EFFECT OF POST FREEZING ON SEMEN OF DIFFERENT ARTIFICIAL INSEMINATION BULL BREEDS.

Transcription

1 EFFECT OF POST FREEZING ON SEMEN OF DIFFERENT ARTIFICIAL INSEMINATION BULL BREEDS. A PROJECT REPORT SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF BACHELOR OF VETERINARY MEDICINE, UNIVERSITY OF NAIROBI. SUBMITTED BY: PAPA EKEYA CONRAD J30/2046/2010 SUPERVISOR : PROF.V.T.TSUMA (BVM, Dip.Anim.Reprod.FRVCS, PhD) FACULTY OF VETERINARY MEDICINE. DEPARTMENT OF CLINICAL STUDIES. UNIVERSITY OF NAIROBI.

2 DECLARATION I hereby declare that this project is my original work and to the best of my knowledge it has never been submitted to any other university or institution of higher learning for the award of any degree. SIGN.. DATE.. NAME... This project has been submitted with my approval as the University of Nairobi supervisor. SIGN DATE.. NAME.. DEDICATION This work is dedicated to my family Thank you for always believing in me and for encouraging me to follow my dreams, whatever it takes. Without your guidance, love and support I would not have been able to achieve my goals. You are the best and am honored to be called your son. To my uncles, thank you for always keeping me in touch with the reality.

3 ACKNOWLEDGEMENT My heartfelt gratitude goes to my supervisor Prof. Tsuma for his guidance and supervision throughout the project. Am equally indebted to the institution (Kenya Animal Genetic Resource Centre) in Kiambu County for granting me an opportunity to utilize their laboratory and record books in this study as well as their willingness to volunteer vital information for my project. I would like to sincerely acknowledge the moral and financial support from my family as well as the well wishers throughout my study period. Above all, I thank God the almighty for giving me strength to handle the pressure and for supportive people around me during the difficult times.

4 TABLE OF CONTENTS Title.1 Declaration..2 Dedication...2 Acknowledgement..3 Abstract...5 Chapter one: Introduction 6 Chapter two: Literature Review 8 Chapter three: Research Justification..11 Chapter four: Design and Methodology..13 Chapter five: Results 16 Chapter six: Discussion 20 Chapter seven: Conclusion and Recommendation..21 Chapter eight: Reference.22 Chapter nine: Appendices..23

5 ABSTRACT The main objective of the study was to determine how post freezing affects viability of semen of different artificial insemination breeds of bulls and how to enhance dairy cattle breeding through artificial insemination by reducing losses associated with semen processing. A total of 120 ejaculates were collected from 60 bulls between 1/10/2014 to 30/3/2015 and were analyzed based on ejaculate volume, sperm concentration, mass motility before and after dilution and the post thaw motility and the number of doses before freezing and after thawing The analysis of semen revealed varied characteristics in terms of concentration, mass and individual spermatozoa motility and the difference between doses after dilution and thawing. Results indicated a significant proportion of semen losses in the Aryshire, About 55% of semen was discarded when OPTIXcell was used in dilution of ejaculates collected from Aryshire bulls. Other breeds had registered low semen losses as 11%, 13% and 20% for the Guernsey, Friesian and Jersey respectively. Negligible percentage semen losses were observed when coconut water was used as semen extender under room temperature, such semen could only stay viable for 4-7 days.

