CRYOPRESERVATION OF SOUTH AFRICAN INDIGENOUS RAM SEMEN by Pfananani Hendrick Munyai Submitted in partial fulfilment of requirements for the degree Magister Scientiae Agriculturae in the Faculty of Natural and Agricultural Sciences Department of Animal, Wildlife and Grassland Sciences University of the Free State Bloemfontein May 2012 Supervisor: Prof. T.L. Nedambale Co-supervisors: Prof. J.P.C. Greyling Dr. L.M.J. Schwalbach Acknowledgements I would like to thank God, Almighty for providing em with guidance and encouragement during hard times This study was jointly funded by the National Rercshe aFoundation, Agricultural Research Council and South African Department of Agricult,u Freorestry and Fisheries Thanks to my family for their unwavering emotionsaulp port. Prof. T.L Nedambale is thanked for his mentorshgiupi,d ance and expertise in the laboratory work. Prof. J.P.C. Greyling is thanked for his supervnis aiond writing of this dissertation. Dr. Luis Schwalbach is thanked for co-supervisihneg wt riting of this dissertation. Me. Cynthia Ngwane of ARC-Biometry unit is thankfeodr doing the statistical analysis. Mr. L. Kruger and Mr. L. Mohale are thanked for itnagk care of the experimental animals during the trials. This project would have not been realised, withtohuet assistance of Colleagues at ARC- Germplasm Conservation and Reproductive Biotechgnieoslo. i Dedication To my father (Freddy) and mother (Tshinakaho) My wife (Lutendo), son (Mulweliwashu) and daugh(tLeor ndani) My late brother (Mafanedza) and Emmanuel My sisters (Ntshimbidzeni and Mashudu) Thank you for your encouragement, love and suptphorortu ghout my studies. ii Declaration I hereby declare that the work in this disserta stiuobnmitted to the University of the Free State for the degre,e Magister Scientiae Agriculturae, is my own independent work and has never been previously submitted to any other universI itcye. de the copyright of this dissertation to the University of the Free State. Pfananani Hendrick Munyai Bloemfontein May 2012 iii Table of contents Page Acknowledgements i Dedication ii Declaration iii Table of contents iv List of Tables xi List of Figures xiii List of Plates xiv List of Abbreviations xvi Chapter 1 General Introduction 1 Chapter 2 Literature review 4 Factors affecting cryopreservation and post thaawb ivlitiy of ram semen 4 2.1 Description of semen 4 2.2 Production of semen 4 2.2.1 Site of production 4 2.2.2 Hormones involved in the control of spermgaetnoesis 4 iv 2.2.2.1 Follicle stimulating hormone (FSH) 5 2.2.2.2 Luteinizing hormone (LH) 5 2.2.2.3 The male sex hormone, testosterone 5 2.2.3 Spermatogenesis 6 2.2.3.1 Spermatocytogenesis 6 2.2.3.2 Meiosis 7 2.2.3.3 Spermiogenesis 8 2.3 Seminal plasma 9 2.4 Semen collection methods 10 2.4.1 Semen collection using the artificial vag in a 10 2.4.2 Semen collection using an electro-ejacu l ator 11 2.5 Semen evaluation 12 2.5.1 The importance of semen evaluation 12 2.5.2 Subjective assessment of semen 12 2.5.2.1 Colour and volume of the ejaculate 12 2.5.2.2 Sperm concentration 13 2.5.2.3 Sperm motility 13 v 2.5.2.4 Sperm morphology 14 2.5.3 Objective semen evaluation 15 2.5.3.1 Introduction 15 2.5.3.2 Semen evaluation with the aid of the comerp aust sisted sperm analyser (CASA) 15 2.5.3.2.1 Advantages of using the computer ass sisptedrm analysis (CASA) system 16 2.5.3.2.2 Disadvantages of using the computer taesds sisperm analysis (CASA) 16 2.6 Effect of environmental factors on sperm prcotidoun and quality 16 2.6.1 Age of the ram 17 2.6.2 Season of the year 18 2.6.3 Daylight length (Photoperiod) 18 2.6.4 Ambient temperature and testicular thermuolraetigon 19 2.6.4.1 Ambient temperature 19 2.6.4.2 Testicular thermoregulation 20 2.6.5 The effect of nutrition on semen quality afenrdtility 21 2.7 Factors affecting the viability of sperm a fstermen collection 22 vi 2.7.1 Temperature 22 2.7.2 Semen pH 22 2.7.3 Osmotic pressure 22 2.7.4 Concentration of sperm per