Anil Bhardwaj, Mr. Ramesh, Mr. Shyam Singh and Mr. Ramkesh Meena for extending a helping hand during the whole of work. My great thank to SRF Amit Singh, JRF Gulshan and Ashwini without their help it was difficult to carry out the research work. I also thank to Sukhvinder, Sachin and Suraj, amit and Sirka for their help in collection samples and AI. It has been my good fortune to have collegeagues and friends like Deepak Kale, Sachin Selke, batchmates, Yajju, Roshan and Biplov, Saroj Roy, and juniors Amrendra, Kumaresh, Suresh, Tapas and Siddharth whose

6 brotherly love, care and help during the hour of need has completely overwhelmed me. I am extremely thankful and profoundly obliged for benevolent help extended by them. I extend my deep sense of appreciation to my loving juniors for their respect, affection and wholehearted cooperation. Time spent with them under the cafe tree will always be remembered. I cannot forget the joyful days shared with my seniors Rohit, Maneesh, Thakur and Amit Singh whose loving company and magnanimous help throughout my stay at N.D.R.I, made it extremely joyful. The moments spend with them is captured in my heart for lifetime. I wish to thank my distant friends Madhav Dhapade, Santosh Kendre, Subhash, Siddarth, Sahebrao, Datta and Datta Narwade for their moral support. I have no words to express my sincere gratitude to my Aai, Baba and mother-in-law, brother-in-law, brothers Hari and Kishor and for their ineffable love, selfless support, inspiration and faith in me which has always made things much simpler and life more worthy to live. It s their immense patience and blessings which made my dream transformed into reality. No words will be sufficient to express my thanks to my dear wife Amrapali and daughters Sanchi and Pari who has made my stay memorable at Karnal. I owe everything to the almighty Lord Buddha and Dr. Babasaheb Ambedkar and thank them for every blessing he has bestowed upon me. Date: 31 st July, 2013 (Narwade Baban Madhavrao)

7 CONTENTS Chapter Title Page No. 1.0 INTRODUCTION REVIEW OF LITERATURE Semen Quality Attributes Semen volume Color and Consistencies of fresh semen Concentration of Spermatozoa Mass motility Progressive motility Post-thaw motility Seasonal variation in breeding activity and sperm production Effect of season on freezability of goat semen Seminal plasma Effect season on seminal plasma and its removal Seasonal variation of seminal plasma proteins (Heparin Binding Proteins) Effect of modified method of semen collection Cryopreservation Diluents Osmolality ph and buffering systems Effect of trehalose incorporation in semen extender Cryoprotectants Methods of Freezing Freezing protocol Thawing of semen samples In-vivo Fertility Test: Conception rate after AI 30 with frozen semen Per cent non-eosinophilic (live) sperm Morphological abnormal sperm In-Vitro Tests Hypo Osmotic Swelling Test (HOST) Acrosomal Integrity 33-34

8 3.0 MATERIALS AND METHODS Location of Farm and Climate Classification of season Selection of bucks Semen collection time and method Fresh semen evaluation of Alpine x Beetal crossbred goat Colour Consistancy Volume Mass Activity Motility estimate (%) Sperm concentration Eosinophilic (dead) and Non-eosinophilic (live) spermatozoa count (%) Hypo-osmotic Swelling Test (HOST) Acrosome Integrity Cryopreservation of semen Dilution of semen Printing of Straws Filling and sealing of Straws Freezing of Straws Semen preservation evaluation: Thawing Evaluation of frozen semen samples EXPT I Freezability of crossbred-goat (AXB) semen in relation to heparin-binding proteins (HBPs) in different seasons. EXPT II Effect of modified method of semen collection on cryopreservation of goat semen EXPT III (A) Effect of trehalose + SDS incorporation in semen extender on characteristics of cryopreserved goat semen

9 3.3.5 EXPT III (B) Effect of different concentration of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen EXPT-III (C) Freezability of crossbred goat semen in tris-soya-milk and trehalose - soyamilk -based extenders EXPT IV (A) In-vitro Fertility Test with Cryopreserved AXB Crossbred goat EXPT IV (A) In-vitro Fertility Test with Cryopreserved AXB Crossbred goat 3.4 Statistical analysis RESULTS AND DISCUSSION Fresh semen evaluation of Alpine x Beetal bucks in different seasons of the year Color and Consistency Volume Mass Motility Progressive Motility Percent Live Sperm Concentration (10 9 /ml) Percent HOST Intact Acrosome Freezability of crossbred buck semen in different seasons in relation to Heparin Binding Proteins (HBPs) 4.3 Effect of modified of method semen collection on semen parameters characteristics in different seasons Effect of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen Effect of different concentration of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen 4.6 Freezability of crossbred goat semen in Trissoya-milk and trehalose - soya-milk -based extenders. 4.7 In-vitro Fertility Test Cryopreserved Crossbred goat (AXB) Semen

10 4.8 In Vivo fertility of semen cryopreserved with TCGEY and TCGEY + Trehalose based extender SUMMARY AND CONCLUSIONS BIBLIOGRAPHY I-XV

11 LIST OF TABLES Table No. Title After page No. Table Semen volume of different breeds of goat 7 Table Grades of semen by colour and consistency 8 Table Sperm concentration of goat (109/ml) 9 Table Mass motility of goat spermatozoa 10 Table Progressive motility (%) estimate of goat spermatozoa 11 Table Post-thaw motility (%) goat spermatozoa 11 Table Freezability of goat semen in temperate region 12 Table Improvement of sperm motility by modified method of 18 collection Table Motion parameters and recovery rates of spermatozoa 18 collected into tubes containing 10 ml of extender Table Fertility rate of buck in different conditions 31 Table-3.1 List of Alpine X Beetal bucks under experiment 36 Table Composition of solutions for HOST 41 Table Mean ± SE fresh semen parameters of Alpine X Beetal 59 crossbred buck semen collected during the different seasons Table Mean ± SE fresh semen parameters of Alpine X Beetal 59 crossbred buck semen collected during different seasons Table Mean ± SE Fresh semen parameters of Alpine X 59 Beetal buck semen in all seasons Table Mean Square for fresh semen parameters of Alpine X Beetal buck semen in different seasons 59 Table Table Table Table Table Expression of Heparin Binding Proteins (HBPs) in seminal plasma of AXB crossbred goat in different seasons in SDS-Page 2D Gel electrophoresis for Heparin binding Proteins (HBPs) of seminal plasma of AXB crossbred goat for different seasons Mean ± SE for Pre-freeze parameters of Alpine X Beetal crossbred buck semen cryopreserved during different seasons Mean ± SE for Pre-freeze parameters of Alpine X Beetal crossbred buck semen cryopreserved during different seasons Mean ± SE for Pre-freeze parameters of Alpine X Beetal crossbred buck semen cryopreserved in two types of extenders during all over seasons

12 Table Table Table Mean ± SE for interaction of Season X Extender of buck semen cryopreserved in two types of extenders during different seasons for prefreez parameters Mean ± SE for interaction of Buck X Extender of buck semen cryopreserved in two types of extenders for prefreez parameters Mean ± SE for interaction of Season X Buck X Extender of crossbred buck (Alpine X Beetal) semen cryopreserved in two types of extenders during different seasons for prefreez parameters Table Mean ± SE for Post-thaw semen parameters cryopreserved in two types of extender during different seasons Table Mean ± SE for Post-thaw semen parameters cryopreserved in two types of extender during different seasons Table Mean ± SE for Post-thaw semen parameters cryopreserved in two types of extender during different seasons Table Table Table Table Table Table Table Table Mean ± SE for post-thaw parameters of season X extender interaction of buck semen cryopreserved in two types of extenders during different seasons Mean ± SE for post-thaw parameters of Buck X Extender interaction of buck semen cryopreserved in two types of extenders during different seasons Mean ± SE for post-thaw parameter of Season X Buck X Extender interaction of buck semen cryopreserved in two types of extenders during different seasons for ANOVA for pre-freeze parameters of semen frozen in different extenders during different season seasons ANOVA for post -thaw parameters of semen frozen in different extenders during different season Mean ± SE for fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during entire seasons (n=60) Mean ± SE for interaction of Season X Method of collection on fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods cryopreserved during different seasons (n=120) Mean ± SE for interaction of Buck X Method of semen collection on fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods cryopreserved during different seasons (n=120)

13 Table Table Table Table Table Table Table Table Table Table Table Mean ± SE for interaction for Season X Buck X Method of collection on initial fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods cryopreserved during different seasons (n=120) Mean ± SE for pre-freeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during entire season (n=120) Mean ± SE for interaction of Season X Method of collection on pre-freeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Mean ± SE for interaction Buck X Extender on prefreeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Mean ± SE for interaction for Season X Buck X Extender on pre-freeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Mean ± SE for post-thaw parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during entire year(n=120) Mean ± SE for interaction of Season X Method of collection on post-thaw parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Mean ± SE for interaction Buck X Method of collection post-thaw semen parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Mean ± SE for interaction for Season X Buck X Method of collection on post-thaw semen parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Mean square for fresh semen parameters collected by different methods of collection (Empty collection tube and with 10 ml TCG buffer) during different seasons Mean square for Pre- freeze semen parameters collected by different methods of collection (Empty collection tube and with 10 ml TCG buffer) during different seasons Table Mean square for Post-thaw semen parameters collected 78

14 Table Table Table Table Table Table Table Table by different methods of collection (Empty collection tube and with 10ML TCG buffer) during different seasons Mean ± SE of freezability parameters of crossbred buck (Alpine X Beetal) semen in TCG-egg Yolk Extender-1 and Trehalose-egg Yolk based extender-2 (n=100) Mean square for pre-freeze and post-thaw parameters for TCG (Extender-1) and TCG+ trehalose based extenders (Extender-2) Mean ± SE of freezability parameters of AXB crossbred goat semen in different concentration of Trehalose. Mean square freezability parameters of AXB crossbred goat semen in different concentration of Trehalose Mean ± SE for freezability parameters of crossbred goat semen in different types of egg yolk and soya extract based extenders (n=10) Mean square for freezability parameters of crossbred goat semen in different types egg yolk and soya extract based extender supplemented with trehalose at initial, pre-freeze and post thaw. In-vitro fertility test cryopreserved crossbred goat AXB semen in different TCGEY and TCGEY+Trehalose extender. (n=30) Pregnancy rate and kidding rate of does after AI with frozen semen cryopreserved with two types of extenders. (n=52)

15 LIST OF FIGURES Fig. No. Title Page no. Figure- 3.1 Monthly THI during the experimet 36 Figure Flow chart for Purification of HBPs for SDS-PAGE 47 and 2D gel Figure Flow chart of effect of modified method of semen collection on cryopreservation of goat semen. 50 Figure Flow chart of Effect of trehalose incorporation in 51 semen extender on characteristics of cryopreserved goat semen Figure Effect of different concentration of trehalose 52 incorporation in semen extender on characteristics of cryopreserved goat semen. Figure Semen volume (ml) of AXB crossbred bucks 59 Figure Mass motility (0-5 Scale) of AXB crossbred buck semen Figure Mass motility (0-5) for AXB buck in different seasons 61 Figure Progressive motility (%) of AXB buck semen Figure Progressive motility (%) of AXB buck semen different seasons 62 Figure Live (%) of AXB crossbred buck semen 63 Figure Live (%) of AXB buck semen in different seasons 64 Figure Sperm Concentration (10 9 /ml) of AXB bucks 65 Figure Sperm Concentration (10 9 /ml) of AXB buck semen in different seasons 66 Figure HOST (%) of AXB buck semen 67 Figure HOST % of AXB buck semen in different seasons 67 Figure Intact Acrosome % of AXB buck semen 68

16 Figure Figure Figure Intact Acrosome of AXB buck semen in different seasons SDS Page of seminal plasma heparin binding proteins in different seasons 2D Gel electrophoresis of Heparin binding Protein in different seasons Figure Pre-freeze parameters of semen in different seasons 71 Figure Pre-freeze parameters of semen of AXB bucks 72 Figure Figure Pre-freeze parameters of semen cryopreserved in different extenders Post-thaw parameters buck of semen cryopreserved in different seasons Figure Post-thaw parameters of different AXB buck semen 73 Figure Post thaw parameters of AXB buck semen cryopreserved in different extenders 74 Figure Figure Figure Figure Figure Figure Figure Fresh semen quality parameters collected by different method of collection Pre-freeze parameters of AXB buck semen by different method of collection Post-thaw semen parameters of AXB buck semen collected by different Method of collection Semen parameters at pre-freeze in two types of extenders Post-thaw of semen parameters in two types of extenders Live per cent of spermatozoa in semen at initial, prefreeze and post-thaw stages of semen preservation Progressive motility per cent spermatozoa in semen at initial, pre-freeze and post-thaw stages of

17 Figure Figure Figure Figure Figure preservation HOST per cent of spermatozoa at initial, pre-freeze and post-thaw stages of preservation Intact Acrosome spermatozoa per cent at initial, prefreeze and post-thaw stages of preservation live per cent at different stages of cryopreservation for different types of extenders Progressive motility per cent at different stages of cryopreservation for different types of extenders HOST per cent at different stages of cryopreservation for different types of extenders Figure Intact acrosome % at different stages of cryopreservation for different types of extenders 86 Figure Figure Cleavage, two cell and 4-8 cell stage embryos per cent Pregnancy rate and kidding rate of does after AI with frozen semen cryopreserved with two types of extenders TCGEY and TCGEY+Trehalose 90 91

18 LIST OF PLATES Plate No. Title After page No. Plate-1 Crate for semen collection and transcervical AI 37 Plate-2 Semen collection in goat with artificial vagina 37 Plate-3 Goat transcervical AI with frozen semen 90 Plate-4 Per rectal ultrasonography at 35 Days for pregnancy 90 diagnosis in Goat Plate-5 In-vitro fertilization 90

19 ABBREVIATIONS < Less than > More than 0 C Degree Celsius AI Artificial insemination cm Centimetre CR Conception rate d.f. Degrees of freedom Fig. Figure H HOUR HBPs Heparin binding proteins HOST Hypo Osmotic Swelling Test i.e. That is IA Intact acrosome (%) Kg Kilogram LIVE Non-eosinophilic count (% LN 2 Liquid Nitrogen Mg Milligram Ml Millilitre mm Millmolar MT Million tones n Number of observations Ng Nanogram No. Number PM Prograssive motility (%) RH % Relative humidity % SE Standard error SM Square means TCG Tris Ctric Acid Glucose buffer viz. Namely vs. versus

20

21 ABSTRACT The present investigation was undertaken to study the freezability of (Alpine X Beetal) crossbred-goat semen relation to heparin-binding proteins (HBPs) as well as fresh semen quality parameters in different seasons like winter, hot-dry summer and hot-humid summer. And to study of trehalose incorporation in semen extender on characteristics of cryopreserved semen. Fresh and frozen semen quality evaluated by collecting semen in 10 ml TCG buffer and without TCG buffer and also to study in-vitro and in-vivo fertilization with frozen semen. The results were found that there was expression of more heparin binding proteins in hot-dry summer and hot-humid summer than winter season. The freezability of crossbred goat semen was significantly higher in winter season than hot-dry summer and hot-humid seasons. There was significant higher fresh semen parameters like mass motility, concentration, live per cent, progressive motility per cent, HOST and intact per cent spermatozoa in semen sample collected during winter than the hot dry summer and hot humid seasons. We found that trehalose replacing of basic TCG buffer have given better results. There was significant higher live per cent, progressive motility per cent, HOST and intact per cent spermatozoa when semen collected in tubes containing 10 ml of TCG buffer solution than without buffer empty collection at fresh semen evaluation parameters, prefreez and post-thaw. There was significant higher per cent of in-vitro fertility for fresh semen than frozen semen. We found that in-vitro fertility was higher semen frozen in extender supplemented with trehalose. There was significant higher in-vivo fertility (61.54 Vs 46.16) for semen frozen in extender supplemented with trehalose than conventional extender. Overall results are suggested that goat semen collected in TCG buffer successfully cryopreserved in extender supplemented with trehalose. Semen harvested during favorable winter season can be used throughout year in crossbred Alpine X Beetal goat for artificial insemination.

22 1. INTRODUCTION Goat, poor man s cow contributes significantly to the economically weak family and to national income. World population of goat is around million (FAO, 2010) of which Asia possesses about 64.09% and India 14.75% of total world population (FAO, 2007). India possesses about million goats and stands second to China. Goat population has increased at the rate 3.1% per annum during last two decades (Census, 2007). Scientific breeding and management practices can improve the productivity of Indian goats. More efficient genetic selection schemes, and the manipulation as well as storage of genetic material are possible only with the help of artificial insemination with frozen semen technology. Artificial insemination (AI) has an important role in goat breeding, especially in intensive system of production to control reproduction in conjugation with accurate progeny testing, to improve the production of milk, meat and hair. AI results an increase in number of offspring per sire, allows a special and temporal (in case of frozen thawed semen) dissociation between collection of spermatozoa and fertilization. AI allows rapid and widespread diffusion of improved genotypes and exchange of genotypes without transmitting diseases. Obviously, deep freezing of buck semen and application of frozen semen technology are the only alternative left for scientific breeding of goats. However, the success of an AI program in goat depends upon proper semen collection, storage, and use. There is increasing interest in use of frozen-thawed buck semen for AI or invitro fertilization (IVF). At the farm level the control of reproduction allows kidding at a precise season of year, a synchronization of kidding over a limited period of time, and facilitates supplementary feeding to meet the demand of lactation. AI with freshly diluted semen has limitations for rapid loss of sperm motility and fertilizing ability over the time. Besides that continuous provision and maintenance of superior bucks in existing network of AI centers all over the country is difficult and expensive. As seminal plasma of goat is a complex mixture containing wide variety of component that has both beneficial (Baas et al. 1983; Azeredo et al., 2001; Perez et 1 Introduction

23 al., 2001) and detrimental (Cross, 1996; Brinsko et al., 2000; Leboeuf, et al. 2000) effects on spermatozoa. These effects have been explored using ejaculated spermatozoa separated from seminal plasma by washing shortly after collection. However, Cross (1996) found that the effect of seminal plasma did not disappeared after spermatozoa are washed and postulated that ejaculated spermatozoa exposed to accessory sex gland secretions were already subjected to harmful modification in their functions, such as motility and fertilizing capacity. De Pauw et al. (2003) reported that when bovine semen was collected in tube containing a small volume of extender, there was improvement in characteristics of spermatozoa and the effect of the seminal plasma on ejaculated spermatozoa after washing was absolutely different from the effects of accessory gland fluid on epididymal spermatozoa during ejaculation. On the other hand, Way et al. (2000) examined the effect of accessory sex gland fluid on the viability of bovine epididymal spermatozoa and found that accessory sex gland fluids decreased the percentage of motile spermatozoa and accelerated cell death. Blash et al. (2000) also reported differences in motility and acrosome integrity between ejaculated and epididymal spermatozoa after incubation in seminal plasma. The percentage of live spermatozoa and acrosomal integrity were higher for epididymal than those for ejaculated sperm. Additionally they compared the freezability of the epididymal and ejaculated spermatozoa, and confirmed that the post-thawed motility of epididymal spermatozoa was higher than that of ejaculated spermatozoa. Furthermore, it has been reported by Leboeuf et al. (2000) that ejaculated washed spermatozoa are incapable of attaining the quality found for epididymal spermatozoa. Yamashiro et al. (2007) showed that goat semen collected into tubes containing extender with BSA, improved the quality of ejaculated spermatozoa, which strongly suggested that in-vitro functional characteristics of spermatozoa were abruptly modified by sperm contact with accessory sex gland fluid at ejaculation in buck. The ejaculated semen employed for liquid storage and deep freezing are adversely affected by seminal plasma component. If the ejaculated spermatozoa can be collected in a way that minimizes the effect of accessory gland fluids, it would be possible to enhance sperm motility and survival for preservation. It is well established that seminal plasma plays an important role in the fertilizing ability of spermatozoa. The molecular composition of seminal plasma is 2 Introduction

24 very complex. Proteins from seminal fluid have been implicated in the regulation of Ca 2+ uptake by sperm, in the modulation of sperm capacitation (Miller et al. 1990) and regulation of acrosome reaction (Cross, 1993). The contribution of seminal plasma to fertility is a matter of continuous debate; because some components acts as fertility factors, while others acts as anti-fertility factors when evaluated in-vitro (Killian, et al.1993). Bovine seminal plasma contains a family of closely related to heparin-binding proteins (HBPs) (Miller et al., 1990; Chandonnet et al., 1990) and represents the major proteins found in seminal plasma. They are secreted by seminal vesicles and bind the spermatozoa after ejaculation (Manjunath et al., 1988). Phospholipids-binding sites have been shown on the plasma membrane, suggesting that HBPs plays important role in membrane modification that occurs during capacitation process (Desnoyers and Manjunath, 1992). HBPs were also separated from boar seminal plasma, where they appeared to participate in sperm capacitation and zona pellucida binding (Sanz et al.1993). Moreover, the measurement of these proteins in bovine seminal plasma appears to be valuable in predicting small differences in relative fertility among the bulls (Killian et al., 1993; Bellin et al., 1994). Phospholipase A (PLA) synthesized and secreted by bulbo urethral gland in goat seminal plasma causes coagulation and toxicity to the sperm in egg yolk diluents (Roy, 1957) and its secretion is under influence of season. Goat seminal plasma presents a relative differentiation factors synthesized and secreted by bulbourethral glands that causes coagulation and toxicity for sperm in egg-yolk diluents (Iritani et al.1964). This enzyme hydrolyses phospholipids of egg-yolk into unsaturated fatty acids and lysophospholipids, which are toxic to spermatozoa. When skim milk was used as diluents, the incubation of epididymal goat sperm with seminal plasma during non-breeding season decreased motility and viability significantly (Nunes et al., 1982). This effect was not observed when sperm were incubated with seminal plasma collected during breeding season. Substances produced by vesicular glands (which are able to eliminate toxic effect of bulbourethral secretion) responsible for decrease of epididymal sperm motility was deactivated by fractions from vesicular secretions (Nunes et al., 1982). Conflicting data regarding the negative effect of goat seminal plasma on cryopreservation and phospholipase activity on semen have been reported (Corteel, 3 Introduction