6 CHAPTER ONE INTRODUCTION Semen processing involves a conventional procedure of evaluating ejaculate which is performed before and after cryopreservation. These includes determining the colour, volume, motility, concentration and sperm morphology (Bhate et al., 2008;Karoui et al., 2011).These parameters mostly detect cases of significantly inadequate reproduction or infertility in bulls (Rodriguez- Martinez, 1998). Macro and microscopic evaluation of semen is followed by dilution of sperm, filling in straws, cooling at 4 degrees celcius in the cold room and freezing at -196degree celcius in liquid nitrogen.these phases of producing artificial insemination doses have a significant effect on sperm ejaculate motility and the effect of the extender used is especially important (Siddique et al., 2006).Every part of an ejaculate processing from its collection to production of artificial insemination doses is very important and affects the final quality. Basic quantitative traits of sperm (volume, motility, density) are evaluated across the animal species (Moce and Graham 2008; Bozkurt et al., 2011). Cryopreservation has been an invaluable technique. In order to extend the life span of the viability of spermatozoa, their metabolic rate has to be slowed down thereby reducing the rate at which substrates are used and toxins produced. As a general rule, cooling of spermatozoa is the simplest method that can successfully depress spermatozoal metabolic rate and therefore prolong sperm survival. Use of carbon dioxide and other inhibitors like proteinase are also known to produce a similar but less successful effect (Colenbrander et al., 2003; Cremades et al., 2000).The techniques for successful cryopreservation of spermatozoa have also slowly progressed over the past several decades (Hammerstedt et al., 1990) and are now fairly standardized. Though useful, cryopreservation has detrimental effects on sperm function and fertility even with the most up to date technique. Generally sperm viability is decreased by 50% whereas fertilizing capacity is affected by a factor of sevenfolds after cryopreservation (Lessard et al., 2000).Extenders used in freezing semen have deleterious effects on semen due to osmotic stress, changes in membrane organization, fluidity and permeability as well as changes in lipid composition. In one study the inclusion rate of 4% glycerol in an extender containing 20% egg yolk was found to be superior to 2% or 6% glycerol as regards to progressive motility (Cochran et al., 1984). Several researchers have established the fact that semen collected from individual bulls show significant differences in post thaw motility regardless of factors like initial semen quality, thawing rate, storage period. Foote 1970 demonstrated that effect of individual bulls on fertility to be much larger than the effect of other factors like the type of extender, glycerolating procedure and motile sperm number per insemination.

7 It has been established that semen quality of bulls can also be affected by season (Haugan et al., 2005). Other factors that can detrimentally affect thawed semen include sunlight and presence of oxygen. Packaged semen kept at 4 degrees celcius has shown decreased sperm motility after exposure to radiation (Coulter and Foote, 1977). Collection procedures such as facilities, handlers, method and number of ejaculates can also affect viability of semen. When comparing electro-ejaculator to the artificial vagina method of collection (Austin et al., 1961) found the electro-ejaculator to result in larger but less concentrated volumes of semen.

8 CHAPTER TWO LITERATURE REVIEW Throughout history man and animals have always competed for nutritional resources. Due to demands from an ever growing and evolving human population livestock producers are constantly under pressure to supply more products from more limited resources. They are therefore continuously searching for ways to improve the production efficiency of their livestock. This includes selecting for animals with better conversion ratios, disease resistance and improved reproductive efficiency. These factors can only be improved by genetic selection, in other words selecting and breeding only superior animals. This is known as genetic improvement (Bailey et al., 2000; Salisbury et al., 1978; Vishwanath 2003). For genetic improvement to take place in any population there has to be an increase in gene frequency and genetic combination of desired genes. This is achieved by selecting individuals possessing the desired traits and then manipulating their breeding. Individual bulls show great variation in their ability to produce semen that will freeze successfully. This means that even under identical conditions semen collected from different bulls will not freeze with the same level of success (O'Dell and Hurst, 1955; Almquist et al., 1979).These differences between individuals can be of either genetic or non-genetic origin and seem to be consistent between ejaculates, so for example, it has been established that semen quality of an individual bull may decline with age (Foote, 1975). Woods et al., 1986 reported a lower correlation between semen characteristics and fertility within bulls compared to between bulls. This implies that predictions will be more reliable regarding bull fertility, compared to the fertility of individual ejaculate. Another factor to consider is the heritability of testicular traits. Testis size has been reported to be an indication of bull fertility. Studies have revealed a positive correlation between bull fertility and testicular size. Due to high heritability of testes size, more emphasis has been placed on this trait during selection and evaluation of bulls for breeding purposes (Foote, 2002).