ejaculate 23 2.7.5 Gas environment 23 2.7.6 Light exposure 23 2.8 Semen extenders or diluents 24 2.8.1 Components of ram semen extenders 24 2.8.1.1 Example of a semen extender 25 2.8.2 Cryoprotective agents 25 2.9 Semen cryopreservation techniques 26 2.10 Thawing of cryopreserved semen 30 Chapter 3 Materials and methods 31 3.1 Study location 31 3.2 Experimental animals 31 3.3 Preparation of Diluents 33 3.3.1 Preparation of the egg yolk- citrate exter n de 33 vii 3.3.2 Protocol for preparing the sperm washingu tsioonl (BO-W) (Brackett & Oliphant, 1975) 34 3.3.3 Preparation of the sperm washing solutioOn -(WB) 35 3.4 Semen collection and quality evaluation 5 3 3.4.1 Semen collection 35 3.4.2 Semen evaluation 36 3.4.2.1 Semen concentration 36 3.4.2.2 Semen pH 38 3.4.2.3 Sperm motility evaluation using the CASAs tseym 38 3.4.2.4 Sperm morphology and viability 40 3.5 Liquid storage of ram semen 42 3.6 Semen cryopreservation 43 3.7 Thawing of semen for the post-thaw semen asneas ly 46 3.8 Statistical analyses 47 Chapter 4 Characterization of South African indigenous ram semen 48 4.1 Introduction 48 4.2 Materials and Methods 49 4.3 Results and Discussion 51 viii 4.4 Conclusions 55 Chapter 5 Effect of storage temperatures on the viability of diluted ram semen stored for different periods of time 57 5.1 Introduction 57 5.2 Materials and Methods 58 5.3 Results and Discussion 59 5.4 Conclusions 62 Chapter 6 The effect of temperature and different storage times, on sperm motility of ram semen diluted with an extender containing glycerol 64 6.1 Introduction 64 6.2 Materials and Methods 65 6.3 Results and Discussion 66 6.4 Conclusions 69 Chapter 7 The effect of different glycerol inclusion levels in the semen diluent on the sperm motility characteristics, following cryopreservation in indigenous South African rams 70 7.1 Introduction 70 7.2 Materials and Methods 71 ix 7.3 Results and Discussion 72 7.4 Conclusions 77 Chapter 8 Comparison of the slow cooling and liquid nitrogen vapour method on ram sperm motility rate following cryopreservation 79 8.1 Introduction 79 8.2 Materials and Methods 80 8.3 Results and Discussion 81 8.4 Conclusions 83 Chapter 9 General Conclusions and Recommendations 84 9.1 General Conclusions 84 9.2 Recommendations 85 Abstract 87 References 91 x List of tables Page Table 2.1 Subjective assessment of semen concentration using colour variation 13 Table 3.1 Preparation of egg yolk extender (g/100mL) 34 Table 3.2 Preparation of 10xBO stock solution A (effective for 30 days) 34 Table 3.3 Preparation of 1xBO working solution B (effective for 2 weeks) 35 Table 3.4 The definitions of sperm motility descriptors when using the CASA system 38 Table 3.5 Sperm Class Analyser® V.4.0.0 settings used atoly asne the ram sperm cell motility and velocity charaicstteicrs 39 Table 3.6 The freezing rates used to cool indigenous rame sne during cryopreservation 44 Table 4.1 Mean (±SD) semen volume, pH and sperm concentr aotfi on different South African indigenous ram breeds 52 Table 4.2 Pearson correlations between bodyweight, scrortcaul mciference, semen volume, sperm concentration, semen pH and total sperm motility in South African indigenoruasm s 53 Table 4.3 Sperm morphology evaluation of raw semen from S o uth African Indigenous rams of different breeds 54 xi Table 4.4 Mean (±SD) sperm motility and velocity rates ofu Sth African indigenous ram breeds, as recorded by CA SA 54 Table 5.1 The mean (±SE) sperm motility characteristics oof lepdo diluted South African indigenous ram semen storte d a 5°C or 15°C following evaluation using the CASA tseyms 63 Table 6.1 The mean (±SE) sperm motility characteristics odfi ginenous ram semen diluted with glycerol, stored at two tempuerreast for different periods of time as measured by the CASysAte sm 68 Table 7.1 The mean (±SE) sperm motility and velocity charraisctiecs for different S.A. indigenous rams, as measuredC AbyS A following dilution, prior to