25 1981 and Chauhan and Anand, 1990). Despite the relevance of seminal plasma in fertilization, no detailed study has been carried out related to the role of goat seminal plasma components in different seasons. The active factors in seminal plasma affecting preservability of goat semen in different seasons are little understood in tropical conditions and especially in exotic crossbred goats. The specific aim of the present investigation is to study the preservability of semen in relation to heparinbinding proteins (HBPs) of crossbred goat seminal plasma in different seasons. Cryopreservation, a technique for storage of goat semen, has advantages but freezing and thawing induces detrimental effect in terms of sperm structural, biochemical damage, resulting in decreased motility, membrane integrity, and freezability. During cooling, freezing and thawing process spermatozoa undergoes physical and chemical stresses, which can be minimized by adding cryoprotectant in freezing medium and preservability of spermatozoa can be improved. Trehalose acted as better cryoprotectant in the Tris-citric acid glucose (TCG) diluents. Washed semen diluted and frozen in the TCG medium containing trehalose resulted insignificantly greater total sperm motility, progressive motility and path velocity. Also flow cytometric evaluation revealed that plasma membranes of goat sperm treated with trehalose were more fluid at 38 o C. It formed hydrogen bond with polar head group of phospholipids. The insertion of these disaccharides into the membrane limits the amount of dehydration, and consequently the physical damage due to cell volume changes associated with freezing and thawing (Liu et al., 1998). Trehalose also causes an increase in membrane fluidity due to protein and phospholipids reorganization, a suppression of the injurious effect of membrane lipid phase transition and dehydration of cells at lower temperature. The cell damage is minimized due to less intracellular ice formation and consequently more viable cells were recovered following cryopreservation (Aboagla and Terada 2003). Eiman et al. (2003) and Aboagla et al. (2004b) reported that goat sperm freezability was significantly improved when sperm were frozen in a trehalose-egg yolk extender containing an adequate concentration (0.1%) of sodium dodecyl sulfate (SDS). Artificial insemination (AI) with fresh, chilled or frozen-thawed semen is a basic tool in goat breeding, allowing the diffusion of caprine semen with high genetic 4 Introduction

26 value. In recent years, artificial insemination with fresh semen and frozen semen has become a common technique in goats at institutional level, however, the commercial use of frozen thawed semen has been relatively limited due to many bottle necks in small ruminants especially lower conception rate compared to natural service, general awareness and necessary qualified inseminator are being the major restrictive factors. So there is a need to overcome these problems, popularize AI, and to develop simple and viable insemination technique that can be imparted to existing inseminators in the country and genetic improvement can be done successfully. In view of the above gaps in knowledge in a holistic manner, the present research was envisaged to critically examine the effect of processing intervention on cryopreservation of crossbred dairy goat semen with the following specific objectives: 1. To study the freezability of crossbred (Alpine X Beetal) goat semen in relation to heparin-binding proteins (HBPs) in different seasons. 2. Effect of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen. 3. To study in-vitro and in-vivo fertilization with frozen semen. 5 Introduction

27 2. REVIEW OF LITERATURE Artificial insemination, using the frozen semen technology is presently the most extensively employed technique for improvement of genetic potential of livestock species especially bovines. However, comparatively less efforts has been made towards caprine due to many constrains like cost effectiveness of technique, success rate and availability of well trained manpower. The idea that the sperm cell will remain in living condition without expenditure of any significant metabolic energy for an indefinite period inspired the scientist to preserve living cell in the frozen state. Further, it was the spectacular discovery of Polge et al. (1949), which showed that the death of spermatozoa on freezing could be avoided by suspending the cell in a medium containing glycerol. Considerable progress has been made in refining and perfecting the technique for maximizing the recovery of spermatozoa after cryopreservation. Detailed studies on cooling rates, equilibrium time, freezing protocol, thaw rates and their interdependence have been studied for increasing the viability of spermatozoa thus maximizing their availability, resulting in optimum prolificacy of germ plasma from a limited number of superior sires. As fresh semen is not practical in number of situation such as storage for a long period, difficulties in identification, transportation, etc., an improvement in cryopreservation method is necessary. With a view to cover the existing information available on the topic, the review of literature is being presented under relevant segments as follows: 2.1 Semen Quality Attributes: Several genetic, nutritional, physiological and disease factors are known to affect seminal attributes in bucks. The semen profile of all Indian bucks seem to improve significantly in rainy or winter season, generally ahead of the major breeding season in does i. e. rainy and winter (Leboeuf et al., 2000). 6

28 2.1.1 Volume: Variation exists in the volume of semen obtained from different breeds, within breed and even the same male at different time of collection. Volume of semen was reported to vary with breed, age, body size, frequency of collection, exercise, teasing, level of nutrition, season and method of collection. The volume of ejaculate of Alpine and Poitevine breeds was more in autumn and winter, during breeding season and decrease to minimum in the spring and summer, non-breeding season. The sperm concentration of the ejaculate followed an opposite trend reflecting seasonal variation in the secretion and release of seminal plasma from the accessory glands, which were in active when the concentration of testosterone was high during breeding season and quiescent when testosterone was low during the non breeding season. In general young buck and those of smaller size within species, produced smaller volume of ejaculates. Table Semen volume of different breeds of goat Exotic Breeds Age (Yrs.) Volume (ml) References Saanen ± 0.50 Peterson et al., 2007 Florida ± 0.06 Dorado et al., 2007 Saanen ± 0.07 Gacitua and Arav, 2005 Boar ± 0.06 Tuli et al., 1991 Indian Breeds Age (Yrs.) Barberi ± 0.07 Prasad et al., 1970 Jamunapari ± 0.02 Sinha et al., 1981 Black Bengal ± 0.33 Sinha and Singh, 1982 Surati ± 0.10 Jankiraman, 1990 Ganjam ± 0.24 Pattanaik et al ND bucks ± 0.02 Singh and Purbey, 1992 Osmanabadi ± 0.03 Puranik et al., 1993 Crossbred bucks ± 0.02 Alpine x Beetal and Saanen x Beeta l 2 to ± 0.03 Kale,

29 2.1.2 Colour and Consistency: The colour and consistency vary in different species and provides information about quality of semen. The colour and consistency of semen correlates positively with sperm concentration and motility. The number of sperm cells in a given volume of seminal fluid affects its appearances. Semen that is opaque and thick is relatively high and which is clear and translucent is relatively low in sperm concentration. Semen with curd like appearance, containing chunk of material indicates infection of the reproductive system. Kale (1995) studied the semen quality of 10 crossbred bucks in different seasons and observed with creamy colour with thick consistency during cold humid season and creamy colour with thin consistency during dry hot, hot humid seasons. Naskar (1995) observed creamy colour and thick consistency of crossbred (Alpine x Beetal and Saanen x Beetal) matured buck semen. Table Grades of semen categorised by colour and consistency in goat (Kale, 1995) Colour and constancy of semen Thick creamy Thin creamy Thick milk Thin milk Watery type Grades of semen Excellent Very good Good Fair Extremely poor Concentration of Spermatozoa: Correct determination of the number of spermatozoa per ml of semen is extremely important, as it is highly variable semen characteristic with methods and instruments used for estimation. Concentration of spermatozoa varies with the growth and maturity of animal, reproductive soundness, amount of testicular parenchymatous tissue, feeding regime and it reflects the status of testicular function and hormonal relationship. 8

30 Table Mean ± S.E of sperm concentration of goat (10 9 /ml) Breeds Mean±SE (10 9 /ml) References Barbari ± Mittal and Pandey, 1972 Jamunapari ± Dabas et al., 1982 Black Bengal ± Raja-Nasir et al.,1982 Pashmina ± Mazumdar and Mazumdar,1983 Surti ± Jankiraman,1990 Alpine x Beetal and Saanen x Beetal Alpine x Beetal and Saanen x Beetal ± ± Kale, 1995 Chavan,1999 Saanen ± Gacitua and Arav, 2005 Florida ± Dorado et al., Mass motility: Examination of fresh ejaculated semen was done under low power microscope and rating was given on the basis of swirling currents. Mass motility is mostly assessed by graded estimation of the vigour of swirls and wave formation in undiluted semen. It is graded under low magnification of microscope according to mass movement of spermatozoa (0 to 5 point scale). Normal buck ejaculates exhibit a wave like motion when viewed in reflected light. The characteristics phenomenon is believed to be due to flow orientation of sperm on ejaculation. 9

31 Table Mass motility of goat spermatozoa. Breeds Mean References Jamunapari x Black Bengal 4.75 Singh et al., 1985 Chanthangi 4.75 Mahemood et al., 1988 Surti x Marwari 3.50 Jankiraman, 1990 Boer 3.50 Tuli et al., 1991 Black Bengal 4.44 Sinha and Singh, 1982 Osmanabadi 3.36 Puranik et al Alpine x Beetal and Saanen x Beetal Alpine x Beetal and Saanen x Beetal Kale, 1995 Naskar, Progressive motility: Percent progressive motility of sperm has long been used for assessing quality of semen. Good quality semen should contain percent progressively motile sperm. A small drop of diluted semen is placed on a dry slide under a cover slip maintained at 37 0 C and examined at 200X magnification. Jankiraman (1990) reported maximum motility in monsoon (81.47 ± 2.94 and 78.90± 5.89) and minimum in summer (71.25± 3.25 and ± 2.25) for Surti and Marwari buck, respectively. The motility percent for Ganjam buck was 75 to 80 percent (Pattanaik et al., 1991). Tuli and Holtz (1992) observed higher values for initial motility during winter (71.23V± 2.52) than in summer (68.66±2.25). However, seasonal influences were not significant. Kale (1995) reported in a study carried out on 10 crossbred bucks (Alpine x Beetal and Saanen x Beetal) that mean progressive motility percentage was 77.75± Season and buck have significantly (p<0.01) influenced the percent progressive motility of the semen. 10

32 Table Mean ± SE progressive motility estimate of goat spermatozoa (%) Breeds Progressive motility % References Saanen ± 0.68 Barbari ± 1.50 Surti ± 2.94 Marwari ± 5.89 Pandey et al Jankiraman, 1990 Boer ± 1.50 Tuli et al., 1991 ND goat ± 0.40 Osmanabadi ± 2.01 Crossbred ± 1.90 Alpine x Beetal and Saanen x Beetal Alpine x Beetal and Saanen x Beetal Alpine x Beetal and Saanen x Beetal Singh and Purbey,1992 Puranik et al., ± 0.77 Kale, ± ± 1.59 Naskar, ± 1.38 Chavan, Post thaw motility (PTM): Table Post thaw motility (%) of goat spermatozoa Breed PTM (%) References Saanen Gacitua and Arav, 2005 Shiba Aboagla and Terada, 2004a Saanen Aboagla and Terada, 2004a Florida Dorada et al., 2007 Black Bengal Singh et al.,1995 Jamunapari Tiwari and Bhatacharya, 1987a Sirohi Sinha, 2000 Barbari Sinha, 2000 Marwari Sinha,

33 Post thaw motility is the main criteria for freezability of semen that differs from individual, breed, use of extender, freezing protocol, thawing process Seasonal variation in breeding activity and sperm production Length of breeding season in goats varies inversely with latitude in temperate climate. At the mid high latitudes (35 o ) goat shows marked seasonality. In tropics and subtropics goat bred all time and kidding is related to environment other than photoperiod (Murugaiyah, 1992). However, in seasonally breeding buck; sexual behavior, testicular size and sperm production influenced by photoperiod. Sexual activity decrease in spring and resumption occurs in late summer and autumn in temperate climate. Semen volume is high in autumn and winter which is breeding season. Semen concentration is high but volume is less in spring and summer-called non breeding season. Quality of semen is also affected by season. Motile sperm percentage is high in breeding season while lower in non breeding season (Leboeuf et al., 2000). In Indian goats volume and concentration decreased with high temperature, relative humidity (RH) and high rainfall (Murugaiyah, 1992) Effect of season on freezability of goat semen The influence of season on post freezing in northern temperate zone in boer goat was reported and no difference was found for motility and live sperm in summer and autumn (p>0.05). However, post thaw motility (45%) and live sperm (49%) were significantly higher (p>0.05) in winter than summer and autumn (Tuli and Holtz, 1995). Table: Freezability of goat semen in temperate region (Tuli and Holtz, 1995). Seasons Motility % Live Sperm % Spring 29 ± 2 a 32 ± 3 a Summer 23 ± 3 a 27 ± 4 a Winter 45 ± 3 b 49 ± 3 b 12

34 2.2.2 Seminal plasma In mammals, seminal plasma is a complex mixture of secretions from the epididymis and various accessory sex glands, as well as their chemical composition and functions varied among the species. Molecular composition of seminal plasma is very complex. Seminal plasma plays an important role in fertilizing ability of sperm. Proteins from fluid have been implicated in the regulation of acrosome reaction, Ca 2+ uptake by sperm and in the modulation of sperm capacitation. Anti-fertility activity is also related to seminal components in variety of animals including rabbits, bull, mice, and humans. The contribution of seminal plasma to fertility factors, while other acts as anti-fertility factors when evaluated in vitro was reported by Bellin et al., (1994). Bovine seminal plasma contains a family of closely related heparin binding proteins (HBPs). These represent the major acid proteins found in seminal plasma secreted by seminal vesicles and bind to spermatozoa after ejaculation. Phospholipid binding sites have been shown on the sperm plasma membrane, suggesting that the HBPs play an important role in the membrane modification that occurs during capacitation process. HBPs in participate in sperm capacitation as well as zona pellucida binding. Moreover, the measurement of these proteins in bovines appears to be valuable in predicting small differences in relative fertility among bulls (Bellin et al., 1996) Effect season on seminal plasma and its removal The reproductive activity of goat is influenced by the season, with photo period being the main environment signal timing the reproductive cycles. And the reproductive activity involves seasonal changes in testicular weight and size, sperm production, testicular secretion, mating activity, fertility and seminal plasma proteins (Leboeuf et al., 2000, Falci et al., 2002). Washing of spermatozoa in a physiological solution increased the percentage of motile spermatozoa and their motility before and after freezing. The improvement in the capacity to withstand freezing thawing was observed in milk and egg yolk based diluents (Corteel, 1974). In buck, removal of seminal plasma by washing the spermatozoa immediately after collection increased the percentage of live cells and their motility during storage in egg yolk and milk diluents but ejaculated and washed semen was unable to attain the quality found for epididymal semen (Leboeuf et al., 13

35 2000). Ejaculates collected in breeding season contain more seminal plasma than those collected in the non-breeding season, and a high negative correlation between the volume and of seminal plasma in ejaculate and the percentage of motile cells after thawing was found on a full year basis. The percentage of motile cells after freeze-thawing was proportional (r= 0.9) to the concentration of sperm cells and conversely proportional (r= - 0.9) to the initial volume of the ejaculate. When the invitro survival of ejaculated sperm and epididymal goat sperm was examined in milk diluents, percentage of live spermatozoa and the motility after cooling to 4 o C, freezing thawing and incubation at +37 o C was found to be higher for epididymal than ejaculated sperm (Chemineau, 1978 cited from Leboeuf et al. 2000). Ritar and Salmon (1991) found that the concentration of egg yolk in diluted semen that allowed acceptable sperm survival rates during the 6 hrs post thawing incubation varied from 0% to 12%, depending on the month of collection. Moreover, the recovery after thawing was similar to unwashed cells, but washing markedly improved survival during incubation at 37 o C for 6 hrs, especially when egg yolk (1.5% and 6%) was present. In spite of the favorable effect of sperm washing, a difference between seasons of collection was observed. Thus, the attitude to withstand freeze-thawing condition was better for spermatozoa obtained during the sexual season than the outside the sexual season (Corteel et al., 1980). The seminal plasma produced in non-breeding season was more detrimental on motility and survival rate of epididymal spermatozoa in skim milk extender than seminal plasma from breeding season. It was suggested that the negative effect of bulbourethral gland secretion partially inhibited by the presence of vesicular gland secretions during breeding season (Nunes et al.1982). Ritar and Salmon (1983) recommended that washing may be avoided in extenders containing small amount of egg yolk concentration less than 1.5 percent. However, washing is required when egg yolk concentration more than 10 percent is used in the extender. Dilution and centrifugation of the semen to remove the seminal plasma before extension and freezing is complicated, time consuming, may cause some loss of sperm and can affect viability. Goat seminal plasma presents a relevant differentiation factor synthesized and secreted by bulbourethral glands that causes coagulation and toxicity to 14

36 spermatozoa. The substance identified as phospholipase A (PLA), and its secretion is under seasonal influence. This enzyme hydrolyzes phospholipids from egg yolk diluents into unsaturated fatty acids and lysophospholipids, which is toxic to spermatozoa. When the skimmed milk was used as diluents, the incubation of epididymal sperm with seminal plasma collected during non-breeding season leads to a significant decrease in motility and viability. This effect was not observed when the seminal plasma collected during breeding season and incubated with epididymal sperm. Substances produced from vesicular gland, which able to eliminate the toxic effect of bulbothral secretion were separated by liquid chromatography on Sephadex G-100. The protein fraction responsible for decrease of epididymal sperm motility was deactivated by fraction from vesicular secretions (Nunes et al., 1982) Seasonal variation of seminal plasma proteins (Heparin Binding Proteins) Kaur (2001) found that Electrophoretic profile of seminal plasma and detergent extracted whole sperm proteins reveals most of common proteins. Lyophilized seminal plasma protein showed inhibitory effect on capacitation. HBPs purified by heparin affinity chromatography were in basic nature (Mol. Wt. 33 & 15.5 kd). HBPs exerted stimulatory effect on capacitation, choline phospholipids efflux. Heparin binding proteins present in the seminal corresponded to Mol. wt 189 Kd 26.9 and 15.8 Kd. Kaur and Sharma (2009a). They isolated the seminal plasma proteins with radiolabelled-i 125 by Lactoperoxidase-catalysed radioiodination method. The iodinated seminal plama proteins were being 158, 95.7, , 28.8, 25.1, 19.0, 15.8, and 8.3 Kd. Most of the iodinated proteins were adsorbed on sperm surface only the proteins of MOL. WT. 45.7, 38.9, and 19 Kd were not adsorbed on the surface of epididymal spermatozoa (Kaur and Sharma 2009b). Falci et al. (2002) investigated seasonal changes in seminal plasma protein of Saanen goat under natural conditions. The pattern of Heparin binding proteins (HBPs) such as a band of 178 kd unique to breeding season; a decrease in 119 Kd proteins; and an increase in protein ranking from 73 to 104 kd. HBPs caused deterioration of sperm motility and acrosome breakage in media containing and not containing skimmed milk; the effect was most remarkable with the protein from nonbreeding season. Furthermore, they found that HBPs presented phospholipase A2 (PLA2) activity, which was 4.4- fold higher in non-breeding season than in breeding 15

37 season. Binding site found for HBPs was identified in the sperm surface, at the middle piece of the spermatozoa. Thus proteins from goat seminal plasma are under seasonal control and associated with sperm function during breeding and nonbreeding seasons. Barbara et al. (2005) purified whole seminal plasma of goat and found to display both lipase and Phospholipase-A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mrna isolated. The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices. From bulbourethral gland DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The most common cryopreservation diluents used for goat semen contain either egg yolk or non fat dried skim milk. However, these are detrimental to goat sperm. The harmful interaction between seminal plasma and egg yolk was first documented by Roy (1957). He observed that when neat semen was added to the egg yolk, it was coagulated and sperm died due to Egg-yolk coagulating enzyme (EYCE). The sperm cell maintained their motility after removal of seminal plasma. Nunes et al. (1982) identified a bulbourethral gland protein which decreased survival of frozen goat sperm diluted in milk based media. It also induced acrosome reaction and subsequent death of spermatozoa incubated in milk medium at 37 0 C. The EYCE was identified as phospholipase A as kda glycoprotein lipase from goat bulbourethral gland (BUSgp60) (Pellicer-Rubio et al., 1997). This hydrolysis causes the sperm membrane more fusogenic thereby inducing the acrsome reaction (Upreti, 1999), and chromatin decondensation which is toxic to sperm (Purdy, 2006). BUSgp60 has structural homology to porcine pancreatic lipase (Carriere at al., 1994) and similar to EYCE. BUSgp60 is responsible for hydrolysis of 16