9 Every part of an ejaculate processing from its collection to production of artificial insemination doses is very important and affects the final quality. Sperm quality is influenced by many factors such as breed, variation between individuals, age of sire (Bezdiceket et al., 2007; Stoic et al., 2009).by feeding ration composition, content of specialized feeding supplements (Cerovsky et al, 2009; Jaycno et al., 2009), environmental conditions, frequency of ejaculate collection (Wolf and Smith 2009; Karoui et al., 2011) and by the season of the year (Hajirezaee et al., 2010). Diluting of semen with an extender, cooling of semen in the cold room, filling of straw with semen and freezing of semen follows macro and microscopic evaluation of semen for colour, volume, motility, concentration. These phases of producing artificial insemination doses have a significant effect on sperm motility and effect of the extender used is especially important (Siddique et al., 2006). Research on improving ejaculates was done earlier and sexual preparation is used to increase the volume as well as the sperm concentration per ejaculation. Almquist et al and Bratton et al., 1979 investigated the effect of sexual stimulation prior to semen collection as well as the intensity of each collection. These studies have resulted in the recognition of the important role that sexual response of individual bulls play in the quality of the semen collected. It was also suggested that these stimuli should be adapted for each individual bull in order to achieve optimal ejaculation results. This was done by washing of the bull and allowing it to dry then the bull was allowed to mount a teaser animal a few times before collection. A collection frequency of six times per week was suggested. The volume of sperm collected is greatly affected by frequency of ejaculation (Foote, 1975; Salisbury, 1978; Mitchell and Doak, 2004) Numerous semen extenders have been utilized as primary and secondary extenders in dilution of the ejaculates collected. These include egg yolk and those based on skimmed milk with egg yolk (Pickett and Amann, 1993).Good success has been reported with the use of trehalose as a cryoprotectant within a skimmed milk-egg yolk extender. It is suggested that trehalose has a stabilizing effect on the spermatozoan plasma membrane (Steinmann, 1996). The initial purpose of an extender was to prolong the life of sperm to allow for transport of semen. Today this is still the main goal of a successful extender but other requirements have been added-such as

10 increasing volume of a single ejaculate so that it can be used to inseminate several female animals (Mitchell and Doak, 2004). Cryopreservation has been an invaluable technique. As a general rule, cooling of spermatozoa is the simplest method that can successfully depress spermatozoal metabolic rate and therefore prolong sperm survival. Though chilling semen provides an efficient and successful means of short term storage, it has yet some adverse effects on the spermatozoa manifested as a depression in viability rate, structural integrity, depressed motility and conception rates (Bateillier et al., 2011; Medeiros et al., 2002; Watson, 2000).The effects of cryopreservation on sperm function and fertility have been widely researched particularly in bovine, various sperm organelles have been known to be affected due to the detrimental effects of cryopreservation such as induction of premature acrosomal reaction, altered mitochondrial function, reduction of motility and failure of chromatin recondensation, all of which influence viability and fertility of sperm cells have been reported by different authors (Chaveiro et al., 2006; Scooter et al, 2005; Watson, 2000; Wongtawan et al., 2006). During chilling the speed of temperature drop are known to have an effect on the spermatozoa due to cold shock and the success rate of freezing semen. The extent of damage to spermatozoa as a result of cold shock depends not only on the drop in temperature but also the speed with which this drop is attained. The rate of temperature drop was found to be most critical over the specific temperature range of 0-5degrees when motility was evaluated later. In general the faster the rate of cooling the more severe is the damage (Kayser, 1990). There is further evidence which suggests that the rate of temperature drop also determines the subsequent active life of spermatozoa (Andrabi, 2007). Frozen spermatozoa are further injured during the thawing process which has been regarded as being due to re-crystallization of ultras microscopic ice crystals to form comparatively large ice crystals (Watson, 2000; Woelders, 1997).