cryopreservation 3 7 Table 7.2 The mean (±SE) effect of different glycerol inclouns irates on the pre-freezing sperm motility and velocity chcateraristics of indigenous ram semen, as measured by CASA 75 Table 7.3 The mean (±SE) effect of different glycerol inclouns ilevels on the post thaw sperm motility and velocity charaicsteicrs of pooled indigenous ram semen, as measured by CASA 77 Table 8.1 Comparison of ram sperm motility and velocity chcateraristics following cryopreservation by two freezing methods as analysed by the CASA system 83 xii List of figures Page Figure 2.1. Spermatogenesis indicating the sequence of evendn ttsim ae involved in spermatogenesis in the ram 8 xiii List of Plates Page Plate 3.1 Damara ram used as a semen donor 31 Plate 3.2 Namaqua Afrikaner ram used as a semen donor 3 2 Plate 3.3 Pedi ram used as a semen donor 32 Plate 3.4 Zulu ram used as a semen donor 33 Plate 3.5 Electro-ejaculator used for semen collection 36 Plate 3.6 Thermo flask used for temporary semen storage caoftlelerction 36 Plate 3.7 SpermaCue® used for the determination of spernmc ecnotration 37 Plate 3.8 Semen pH meter used in this study 37 Plate 3.9 Incubator used for semen incubation prior to msp merotility evaluation 39 Plate 3.10 Sperm Class Analyzer® used for sperm motilityl ueavtaion 40 Plate 3.11 Fluorescent microscope (BX 51TF) used for speromrp mhology and viability 41 Plate 3.12 Eosin/nigrosin stained ram sperm cells 42 Plate 3.13 Walk-in refrigerator used during semen storagde parnocessing 43 xiv Plate 3.14 Defy VT60 cooler used during liquid semen sto r age 43 Plate 3.15 The programmable freezer used for semen free zing 45 Plate 3.16 Freezing of semen in liquid nitrogen vapour 45 Plate 3.17 Liquid nitrogen tanks used for semen storage 46 Plate 3.18 Water bath used during thawing of the semen str aws 47 xv List of Abbreviations ADP Adenosine diphosphate AI Artificial insemination ALH Amplitude of lateral head movement ANOVA Analysis of variance ARC Agricultural Research Council ART Assisted reproductive technologies ATP Adenosine triphosphate AV Artificial vagina BCF Beat cross frequency BO Bracket and Oliphant BSA Bovine serum albumin CASA Computer assisted sperm analysis CPA Cryoprotective agent DMSO Dimethylsulfoxide EE Electro-ejaculation EDTA Ethylenediaminetetraacetic acid EYC Egg yolk citrate FBS Fetal Bovine Serum FSH Follicle stimulating hormone GnRH Gonadotropin releasing hormone ICSH Interstitial cell stimulating hormo ne IU International unit IVEP In Vitro Embryo Production xvi IVF In Vitro Fertilization LH Luteinizing hormon e LIN Linearity LN2 Liquid nitrogen LSD Least significant difference MOET Multiple Ovulation and Embryo Transfer PVC Polyvinyl chloride ROS Reactive Oxygen Species SEM Standard error of means SSH Spermatogenic stimulating hormo ne STR Straightness VAP Average path velocity VCL Curvilinear velocity VSL Straight line velocity WOB Wobble xvii Chapter 1 General Introduction The South African sheep population is consistenbtelyin g improved as result of local and international trade of superior genetic materiahle. Ttwo major systems that are used for this purpose are the transport of live animals and et xopfo frrozen ram seme nF.or decades there has been speculation regarding the exploitations hoefe p breeds indigenous to Southern Africa regarding food security. It has been alle gtehdat these indigenous sheep breeds (including the Damara, Namaqua Afrikaner, Pedi aZnudlu breeds) are specially adapted to the South African arid environmental conditions apnodssess certain favourable traits (excellent mothering ability, natural tolerancee txote rnal parasites and diseases, high fertility, etc.), which could be incorporated into a viabled apnrofitable crossbreeding programme (Ramsaye t al., 2001). In order to exploit the productive tr aoitfs the Damara, Namaqua Afrikaner, Pedi and Zulu sheep, it is however etsiasel tnhat the genetic material (in this case the male) firstly