38 plasma membrane triglycerides and triglycerides in the milk that result in fatty acid production (lysolecithine production with egg yolk and olic acid with milk triglycerides) which is toxic to the sperm (Pellicer-Rubio et al., 1997; Pellicer-Rubio and Combarnous, 1998). The conventional method of overcoming the harmful interaction of seminal plasma and egg yolk or milk protein is to dilute the goat semen in a buffered diluent and then separate the seminal plasma from the sperm by centrifugation. While other reports positive results without washing (Azeredo et al., 2001). Alternative diluents that minimize the sperm and lipase interactions have been proposed, including adding BUSgp60 lipase inhibitors, using lipid free cow milk, a triglycerides-free diluent containing the milk protein casein, or using milk other than cow where fatty acid and triacylglycerol structure differ so that enzymatic reaction do not occur (Pellicer-Rubio and Combarnous, 1998) Effect of modified method of semen collection From above discussion it can be stated that seminal plasma is a complex mixture containing a wide variety of components that have both beneficial and detrimental effects on spermatozoa. Cross (1993) found that the effect of seminal plasma on ejaculated spermatozoa did not disappear after spermatozoa were washed, postulating that ejaculated spermatozoa exposed to accessory sex gland secretions were already subject to harmful modifications in their functions. De Pauw et al. (2003) reported that when bovine semen was collected in tubes containing a small volume of extender supplemented with egg yolk, the characteristics of ejaculate spermatozoa were improved. Thus, it is conceivable that the effects of seminal plasma on ejaculated spermatozoa after washing are absolutely different from the effect of accessory gland on epididymal spermatozoa during ejaculation. On the other hand Way et al. (2000) examined the effect of accessory gland fluid on viability of bovine epididymal spermatozoa and demonstrated that exposure of epididymal sperm to accessory gland fluids decreased the percentage of motile spermatozoa and accelerated cell death. Blash et al. (2000) also reported that when differences in motility and acrosome integrity between the ejaculated and epididymal spermatozoa after incubation in seminal plasma were assessed, the percentage of live sperm and acrosome integrity for epididymal sperm were higher than those for ejaculated sperm and freezability also were higher for epididymal spermatozoa than 17

39 the ejaculated spermatozoa. Furthermore, it is has been reported that ejaculated even washed spermatozoa unable to attain the quality found for epididymal spermatozoa (Lebouef et al., 2000). In case of practical application in animal production, it is possible that ejaculated spermatozoa employed for liquid and deep freezing can be collected in a way that minimizes the effect of accessory reproductive fluids and it would be possible to enhance sperm motility and survival of preservation. Yamasiro et al. (2007) found that goat semen collection in tubes with large volume of extender improved motility, progressive motility and acrosome integrity of frozen-thawed spermatozoa (p<0.05) significantly. Table: Improvement of sperm motility by modified method of collection (Yamasiro et al. 2007). Goat Treatment (TCG) Motility % Progressive Motility % Path velocity u/s A 0 ml 83.0± ± ±6.8 1 ml 82.3± ± ± ml 87.3± ± ±7.0 B 0 ml 54.3± 2.4 a 37.0±2.0 a 110.0±4.0 1 ml 54.6±2.4 a 41.6±1.7 a 98.0± ml 72.3±1.4 b 51.6±4.2 b 99.3±8.2 Table: Motion parameters and recovery rates of spermatozoa collected into tubes containing 10 ml of extender supplemented with BSA (Yamasiro et al. 2007). Motility % Progressive motility % (P<0.05) Treatment TCG + After collection Postthaw Recovery rate 0 ml 82.0± 2.0 a 56.6±5.9 a 68.3± ml 86.6±1.8 ab 68.0±5.5 ab 79.3± ml + BSA 0.1% 84.3±2.8 ab 67.3±5.4 ab 75.3± ml + BSA 1% 85.5±0.3 ab 68.3±4.6 ab 75.3± ml + BSA 5% 91.6±0.3 b 77.3±3.7 b 89.0±7.0 0 ml 55.0±1.7 a 42.0± ± ml 59.6±1.3 ab 47.3± ± ml + BSA 0.1% 58.0±3.5 ab 48.6± ± ml+ BSA 1% 63.3±2.6 bc 48.0± ± ml + BSA 5% 67.6± 2.3 c 48.6± ±7.0 18

40 Cryopreservation Diluents The purpose of a cryopreservation diluent is to supply the sperm cell with source of energy, protect the cell from temperature related damage and maintain a suitable environment for the spermatozoa to survive temporarily. Each of different components comprising the media has been investigated separately and in combination, to maximize the post-thaw sperm viability and fertility. In general goat sperm cryopreservation medium includes non penetrating cryoprotectant (milk or egg), a penetrating cryoprotectant (glycerol, ethylene glycol, or dimethyl sulfoxide), a buffer (Tris), one or more sugars (glucose, lactose, raffinose, sacchrides, or trehalose), salt (Sodium citrate, citric acid) and antibiotics (penicillin, streptomycin) A non fat dried skim milk diluent or Tris-glucose diluents are most commonly used for cryopreserving goat sperm. Modifications of these diluents have been investigated with variable results (Singh, et al., 1995; Keskintepe et al., 1998; Blash et al., 2000). To address these issues specific about goat semen cryopreservation the following media (osmolality, ph and buffers, sugars, cryoprotectants) review are presented Osmolality Goat spermatozoa survive cryopreservation and remain fertile in media composed of a wide range of constituents. Different sugars, salts and buffers, for example, can be included in varying molar concentration in the cryopreservation medium, without injuring the spermatozoa. While the osmolality may vary within the limits, goat spermatozoa prefer a hyper osmotic medium for cryopreservation. Bowen et al. (1988) reported that less damage occurred to goat sperm when frozen in diluent having osmolaties ranging between 425 and 525 mosm compared to media having an osmolality of either 325 or 625 mosm ph and buffering systems Large changes in semen ph can result in sperm damage, infertility, or sperm mortality. Therefore, in order to sustain viability and freezability of sperm, it is essential to maintain a proper environment by controlling the ph fluctuations in cryopreservation media. One of the functions, seminal plasma plays, is to buffer the sperm cells against changes in ph, but this is meant to occur in-vivo and not under 19

41 in-vitro conditions which occur during cryopreservation. For this reason, substances that buffer ph changes are routinely included in cryopreservation medium to minimize the changes. In general buffering solution should have a ph of 7.0 (ph range 6.0 to 8.0), be water soluble, membrane impermeable, have minimum interactions with salts, be minimally affected by buffer concentrations, temperature and ionic content on dissociation of buffer metal complexing properties, be able to withstand enzymatic and non enzymatic degradation (Purdy, 2006). Although, many investigators have determined the effect of ph on buck sperm, the ph of cryopreservation medium is not frequently reported. However, the ph of egg yolk or milk based medium goat sperm diluent should range between 6.75 and 6.8 (Ritar and Salamon, 1982). Jaiswal and Mazumdar (1998) observed that altering the extra cellular ph increases the intracellular ph and indicate forward motility in caput-epididymal sperm. Increasing the medium ph 7 to 8 stimulates about 50% of cauda epididymal sperm to become motile, indicating a sensitivity of sperm to ph fluctuation. In addition, increasing intra cellular ph activates downstream or parallel pathways that activates protein kinase A, indicator of capacitation in mammalian sperm cell. Salamon and Ritar, (1982) investigated Tris buffer across a range of concentration and recorded an interaction between Tris concentration and the sugars present in the medium. Post-thaw motility was greater with Tris buffer, at any concentration (300, 375, or 450 Mm), being combined with 21, 42, or 62 Mm glucose (mean total motility of all concentrations, 33% or fructose (33%). The addition of lactose (29%) or raffinose (17%) resulted in lower percentages of motile sperm cells. It was concluded that under condition tested, buck semen prefer fructose or glucose, when combined with Tris, and that the concentration of the sugars required is very small ( mM) for cryopreservation. Molinia et al. (1994) reported greater cryoprotective effects for monosaccharides than disaccharides, when used in combination with Tris. Many theories has been proposed, but it is generally believed that buffer aid in the cellular dehydration process by creating an osmotic force, thereby increasing the physical stability of the sperm cell membrane, and neutralizing acid during invitro storage. Tris is buffer choice for use with buck semen, but this is not in case of 20

42 other species where negative aspect of buffer, such as increased capacitation and acrosome reaction rates, and swelling of apical ridge of sperm cell, have been documented (Ijaz et al., 1989). In other mammalian species (bovine and ovine) Tris is a poor buffer particularly below a ph of 7.5, where it is unable to withstand temperature fluctuations. The ph of the media can decrease as much as 1 ph unit when Tris media was warmed from 0 to 37 0 C (Purdy, 2006) Effect of trehalose incorporation in semen extender It is logical to include sugar in cryopreservation diluents, as seminal plasma contains sugars. Goat semen readily utilizes fructose, glucose lactose and other sugars for respiration and these sugars also provides osmotic balance and cryoprotection, but of all the sugars, fructose has greatest molar concentration in neat semen (Salamon and Ritar, 1982; Aboagla and Terada, 2003). Sucrose and glucose are low molecular weight molecules that can pass through the plasma membrane of a sperm. Various effects can be observed when sugars such as lactose, sucrose raffinose, trehalose, or dextrans are added to diluents. In these instances, sugar creates an osmotic pressure, inducing cell dehydration and therefore, a lower incidence of intracellular ice formation. These sugars also interacts with phospholipids in a plasma membrane, reorganizing the membrane which results in sperm that is better suited to surviving the cryopreservation process (Aisen et al., 2002) unlikely the simple sugar glucose and fructose, these disaccharides acts primarily as cryoprotectants. Eiman et al. (2003) reported that trehalose acted as cryoprotectant in the Triscitric acid glucose (TCG) diluent developed by Salamon and Ritar, 1982). Washed semen samples were diluted and frozen in the TCG medium containing 0, 93.75, 187.5, or 375 mm trehalose. They observed that post-thaw motility of sperm frozen in the presence of 375 mm trehalose resulted in significantly greater total sperm motility (78%), progressive motility (61%) and path velocity (96u/s), compared to control 0 mm trehalose treatment (62%, 49%, 81u/s respectively) (p<0.05). Flow cytometric evaluation revealed that plasma membranes of goat sperms treated with trehalose to be more fluid at 38 0 C and forms hydrogen bond with polar head group of phospholipids. The insertion of these disaccharides into the membrane limits the amount of dehydration that can occur, and consequently the physical damage due to 21

43 cell volume changes associated with freezing and thawing (Liu et al., 1998). Trehalose also causes an increase in membrane fluidity due to protein and phospholipids reorganization, a suppression of the injurious effect of membrane lipid phase transition; accordingly a dehydration of cells at temperatures lower than Cell damage is minimized due to less intracellular ice formation and consequently more viable cells being recovered following cryopreservation (Eiman et al. 2003). In spite of this evidence in favour of the beneficial effects of trehalose, not all studies have found the same results. Chen et al. (1993) reported that trehalose caused only minor improvement in bull sperm survival. Liu et al. (1998), in a study on the freezability of bull spermatozoa in TCG extender containing up to 25% (v:v) trehalose or sucrose, concluded that replacing part of TCG-containing egg yolk with these sugars had no beneficial effects. The low concentrations of trehalose (0.05, 0.1 M) used by these researchers may account for the low improvement in sperm survival because trehalose in high concentrations was used by the many authors who found significant protection against freeze damage (Iwakiri et al., 2000). The addition of high concentrations of trehalose to sperm extender provide the best protection with regard to post-thaw motility parameters, recovery rates, thermal resistance, and acrosome integrity, with the best results obtained for the 100% trehalose extender. However, supplementation of the diluent by trehalose has been shown to have varying effects on freeze tolerance (Molinia et al., 1994; Yildiz et al., 2000). Woelders et al. (1997) demonstrated that an isotonic sugar medium in which Tris-citrate components were substituted with sucrose and trehalose was significantly superior to a Tris-citrate egg yolk medium in preserving the motility and acrosome integrity of bovine spermatozoa. Moreover, in a comparison of raffinose and trehalose, Storey et al. (1998) showed that trehalose resulted in a significantly better recovery rate in intact mouse spermatozoa. In addition, Aisen et al. (2000) observed that trehalose significantly improved the viability of ram spermatozoa assessed for motility and acrosome integrity, with the best results obtained for a trehalose + EDTA extender. Molinia et al. (1994) concluded that the motility of frozen-thawed ram spermatozoa was higher in the presence of sucrose or trehalose than in the presence of glucose when glycerol was not incorporated in the diluent; whereas when glycerol was employed in the diluent, no differences were observed among the 22

44 various sugar types. Frozen-thawed dog spermatozoa have also been found to be protected by the supplementation of trehalose in the diluent (Iwakiri et al., 2000) Cryoprotectants A cryoprotectant is included in cryopreservation medium to minimize the physical and chemical stresses resulting from the cooling, freezing and thawing of sperm cells. Cryoprotectants are classified as either penetrating or non-penetrating. A penetrating cryoprotectants is membrane permeable and acts intra and extracellularly. Penetrating cryoprotectants are solutes, which causes dehydration of spermatozoa due to osmatically driven flow of water, which varies according to compound. After short periods of time the cryoprotectant and water equilibrate and results in similar intracellular and extracellular concentrations. As the sperm cell has less intracellular water, the freezing point of cell decreased and less intra cellular ice formation; which is beneficial, because intracellular ice results in cell death, and consequently reduced fertility of semen sample (Purdy, 2006). Holt (2000) reported that the penetrating cryoprotectant also caused membrane lipid and protein rearrangement, which increased membrane fluidity, greater dehydration at lower temperatures, therefore, an increased ability to survive cryopreservation. Additionally, penetrating cryoprotectant are solvents that dissolve sugars and salts in the cryopreservation medium. A non-penetrating cryoprotectant cannot cross the sperm plasma membrane and therefore, only acts extracellularly. Therefore, a non-penetrating cryoprotectant may modify the plasma membrane of a cell, or act as a solute and lower the freezing temperature of the medium (Purdy, 2006). Many membrane-permeable cryoprotectants (glycerol, dimethyl sulfoxide, ethylene glycol, and propylene glycol), and their combinations, have been tested with buck sperm, but the most frequently used penetrating cryoprotectant is glycerol (Purdy, 2006). The addition of glycerol may be performed in a 1-, 2-, or 3- step methodology at either 37 0 C or 5 0 C (Leboeuf et al., 2000). The final concentration (v/v) of penetrating cryoprotectant in a medium varies but is determined by the toxicity of chemical, and its beneficial effect on the spermatozoa. Glycerol, dimethyl sulfoxide and ethylene glycol are generally used in the range of 1-8 % but greatest post-thaw recovery of sperm has been achieved with glycerol. Combination of 23

45 cryoprotectants, such as glycerol and dimethyl sulfoxide have been used and yielded positive results (Purdy, 2006). Kundu et al. (2000) observed that using glycerol (6%) alone as cryoprotectant resulted in higher percentage of motile sperm following cryopreservation (35%), compared to sperm frozen using ethylene glycol (13%) or dimethyl sulfoxide (21%). Kundu et al. (2001) further reported that the use of both glycerol (6%) and dimethyl sulfoxide (5.9%) achieve synergistic effect of cryoprotectants. The post thaw motility using glycerol and dimethyl sulfoxide media separately was 33% and 15%, respectively, while combination of the cryoprotectant resulted in 45% progressively motile spermatozoa. Addition of glycerol can induce osmotic damage to spermatozoa but the extent of damage varies according to species. However, goat spermatozoa are reasonably tolerant to these osmotic conditions and can withstand a rapid exposure to glycerol. Trials testing stepwise addition of glycerol have yielded variable results, depending on temperature at which the glycerol was added. Salamon and Ritar (1982) reported a single step glycerol addition to semen sample at 30 0 C to resulted in more motile spermatozoa, compared to a 2-step glycerolization at 30 0 C, or a 2-step glycerolization at decreasing temperatures (glycerol dilution at 30 0 C and dilution at 5 0 C). Tuli and Holtz (1994) reported a step-wise method of glycerolization at 37 0 C to results in similar proportions of progressively motile and live sperm, compared to a step-1 glycerolization at 37 0 C, or a step-wise dilution at 5 0 C, but the step-wise method of glycerolization at 37 0 C had significantly less GOT (glutamic oxaloacetic transaminase) release (99 units/ml) after thawing compared to other method (105 and 108 units/ml, respectively; p<0.05). From these results, it appeared that glycerol could be added to sperm in a single step. The most common non penetrating cryoprotectants used are egg yolk (2-20%) (Ritar and Salamon, 1982; Tuli and Holtz, 1994) and non-fat skim milk (10%, w/v) (Leboeuf et al, 2000). Recent research also identified other membrane impermeable chemicals that may be used as cryoprotectant. Kundu et al. (2001), using cauda epididymal sperm 24

46 and egg yolk and glycerol free medium, demonstrated that amino acids (L-proline, glyceine, or L-glutamine; mM) could be used as cryoprotectant in a goat cryopreservation media and yield better post-thaw results (8-14% forward motility and 11-19% total motility recovery), compared to control group (0% motile cells with 0 Mm amino acids). The amino acid exerted even greater protection when combined with glycerol (0.87M) and dimethyl sulfoxide (0.76M), which resulted in post-thaw progressive motility of 50-55%. Kundu et al. (2002) reported that dextran ( kDa) may be added as non-penetrating cryoprotectant in the same egg yolk and glycerol-free medium, and yield 23 and 25% total and progressive motility, after post thawing. The greatest percentages of forward (58%) and total sperm motility (60%) was recorded with 6.27 mm of 10 kda dextran in combination with glycerol (0.87M) and dimethyl sulfoxide (0.76M) compared to control (22% forward motility and 25% total sperm motility). Kundu, et al. (2002) hypothesized the combination of glycerol, dimethyl sulfoxide and the dextran to result in additive beneficial effect during cryopreservation, because of molecular masses of the different chemicals. Glycerol and dimethyl sulfoxide are membrane permeable and therefore, will dehydrate the spermatozoa and minimize the intracellular ice formation. As dextran is membrane impermeable, its effect will be greatest outside the sperm cell, most probable by interrupting ice formation extra cellular. Consequently, this combination of chemical substances results in cryoprotective action that is both intracellular and extracellular. Kundu et al. (2000, 2001 and 2002) demonstrated that goat sperm can be cryopreserved in egg yolk and skim milk free media, but these experiments were carried out on only the cauda epididymal sperm. Aboagla and Terada (2004a) conducted experiments to find out effects of egg yolk on freezing step of cryopreservation on viability of goat spermatozoa. Post-thaw motility and progressive motility and recovery rate was significantly higher in frozen semen with egg yolk than without egg yolk. The intact acrosome were significantly (p<0.05) higher in trehalose extender without egg yolk than with egg yolk (74.0±3.9 Vs 49.0±1.3). 25

47 They suggested that incubating the spermatozoa in a diluent containing egg yolk and glycerol before freezing could have been played a part in increasing the percentage of sperm undergoing the acrosome reaction, resulted in a lower post-thaw acrosome integrity percentage after freezing and thawing.the recovery rates of frozen thawed sperm acrosome were higher (P<0.05) for the semen samples frozen in trehalose extender containing no egg yolk than those frozen in extender containing egg yolk. They found that addition of egg yolk played major role during freezing steps of goat spermatozoa, and that the addition of trehalose increased significantly its cryoprotective effects. They concluded that neither glycerol nor egg yolk alone reduced the intact acrosome percentage; however, combination of these two major cryoprotectants significantly reduced the percentage of intact acrosome spermatozoa. Hidalgo et al. (2007) found that the changes in sperm head morphometry of cryopreserved samples might be a consequence of chromatin over condensation or to acrosomal damage which caused reduced fertility Methods of Freezing Comparison between methods and protocols for freeze-thawing of semen are difficult because different parameters involved and lack of uniformity in methodology. The various procedures may differ by following points: a) Males selected on freezability of their semen. b) Use of ejaculates devoid of or containing seminal plasma, washing method, composition of washing solution, intensity of washing: dilution rate and centrifugation. c) Composition of freezing diluents and presence or absence of egg yolk. d) Method of dilution, glycerol addition and its concentration. e) Inclusion or avoidance of equilibration time and its duration before freezing. f) Packaging methods for semen in mini (0.25), medium (0.5) straws, or in pellets. Leboeuf et al. (2000) reported that goat spermatozoa were first frozen (-79 0 C) by Smith and Polge (1950). They stated that fertility of frozen-thawed semen was too low to be of practical value. The method of freezing and diluents used by earlier workers were those that proved successful in bull. They reported fertility results varied from poor (3-15%), moderate (20-48%) to satisfactory (up to 70%) in goat. They processed semen with seminal plasma, despite the finding detrimental effect of goat seminal plasma. 26