11 CHAPTER THREE RESEARCH JUSTIFICATION There are several factors that can have an effect on the final number of sperm that survive the collection, processing, freezing and thawing procedures. These factors include: initial motility of sperm prior to any processing or freezing procedures, the quality of the extender added to the semen, freezing curve applied, method of storage and the thawing procedure. Initial sperm motility can be affected by individual bull, environment as well as the available semen collection facilities. The extent to which semen collected is diluted during the processing procedures could in turn be influenced by the type of extender used including the source of cold shock protection, the buffering agent, the amount of glycerol added, antibiotics added and the PH of extender. These factors compromise the quality of semen and have led to semen losses post thaw due to discarding. Semen can be invalid both pre-dilution and during thawing due to poor characteristics such as abnormal colour, low motility and low concentration that do not meet the required standards. In Kenya semen produced does not meet the demand from livestock producers who are constantly under pressure to supply more animal products even from more limited resources. These livestock producers have continuously searched for ways to improve the production efficiency of their livestock to the extent of importing semen from various countries. Therefore this project focused on exploiting this loop hole by reducing the losses encountered during semen collection, processing and freezing which would help in ensuring availability of quality semen to the livestock producers who are willing to maximize production of their animals. This would meet the booming demand from increasing Kenyan population for animal products. Improvements of semen quality would be one step in alleviating those losses incurred in the artificial insemination centre and these would ensure that most semen sustain the freezing temperatures.

12 Semen traits could only be improved by genetic selection. For genetic improvement to take place in any population there has to be an increase in both the gene frequency and gene combination of the desired genes. This is achieved by selecting individual animals possessing the desired traits and then manipulating their breeding. For example heritability of testicular traits. Testes size has been reported to be an indication of bull fertility. Due to high heritability of testes size, more emphasis has been placed on this specific trait during selection and the evaluation of bulls for purposes of breeding (Foote. 2002) Trial of other extenders to establish one with better sustainability of spermatozoa in freezing temperature could also be a solution to semen losses. For example coconut water was found to sustain semen for 4-7 days without major losses though at room temperature. In some studies trehalose has been used as a cryoprotectant within skimmed milk-egg yolk extender. It was suggested that trehalose had a stabilizing effect on the spermatozoa plasma membrane (Steinmann, 1996). Freezing curve applied during cryopreservation determines the extent of damage to spermatozoa due to cold shock. Temperature drop as well as the speed with which the drop is attained is critical. In general the faster the rate of cooling the more severe is the damage (Kayser, 1990).There is further evidence which suggests that the rate of temperature drop also determines the subsequent active life of spermatozoa (Andrabi, 2007). OBJECTIVES The general aim of this study was to enhance dairy cattle breeding through artificial insemination by reducing semen losses associated with processing and freezing. SPECIFIC OBJECTIVES 1. To evaluate processing and post freezing semen losses encountered at Kenya Animal Genetic Resource Centre. 2. To evaluate semen losses in four bull breeds: Friesian, Guernsey, Aryshire and Jersey. 3. To compare semen losses among the four bull breeds.

13 CHAPTER FOUR MATERIAL AND METHODS The study was conducted at Kenya Animal Genetic Resource Centre from 1/10/2014 to 30/3/2015.The information required was prospectively collected from routine semen evaluation, semen processing laboratory results and retrospectively from register books maintained at Kenya Animal Genetic Resource Centre. A total of 120 ejaculates analyzed were obtained from 15freshians, 15aryshires, 15 jerseys and 15guernseys. Age of collection was estimated at 2 and half years. Bulls were managed uniformly by individual housing and were maintained on a diet of hay, approximately 3kgs of concentrate, vitamins and mineral mix. All bulls were periodically vaccinated against foot and mouth disease. EVALUATION OF BREEDING SOUNDNESS Selection of calves as trial bulls was mostly on milk production of their mothers At KAGRC, male calves were selected at the age of 6months to 1 year. Breeding soundness was evaluated by examining their body conformation, legs, hooves, joints, stepping of bull, testes, scrotum and eye After certification as trial bulls they were tested for their libido and the characteristics of semen (volume, mass motility, concentration and individual motility). SEMEN COLLECTION Semen was collected in the morning after the bulls had been washed and left to dry. Collection was then preceded by a controlled sexual preparation. Two false mounts with an interval of almost 1minutes was observed. At the 3 rd mount using a teaser bull, an ejaculate was obtained with an artificial vagina. Two ejaculates were collected from each bull on each collection day. The interval between 2 consecutive collections varied from 2 or 4days.