48 Conventionally semen cooled at C and diluted and subsequently inclusion or avoidance equilibration, freezing is done in straws and pellet form. Straws containing the semen are suspended horizontally in liquid nitrogen vapor 4-5 cm above the liquid nitrogen (LN) level for 4-5 minutes, and then are immerged into the liquid nitrogen. In France, it is recommended to suspend the straws above the LN in two steps, first at 16 cm for 2 minutes, then at 4 cm for 3 min. before immersion into LN. The velocity of cooling can be regulated by the distance of straws from the level of LN and the size of straw: fine (0.25 ml) or medium (0.5ml) (Leboeuf, 1989). Pellet freezing on dry ice at 79 0 C and immersion of pellets in LN at C is a rapid and simple method. The velocity of cooling to 79 0 C can be regulated by the volume of pellet. The volume of semen in the straw and pellet had a marked effect on packaging method. However, freezing of straws on a pre-cooled 1 cm wire mesh or perforated plate gave better viability results than freezing on racks giving only two contact points with straws at their ends. This has been attributed to earlier seeding by multiple contacts with the rack which most probably minimizes super-cooling. Although, post-thawing viability (Ritar et al., 1990b) and fertility were better following pellet than straw freezing. Straw freezing is time consuming. Freezing of sperm in pellets is rapid and inexpensive, but inventory management is problematic because the actual semen samples cannot be labelled. However, straws are more preferred by most commercial traders, as it allows more accurate identification of semen doses. In pellet freezing method, semen sample is cooled, semen aliquots of ml are dispensed into indentations on a block of dry ice (solid carbon dioxide; C) and frozen for 2-4 min. The pellets are then plunged into liquid nitrogen for storage. In straw freezing method diluting and cooling the samples, the sperm are loaded into 0.25 or 0.5 ml straws, placed on racks and frozen in liquid nitrogen vapor; the temperature of which varies by height above liquid nitrogen or a programmable freezer that controls the cooling rate have both been successfully used to cryopreserve the goat sperm. When using Styrofoam box, the rack containing the samples is placed into the liquid nitrogen vapor at a height of 3-4 cm above the liquid for 7-8 minutes and the straws are then plunged into liquid nitrogen for storage. Alternatively, size of straw determines the freezing height above the 27

49 liquid nitrogen. It was suggested that 0.5 ml straw should be frozen at 4 cm above liquid nitrogen for 5 min, while 0.25ml straws should be lowered to 4cm for 3 min, and plunged into liquid nitrogen for storage, however, other freeze heights and time have been quoted e. g., 4-5cm above liquid nitrogen for 4-5 minute with acceptable results (Gravance, et al., 1997; Leboeuf et al., 2000). Blash et al. (2000) reported that programmable freezers are convenient for freezing the large quantity of semen straws and for controlling the rate of freezing. The freezer may replicate pellet freezing by placing the semen straws in freezer at 80 o C for 7-15 min and then plunging into liquid nitrogen. The benefit of programmable freezer with customized freezing curve e.g., 4 to 5 0 C at 4 0 /min, -5 to C at 25 0 /min and 110 to C at 35 0 / min, and then semen straws can be plunged into liquid nitrogen was standardized by IMV Corporation, USA. The notable variation in fertility rates of goat semen before introduction of Corteel s washing method, apart from other factors, could have been partly attributed to different equilibration period which varied from 1 to 24 h. the most acceptable equilibration time was 1 h and 3 h (Corteel, 1974, Das and Rajkonvar, 1995, Singh et al., 1995). Washing of goat semen consist of dilution of freshly collected ejaculates with a washing solution (1:5-1:10) and centrifugation for minutes at 1000g. Different washing solutions were used such as Krebs-Ringer phosphate buffer (with or without glucose) or the freezing diluent (skim milk, tris or other media) without glycerol. Corteel (1974, 1975) and Corteel and Lebouf (1990) developed a method in which washed spermatozoa were re-suspended with non-glycerated diluent to half the final concentration and cooled 30 0 C to 4 0 C in 1 h and further diluted with glycerol containing diluent portion (14%) to a cell concentration of 400 to 500 X 10 6 ; (the final glycerol concentration is 7%). Then they followed a 1 to 3 h equilibration period at 4 0 C before freezing. The washed spermatozoa can be re-suspended also with the glycerol- containing diluent to final cell concentration or dilution rate, cooled in 1 h to C, further equilibrated and then frozen Freezing protocol: Freezing is lethal to most of the living system and spermatozoa are no exception. Yet it can also preserve cells and their constituents, and it may someday 28

50 permit the long-term storage of whole viable organs. It can slow or stop some biochemical reactions, but it accelerates others. Contrary to usual impression, the challenge to cell during freezing is not their ability to endure storage at very low temperature; rather it is the lethality of intermediate zone of temperature (-15 to C) that a cell must traverse twice- once during cooling and once during warming (Aleandri et al., 1998). Down to -5 0 C, the cells and their surrounding medium remain unfrozen both because of super-cooling and because of the depression of the freezing point by the protective solute that are frequently present, between -5 and C ice forms in the external medium (either spontaneously or as a result of seeding the solution with an ice crystal), but the cell contents remain unfrozen and super cooled, presumably because the plasma membrane block the growth of ice crystal into the cytoplasm. The super cooled water in the cell has a higher chemical potential than that of water in partly frozen solution outside the cell, and the response to this difference in potential, water flows out of the cell and freezes externally (Gilmore et al., 2000) The optimum freezing protocol for cauda epididymal sperm cells of goat in computer controlled biofreezer (room temperature (30 ± 2 0 C) to 5 0 C at C per minute; 5 0 C to C at 5 0 C per minute ; and C to C at 20 0 C per minute) was reported by Kundu et al. (2000; 2001 and 2002) Thawing of semen samples Thawing sperm samples is determined by the method used to freeze the sperm. Sperm pellet should be thawed in a dry test tube at 37 0 C while thawing of straws may be performed using various methods. Traditionally a straws thawed by placing it in a 37 0 C water bath for 12-30s. This method is superior (36.1% mean motility across four diluents after 4 h of incubation) to a slow thaw methods (18.9% motility), where the semen straw placed in a 5 0 C water bath for two minutes (Deka and Rao, 1987). Tulli et al., (1991) found that increasing the thawing temperature to 70 0 C and thawing the straw for only 7 second resulted in significantly higher progressive motility (36.9%) and plasma membrane integrity (39.8%) compared to thawing straws at 37 0 C for 2 min (31.5%, 33.7%; progressive motility and membrane integrity, respectively) or 40 0 C for 20s (32.4%, 33.5%; progressive motility and 29

51 membrane integrity, respectively). Attention to temperature and timing becomes much more critical as temperature can result in tremendous sperm mortalities if performed improperly In-vivo Fertility Test: Conception rate after AI with frozen semen Frozen thawed semen is used for insemination in natural, synchronized or induced estrus in which several factors can influence the fertility. Conception rate with frozen buck semen varied between percent. Differences may be due to age, breed, and stage of heat and insemination technique. Conception was poor (35-45%) due to deposition of semen in over the opening of cervix (Sinha, et al., 1987; Tiwari and Bhattacharya, 1987b). Improvement in fertility was observed due to deep cervical insemination technique (Deka and Rao, 1989; Deshpande and Mehata, 1991). 30

52 Table:2.5.1 Fertility rate of buck in different conditions Reed Conception rate % Conditions References Jamunapari Sinha et al., 1987 Alpine Deep cervix Leboeuf, 1989 Beetal TYG + DMSO TYGL+DMSO Singh et al., 1995 Black Bengal Angora Cashmere Boer Saanen Florida Tris Tris+glutathione Single AI Double AI Sinha et al.,1996 Ritar and Salomon, Ritar et al., 1990a Ritar and Ball, Trans cervical Laparoscopic Wash semen Unwashed Tris Skim milk Sohnrey and Holtz, 2005 Gacitua and Arav, 2005 Hidalgo et al., 2007 Sirohi Deep cervical Sinha et al., 2007 Majorera Florida LN C Batisa et al., Laparoscopic Amoah and Gelaye, Tris Dorado et al., Milk Young Old Pluriparous 31

53 Percent Non-eosinophilic (live) sperm: The live percent of spermatozoa can be predicted by initial motility of semen (Tomar, 1984) yet the actual evaluation of live percentage is desirable. Semen samples having less than 50% live spermatozoa are of questionable fertilizing capacity, where as sample containing 50 to 90% live spermatozoa showed no difference in fertilizing capacity. Semen with more than 30% initial dead spermatozoa is not good for preservation. Lasley and Bogart (1942) showed that live and dead spermatozoa could be differentiated by their ability to get stained by eosin dye Morphological abnormal sperm: Buck fertility depends on morphologically normal spermatozoa present in the ejaculates. The abnormality is classified as head, middle-piece and tail abnormality (Kumar et al., 1992). The fertility is hardly affected if the abnormal spermatozoa do not exceed percent (Pant et al., 2002) In-Vitro Tests: The fertility of any semen sample is usually predicted by routine method of semen analysis like concentration, motility, viability, acrosomal integrity etc. However, these visibly measurable sperm characteristics have low correlation with predictive values to male fertility (Lunford et al., 1976). The ability to evaluate sperm characteristics in-vitro is essential to establish a correlation between the importance of any characteristics and overall sperm fertilizing abilities. In-Vitro Fertility tests includes HOST, sperm capacitation test and in-vitro fertility with in-vitro matured oocytes Hypo-Osmotic Swelling Test (HOST): The membrane integrity is not only important for sperm metabolism, but also play significant role in fertilization because a correct change in the properties of membrane is required for sperm capacitation, acrosome reaction and binding of spermatozoa to the egg surface for which a biochemically active membrane is required (Jeyendran et al., 1984; Chan et al., 1985). The assessment of the membrane may be a useful indicator of the fertilizing ability of the spermatozoa (Barratt et al., 1989; Avery et al., 1990; Azeredo et al. 2001). 32

54 Principle of HOST: This test is based on the observation of the morphological alteration in spermatozoa, when exposed to the hypo-osmotic solutions. Morphological changes in sperm tail take place when spermatozoa are subjected to hypotonic solution (Kumi-Diaka, 1993) and are indication of membrane integrity and normal functional activity. Jeyendran et al. (1984) reported that the sperm tail was particularly more susceptible to the hypo-osmotic medium. Viable sperm in a hypotonic solution has bent coiled tail whereas dead sperm had straight tails probably associated with cell lysis. Therefore, it was hypothesized that the ability of the sperm to swell in hypotonic solution indicates its membrane integrity and normal function activity (Takahasi et al., 1990). Aisen et al. (2005) reported that the HOS test for goat spermatozoa significantly higher for the hypertonic extender compared to isotonic Tris extender (p < 0.05) Acrosomal Integrity: After being deposited in the female genital tract, the spermatozoa traverse a long distance (before fertilization) during which some important functional and structural changes of spermatozoa takes place, of which capacitation is most important. Capacitation is a biochemical change in the plasma membrane that renders the spermatozoa capable of undergoing acrosome reaction (Yanagimachi, 1981). The acrosome is a large secretary granule, lying beneath the plasma membrane and it consists of a matrix bounded by an outer and inner acrosomal membrane. During acrosome reaction, due to fusion of plasma membrane and outer acrosomal membrane (Berger et al.,1985), acrosomal cap gets perforated which leads to the release of the acrosomal content viz. hyaluronidase, acrosine etc. (Brucker and Lipford, 1995) which help to dissolve the zona pellucida at the site of sperm entry (Galli et al., 1994). Actually, the acrosome reaction is an exocytotic process, which is triggered by the influx of Ca ++ ions following the modification of the plasma membrane surface during capacitation of sperm (Chauhan, et al., 1994; First and Parrish, 1987). 33

55 Abnormal acrosome may cause leakage of membrane bound enzymes found in the acrosome thus impairing fertilizing ability of spermatozoa. Simultaneously whiplash type sperm motility is observed as a result of changes in the sperm membrane, which is also, demonstrated in bovine spermatozoa abnormalities by using the geimsa stain technique (Rao et al., 1990; Raizada et al., 1990; Bhosrekar et al., 1994). They noticed that the incidence of acrosomal abnormality was significantly higher after freezing than before. 34

56 3. MATERIALS AND METHODS 3.1 LOCATION OF FARM AND CLIMATE The present investigation was conducted on bucks maintained at goat farm and Artificial Breeding Research Centre, National Dairy Research Institute (NDRI), Karnal, India. The institute is situated at an altitude of 250 meters above the mean sea level on N latitude and E longitude. The maximum ambient temperature goes up to 45 0 C during summer and minimum about 2 0 C during winter. The annual rainfall is about 760 to 960 mm, most of which is received during July and August. Relative humidity ranges from 41 to 85 per cent. The THI is given in figure-3.1. Bucks were kept in individual pens (15 5 ) under loose housing system on concrete floor with the orientation of east-west direction through its long axis. The pens were separated by wall and wire partitions that restricted direct physical contact but maintained eye contact in adjacent pens and a free movement within the shed. One third of the area was covered with concrete slab. Bucks were fed concentrate ration with 21 percent CP and 70 percent 1.0 kg per buck at 9.00 AM daily. Institute farm grown seasonal green fodder such as maize, cowpea, berseem, Lucerne etc., depending on their availability, was fed ad libel to the animals. The buck had free access to clean drinking water throughout the day. Buck sheds were cleaned once daily early in the morning. Vaccination, de-worming and other herd-health programs were followed as per the farm schedule, to ensure good health and vigour CLASSIFICATION OF SEASONS For the purpose of experiment, seasons were classified on the basis of monthly average maximum, minimum temperatures (Figure-3.1) and relative humidity %. According to these climatic factors, Karnal location has three distinct seasons namely: i) Winter (cold Humid) (S1) - November, December, January, February and March ii) Hot-dry Summer (S2) April, May and June iii) Hot-humid Summer (S3) July, August, September and October 35

57 Figure-3.1 Monthly THI during the experimet 3.2. SELECTION OF BUCKS Five crossbred bucks (Alpine X Beetal) maintained at institute s flock, their ejaculate having mass motility +3 and above 80% individual motility, sperm abnormality less than 10% were selected for primary study and more than 50% post thaw motility in winter season were selected for further study. The details of the bucks used for study are presented in Table 3.1. Table: 3.1. List of Alpine X Beetal bucks under experiment Breed / Strain Alpine X Beetal Buck No. Date of birth Age (years) on 1/11/2009 Body Wt. (Kg) AB /11/ AB-36 02/11/ AB-64 08/11/ AB-66 08/11/ AB /11/

58 Plate- 1 Crate for semen collection and transcervical AI Plate- 2 Semen collection in goat with artificial vagina

59 3.2.2 SEMEN COLLECTION TIME AND METHOD Weekly three to four ejaculates in Winter (cold Humid) (S1) season and three ejaculations in rest of seasons namely, Hot-dry Summer (S2) and Hot-humid Summer (S3) Hot-humid Summer (S3) were collected. The bucks were taken to the site of collection in the morning hours at 7.00 AM in winter season and 6:30 AM in rest of seasons. The bucks were trained initially before regular collection for experiments to donate semen in artificial vagina (AV) on dummy buck or teaser buck. The dummy buck was restrained in the wooden crate (Plate-1) and other buck was allowed to mount on it. Semen was collected (Walton, 1945) using goat artificial vagina (AV) of 6 inches size Danish Model with smooth lining (IMV Pvt. Ltd.), on a dummy buck in different seasons of the year 2009 to 2010 (Plate-1). The temperature of AV was maintained at 42 C with warm water and sufficient pressure and proper lubrication with sterilized a vaseline jelly. On each collection, only one ejaculates was taken. A period of sexual preparation was given which consisted of at least two false mounts separated by about one minute restraint before collection of semen sample. Immediately after collection, each sample was placed in a water bath at 30 C and transported to laboratory within 15 minutes for various standard laboratory tests. CHEMICALS: Trehalose (SLR), Sodium laurel sulphate (SDS), TRIS (Tris hydroxymethyl aminomethane) (SLR), citric acid (SLR), glucose, Fructose (SLR), Heparin (SRL) Sephrose (Sigma). EXPERIMENTAL DESIGN FRESH SEMEN EVALUATION: Fresh semen evaluation of Alpine x Beetal bucks in different seasons In order to study fresh semen parameters five AXB bucks were selected. Three semen samples from each buck in a week for 52 weeks were evaluated. A total of 780 ejaculates were collected from 5 bucks in a year from November, 2009 to October,

60 Following physical parameters of semen were recorded: Colour Consistency Volume Microscopic semen parameter evaluation: Mass activity Individual motility Sperm concentration Non-eosinophilic sperm Morphological abnormality (only for selection purpose of bucks) In vitro evaluations: Hypo-osmotic swelling test (HOST) (Jayendran et al., 1984) Acrosome integrity Color: creamy or lemon yellow was recorded Consistency: thick, thin, opaque or watery was recorded Volume The semen was collected in 15 ml graduated metal free glass tube (0.1 ml accuracy) and recorded volume of semen samples Mass Activity Mass motility was assessed just after the semen collection. Gross swirl rating (GSR) of undiluted semen was performed within 15 min of collection. Two 10 µl of undiluted semen was placed on two places on warmed slide placed stage warmer (37 C) and scored on a scale of 0-5 using 10X objective lens on the phase contrast microscope (Nikon Eclipse E600, Tokyo, Japan). Motility was expressed qualitatively in a motility scale (0-5) as per the description given by Matharoo et al. (1985). The average of the two scores was recorded. The scale of 0-5 represents the following: 38

61 0 = No movement present 1 = Sluggish swirls and spermatozoa show weak-laboured movement 2 = Swirls of intermediate speed 3 = Swirls of rapid speed 4 = Many swirls showing rapid and vigorous speed 5= uniform single swirl showing rapid and very vigorous speed Motility estimate (%) Manual progressive motility and percentage motile spermatozoa were determined by placing 100 µl of undiluted semen into pre-warmed tubes containing 900 µl of Tris buffer and mixing. Five micro-litters of diluted semen was placed on a warmed glass slide (37 C) and allowed to spread uniformly under the cover slip. Strength of motility rating was scored on a scale of 0 to 5 using 200X magnification with phase contrast microscope (Nikon Eclipse E600, Tokyo, Japan) equipped with a 37ºC-heated stage and represents the following: Percent progressive motility (0 100%) was measured on at five representative areas of the slide. The average of the five scores for each category was recorded. If the difference between two consecutive counts exceeded 10 percent, two new counts were performed Sperm concentration Sperm concentration was estimated by Haemocytometer (Neubauer s chamber) method. The Neubauer slide of the haemocytometer has two counting chambers, RBC and WBC chamber. RBC chamber is at the centre and the WBC chambers are at the corners. Each RBC Chamber (Primary Square) is divided into 25 (5x5) secondary squares. Each secondary square is further divided into 16 (4x4) tertiary squares. Thus each primary square is divided into 400 (25x16) tertiary squares. These total 400 tertiary squares measure a total area of 1mm 2. Likewise each WBC chamber is divided into 16 secondary squares. Area wise each WBC chamber is equal to the RBC chamber. When a drop is placed under the cover slip in a Neubauer cell counting 39

62 chambers, the thickness of the film on chamber is 0.1mm. Thus the total volume of the semen covering 400 tertiary square in RBC chamber or 16 secondary squares in WBC chamber is 0.1mm 3. Preparation of Diluting Fluid Eosin-Y (Water soluble) 0.05 gm and 1.0 gm of Sodium Chloride were dissolved in 100 ml distilled water. Steps for examining the sample - 0.1ml of well mixed semen was taken ml of the diluting fluid was added to it and mixed well (now the dilution 1:100) - 5 ml of the above-diluted semen was taken and 5 ml of diluting fluid was added to it (now the dilution is 1:200). - One drop of diluted semen as placed under the cover slip in a Neubauer cell counting chamber. Over flowing was avoided. Allowed settling for five minute. - Examined under high power objective of the microscope (200X). - Spermatozoa were counted in 16 secondary squares in each WBC chamber. - Counting is done in all four WBC chamber i.e., a total of 64 secondary squares were counted and average was taken. Calculation No. of sperms counted in 16 secondary squares = n No. of sperms in 0.1mm 3 of diluted semen = n x (dilution rate 1:200) No. of sperms in 0.1mm 3 of undiluted semen = n x 2 x10 2 No. of sperms in 1mm 3 of undiluted semen = n x 2 x10 3. So concentration is n x 2 x 10 6 per ml Eosinophilic (dead) and Non-eosinophilic (live) spermatozoa (%) The staining solution for the one-step technique contained 0.67 per cent eosin-y and 10 per cent nigrosine. Thus, 0.67g of eosin-y yellow and 0.9g sodium chloride were 40

63 dissolved in 100 ml distilled water under gentle heating. Then 10g of nigrosine was added. The solution was brought to the boil and allowed to cool to room temperature (20 C) after which it was filtered through quality filter paper (Whatman filter paper no. 40) and stored in a dark and sealed glass bottle. Before use, the staining solution was brought to room temperature. Semen samples were kept at 37 C for 30 min before analysis. Forty microlitre (µl) of neat semen was mixed in a micro-centrifuge tube with 400 µl eosin-nigrosine staining solution. The suspension was kept for one minute at room temperature (27 C). Then, a 12 µl droplet was transferred with the pipette to a labelled microscope slide (pre-warmed to 37 C) where it was smeared. Duplicate smears were made from each sample. After smearing it was allowed to air dry at room temperature. The smears were air dried and examined directly. About 200 sperms were assessed at a magnification of 100 X under oil immersion with a high-resolution 100 X bright field objective (not phase contrast). Sperms that were white (unstained) were classified as non-eosinophilic and those that showed any pink or red colouration were classified as dead, with the sole exception for sperm with a slight pink or red appearance restricted to the neck region ( leaky necks ), which were assessed as noneosinophilic Hypo-osmotic Swelling Test (HOST) The hypo-osmotic swelling test was performed according to the methods described by Correa et al. (1994) for goat fresh semen. Hypo-osmotic solutions of 100 mosmol l -1 were prepared as follows: Table Composition of solutions for HOST: Ingredients HOST solution Control solution Sodium citrate (g) Fructose (g) Millipore water upto (ml) Osmolality (mosm Kg -1 )

64 Procedure: One ml of hypo-osmotic solution, having an osmotic strength of 100 mosm Kg -1 was mixed with 0.1 ml of semen and incubated at 37 C for one hour. An aliquot under test was similarly incubated in an isotonic solution (control) with an osmotic strength of 375 mosm Kg -1. Following incubation, a drop of well mixed solution was taken on a clean dry glass slide and covered with a cover-slip. Sperm tail curling was recorded as an effect of swelling due to influx of water. A total of about 200 spermatozoa were counted in different fields at 400X magnification under DIC microscope. The total proportion of swollen spermatozoa was calculated by dividing the number of reacted cells by the total spermatozoa counted in the same area and multiplying the figure by 100. The proportion of swollen spermatozoa from a control sample was subtracted from this value. These spermatozoa were classified in four different classes according to the presence of following swelling pattern (Takahasi et al., 1990). Observation/pattern: A. No swelling, no membrane reaction B. Swelling of tip of tail C. Different type of hair pin like swelling pattern or swelling of mid-piece D. Complete tail swelling Spermatozoa displaying B, C or D were considered positive for the HOST test. 42

65 Acrosome Integrity Staining was carried out as described by Hancock (1952). Giemsa stain grounded with absolute methanol in a pastle and mortar. To this glycerol was added. Stain mixture was stored at 37 0 C for one week. During this storage period, it was shaken for 2 minutes each day. 2.) Preparation of Soreson s phosphate buffer: Composition: Solution A: Sodium phosphate dibasic (Na 2 HPO 4.2H 2 O) Distilled water gm 1000 ml Solution B: Potassium phosphate monobasic (KH 2 PO 4.2H 2 O) Distilled water 9.08 gm 1000 ml The desired ph (7.0) was obtained by adding enough solution B to solution A (61.2 ml) given below to make a volume of 100 ml. 3.) Preparation of final stain solution: 3.0 ml of Giemsa stain diluted with 2.0 ml Sorenson s M/15 phosphate buffer (ph=7.0) and distilled water 35 ml. The staining solution was stored in refrigerator. Fresh stain was prepared every week. Staining Procedure A thin smear of extended semen was prepared on a non-greasy, clean and dry slide. The smear was air-dried at room temperature for at least 10 minutes in a current 43

66 of warm air. Smear was fixed by immersion in buffer formal saline (10 percent) for 15 minutes. Then it was washed in running tap water for minutes and dried. Again the slide was immersed in buffered Giemsa solution for 90 minutes and rinsed briefly in distilled water and dried. The dried smears were studied at 1000X under a light microscope using oil immersion without cover glass. Each time about 200 spermatozoa were counted for acrosome status. 3.4 CRYOPRESERVATION OF SEMEN: The processing method for freezing of semen was done at Artificial Breeding Research Centre of National Dairy Research Institute, Karnal. The method followed is described below Dilution of semen Optimum number of motile sperm per dose at the time of insemination was fixed at about 100 million sperm assuming 50 percent post thaw motility. Post-thaw sperm motility of an individual buck can be determined by post-thaw evaluations on several ejaculates of the buck. A standard of minimum 40 percent post thaw motility was considered as freezable semen. Expected dose of semen sample (0.25 ml 100X10 6 motile sperm) = {semen volume (ml) X sperm concentration (10 6 ) X (individual motility/100)) /100(Per dose 10 6 in 0.25 ml). Four doses of semen were produced from one ml of diluted semen (volume of French mini straw is 0.25 ml). Total volume of diluted semen (total volume of diluter to be added + semen volume) = expected dose of semen sample/4 (0.25 ml for 0.25 ml straw packaging). Semen samples showing more than +3.0 mass activity and more than 80% progressive motility were considered for freezing. Before dilution concentration of spermatozoa was determined. Till the final dilution rate was decided, semen was prediluted to a low volume of extender and stored in water bath (30 C). The individual motility of the sperm was assessed with the diluted semen using phase contrast microscope. Sample showing more than 80 percent motility was taken for further processing. Dilution rate was decided based on the motility rate and sperm concentration. A minimum of 100 million motile sperm in each dose was used. As soon 44

67 as the final volume of diluted semen had been determined the remainder of the diluent was added. After dilution rate was decided on the basic of sperm concentration and initial motility (%). Different extenders were kept in water bath at 30 C. i) Addition of glycerol to extenders: Glycerol was mixed gently with extender and kept in water bath at 30 C. Four per cent (v/v) glycerol was added in each type of extender and the final concentration was maintained at 4 percent (v/v) of each extender Printing of Straws The required number of straws was labelled by printing through Domino Ink Jet Printer A200 Pinpoint (Domino UK Ltd, Bar Hill, Cambridge, CB3 8TU, England) Filling and sealing of Straws French Top Bull mini straws (0.25 ml; 135 mm length and 2 mm diameter, (IMV) of different colours were used. Automatic straw filling and sealing machine (IMV, France) was used for filling of semen into the straws and sealing it. Filling and sealing were done at room temperature (23 0 C). Straws were placed on the single layered horizontal position in the straw rack and kept for 2.5 to 3 h at cold room (5 C) to give sufficient equilibration period. At the end of equilibration period, the pre-freeze motility, HOST and Acrosome integrity were again recorded Freezing of Straws Freezing Method: Diluted semen samples were filled in French straw (0.25 ml) quickly at room temperature (23 0 C) and equilibrated at 5 0 C for 2.5 to 3 hours. Freezing was done by keeping straws on rack 8-10 cm (3-4 inch) above liquid nitrogen for 5 minute and subsequently transferred to liquid nitrogen. Thawing was carried out at 37 0 C for 30 seconds. All parameters for fresh, pre-freeze and post freezing were recorded for evaluation of semen for each buck. 45

68 3.4.5 Semen preservation evaluation: Pre freezing microscopic parameters: Progressive motility liveability HOST (Jayendran, et al., 1984) Acrosome-integrity (Aboagla and Terada, 2003) All prefreez parameters were evaluated by above described method Thawing: The straws were thawed in a 37 C water bath for 30 seconds after removing from the LN 2 container. Immediately after thawing, motility of semen was checked. Semen was taken into micro-centrifuge tube and kept at 37 C till further examination Evaluation of frozen semen samples Post freezing microscopic parameters: Progressive motility Liveability HOST (Jayendran, et al., 1984) Acrosome-integrity (Aboagla and Terada, 2003) In vitro and In vivo fertilization test for 5 bucks as described in Expt Experiment I Freezability of crossbred goat (Alpine X Beetal) semen in relation to heparinbinding proteins (HBPs) variation in seminal plasma of goat in different seasons. For this purpose five Alpine X Beetal bucks (AB-36, AB-64, AB-66, AB-153 and AB-986) were selected on the basis of semen quality. Four ejaculates in a moth were collected for seminal plasma collection and four ejaculates for cryopreservation (total 8 ejaculates) from each buck in every month for one year from November, 2009 to 46

69 October, A total of 220 ejaculates were collected for seminal plasma collection and 220 ejaculates for cryopreservation. Seminal plasma processing for SDS-PAGE and 2D: Seminal plasma was removed by centrifugation at rpm for 20 min. at 4 o C and stored at -70 o C until used. All the stored seminal plasma samples were pooled for each season such as Winter (S1) (November, December, 2009, January, February and March 2010), Hot-dry Summer (S2) (April, May and June 2010) and Hot-humid Summer (S3) (July, August, September and October, 2010). All the three pooled samples were taken into test tube and centrifuged at rpm for 20 min at 4 0 C. These samples were prepared for SDS-PAGE as shown in following flow chart. Figure Flow chart for Purification of HBPs for SDS-PAGE and 2D Pooled Samples of Each Season Centrifuged at rpm for 20 min at 4 0 C Supernatant precipitated by 60% Ammonium sulphate Centrifuged at rpm for 20 min at 4 0 C Supernatant removed and precipitate was dissolved in 20 mm Phosphate buffer of ph =7 Diluted precipitation dialysis in 20 mm Phosphate buffer of ph= 7 Dialysis with phosphate buffer 20 mm Changed buffer for every 6 hours for 4 times Purification of protein Quantification of protein SDS page 2D Gel 47

70 Seminal plasma were pooled from every month samples from different bucks and purified. In winter 2.58 µgm/µl and in Hot dry 3.38 µgm/µl and in Hot humid it was found to be 4.4 µgm/µl were harvested in the heparin binding column. These HBPs were used for SDS-page and 2D gel electrophoresis. Cryopreservation: For cryopreservation two types of extenders were prepared. First Tris-Citric acidegg Yolk based (TCGEY) which containing TCG (Tris, Citric acid and Glucose) buffer with 20% egg yolk and 4% glycerol (Salmon and Ritar, 1980) and Second TCG buffer was diluted with 375 mμ solution of trehalose by replacing 35% of basic TCG buffer which was developed and used as second extender. TCG buffer: The buffer solution was prepared using Tris (hydroxymethylaminomethane)-citric acid-glucose (TCG) buffer which contained mm tris (SLR), mm citric acid and 41.0 mm glucose. This solution was adjusted to ph 7.0 with 0.01N HCl and adjusted to desired volume with mill-q water to 375 mosm. The temperature of buffer was maintained at 30 0 C. As at higher temperature 37 0 C there is fluctuation in the ph. TCG buffered was also used for semen collection. Each ejaculate divided into two aliquots and cryopreserved in two types of extenders namely Extender-I (TCGEY) and Extender II (TCGEY+Trehalose) during all S1, S2, and S3 seasons. All semen parameters were recorded before and after freezing of semen samples collected in different seasons EXPERIMENT II Effect of modified method of semen collection on cryopreservation of goat semen The study was conducted to find the significance of reducing contact between seminal plasma components and the sperm membrane at ejaculation on cryopreservation in different seasons. The experiment was carried out in Winter (December, January), Hot-dry Summer (April and May) and Hot- humid Summer- (July, August). 48

71 Five bucks AB-36, AB-64, AB-66, AB-153 and AB-986 were selected (Table 3.1) on the basis of best seminal quality attributes. In preliminary studied semen samples were collected in tubes without TCG buffer and semen quality was evaluated and buck number having high quality semen attributes such as average volume, live cells, motility and concentration were selected. Four ejaculates in each month from each buck were collected into semen collecting tubes; 0 ml (empty), and 4 ejaculates in 10 ml of basic buffer TCG. A total of 8 semen samples from each buck for each season were collected. 60 samples for each treatment and a total of 120 samples were processed. To avoid variations, semen sample were firstly collected in empty tubes at start day of week and second day of the week semen sample was collected in tubes containing 10ml of TCG buffer solution at 30 0 C temperature in the same. Semen samples were transported within 15 minutes of collection to the laboratory for various tests. Immediately after reaching the laboratory semen samples were centrifuged at 1200 rpm for 10 minutes and seminal plasma was removed. The sperm pellets were re-suspended again with buffer for removal of seminal plasma as above and split sample was prepared in very small quantity of TCG buffer, which were diluted in TCGY+Trehalaose buffer for freezing. Freezing extender: Tris-Ciric Acid-Glucose-Egg Yolk (TCGEY) +Trehalose extender defined by Salmon and Ritar (1980) and second extender used was TCGEY which containing 35% solution of 375 mm Trehalose replacing basic TCG extender. The dilution of semen palate was done two step procedure to keep the egg yolk 20% and glycerol 4%. Antibiotic were added to each extender as Annexure-II this new procedure of dilution of trehalose was developed to reduce the hectic procedure addition of SDS, stirring and centrifugation and overnight storage as described by Aboagla and Terada (2003). Firstly adding the semen sample in freezing extender without glycerol and cooling to room temperature i.e C. Final dilution was done by adding half of total diluents with 8% glycerol to a final sperm concentration of 400 X10 6 /ml. Semen samples were cryopreserved by conventional method of freezing by equilibrating at 5 0 C for 2.5 to 3.0 hrs as described in above paragraph 49

72 3.3.1 Freezing method Figure Flow chart of effect of modified method of semen collection on cryopreservation of goat semen. 5 BUCKS Collected in TCG buffer Collected without buffer Washed by Centrifugation at 1200 rpm Washed by Centrifugation at 1200 rpm Cryopreserved in (TCG+T) Egg Yolk Extender Cryopreserved in (TCG+T) Egg Yolk Extender EXPERIMENT III (A). Effect of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen To find out the effect of replacement of basic buffer (TCG) solution with M trehalose solution for cryopreservation of crossbred goat semen. Five bucks (AB-36, AB-64, AB-66, AB-153, AB-986) were selected for experiment and used for freezability of semen in Winter- December, January, February and March with (100% trehalose) and without trehalose (only TCG). Twenty semen samples from each buck and total 100 semen samples were collected in tubes containing 10ml of (TCG) extender. Seminal plasma was removed by washing at 1200 rpm for 10 min twice. After removal of seminal plasma sperm palate were divided into two equal parts. Sperm pellets were diluted with different concentrations of trehalose extender (0, 100%) containing 0.05% SDS and egg yolk 20%. Trehalose solution (0.375mM) was added to TCG solution to give the following trehalose extenders: 0% (only TCG) and 100% (v/v) (only trehalose). These extenders 50

73 were prepared hours before use and kept at 4 0 C. Each extender was added with SDS 0.05% (w/v) stirred for one hour and centrifuged at 10,000 rpm for 1 hour, supernatent was aspirated and filtrated with 0.45µm membrane filter (Millipore, S.A., Molsheim, France). Antibiotic amicacin, neomycin and tylocin were added to each extender at the rate given in Annexure-II. Diluted semen samples were further diluted with each trehalose extender containing 8% glycerol to a final concentration of 4% glycerol and a sperm concentration of 100 x 10 6 /dose (0.25 ml) or 400 X 10 6 ml -1. After final dilution prefreeze parameters were checked. French straw 0.25 ml were filled automatically by machine (IMV MRS 3) and equilibrated at 5 C for 2.5 to 3 hrs in the cooling chamber and freezing was carried out as described in above paragraph Thawing of straw was carried out at 37 C temperature for 30 seconds. All freezing parameters were recorded as described in above paragraph. The flow chart of the experiment is given bellow: Figure Flow chart of Effect of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen 5 BUCKS 5 bucks 10 ejaculates from each buck Collected in TCG buffer Pooled 0% T 100 % T 51

74 3.3.5 EXPERIMENT III (B). Effect of different concentration of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen. Semen samples from 5 AXB bucks were collected in semen collection tubes containing 10ml of TCG buffer were initially evaluated for individual motility and pooled together and divided into four equal parts. Seminal plasma was removed by washing at 1200 rpm for 10 min twice. Sperm pellets were diluted with different concentrations of trehalose extender (0.0%, 30% 70% and 100%) containing 0.05% SDS and egg yolk 20% and further processed as per EXPT. 3 A. The flow chart of the experiment is given bellow: Figure Effect of different concentration of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen. 5 bucks 10 ejaculates from each buck Collected in 10 ml TCG buffer Pooled Split 0% T 35% T 70% T 100% T EXPERIMENT-III (C) Freezability of crossbred goat semen in tris-soya-milk and trehalose - soya-milk -based extenders. AXB bucks were selected for this experiment. This experiment carried out in September and October, Semen samples collected tubes containing Tris buffer (TCG), pooled after evaluation of progressive motility estimate % and washed twice by centrifugation at 1200 rpm for 10 minutes within 15 minutes of collection and split into 4 52

75 Flacon tubes and diluted with following 4 types of extenders at room temperature (23 0 C). Trehalose Soya based Extender: For cryopreservation different extenders were prepared i.e Tris-Citric acid-egg Yolk based (TCGEY) (Salmon and Ritar, 1980) (1) Tris (375mM)- Citrate (124mM) Glucose(41mM) + 20% egg yolk (EY) (C1:TCG), (2) TCG containing mM trehalose + 20%EY(C2 :TCG+T) (3) TCG+ 25 % Soymilk and (4) TCG with mM trehalose 25 % (TCG+T) + Soya milk based and 54.83mM glycerol. This buffer was adjusted to ph 7.0 with 0.01N Hcl before addition of egg yolk or soymilk. Prepared soya based was used at 25 % of total volume of extender (Singh, et al., 2012). Trehalose Soya based Extender (TSME) was prepared by Trehalose 0.375M, 41.0 mm glucose (SLR) and Tris was added to adjust the ph at 7. Soya extract was prepared by method described by Nelson et al., 1976). Prepared soya based was used at 25% of total volume of extender (Singh, et al., 2012). Dilution and freezing was carried by earlier described method. Pre freez and post-thaw quality of semen samples in all extender were recorded as per paragraph EXPERIMENT IV (A). In-vitro Fertility Test with Cryopreserved Crossbred goat (AXB and SXB) Semen Post thaw semen straws were used for determination of in-vitro fertility with goat in-vitro matured oocytes. In Vitro Fertilization Test Collection and transport of ovaries: Goat ovaries along with the reproductive organs were purchased from Delhi slaughter house and washed 2-3 times with C normal saline (0.9%) containing antibiotics penicillin (400 IU) and streptomycin (50 µg/ml). Then they were transported to laboratory within 3-4 h in a thermo flask containing warm normal saline solution ( C) supplemented with antibiotics. The ovaries were trimmed with sterile scissors and washed 4-5 times with warm normal saline fortified with antibiotics. The ovaries were ready for further processing. 53

76 Oocyte recovery: For collection of oocytes, oocyte collection medium (annexure-i) consisting of TCM-199, 25 mm HEPES, L-glutamine, BSA, Gentamycin and 10% FBS was used. Fresh medium (100 ml) was prepared, filtered through 0.22 m membrane filter (MillexG) and ph was adjusted to Prior to use it was kept in 5% CO 2 incubator at C for at least 2 h for equilibration. Puncturing as well as slicing method was used for sufficient recovery of oocytes. Ovaries were placed one by one in a oocyte searching dish containing oocyte collection media. Then ovaries were grasped by sterilized forceps and the surface follicles (2-5 mm diameter) were punctured with the help of a sterilized needle. Then the ovaries were subsequently sliced by sterile scalpel. At a time ovaries were processed in this way and the dish was observed under zoompipettes were used. They were pulled over the flame so as to have inner diameter of approximately 300µm. Oocytes along with the cumulus cells (COCs) were picked up gently without damaging the cumulous cells and were kept in a 35 mm Petri dish containing media for further washing. Grading of oocytes: The collected oocytes were graded A, B, C, and D according to the number of cumulus layer surrounding the oocytes and the homogeneity of ooplasm. Grade A: the oocyte with cumulus oocyte complexes (COCs) having more than five layer with evenly granulated cytoplasm. Grade B: the oocytes with cumulus complexes (COCs) having 3-5 layers with evenly granulated cytoplasm. Grade C: the oocytes with fibrous (much expanded) cumulus with dense and irregular cytoplasm. Grade D: the oocytes with irregular cytoplasm and without cumulus layer. Only Grade A and B oocytes were taken for further maturation. 54

77 In vitro maturation of goat oocytes: Collected COCs were washed 3-4 times in oocyte collection medium and 2 times in maturation medium ( Annexure) containing TCM199 (HEPES modified), FSH, LH, Estradiol- 17β, BSA and EGS. Four drops of 100µl maturation medium were made in 35 mm dishes and covered with mineral oil. These dishes were placed in the 5% CO 2 incubator prior to use for the equilibration. After washing oocytes, the dishes were taken out from the incubator and A and B grade oocytes were placed in each drops of maturation medium. Then the petri dishes were incubated at C under 5% CO 2 with maximum humidity for 27 h (De Smedt et al., 1992). Preparation of goat oviductal epithelial cells (GOES) Along with the ovaries 2-3 goat oviducts were trimmed from the reproductive organs. They were then washed 4-5 times in normal saline fortified with antibiotics. Two clear glass slides were taken. The oviducts were placed on a glass slide and with the other slide they were pressed so that the oviductal content came out. Then they were collected in a 15 ml graduated tube containing 5 ml embryo development media with the help of a 1 ml micro pipette and mixed properly. Then the tube was allowed to stand 2-3 minute so that the cells got settled. The supernatent was thrown out and 5 ml fresh media was added and the cells were mixed and again allowed to stand for 2-3 minutes. Likewise cells were given 4-5 washings in embryos developing medium. Then motility of cells was checked under microscope before culturing them in embryo developing medium in a 4-well tissue culture dish. The 4 well culture dishes with GOEC were incubated at C in CO 2 incubator with 5% CO 2 with maximum humidity. Semen collection and semen processing: Frozen semen samples with more than 55% post thaw progressive motility and fresh semen was collected from the experimental buck and semen having more than 80% progressive motility was taken for IVF. Fifty micro litre of neat semen was placed in 2 ml sp-talp medium (Annexure) (Parrish et al., 1989) in two tubes and allowed to spermatozoa to swim-up during incubation for 15 min at C under 5% CO 2. After incubation, the top 1.5 ml was taken, mixed with 12 of sp-talp media in a 15 centrifuge tube and centrifuged at 1400 rpm for 7 min. Supernatent was discarded and sperm 55