14 SEMEN EXAMINATION After collection, Tubes of semen are placed in water bath at degree celcius. Macroscopic evaluation of the ejaculate was done. Some ejaculates were discarded due to abnormal colour, presence of blood or dirt, poor density and poor PH (alkaline) Semen was observed after collection. The colour of semen was whitish creamy to yellowish. Greenish tinge in jersey breeds was normal. Any abnormal colour of semen led to the discard of the semen. Volume was measured on graduated collection tube (ml). Mass motility was estimated by placing a large drop of semen on a glass slide and observation done under low objective lens. Wave motion or cloud movement indicated life and motion of spermatozoa. It was usually scored between Meant slight swirling motion. Very poor to fair motility motility 4-6 meant dark prominent waves in very rapid motion. Good to very good Progressive forward motility was estimated by placing a small drop of semen on glass slide, putting a cover slip on the drop and observing it under high power objective lens. Morphological deformities could be observed. It was usually scored in terms of percentage very good good fair poor 0-20 very poor

15 Concentration was estimated using a photometer but sometimes haemocytometer was used to determine the concentration of semen. DILUTION Semen was collected and placed in a water bath at degree celcius during evaluation. Raw semen was buffered using OPTIXcell in a ratio of 1:1 in a water bath. It was gently swirled and incubated for 10 minutes in water bath at degree celcius Having calculated the required extender, it was poured on a separate flask as 10mls were reserved for rinsing off the semen collection tube. Buffered semen was then slowly poured into the extender. Collection tube was rinsed off with the 10mls of extender that had been kept aside. STABILIZATION Extended semen was then taken to the cold room (4degrees celcius). It was left in the room for 3-5 hours in order to equilibrate the exchange between media and semen to prevent cold shock. PACKAGING While in the cold room extended semen was filled in straws mostly 0.5mls straws automatically. End of the straws had been sealed with polyvinyl material and were then frozen. Straws had labels: sire name, breed code, registration number, processing location and date frozen.

16 FREEZING After stabilizing semen for 3-5 hours to avoid cold shock, straws were placed on horizontal freezing rack and placed 3cms above the surface of liquid nitrogen. This was allowed for about 7-10 minutes for the temperature to get to -100 degree celcius. Once the temperature reached degree celcius straws were plunged into liquid nitrogen at -196 degree celcius. POST THAWING EVALUATION Post thaw motility was measured 24hours after storage in liquid containers. Bull semen from the frozen straws were thawed in a water bath at 37degrees celcius for 30seconds and evaluated individually for the spermatozoa percentage progressive motility. Sperm was evaluated for motility, morphology and viability. Good semen had 40-50% progressive motility immediately post thawing. SEMEN STORAGE AND HANDLING Straws are stored frozen in liquid nitrogen in tanks at -196 degrees celcius. At these temperature spermatozoa remains inactive but viable.