78 pallet was washed suspended in TALP medium containing 50 µl /ml heparin (Katska et al., 2003) and incubated at at C under 5% CO 2 for 1.5 hours for capacitation. A drop of sperm suspension was monitored at every step of processing under microscope to check the motility. Progressive movement, head to head attachment and glass surface attachment indicated the capacitation of spermatozoa. Preparation of oocyte and in vitro fertilization Matured oocytes after 27 h of maturation were taken out from maturation medium drops and expanded cumulus cells were removed by pasture pipetting in fert-talp medium (Annexure-II) using sterilized pasture pipette. The oocytes were washed 5-6 times in fert-talp medium. About denuded oocytes were placed in 50 µl droplet of fert-talp and covered with mineral oil and incubated at C under 5% CO 2 for equilibration of medium prior to in vitro fertilization. About 50 µl (2x10 6 to 4x10 6 ml -1 ) of capacitated sperm suspension was added to each 50 µl drop fert-talp medium containing denuded oocytes and co-incubated for 15h at C under 5% CO 2 in incubator. Different stages of cleavage were recorded (Plate-5) EXPERIMENT IV (B). In Vivo fertility of semen cryopreserved with TCGEY and TCGEY + Trehalose based extender Cryopreserved semen samples (Control Tris citric acid based extender) 0% and (TCG+Trehalose based extender) from winter season were used for Artificial insemination in natural heat in does for in vivo fertility status of semen. Twenty-six does for each extender and a total 52 does were used for trans cervical artificial insemination. Selection of does: All female goats selected for AI were of second parity. Detection of heat: Usually does were coming in oestrus or heat at the end of May and start of June months naturally. The natural heat was utilized for AI in does. Heat was detected by 56

79 moving the vasectomised buck at early morning at 6:00 AM and evening 6:00 PM after milking the does and visualization. Artificial Insemination (AI) time and methodology: Artificial Insemination (AI): Does in oestrus were separated from the flock and kept in separate enclosure. Does were inseminated in morning 8:30 AM and evening 6:30 PM and left in the flock. The doe was held tightly by holding two legs up on the crate made up of wooden with the help of two assistant. The hind legs of goat were lifted so the rear part is kept higher (45 0 ) than the fore part of the body as shown in Plate-3. A lubricated speculum (IMV Ltd.) was used to open the vagina and a flash-light to illuminate the cervix. The AI gun was inserted slowly and deeply manipulating cervix in to the cervix and bypassing the cervical fold and semen was released slowly. After insemination doe was kept in this slanting position for five minutes before releasing in the flock. All the inseminated does were watched for return of heat around 21 days (± 2 days) after insemination. Preparation of AI: Random semen sample from frozen straws from each treatment group was selected and thawed at 37 o C for 30 seconds before the day of insemination. Post-thaw motility, progressive motility and acrosome integrity was recorded for the semen samples and semen samples with more than 50% progressive motility, more than 55% live intact acrosome for the buck were used for AI. Pregnancy Diagnosis: Pregnancies were ascertained by using trans-rectal ultra sonography with linear probe 3.5 to 7.5 Hertz on 35 th to 48 th (Plate-4) day post AI and actual kidding and kidding rate was recorded. 3.4 Statistical analysis: Data were analyse using general linear model by the help of SPSS Statistical software

80 4. RESULTS AND DISCUSSION The present investigation was undertaken to study the freezability of crossbred (Alpine X Beetal) goat semen in relation to heparin-binding proteins (HBPs) in different seasons and with trehalose incorporation in semen extender on characteristics of cryopreserved goat semen, to know the effect of modified method of semen collection and also to study in-vitro and in-vivo fertilization with frozen semen. Firstly semen from Alpine X Beetal were collected and evaluated for fresh semen parameters. In first experiments semen from five bucks frozen in two types of extender, Extender-1 and Extender-2 whole year and simultaneously seminal plasma collected, categorized into three seasons and processed for SDS page and 2D. In second experiment fresh and frozen semen quality evaluated by collecting semen in 10 ml TCG buffer and without TCG buffer. And in third experiment trehalose was used in various quantities in extenders of egg yolk and soya extract based. In fourth experiment semen frozen in winter season used for in-vitro and invivo AI in does. Fresh semen parameters were also recorded for Alpine X Beetal semen samples in all three seasons. Results of the different experiments conducted during the study are presented and discussed. Tables for Mean ± S.E. and Squares Means (SM) of the parameters have been presented along with discussion. 4.1 FRESH SEMEN EVALUATION OF ALPINE X BEETAL BUCKS IN DIFFERENT SEASONS OF THE YEAR In order to study fresh semen parameters of two breeds, five bucks from each breed were selected. A total of 125 semen samples were collected from each buck in all the seasons and total 625 samples from 5 bucks were analysed and results are presented in Table to Colour and Consistency: It was observed from the results that the colour of the semen samples collected from all the bucks was creamy with thick consistency during S1 season (winter) slightly thin creamy during S2 (Hot dry summer) and S3 (Hot humid summer) 58

81 seasons. Creamy colour and thick consistency of crossbred (Alpine x Beetal) matured buck semen have been reported by Kale (1995) and Naskar (1995), with no difference in colour and consistency in different season were found for same crossbred breed goat. However, in our study in non breeding season there is thin consistency and creamy colour was observed. This may be due to frequency of samples collection. However, Ahmad and Noakes, (1996) reported physical appearance of the ejaculates, which varied from a yellow or whitish-yellow colour during September-December in breeding season to a creamy-white colour during the remainder of the year, which is similar to our finding. Similar colour of semen was also reported by Igboeli (1974) in Boer and native Zambian goats Volume: The variation in semen volume is known to exist between breeds and between the animals, even at different time of collection. The means for volume of semen produced and analysis of variance of the volume in different seasons are given in the Table and Figure AB1 AB2 AB3 AB4 AB5 Figure Semen volume (ml) of AXB crossbred bucks It was observed that the volume (ml) for buck AB5 was (1.47± 0.02) significantly more than the other bucks, AB1 (1.20 ± 0.02), AB2, AB3 and AB4. There was no significant difference between AB2 and AB3 for volume. The semen volume (ml) for AXB crossbred buck in Winter (S1) season was 1.00 ± 0.02, in Hot dry Summer (S2) season 1.22 ± and in Hot-humid Summer (S3) was

82 Table Mean ± SE fresh semen parameters of Alpine X Beetal crossbred buck semen collected throughout the year. Progressiv e motility Conc. X10 9 / Intact Acrosom Volume MM Bucks Live % HOST % (ml) (0-5) (n=125) % ml e % Mean 1.20 a 3.39 a a a 3.41 a a a AB1 ±S E Mean 0.86 b 3.38 a b a 2.55b a b AB2 ±S E Mean 0.88 b 3.16 b b b 2.68b bc b AB3 ±S E Mean 1.02 c 3.28 a b b 3.30 a a c AB4 ±S E Mean 1.47 d 3.22 a b b 2.93 c d c AB5 ±S E Over all Mean (625) ±S E Means bearing different superscript in a column differ significant *P<0.05, **P<0.01 Table Mean ± SE fresh semen parameters of Alpine X Beetal crossbred buck semen collected during different seasons Intact Buck Volume MM Progressive Conc. Seasons Live % No. (ml) (0-5) motility % X10 9 HOST % Acrosome /ml % S1 Mean 1.00 a 3.61 a a a 3.87 a a a (n=265) ±S E Over S2 Mean 1.22 b 2.81 b b b 2.58 b b b all (n=130) ±S E S3 Mean 1.11 c 3.17 c c c 2.72 c c b (n=230) ±S E Means bearing different superscript in a column differ significant *P<0.05, **P<0.01

83 Table Mean ± SE Fresh semen parameters of Alpine X Beetal buck semen in all seasons Buck No. AB1 (n=125) AB2 (n=125) AB3 (n=125) AB4 (n=125) AB5 (n=125) Seasons S1 S2 S3 Over all S1 S2 S3 Over all S1 S2 S3 Over all S1 S2 S3 Over all S1 S2 S3 Over all Volume (ml) MM (0-5) Progressive motility % Live % Conc. X10 9 /ml HOST % Intact Acrosome % Mean ± S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E Mean ±S E

84 ±0.02. It was significantly higher (P<0.01) in Hot-dry Summer (S2) than the other seasons S1 and S3. The semen volume was higher in the crossbred buck was higher than the Indian goat breeds reported by different authors (Prasad et al., 1970, Barberi; Sinha et al., 1981, Jamunapari; Sinha and Singh, 1982, Black Bengal; Jankiraman, 1990; Surti; Pattanaik et al., 1991, Ganjam; Puranik et al., 1993, Osmanabadi). However, Kale (1995) and Puranik et al., (1993) reported very less volume in crossbred buck. Seasonal variation of volume was found in Bagdad Hussain et al. (2012), and the best semen was found in September and October months which has similar climatic variable Mass Motility: The mass motility measured on 5 scale point. The mass motility of AXB buck semen given in Table and Figure The mass motility of semen was significantly different among the bucks. The mass motility of semen of AB1 was highest (3.39) than the other bucks AB1 AB2 AB3 AB4 AB5 Figure Mass motility (0-5 Scale) of crossbred AXB bucks semen The mass motility % was highest for buck AB1 (3.39), than AB2 (3.38), AB3 (3.16) AB4 (3.28) and AB5 (3.22). There was significant difference (P<0.05) between AB1 and AB3. However, there was no significant difference (P<0.05) for buck AB1 and AB2 for mass motility percent. However, there was significant difference (P<0.05) in mass motility for the seasons. The highest mass motility % found in winter season (S1) (3.61±0.03) than 60

85 S2 (2.81±0.06) and S3 (3.17±0.04) seasons. The mass motility differ (P<0.01) significantly for different seasons. The all over motility for AXB bucks was 3.29 ± S1 S2 S3 Figure Mass motility (0-5) for AXB bucks in different seasons However, mass activity was reported higher in indigenous breed (Singh et al., 1985, Jamunapari x Black Bengal; Mahemood et al., 1988, Chanthangi; Jankiraman, 1990, Surti x Marwari; Tuli et al., 1991, Boer and Singh, 1982, Black Bengal; Puranik et al., 1993, Osmanabadi). There may be lower mass motility in Alpine X Beetal crossbred goat because Kale (1995) and Naskar (1995) also reported similar values for Alpine X Beetal and Saanen X Beetal crossbred goat Progressive Motility: Progressive motility % was estimated for fresh samples of AXB bucks and the results found are presented in Table totable and Figure to Figure The percent progressive motility was 77.72±0.59, 75.76±0.73, 75.80±0.74, 76.40±0.58 and 76.60±0.36 for AB1, AB2, AB3, AB4 and AB5 bucks, respectively, and the overall progressive motility percent was 76.46± The progressive motility for Buck AB1 semen was significant (P<0.05) more than other bucks. However, it was found that there was no significant difference among rest of the bucks for percent progressive motility of semen. There was significant difference (P<0.01) among the seasons. The progressive motility % was 81.23±0.27 in S1 season, 71.58±0.70 in S2 season and 73.72±0.45 in S3 season for all over bucks. 61

86 AB1 AB2 AB3 AB4 AB5 Figure Progressive motility (%) of AXB buck semen S1 S2 S3 Figure Progressive motility (%) of AXB buck semen in different seasons The all over season progressive motility was 76.46±0.31. Buck wise difference for seasons is given in Table The hot dry and hot humid seasons are detrimental to the sperm quality due to long day length in the Karnal (Latitude 28 16' N 77 05' E Longitude) having day length of more than 13 hrs and it reaches maximum around 14 hrs in June and July and again reaches 12 hrs in September. Jankiraman (1990) reported maximum motility in monsoon (81.47 ± 2.94 and 78.90± 5.89) and minimum in summer (71.25± 3.25 and ± 2.25) for Surti and Marwari buck, respectively may be the home tract of these breed have longer monsoon and has less sun intensity due to cloud cover. There is a need to do further 62

87 study on light intensity and day length hour in tropical and sub tropical climate effect on semen quality of bucks as they are affected much due to season. Kale (1995) reported that in crossbred bucks (Alpine x Beetal and Saanen x Beetal) mean progressive motility percentage was 77.75± 0.77 and season and buck have significantly (p<0.01) influenced the progressive motility percent of the semen, which is similar to our study. Similar progressive motility was reported by other worker in India (Pandey et al.,1985, Jankiraman, 1990, Tuli et al., 1991, Singh and Purbey,1992; Puranik et al., 1993) Live Percentage: Non-eosinophilic (live) spermatozoa count % for AXB bucks were 81.18±0.56, 79.84±0.49, 77.62±0.37, 76.83±0.47 and 77.77±0.36 for AB1, AB2, AB3, AB4 and AB5 bucks respectively.table and Figure The live spermatozoa count % of buck AB1 found significant (P<0.01) different from AB3, AB4 and AB5 bucks, respectively. However, there was no difference between buck AB1 and AB2. And also no difference among buck AB3, AB4 and AB5 for live spermatozoa count % AB1 AB2 AB3 AB4 AB5 Figure Live (%) of AXB crossbred buck semen The live spermatozoa count % was significantly different in different seasons for each buck. The results are shown in Table and Figure All AXB bucks spermatozoa live count % in different seasons were 81.61±0.27, 74.32±0.36 and 77.68±0.23 for S1, S2 and S3 seasons respectively. There was significant difference (P<0.01) among the seasons. Highest live spermatozoa count 63

88 % observed in S1 than the rest of seasons, however, there was improvement in the live spermatozoa count in S3 season. Overall live spermatozoa count % was 78.65±021. Such variation in live sperm count was also reported in pure bred Alpine and Saanen buck in Greece by Karagiannidis, et al. (2000) S1 S2 S3 Figure Live (%) of AXB buck semen in different seasons Sperm Concentration (10 9 /ml): The spermatozoa concentration X10 9 /ml for AXB bucks was calculated and presented in Table-4.1. It was found that total spermatozoa concentration per ml of semen was 3.41±0.07, 2.55±0.06, 2.68±0.06, 3.30±0.05 and 2.93±0.08 for AB1, AB2, AB3, AB4 and AB5 bucks, respectively. The overall concentration of spermatozoa for AXB was 2.97±0.03 X10 9 /ml. The highest spermatozoa concentration was found in the semen of buck AB1 than the other, however, there was no significant difference (P<0.01) from buck AB4. Buck AB2 and AB3 also didn t significantly different from each other. Buck AB1 and AB4 were significantly different (P<0.01) from AB2, AB3 and AB5. The spermatozoa concentration the buck significantly (P<0.01) varied with seasons (Table-4.1.2). All over buck spermatozoa concentration for different seasons were 3.87±0.02, 2.58±0.03 and 2.72±0.02 in S1, S2 and S3 respectively. All seasons were significantly different (P<0.01) from each other for sperm concentration. 64

89 AB1 AB2 AB3 AB4 AB5 Figure Sperm Concentration (10 9 /ml) of AXB bucks S1 S2 S3 Figure Sperm Concentration (10 9 /ml) of AXB buck semen in different seasons The highest sperm concentration was observed during S1 season than the other two seasons. It may be due to less volume in S1 season and more volume in S2 and S3 season. The overall concentration of spermatozoa for AXB was 3.06±0.15. The highest spermatozoa concentration found in the semen of Buck AB2 and AB3 also didn t significantly different from each other. Similar findings are reported by Ahmad and Noakes (1996). In our study semen was collected for whole year and the average concentration value was lower in S2 and S3 season it may be due to more volume than S1 season. In the literature there 65

90 is wide variation of concentration was reported as most of the studies have not reported the season of evaluation. In Indian goats volume and concentration decreased with high temperature, relative humidity (RH) and high rainfall (Murugaiyah, 1992). Length of breeding season in goats varies inversely with latitude in temperate climate. At the mid high latitudes (35 o ) goat shows marked seasonality. In tropics and subtropics goat bred all time and kidding related to environment other than photoperiod (Murugaiyah, 1992). Effects of season on sperm concentration, our results were in agreement with those obtained by Ahmed, et al. (1997) in which they reported that highest values of sperm concentration in summer and autumn compared with spring HOST Percentage: The HOST positive spermatozoa % was 69.72±0.48, 68.88±0.56, 68.34±0.47, 69.61±0.42 and 70.02±0.41 for buck AB1, AB2, AB3, AB4 and AB5 respectively. There was significant difference (P<0.01) in HOST positive sperm % between buck AB1 and buck AB3, However there was no difference between AB2, AB4 and AB5. And also AB5 was significantly different (P<0.01) from AB3 for percent HOST positive sperm AB1 AB2 AB3 AB4 AB5 Figure HOST% of AXB buck semen The HOST % was 73.23±0.24, 64.45±0.31 and 67.55±0.20 in S1, S2 and S3 seasons. Over all HOST% was 68.41± 0.16 in AB bucks. The seasonal differences 66

91 were observed among the three seasons for individual buck as well as all over bucks in different seasons. Percentage of HOST reacted spermatozoa were highest in S1 season than the other two seasons. There was significant (P<0.01) difference among the seasons HOST per cent. The HOST % was improving in S3 season S1 S2 S3 Figure HOST % of AXB buck semen in different seasons Intact Acrosome: Intact acrosome % was 93.02±0.23, 93.66±0.18, 93.68±0.17, 94.49±0.21 and 94.62±0.15 for AB1, AB2, AB3, AB4 and AB5 bucks respectively. The overall intact acrosome % for AXB breed was 93.89±0.09. the intact acrosome % of buck AB1 was significant different (P.0.05) from buck AB2, AB3, and AB5; however, there was no significant difference between buck AB2 and AB3 and also between AB4 and AB5. Intact acrosome % for AXB breed was 95.13±0.11, 92.21±0.16 and 92.84±0.14 for S1, S2 and S3 seasons respectively. 67

92 AB1 AB2 AB3 AB4 AB5 Figure Intact Acrosome % of AXB buck semen S1 S2 S3 Figure Intact Acrosome of AXB buck semen in different seasons. Intact acrosome % was 95.57±0.11, 94.47±0.18, 94.10±0.12 for S1 S2 and S3 season respectively. 4.2 Freezability of crossbred buck semen in different seasons in relation to Heparin Binding Proteins (HBPs) Heparin binding Protein expression in different seasons Seminal plasma were pooled from every month samples from different bucks and purified. In winter 2.58 µgm/µl and in Hot dry 3.38 µgm/µl and in Hot humid it 68

93 was found to be 4.4 µgm/µl were harvested in the heparin binding column. These proteins were subjected to SDS page and the page photo is given in Fig Figure SDS Page of seminal plasma heparin binding proteins in different seasons Table Expression of Heparin Binding Proteins (HBPs) in seminal plasma of AXB crossbred goat in different seasons in SDS-Page Season Spot No. Winter (S1) 1 2 Hot-Dry Summer (S2) Hot humid Summer (3) The number of HBPs expressed in seminal plasma of crossbred goat. There was variation in the protein expression in different seasons, however, the prominent HBP spot no.3 was prominent in Hot-Dry Summer (S2) and Hot Humid Summer 69

94 (S3) than the Winter (S1) season. During S2 and S3 season, there was maximum deleterious effect on fresh and frozen semen quality. To know which protein causing the reduced freezability during S2 and S3 season further study done with 2D gel electrophoresis of purified HBPs and results are presented in Figure D Gel electrophoresis of Heparin binding Protein in different seasons Seminal plasma were pooled from every month samples from different bucks and purified. In winter 2.58 µgm/µl and in Hot dry 3.38 µgm/µl and in Hot humid it was found to be 4.4 µgm/µl were harvested in the heparin binding column. These heparin binding proteins were subjected to 2D Gel electrophoresis and some of the new proteins were identified in S2 and S3 season such HBPs were not visible in the winter (S1) season. The HBPs spot no. 6, 9, 10 and 11 were expressed in S2 and S3 seasons where as these proteins were absent in S1 season. In the same from freezing data there was reduced freezability in S2 and S3 seasons than the S1 season. The post thaw progressive motility % was in S1 season which was reduced to and in S2 and S3 seasons, respectively. Live % was in S1 season reduced to and in S2 and S3 season, respectively. The HOST % in S1 reduced to and in S2 and S3 seasons, respectively. The acrosome intact% was reduced to and in S2 and S3 seasons, respectively. This data indicated that there was reduction in freezability of crossbred goat semen in S2 and S3 seasons at the same time there was expression of spot no. 6, 9, 10 and 11 HBPs in these seasons than the winter S1 season. Among these spots some may be responsible for reduction of freezability in S2 and S3 seasons. On this data it may be concluded that HBPs played important role in freezability of crossbred goat semen. Identification and characterization of these spot may clear the picture and the importance of HBPs in freezability of crossbred goat semen. 70

95 Figure D Gel electrophoresis of Heparin binding Proteins of seminal plasma of AXB crossbred goat in different seasons Table D Gel electrophoresis for Heparin binding Proteins (HBPs) of seminal plasma of AXB crossbred goat for different seasons Season Expression of Protein Spot No. Winter (S1) Hot-Dry Summer (S2) Hot humid Summer (3) The freezability of AXB crossbred goat semen was improving during S3 seasons. Similar results for freezability of Boar goat semen in different seasons were reported by Tuli and Holtz (1995) in northern temperate zone. Falci et al. (2002) investigated seasonal changes in seminal plasma protein of Saanen goat under natural conditions. Similar protein was found in the breeding season in our study (S1 band). The pattern of Heparin-Binding Proteins (HBPs) 178 KD was unique to breeding season. HBPs caused deterioration of sperm motility and acrosome breakage. These data showed proteins from goat seminal plasma are 71