17 RESULTS AND DATA ANALYSIS CHAPTER FIVE PERCENTAGE SEMEN LOSSES WITH OPTIXcell AS THE SEMEN EXTENDER BREED FRIESIAN GUERNSEY ARYSHIRE JERSEY EJACULATES TOTAL DOSES DOSES AFTER FREEZING DOSES DISCARDED % DISCARDS % DISCARDS OF TOTAL DOSES FRIESIAN 2995 Total % DISCARDS % DISCARDS OF TOTAL DOSES DOSES AFTER FREEZING 296 DOSES DISCARDED 30 EJACULATES TOTAL DOSES

18 GUERNSEY Total % DISCARD OF TOTAL DOSES % DISCARDS DOSES AFTER FREEZING DOSES DISCARDED EJACULATES TOTAL DOSES ARYSHIRE Total % DISCARDS % DISCARDS OF TOTAL DOSES DOSES AFTER FREEZING DOSES DISCARDED 30 EJACULATES TOTAL DOSES

19 JERSEY Total % DISCARDS % DISCARDS OF TOTAL STRAWS DOSES AFTER FREEZING DOSES DISCARDED EJACULATES TOTAL DOSES PERCENTAGE SEMEN LOSSES WITH COCONUT WATER AS THE EXTENDER BREED FRIESIAN GUERNSEY ARYSHIRE JERSEY EJACULATES TOTAL DOSES DOSES AFTER DILUTION DOSES DISCARDED % DISCARD % DISCARDS OF TOTAL

20 DOSES FRIESIAN 649 Total % DISCARDS % DISCARDS OF TOTAL DOSES DOSES AFTER DILUTION DOSES DISCARDED EJACULATES TOTAL DOSES GUERNSEY 540 Total % DISCARDS % DISCARDS OF TOTAL DOSES DOSES AFTER DILUTION 0 DOSES DISCARDED 30 EJACULATES TOTAL DOSES

21 ARYSHIRE 561 Total % DISCARDS % DISCARDS OF TOTAL DOSES DOSES AFTER DILUTION 0 DOSES DISCARDED 30 EJACULATES TOTAL DOSES 543 JERSEY Total % DISCARDS % DISCARDS OF TOTAL DOSES DOSES AFTER DILUTION DOSES DISCARDED EJACULATES TOTAL DOSES

22 CHAPTER SIX DISCUSSION The present study revealed a higher proportion of semen discards in the Aryshire breeds, about 55.6% of semen losses from Aryshire breeds was encountered when ejaculates were diluted with OPTIXcell. Other breeds registered relatively low proportions of semen discards as 11.1%, 13% and 20.3% for Guernsey, Friesian and Jersey respectively. Not much study has been done to compare post thaw semen losses in the four exotic bull breeds. These losses could be attributed mainly to the sire effect. Individual bulls have great variation in their ability to produce semen that will freeze successfully even under identical conditions. Studies have revealed a significant variation of insemination bull breeds in the volume and concentration of semen per ejaculate (Graffer et al.,1988; Seidel and Foote, 1969; Shelke and Dhami,2001). Mass motility and individual progressive motility of spermatozoa in both raw and extended semen proved to be significantly varied between different bull breeds. Study of semen extended with coconut water at room temperature revealed negligible percentage semen losses in all the bull breeds. 0%, 0%, 0.15% and 0.37% of the discards for Guernsey, Aryshire, Friesian and Jersey respectively were the only semen losses encountered when coconut water was used as semen extender. The number of semen doses pre-freezing and after thawing were compared amongst the artificial insemination bull breeds selected and the most affected bull semen was from Aryshire as shown by the analyzed results. Freezing temperature has adverse effects on the sustainability of spermatozoa as shown by the number of doses that was being discarded from each breeds of bull. Cold temperatures have detrimental effects on sperm cells and generally sperm viability is decreased by 50%, whereas fertilizing capacity is affected by a factor of sevenfold after cryopreservation (Lessard et al., 2000)

23 Utilizing coconut water as semen extender significantly reduced semen wastage caused by discarding as shown by the study results. However this semen was processed at room temperature and therefore their viability was only for about 4 to 7 days. When coconut water was used as extender, collection frequency of semen ejaculates from insemination bulls needed to be increased to meet the daily expected doses. With such semen there was expected increase in production of livestock products to meet the booming demand from the increased Kenyan population.

24 CHAPTER SEVEN CONCLUSION AND RECOMMENDATION More studies have to be conducted to properly establish the cause of post thaw semen losses in the four exotic bull breeds. However semen collection, processing and cryopreservation affects viability of the spermatozoa and this has contributed to losses that are normally encountered through discards. Freezing curves applied during cryopreservation determines the extent of spermatozoa damage. In general the faster the rate of cooling the severe is the damage (Kayser, 1990) Use of coconut water as semen extender and storage under room temperature has been a milestone in the industry in that wastage of semen through discarding has been significantly curbed.

25 CHAPTER EIGHT REFERENCES Amaron P,Christianelli M J, E L Squiresand B W Pickett 1984.fertility of stallion semen processed, frozen and thawed by a new procedure.theriogenology 23:39-45 Bailey J L,Cermier N and M A Sirard 1997.premature capacitation of bovine spermatozoais initiated by cryopreservation Andro.18: Curry M R, 2000.cryopreservation of semen from domestic livestock.rev Reprod 5:46-52 Deneke N, A Lemma and T Yilma 2010-study on the efficiency of conventional semen evaluation procedure employed at Kaliti National artificial insemination centre and fertility of frozen, thawed bull semen. As part of MSc thesis, Faculty of Veterinary Medicine, Adidis Ababa University Desjardins C and H D Hafs 1962 motility and during post thawing, storage of bovine spermatozoa spermatozoa frozen concentrated thawed and re-extended.j Dairy Sci 45:1242 Goffaux M 1965, effect of the number of motile spermatozoa used per insemination on the fertilizing ability of frozen bull semen.ann Zootech 14:409(citation from animal breed.abstr 35:78,1967) Genetic and non genetic factors affecting the quality of bulls semen.pakistan journal of biological sciences. Foote R H and J E Parks 1993.Factors affecting preservation and fertility of bull semen; a brief review on reprod. Fertile.Dev; 5:665 H B Bean, Pickett B W and Martig R C 1963.influence of freezing methods, extenders and storage temperatures on motility and PH of frozen bovine semen Dairy Sci 46:2 p p k

26 J H Bearden, Fuguay JW and Willard S T 2004.Applied animal reproduction. Sixth Edition Pearson Prentice Hall, New Jersey R H Foote and Berndtson W E1972. Freezability of spermatozoa after minimal pre-freezing exposure to glycerol or lactose. Cryobiol 9 p57-60 R H Foote and Coutter G H 1977.Effects of package, extender and light on stored frozen bull spermatozoa.j Dairy Sci 60p R H Foote 2002.The history of artificial insemination; selected notes and notables. J Animal Sci 80p1-10 Pickett B W, R C Hall, Jr. J J Lucas and E W Gibson 1964.influence of sperm number on fertility of frozen bovine semen.j Dairy Sci 47:916

27 CHAPTER NINE APPENDICES APPENDIX 1: SPERM PERCENTAGE MOTILITY BREEDS NUMBER EJACULATES UNDILUTED DILUTED POST THAWING FRESIAN 1 A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B

28 GUERNSEY 1 A B A B A B A B A B A B A B A B A B A B A 65 discard B 70 discard 12 A B A B A B A B ARYSHIRE 1 A 60 discard B 65 discard 2 A B A B A 20 discard B 55 discard 5 A

29 B A B A B A B A 10 discard B 40 discard 10 A B A B 50 discard 12 A B A B 65 discard 14 A B A B JERSEY 1 A B A 60 discard B 70 discard 3 A B A B A B A B A B A B A B A B

30 11 A 60 discard B 65 discard 12 A B A B A B A B APPENDIX 2: ARTIFICIAL INSEMINATION DOSES BREED DOSES B4 THAWING DOSES AFTER THAWING FRIESIAN

31 GUERNSEY

32 ARYSHIRE JERSEY

33

34

35

36

37

38