96 under seasonal control and associated with sperm function during breeding and nonbreeding seasons. A significant increase protein in non breeding season than observed the breeding season (S1). In hot dry season (S2) season there was over expression of some protein which gradually decreased in Hot-Humid Suumer season (S3). Two types of extender were used Extender-I (TCG-Egg yolk) and Extender-II (TCG+Trehalose-Egg Yolk). The Extender-II was developed and used in this experiment. Effect of trehalose addition to TCG extender was evaluated in this experiment with freezability of AXB crossbred goat semen in relation to heparin binding proteins in different seasons Live % Prograssive Motlity % HOST% Intact Acrosome % S1 S2 S3 Figure Pre-freeze parameters in different seasons. The pre-freeze parameters are presented in Table to Table and ANOVA Table There was significant (P<0.01) difference between seasons. All parameters like live%, progressive motility %, HOST % and intact acrosome % were significantly (P<0.01) higher in S1 than S2 and S3 seasons. Lowest found in S2 season an d during S3 season there was improvement in these parameters. 72

97 Live % Prograssive Motlity % HOST% Intact Acrosome % 10 0 AB1 AB2 AB3 AB4 AB5 Figure Pre-freeze parameters of semen of AXB bucks Ext 1 Ext Live % Prograssive Motlity % HOST% Intact Acrosome % Figure Pre-freeze parameters of semen cryopreserved in different extenders There were two types of extender used for cryopreservation of semen (TCEG and TCEG+ trehalose). For during pre-freeze stage there was significant (P<0.05) difference between the extenders for all parameters like live per cent, progressive motility per cent, HOST per cent, intact acrosome per cent. 73

98 S1 S2 S Live % Prograssive Motlity % HOST% Intact Acrosome % Figure Post-thaw parameters of in different seasons. The results of post thaw semen parameters are presented in Table to and ANOVA Table to 14. There was significant difference (P<0.05) between the seasons for post-thaw live per cent of semen. The live per cent concentration for S1 was more than the other two seasons. There was no significant difference between rest of seasons S2 and S3. Progressive motility, HOST per cent and intact acrosome per cent also were higher for S1 than the other seasons Live % Prograssive Motlity % HOST% Intact Acrosome % AB1 AB2 AB3 AB4 AB5 Figure Post-thaw parameters of different AXB bucks There was significant (P<0.01) difference between the extenders for post thaw live per cent, progressive motility per cent, HOST per cent and intact acrosome per cent (Figure-4.2.6). Similar findings are also reported by Dorado et al. (2010) in 74

99 Trehalose extender. However, there was reduction in motility and acrosome integrity after freezing and thawing and trehalose extender was better preserving the quality of post thaw character of spermatozoa Ext 1 EXt Live % Prograssive Motlity % HOST% Intact Acrosome % Figure Post thaw parameters of semen cryopreserved in different extenders Eiman et al. (2003) reported that trehalose acted as cryoprotectant in the Triscitric acid glucose (TCG) diluent developed by Salamon and Ritar, (1982). The insertion of these disaccharides into the membrane limits the amount of dehydration that can occur, and consequently the physical damage due to cell volume changes associated with freezing and thawing (Liu et al., 1998). Trehalose also causes an increase in membrane fluidity due to protein and phospholipids reorganization, a suppression of the injurious effect of membrane lipid phase transition. Cell damage is minimized due to less intracellular ice formation and consequently more viable cells being recovered following cryopreservation (Eiman et al. 2003). In spite of the evidence in favour of the beneficial effects of trehalose, not all studies have found the same results. Chen et al. (1993) report that trehalose caused only minor improvement in bull sperm survival, and Liu et al.(1998), in a study on the freezability of bull spermatozoa in TCG extender containing up to 25% (v:v) trehalose or sucrose, concluded that replacing part of TCG-containing egg yolk with these sugars had no beneficial effects. The low concentrations of trehalose (0.05, 0.1 M) used by these researchers may account for the low improvement in sperm survival because trehalose in high concentrations was used by many authors who found significant protection against freeze damage (Iwakiri et al., 2000). The addition of 75

100 Table Mean ± SE for Pre-freeze parameters of Alpine X Beetal crossbred buck semen cryopreserved during different seasons Overall (220) Means bearing different superscript in a column differ significant *P<0.05, **P<0.01 Table Mean ± SE for Pre-freeze parameters of Alpine X Beetal crossbred buck semen cryopreserved during different seasons Buck Live % Live % Progressive motility % HOST % Intact acrosome % Season Mean ±SE Mean ±SE Mean ±SE Mean ±SE S1 (100) a a a a 0.36 S2 (60) b b b b 0.46 S3 ( c c c c 0.40 Progressive motility % HOST % Intact acrosome % Mean ±SE Mean ±SE Mean ±SE Mean ±SE AB1 (44) a a a ac 0.87 AB2 (44) b a b b 0.73 AB3 (44) a b b bc 1.23 AB4(44) a a c abc 0.61 AB5(44) a a c c 0.79 Means bearing different superscript in a column differ significant *P<0.05, **P<0.01 Table Mean ± SE for Pre-freeze parameters of Alpine X Beetal crossbred buck semen cryopreserved in two types of extenders during all over seasons Live % Progressive motility % HOST % Intact acrosome % Extender Mean ± SE Mean ±SE Mean ±SE Mean ±SE Ext 1 (110) a a a a 0.34 Ext 2 (110) b b b b 0.26 Means bearing different superscript in a column differ significant *P<0.05, **P<0.01

101 Table Mean ± SE for interaction of Season X Extender of buck semen cryopreserved in two types of extenders during different seasons for prefreez parameters. Live % Progressive motility % HOST % Intact acrosome % Season X Extender Mean ±SE Mean ±SE Mean ±SE Mean ±SE Ext S1 Ext Ext S2 Ext Ext S3 Ext Table Mean ± SE for interaction of Buck X Extender of buck semen cryopreserved in two types of extenders for prefreez parameters. Live % Progressive motility % HOST % Intact acrosome % Buck X Extender Mean ±SE Mean ±SE Mean ±SE Mean ±SE Ext AB1 Ext Ext AB2 Ext Ext AB3 Ext Ext AB4 Ext Ext AB5 Ext

102 Table Mean ± SE for interaction of Season X Buck X Extender of crossbred buck (Alpine X Beetal) semen cryopreserved in two types of extenders during different seasons for prefreez parameters (n=220) Live % Progressive motility % HOST % Intact acrosome % Season X Buck X Extender Mean ±SE Mean ±SE Mean ±SE Mean ±SE Ext AB1 Ext Ext AB2 Ext Ext AB3 Ext Ext AB4 Ext Ext AB5 Ext Ext AB1 Ext Ext AB2 Ext Ext AB3 Ext Ext AB4 Ext Ext S2 AB5 Ext Ext AB1 Ext Ext AB2 Ext Ext AB3 Ext Ext AB4 Ext Ext S3 AB5 Ext

103 Table Mean ± SE for Post-thaw semen parameters cryopreserved in two types of extender during different seasons. Live % Progressive motility % HOST % Intact acrosome % Season Mean ±SE Mean ±SE Mean ±SE Mean ±SE S1 (100) a a a a 0.67 S2 (60) b b b b 0.78 S3 ( b b b b 0.60 Overall (220) Means bearing different superscript in a column differ significant *P<0.05 Table Mean ± SE for Post-thaw semen parameters cryopreserved in two types of extender during different seasons. Live% Progressive motility % HOST% Intact acrosome % Buck Mean ±SE Mean ±SE Mean ±SE Mean ±SE AB1 (44) ac a a af 1.30 AB2 (44) ac adf ab ade 1.23 AB3 (44) bd bd bc cde 1.39 AB4(44) ac ae ac def 1.39 AB5(44) bd bef bc e 1.26 Means bearing different superscript in a column differ significant *P<0.05, Table Mean ± SE for Post-thaw semen parameters cryopreserved in two types of extender during different seasons. Live % Progressive motility % HOST % Intact acrosome % Extender Mean ±SE Mean ±SE Mean ±SE Mean ±SE Ext 1 (110) a a a a 0.48 Ext 2 (110) b b b b 0.67 Means bearing different superscript in a column differ significant **P<0.01

104 Table Mean ± SE for post-thaw parameters of season X extender interaction of buck semen cryopreserved in two types of extenders during different seasons. Live % Progressive motility % HOST % Intact acrosome % Season X Extender Mean ±SE Mean ±SE Mean ±SE Mean ±SE S1 EXT a a a a 1.05 EXT b b b b 0.89 S2 EXT a a a a 0.95 EXT b b b b 0.69 S3 EXT a a a a 1.07 EXT b b b b 0.79 Means bearing different superscript in a column for each season differ significant *P<0.05 Table Mean ± SE for post-thaw parameters of Buck X Extender interaction of buck semen cryopreserved in two types of extenders during different seasons. Live % Progressive motility % HOST % Intact acrosome % Buck X Extender Mean ±SE Mean ±SE Mean ±SE Mean ±SE AB1 AB2 AB3 AB4 AB5 EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT

105 Table Mean ± SE for post-thaw parameter of Season X Buck X Extender interaction of buck semen cryopreserved in two types of extenders during different seasons. S1 S2 S3 Season X Buck X Extender AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 Live % Progressive motility % HOST % Intact acrosome % Mean ±SE Mean ±SE Mean ±SE Mean ±SE EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT EXT

106 Table ANOVA for pre-freeze parameters of semen frozen in different extenders during different season seasons Source d.f. Live % Progressive motility% HOST% Intact Acrosome % SEASON ** ** ** ** BUCK ** ** ** EXTENDER * ** * * SEASON * BUCK * SEASON * EXTENDER BUCK * EXTENDER SEASON * BUCK * EXTENDER Error *Significant P<0.05 ** P<0.01 Table ANOVA for post -thaw parameters of semen frozen in different extenders during different season Source d.f. Live % Progressive motility % HOST % Intact Acrosome % SEASON ** ** ** ** BUCK * ** ** ** EXTENDER ** ** ** * SEASON * BUCK SEASON * EXTENDER BUCK * EXTENDER SEASON * BUCK * EXTENDER Error *Significant P<0.05 ** P<0.01

107 high concentrations of trehalose to sperm extender provide the best protection with regard to post-thaw motility parameters, recovery rate, thermal resistance, and acrosome integrity, with the best results obtained for the 100% trehalose extender. However, supplementation of the diluent by trehalose has been shown to have varying effects on freeze tolerance (Molinia et al., 1994; Yildiz et al., 2000). Woelders et al. (1997) demonstrated that an isotonic sugar medium in which Triscitrate components were substituted with sucrose and trehalose was significantly superior to a Tris-citrate egg yolk medium in preserving the motility and acrosome integrity of bovine spermatozoa. Moreover, in a comparison of raffinose and trehalose, Storey et al. (1998) showed that trehalose resulted in a significantly better recovery rate in intact mouse spermatozoa. In addition, Aisen et al. (2000) observed that trehalose significantly improved the viability of ram spermatozoa assessed for motility and acrosome integrity, with the best results obtained for a trehalose + EDTA extender. Molinia et al. (1994) concluded that the motility of frozen-thawed ram spermatozoa was higher in the presence of sucrose or trehalose than in the presence of glucose when glycerol was not incorporated in the diluent; whereas when glycerol was employed in the diluent, no differences were observed among the various sugar types. Frozen-thawed dog spermatozoa have also been found to be protected by the supplementation of trehalose in the diluent (Iwakiri et al., 2000). In our study we have found that addition of trehalose has beneficial effect in semen preservation in all the seasons in all the parameters studied in pre-freeze and post thaw semen. 4.3 Effect of modified of method semen collection on semen parameters characteristics in different seasons The study was conducted to find the significance of reducing contact between seminal plasma components and the sperm membrane at ejaculation on cryopreservation of semen in different seasons. The experiment was carried out in winter (December, January) (S1), Hot-dry summer (April and May) (S2) and Hot- humid Summer- (July, August) (S3). The results are depicted in Table to Table

108 ml 10 ml Live % Prograssive Motlity % HOST% Intact Acrosome % Figure Fresh semen quality parameters collected by different method of collection. There was significant (P<0.05) difference between the methods of collection on fresh semen quality. Live per cent was significantly (P<0.05) high when semen collected in tubes containing 10 ml TCG buffer solution and also other parameters such as progressive motility per cent, HOST per cent and intact acrosome were high at fresh semen evaluation (Table and Figure-4.3.1). Also there was significant (P<0.05) difference within season for semen collection method. Fresh semen parameters were better for semen collected in collection tubes with 10ml of TCG buffer than without TCG buffer for each season Live % Prograssive Motlity % HOST% Intact Acrosome % 0 ml 10 ml Figure Pre-freeze parameters of semen collected by different method of collection 77

109 There was significant (P<0.05) difference for live per cent, progressive motility per cent, HOST per cent and intact acrosome per cent of pre-freeze semen when semen samples were collected by two different methods of semen collection. There significantly higher pre-freeze semen quality observed when semen collected in tubes containing 10 ml of TCG buffer solution than the semen collected without buffer (with empty collection tubes). Also there was significant (P<0.05) difference within season for semen collection method. Pre-freez semen parameters were better for semen collected in collection tubes with 10ml of TCG buffer than without TCG buffer for each season ml 10 ml 10 0 Live % Prograssive Motlity % HOST% Intact Acrosome % Figure Post-thaw parameters of semen collected by different Method of collection There was significant (P<0.01) difference between the collection methods for post-thaw semen parameters. Live per cent, progressive motility per cent, HOST per cent and intact acrosome per cent were significantly (P<0.01) higher when semen samples collected in 10 ml TCG buffer solution than the semen collected without TCG buffer or in empty collection tubes. There was significant (P<0.05) difference within season for semen collection method. Post thaw semen parameters were better for semen collected in collection tubes with 10ml of TCG buffer than without TCG buffer for each season. The semen collected in collection tubes containing TCG buffer proved better for freezing crossbred goat semen. Similar results were reported by De Pauw et al. (2003) that when bovine semen was collected in tubes containing a small volume of extender supplemented with egg yolk, the characteristics of 78

110 ejaculated spermatozoa were improved. Thus, it is conceivable that the effects of seminal plasma on ejaculated spermatozoa after washing are absolutely different from the effect of accessory gland on epididymal spermatozoa during ejaculation. On the other hand Way et al. (2000) examined the effect of accessory gland fluid on viability of bovine epididymal spermatozoa and demonstrated that exposure of epididymal sperm to accessory gland fluids decreased the percentage of motile spermatozoa and accelerated cell death. Blash et al. (2000) also reported differences in motility and acrosome integrity between the ejaculated and epididymal spermatozoa after incubation in seminal plasma. The percentage of live sperm and acrosome integrity for epididymal sperm were higher than those for ejaculated sperm and freezability also were higher for epididymal spermatozoa than the ejaculated spermatozoa. Furthermore, it has been reported that ejaculated even washed spermatozoa were unable to attain the quality found for epididymal spermatozoa (Lebouef et al., 2000). If the ejaculated spermatozoa can be collected in a way that minimizes the effect of accessory reproductive fluids, it would be possible to enhance sperm motility and survival of preservation. Yamashiro et al. (2007) found that goat semen collected in tubes with large volume of extender improved motility, progressive motility and acrosome integrity of frozen-thawed spermatozoa (p<0.01) significantly. We have employed the same method at a dilution rate of 10 times the volume of ejaculates for semen and washed twice in TCG buffer and our results in all season have improved from the control samples without any adverse effect. 79

111 Table Mean ± SE for fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during entire seasons. Method of collection Live% Progressive motility % HOST% Intact acrosome % Mean ±SE Mean ±SE Mean ±SE Mean ±SE 0 ml (60) a a a a ml (60) b b b b 0.42 Over all (120) Means bearing different superscript in a column differ Significant *P<0.05 ** P<0.01 Table Mean ± SE for interaction of Season X Method of collection on fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods cryopreserved during different seasons. Season X Method of collection Winter (S1) Hot-dry Summer (S2) Live% Progressive motility % HOST% Intact acrosome % Mean ±SE Mean ±SE Mean ±SE Mean ±SE 0 ml a a a a ml b b b b ml a a a a ml b b b b 0.41 Hot-humid 0 ml a a a a 0.29 Summer (S3) 10 ml b b b b 0.41 Means bearing different superscript in a column differ Significant P<0.05 for each season Table Mean ± SE for interaction of Buck X Method of semen collection on fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods cryopreserved during different seasons. Buck X Method of Collection AB1 AB2 AB3 AB4 AB5 Live% Progressive motility % HOST% Intact acrosome % Mean ±SE Mean ±SE Mean ±SE Mean ±SE 0 ml a a a a ml ml a a a a ml ml a a a a ml ml a a a a ml ml a a a a ml

112 Table Mean ± SE for interaction for Season X Buck X Method of collection on fresh semen parameters of Alpine X Beetal crossbred buck semen collected by different methods cryopreserved during different seasons. Season X Buck X Method of collection S1 S2 S3 AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 Live% Progressive motility % HOST% Intact acrosome % Mean ±SE Mean ±SE Mean ±SE Mean ±SE 0 ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml

113 Table Mean ± SE for pre-freeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during entire season (n=120) Live % Progressive motility % HOST% Intact acrosome % Method of collection Mean ±SE Mean ±SE Mean ±SE Mean ±SE 0 ml (60) a a a a ml (60) b b b b 0.65 Over all (120) Means bearing different superscript in a column differ Significant *P<0.05 ** P<0.01 Table Mean ± SE for interaction of Season X Method of collection on pre-freeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Season X Method of collection Live% Progressive motility % HOST% Intact acrosome % Mean ±SE Mean ±SE Mean ±SE Mean ±SE 0 ml a a a Winter (S1) 10 ml b b b Hot-dry Summer 0 ml a a a (S2) 10 ml b b b Hot-humid Summer 0 ml a a a a 0.81 (S3) 10 ml b b b b 0.65 Means bearing different superscript in a column for each season differ Significant *P<0.05 ** P<0.01 Table Mean ± SE for interaction Buck X Extender on pre-freeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Progressive motility % Intact acrosome % Live% HOST% Buck X Method of collection Mean ±SE Mean ±SE Mean ±SE Mean ±SE AB1 AB2 AB3 AB4 AB5 0 ml ml ml ml ml ml ml ml ml ml

114 Table Mean ± SE for interaction for Season X Buck X Extender on pre-freeze parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons (n=120) Progressive motility % HOST % Intact acrosome % Live % Season X Buck X Method of collection Mean ±SE Mean ±SE Mean ±SE Mean ±SE S1 S2 S3 AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 0 ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml

115 Table Mean ± SE for post-thaw parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during entire year. Live % Progressive motility % HOST % Intact acrosome % Method of collection Mean ±SE Mean ±SE Mean ±SE Mean ±SE 0 ml (60) a a a a ml (60) b b b b 0.64 Over all (120) Means bearing different superscript in a column differ Significant *P<0.05 ** P<0.01 Table Mean ± SE for interaction of Season X Method of collection on post-thaw parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons. Progressive motility % HOST % Intact acrosome % Live % Season X Method of collection Mean ±SE Mean ±SE Mean ±SE Mean ±SE S1 S2 0 ml a a a a ml b b b b ml a a a a ml b b b b ml a a a a 0.78 S3 10 ml b b b b 0.79 Table Mean ± SE for interaction Buck X Method of collection post-thaw semen parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons. Progressive motility % HOST % Intact acrosome % Live % Buck X Method of collection Mean ±SE Mean ±SE Mean ±SE Mean ±SE AB1 AB2 AB3 AB4 AB5 0 ml ml ml ml ml ml ml ml ml ml

116 Table Mean ± SE for interaction for Season X Buck X Method of collection on post-thaw semen parameters of Alpine X Beetal crossbred buck semen collected by different methods and cryopreserved during different seasons. Progressive motility % HOST % Intact acrosome % Live % Season X Buck X Method of collection Mean ±SE Mean ±SE Mean ±SE Mean ±SE S1 S2 S3 AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 AB1 AB2 AB3 AB4 AB5 0 ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml

117 Table Mean square for fresh semen parameters collected by different methods of collection (Empty collection tube and with 10 ml TCG buffer) during different seasons Source d.f. Live % Progressiv e motility % HOST % Intact Acrosome % Season ** ** ** ** Buck * Method of collection ** ** ** Season * Buck Season * Method of collection * Buck * Method of collection Season * Buck * Method of collection Error Significant *P<0.05 ** P<0.01 Table Mean square for Pre- freeze semen parameters collected by different methods of collection (Empty collection tube and with 10 ml TCG buffer) during different seasons Sources D.f. Live% Progressiv e motility% HOST% Intact acrosome % Season ** ** ** ** Buck * * Method of collection ** * ** * Season * Buck Season * Method of collection Buck * Method of collection Season * Buck * Method of collection Error Significant *P<0.05 ** P<0.01

118 Table Mean square for Post-thaw semen parameters collected by different methods of collection (Empty collection tube and with 10ML TCG buffer) during different seasons Source d.f. Live % Progressive motility % Significant *P<0.05 ** P<0.01 HOST % Intact Acrosome % Season * ** *** ** Buck * * * Method of collection * ** ** * Season * Buck Season * Method of collection Buck * Method of collection Season * Buck * Method of collection Error

119 4.4 Effect of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen. Effect of trehalose extender (0, 100) containing 0.05% SDS on characteristics of cryopreserved AXB goat semen. Results are presented in Table to and Figure to Live % Prograssive motility % HOST % Intact acrosome % Ext-1 Ext-2 Figure Semen parameters at pre-freeze in two types of extenders Ext-1 Ext Live % Prograssive motility % HOST % Intact acrosome % Figure Post-thaw of semen parameters in two types of extenders It was observed that pre-freeze and post thaw semen quality was significantly (P<0.05) higher for Extender1 than Extender2 in terms of percent liveability, progressive motility, HOST and intact acrosome. Sperm frozen in the presence of 80

120 375 mm trehalose resulted in significantly (P<0.05) greater total sperm liveability, progressive motility, HOST and intact acrosome than without trehalose. 4.5 Effect of different concentration of trehalose incorporation in semen extender on characteristics of cryopreserved goat semen Sperm pellets were diluted with different concentrations of trehalose extender (0.0, 30, 70 and 100 per cent) containing 0.05 per cent SDS and egg yolk 20 per cent and cryopreserved. Results are presented in Table to and Figure to There was significant (P<0.05) difference for live per cent at pre-freeze and post thaw of semen. At pre-freeze Live per cent for T4 extender (100 per cent trehalose) was significantly (P<0.01) higher than Extender T1 (without trehalose). However there was no significant (P<0.05) difference between T1 and T2 and also there was no significant (P<0.05) difference between T3 and T4 for live per cent Initial Pre-freez Post-thaw TCG ( C ) T 35% T 70% T100% Figure Live per cent of spermatozoa in semen at initial, pre-freeze and post-thaw stages of semen preservation 81

121 Table Mean ± SE of freezability parameters of crossbred buck (Alpine X Beetal) semen in TCG-egg Yolk Extender-1 and Trehalose-egg Yolk based extender-2. Buck No. Live % Progressive Motility % Host % Intact acrosome % Extender 1 Extender 2 Extender 1 Extender 2 Extender 1 Extender 2 Extender 1 Extender 2 AB1 (20) AB2 (20) AB3 (20) AB4 (20) AB5 (20) All bucks (100) Pre-freez ± 1.22 a ± 0.81 b ± 1.23 a ± 0.75 b ± 0.77 a ± 0.56 b ± 1.16 a ± 0.72 b Post-thaw ± 1.72 a ± 1.09 b ± 1.43 a ± 0.94 a ± 1.62 a ± 1.16 b ± 1.62 a ± 1.10 b Pre-freez ± 0.60 a ± 0.44 b ± 0.75 a ± 0.61 a ± 0.50 a ± 0.52 a ± ± 0.45 Post-thaw ± ± ± ± ± ± ± ± 1.40 Pre-freez ± ± ± ± ± ± ± ± 0.42 Post-thaw ± ± ± ± ± ± ± ± 1.35 Pre-freez ± ± ± ± ± ± ± ± 0.43 Post-thaw ± ± ± ± ± ± ± ± 0.64 Pre-freez ± ± ± ± ± ± ± 0.90 a ± 0.55 b Post-thaw ± ± ± ± ± ± ± ± 1.93 Pre-freez ± 0.42 a ± 0.23 b ± 0.50 a ± 0.31 b ± 0.53 a ± 0.33 b ± 0.38 a ± 0.26 b Post-thaw ± 1.02 a ± 0.61 b ± 0.89 a ± 0.60 b ± 0.82 a ± 0.77 b ± 0.92 a ± 0.60 b Means bearing different superscript in a row for each parameter differ Significant *P<0.05 ** P<0.01

122 Table Mean square for pre-freeze and post-thaw parameters for TCG (Extender- 1) and TCG+ trehalose based extenders (Extender-2) Parameters d.f. Pre- freeze Post- thaw Buck Live % * * Progressive motility % * HOST% * ** Intact acrosome % * * Extender Live% ** ** Progressive motility % ** ** HOST% ** ** Intact acrosome % ** ** Buck * Extender Live % Progressive motility % HOST% Intact acrosome % Means bearing different superscript in a column differ Significant *P<0.05 ** P<0.01**

123 After post-thaw live per cent for extender T4 was significantly (P<0.01) high than other extenders. There was significant (P<0.01) difference among extenders T1, T2, T3 and T4 for live per cent. There was difference for progressive motility per cent at pre-freeze but differences were not significantly. However, post-thaw of semen samples were significantly (P<0.01) different for progressive motility per cent TCG ( C ) T 35% T 70% T100% Initial Pre-freez Post-thaw Figure Progressive motility spermatozoa in semen at initial, prefreeze and post-thaw stages of preservation Post thaw progressive motility per cent for extender T4 was significantly (P<0.05) highest than other extender and there was significant (P<0.01) difference between extender T1 and T3. However, there was no significant difference betweent1 and T2 and also between T2 and T3 for progressive motility per cent. There were differences among the extenders for HOST per cent at pre-freeze but those were not (P<0.05) significantly. 82

124 Initial Pre-freez Post-thaw 10 0 TCG ( C ) T 35% T 70% T100% Figure HOST per cent of spermatozoa at initial, pre-freeze and post-thaw stages of preservation Post-thaw HOST per cent for T4 extender (100 per cent trehalose) was significantly (P<0.01) higher than Extender T1 (without trehalose). However, there was no significant (P<0.01) difference between T2 and T3 extenders for HOST per cent TCG ( C ) T 35% T 70% T100% Initial Pre-freez Post-thaw Figure Intact Acrosome spermatozoa per cent at initial, pre-freeze and post-thaw stages of preservation There was difference for progressive motility per cent at pre-freeze but differences were not significantly. However, post-thaw of semen samples were significantly (P<0.01) different for progressive motility per cent. 83

125 Table Mean ± SE of freezability parameters of AXB crossbred goat semen in different concentration of Trehalose. Pre-freez TCG ( C ) T 35% T 70% T100% Live% ± 1.63 c ± 1.28 cb ± 0.94 b ± 0.76 a Progressive motility % ± ± ± ± 1.30 HOST % ± ± ± ± 1.42 Intact acrosome % ± 1.36 c ± cb ± 0.76 b ± 0.79 a Post thaw TCG ( C ) T 35% T 70% T100% Live% ± 1.21 d ± 1.20 c ± 0.61 b ± 0.91 a Progressive motility % ± 1.16 a ± ab ± b ± 1.38 c HOST % ± 1.20 a ± 0.84 ab ± 1.03 b 59.0 ± 1.09 c Intact acrosome % ± 1.48 a ± bd ± 0.74 b ± 0.94 c Means bearing different superscript in a row for each parameter differ Significant *P<0.05 ** P<0.01

126 Table Mean square freezability parameters of AXB crossbred goat semen in different concentration of Trehalose Sources d.f. Initial Per-freeze Post-thaw Live% Progressive motility % HOST% Intact acrosome % Between Groups Within Groups Between Groups Within Groups Between Groups Within Groups Between Groups Within Groups * ** ** ** * ** Means bearing different superscript in a column for each parameter differ Significant *P<0.05 ** P<0.01

127 From above results it was observed that higher concentration of trehalose with presence of 0.05% SDS in extender preserved better the lower concentration of trehalose with presence of 0.05% SDS. Similar results were reported by Eiman et al. (2003). This beneficial effect of trehalose was due to the insertion of these disaccharides into the membrane. Trehalose limited the amount of dehydration and consequently the physical damage due to cell volume changes associated with freezing and thawing (Liu et al., 1998). Trehalose also caused an increase in membrane fluidity due to protein and phospholipids reorganization, a suppression of the injurious effect of membrane lipid phase transition. Cell damage was minimized due to less intracellular ice formation and consequently more viable cells being recovered following cryopreservation (Eiman et al. 2003).. However, supplementation of the diluent by trehalose has been shown to have varying effects on freeze tolerance (Molinia et al., 1994; Yildiz et al., 2000). Woelders et al. (1997) demonstrated that an isotonic sugar medium in which Triscitrate components were substituted with sucrose and trehalose was significantly superior to a Tris-citrate egg yolk medium in preserving the motility and acrosome integrity of bovine spermatozoa. Moreover, in a comparison of raffinose and trehalose, Storey et al. (1998) showed that trehalose resulted in a significantly better recovery rate in intact mouse spermatozoa. In addition, Aisen et al. (2000) observed that trehalose significantly improved the viability of ram spermatozoa assessed for motility and acrosome integrity, with the best results obtained for a trehalose + EDTA extender. Molinia et al. (1994) concluded that the motility of frozen-thawed ram spermatozoa was higher in the presence of trehalose than in the presence of glucose when glycerol was not incorporated. Frozen-thawed dog spermatozoa have also been found to be protected by the supplementation of trehalose in the diluent (Iwakiri et al., 2000). In our study we have found that addition of trehalose has beneficial effect in semen preservation in all the seasons in all the parameters studied at prefreeze and post thaw semen. 84

128 4.6 Freezability of crossbred goat semen in Tris-soya-based and trehalose - soya-based extenders. The results are presented in Table to and Figure to At pre-freeze, there was higher live %, HOST % for TCG+T Extender than the other extender; however, there was no significant difference among the different extenders for progressive motility % and intact acrosome % EXT-1 EXT-2 EXT-3 EXT-4 Initial Pre-Freez Post-thaw Figure live per cent at different stages of cryopreservation for different types of extenders EXT-1 EXT-2 EXT-3 EXT-4 Initial Pre-Freez Post-thaw Figure Progressive motility per cent at different stages of cryopreservation for different types of extenders 85

129 Table Mean ± SE for freezability parameters of crossbred goat semen in different types of egg yolk and soya extract based extenders. (n=10) Types of extender C1: TCG EY C2 : TCG+35%T EY TCG Soya extract TCG+35%T Soya extract Means bearing different superscript in a column differ Significant *P<0.05 ** P<0.01. Initial ± ± ± ±0.19 Live % Progressive motility % HOST % Intact acrosome % Pre- Postthaw Pre- Post- Pre- Pre- Post- Freeze Initial Freeze thaw Initial Freeze Post-thaw Initial Freeze thaw ± ± 0.82 ± 1.52 ad ±0.82 ± 1.13 ± 2.10 a ±1.74 a ± 1.35 a ±0.38 ± 0.80 ± 1.77 a ± ± ± ± 1.70 b ± ± 0.82 c ± ± 1.17 d ± ± ± ± ± 1.83 b ±1.82 a ± a ± ± 1.64 ±1.13 b ± ± 1.75 c ±1.03 b ± 1.44 b ± ± 0.26 ac ± ± 1.22 d ± ± ± ± ± 1.58 b ± 0.80 c ± 1.26 ad

130 Table Mean square for freezability parameters of crossbred goat semen in different types egg yolk and soya extract based extender supplemented with trehalose at initial, pre-freeze and post thaw. Source d. f. Initial Per-freeze Post- thaw Live % ** Progressive motility % ** HOST% * ** Intact acrosome % * ** Significant *P<0.05 ** P<0.01

131 Initial Pre-Freez Post-thaw 10 0 EXT-1 EXT-2 EXT-3 EXT-4 Figure HOST per cent at different stages of cryopreservation for different types of extenders EXT-1 EXT-2 EXT-3 EXT-4 Initial Pre-Freez Post-thaw Figure Intact acrosome % at different stages of cryopreservation for different types of extenders There was significant difference for freezing parameters among different extenders. There was significantly higher live%, progressive motility%, HOST% and intact acrosome % in extender (TCG+T) EY (Ext.1) than Ext.1, Ext.3 and Ext.4. Soya based showed comparable results with egg yolk based extender. Supplementation with trehalose enhanced the freezability in terms of freezing parameters. 86

132 The Tris Citrate and glucose based extender with 20% Egg yolk and 4% glycerol (Ext.1) was used as the control group because this is the conventional standard extender for goat semen cryopreservation. The semen parameters observed in the pooled fresh ejaculate of goats after collection and washing, after equilibration and after thawing of all four extenders TCG with egg yolk (Ext.1) TCG extender replaced with 35% Trehalose (Ext.2) or egg yolk replaced with 25% Soya extract (Ext.3) and 35%Trehalose and 25% soya extract combination (Ext.4) either fresh or frozen are shown in Table to and Fig to After thawing, it was observed that the sperm motility values of the frozen semen were lower (P<0.05) than those of the fresh semen; however, the live sperm, progressive motility, host as well as intact acrosome differed significantly (P>0.05) between the samples frozen with TCG (Ext.1or trehalose (Ext.2). Only soya extract performed lowest among all the dilutors. There was significant lower host positive and intact acrosome sperm in both soya extract extenders with or without trehalose. However addition of trehalose to soya extract in extender 4, had improved all sperm parameters and are at par or little higher without any significant difference (P<0.05) with the TCG conventional extender. All the post-thaw parameters were comparable to the percentages live sperm was also significantly intact acrosome did not differ (P > 0.05) between the samples of fresh or frozen semen (Fig to 4.6.5). Our analysis of the plasma membrane integrity (HOST) and intact acrosome in trehalose incorporated extender (Ext.2 and Ext. 4) showed that the semen samples had significant better preservability (P>0.05) from the other two extender without trehalose. The sperm motility decreased after the freezing/thawing process when compared to the fresh samples because the sperm plasma membrane is the first structure to suffer damage during cryopreservation (Watson, 1995). When improperly performed, cryopreservation causes thermal shock as well as the stresses induced by ice crystal formation are mainly associated with the accompanying osmotic pressure changes in the unfrozen fraction (Watson, 2000), resulting in irreversible damage to the sperm cells, which is characterized by abnormal movement patterns, the rapid loss of motility, the reduction of metabolic activity, the loss of intracellular components, and injury to the plasma membrane, acrosome, mitochondrial sheath and atonement (Melo et al., 2007). Eiman et al. (2003) reported that trehalose acted as cryoprotectants in the Tris-citric acid glucose 87

133 (TCG) diluent developed by Salamon and Ritar, (1982). The insertion of these disaccharides into the membrane limits the amount of dehydration that can occur, and consequently the physical damage due to cell volume changes associated with freezing and thawing (Liu et al., 1998). Significant differences were observed among groups for acrosome integrity. Acrosomal injuries observed are generally caused by the formation of large ice crystals within the cell, damaging the internal structures and also plasma membrane (Watson, 1995; Oliveira, 2002; Melo et al., 2007; Futino et al., 2010). Although the mechanism by which trehalose protects sperm during freezing /thawing remains unknown completely, there are two hypotheses to explain the phenomenon trehalose can be expected to work as a universal stabilizer of protein. It may be due to its exceptional effect on the structure and properties of solvent water compared with other sugars Kausik and Bhat (2003). Trehalose also causes an increase in membrane fluidity due to protein and phospholipids reorganization, a suppression of the injurious effect of membrane lipid phase transition; accordingly a dehydration of cells at lower temperature. Cell damage is minimized due to less intracellular ice formation and consequently more viable cells being recovered following cryopreservation (Eiman et al. 2003). As in this study we have used semen diluted at collection, may be the effect seminal plasma protein was reduced and had very good post thaw parameters with egg yolk as well as soya extract extender, which is giving similar results for buffalo semen in liquid storage (Pankaj, 2006; Bishist, 2009; Singh et al., 2012). The total soya extract has natural soya lecithin which is protecting the sperm in the absence of egg yolk lecithin. Soya extract was able to maintaining semen quality, which is likely due to heating and blenching of soya beans during soya extract preparation, which inactivates various lipoxygenase enzymes and trypsin inhibitors (Nelson et al. 1976). Heating and blenching process also induces colloidal stability to soya extract and produce uniformity in protein and lipid molecule (Nelson et al., 1976). Some studies have used pure soya lecithin at high concentration, which may be harmful to sperm due to other potential toxicity in (Futino et al., 2010). We emphasize that the concentration of soy lecithin used in this experiment was based on the studies of Singh et al. (2012). They used soya extract for storage of buffalo semen at refrigeration temperature. As cooling is not as deleterious to the structure of the sperm plasma membrane as the freezing process, which causes more significant rearrangements in the structure of the plasma membrane (Barbas and Mascarenhas, 2009). 88

134 Phospholipids, being the major component of membranes, play important physiological functions in reducing the freezing point, thus avoiding the formation of large ice crystals, and in minimizing the replacement of plasmalogens to reduce the possible mechanical damage to the sperm membrane (Graham and Foote, 1987; Giraud et al., 2000; Waterhouse et al., 2006). Therefore, the exogenous phospholipids present in extenders can replace some of the sperm membrane phospholipids to maintain plasma membrane structure and function (Graham and Foote, 1987; Trimeche et al., 1997 and Zhang et al., 2009). Another possibility, also widely accepted by many researchers, is that the egg yolk phospholipids or soya extract lecithin do not enter the membrane to alter the phospholipids concentration but may form a protective film around the cell to prevent the formation of intracellular ice crystals and to protect the sperm membrane from mechanical damage during freezing/thawing (Zhang et al., 2009). Bittencourt et al. (2008) compared two extenders for freezing goat semen, lecithin (Bioexcell) and another based on Trisyolk. These authors reported that the mean values of sperm motility provided by the frozen/thawed semen diluted with Bioexcell were below the minimum recommended for post-thawed goat semen (30%). The Bioexcell and control extender resulted in higher (P < 0.05) rates of acrosomal defects when compared to the fresh semen. However, in our study we have very good comparable acrosomal status of sperm. These findings differ from those obtained in the present study in which the extender based on soya lecithin (Salmani, et al. 2013; Vidal, et al. 2013) at various concentrations was equally effective as skim milk in the preservation of the sperm parameters. Thus, from our study it can be concluded that soya extract for goat semen cryopreservation gives a better alternatives to egg yolk. On the basis of the results obtained in our study, we conclude that soya extract based extender shows potential to maintain semen quality like individual motility, viability, membrane integrity and acrosome integrity, and replacement of trehalose 35% is improving the post thaw quality to a significant to a higher level. Further studies on in vitro and in vivo fertility will confirm these above findings for alternative extender for cryopreservation of goat semen. 89

135 4.7 In-vitro Fertility Test Cryopreserved Crossbred goat (AXB) Semen Post thaw semen straws were used for determination of in-vitro fertility with goat in-vitro matured oocytes (Plate-5). Fresh and frozen semen were compared for in-vitro test. The results are presented in Table and Figure Table In-vitro fertility test cryopreserved crossbred goat AXB semen in different TCGEY and TCGEY+Trehalose extender. (n=30) 4-8 No. 2 cell cell Semen Oocytes Cleavage % stages % stages % FRESH TCG Frozen TCG+T The overall in in-vitro fertility was higher for fresh semen compared to frozen semen. The frozen semen cryopreserved with supplemented trehalose found better than the conventional extender without supplemented with trehalose. The higher per cent of cleavages, two cell stages and 4-8 celled stage embryos were found for fresh semen samples than the frozen semen samples of all bucks FRESH TCG TCG+T 10 0 Cleavage 2 Cell stages 4-8 Cell stages Figure cleavage, two cell and 4-8 cell stage embryos per cent 90

136 Keskintepe et al. (1998) found better cleavages (80%) in semen frozen in skimed milk extender than commercial egg yolk (60%) and egg yolk (25%) based extenders outside breeding season. They also found that highest blatocysts produced with the use of semen frozen in skim milk extender (30%).than commercially egg yolk based (17%) extender and egg yolk (11.3%) based extenders. In our experiment spermatozoa were washed and trehalose use enhanced the in-vitro fertility of goat oocytes with frozen semen. Similar results for were found by Chauhan and Anand (1991). Rho et al.(2001) reported that frozen semen treated with different method of preparations on cleavage % and blasocyst % and they found that at post-insemination cleavages % production for percoll-density (62.4%), swim-up (49.6%) and glass-wool (44.5%) and blasocyst production for percoll-density (18.3) swim-up (11.1%) and glass wool (7.6%). We followed swim-up method found comparable results this study. In another study Martino et al. (1995) observed for fresh semen that low percentage of normal fertilization (24.4%) was observed for pre-pubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro matured (48.7%) oocytes from adult goats. De et al. (2011) supplemented with cysteamine of IVM mediumor IVCmediumor both media improves cleavages to and the blastocyst to production rates in goats, without compromising their quality. We observed in matured goat oocytes and comparable results were found. 4.8 In vivo fertility of semen cryopreserved with TCGEY and TCGEY + Trehalose based extender Cryopreserved semen samples in TCG-EY extender and TCG +Trehalose extender from winter season were used for Artificial insemination in natural heat in does for in vivo fertility status of semen. Twenty-six does for each extender and a total 52 does were used for Transcervical artificial insemination. Result are presented in Table-4.8.1,Figure and Plate-3 to 4. 91

137 Plate- 3 Goat transcervical AI with frozen semen Plate- 4 Per rectal ultrasonography at 35 Days for pregnancy diagnosis in Goat

138 Immature oocytes Mature oocytes 2 cell stage embryo 4-8 cell stage embryo Plate-5 In-vitro fertilization