be characterized and gametese ctoeldl and preservedin -(situ and ex-situ), for future use and incorporation in sheep breedpirnog rammes. For improving reproductive performance, severails atesds reproductive technologies (ART’s), such as artificial insemination, multiple ovulat ioan d embryo transfer (MOET)in, vitro embryo production (IVEP) and semen cryopreserva ctiaon be used. Artificial insemination is the most widely used ART and has made the miogsnti fiscant contribution to genetic improvement worldwide (Evans & Maxwell, 1987; Lebuof,e 2000). For successful artificial insemination, ram semen specific cryopreservatioront opcols should be developed. The cryopreservation technique includes temperatureu crteiodn, cellular dehydration, eventual freezing and subsequent thawing (Medeireot s al., 2002). The lowering from room temperature to 4°C reduces cellular metabolic iatyc taivnd increases the life span of the sperm cells. Cryopreservation has been shown to stopc ealllul lar activities, restarting its normal metabolic functions, after thawing (Mazur, 1984). Sperm cryopreservation usually induces the formna otifo intracellular ice crystals, osmotic and chilling injury, which causes sperm cell dam, acgyetoplasmic fracture, or even effects on the cytoskeleton or the genome related structuIsreasc h(enko, 2003). The main changes that occur during the freezing of gametes are mainlay teredl to ultra-structural, biochemical and 1 functional activities, which may ultimately impasipr erm transport and decrease the survival rate in the female reproductive tract, thereby creindgu fertility. Ultra-structural sperm damage is generally greater in the ram than in the budll athnus seems to be species-related (Salamon & Maxwell, 2000). Sperm preservation protocols differ between anismpaelc ies, due to their inherent abilities to accommodate variations in semen extenders usedh ei nc otoling and freezing processes (Barbas & Mascarenha s2,009). These differences between species regarding thseit isveityn of their sperm to cooling are then largely attreibdu tto the compositional variation of the sperm plasma membranes (Bailety a l., 2000). Differences in fatty acid composition and sterol levels of the cell membrane have also besesno caiated with the tolerance of sperm to cold shock and cryopreservation. Thus, the obse rvvaerdiation between species in sperm survival rate, after freezing and thawing, has b aetetrnibuted to these differences. There may then also be considerable differences between sb reaendd between individual males, regarding the ‘freezability’ of their semen (Hiemras ett al., 2005). A thorough knowledge of the sperm physiology fosr pae cific species is thus essential to maximize post-thaw sperm survival and subsequerntitl itfye (Purdy, 2006). Protocols for different species, including the ram have been ldoepveed and tested over time on various exotic breeds. There is however a need to stud yc haanrdacterize the quality of indigenous (in this case, South African) ram semen, as it ultimly adtetermines the fertility rate achieved. It is deemed necessary to cryopreserve indigenouss eramme n and to develop extenders that may optimise the sperm cryosurvival and guaranhteeeir tsurvival and viability. Sperm quality and its relationship to male fertility aoref utmost importance in animal breeding. Moreover, standard sperm analyses are routinelyle imepnted to determine the acceptability of processed semen for breeding purposes. In thuidsy ,s the Computer Assisted Sperm Analysis (CASA) system has been used to accuramtelays ure the motility characteristics of the indigenous ram sperm cells, as it gives reeli abnld repeatable results. Semen evaluation and cryopreservation studies hbaeveen done in the past, using different cryoprotectants and ram breeds of different agens ,d iofferent nutritional regimes and at various time of the year (season) – all factorst cthoauld affect the semen quality and quantity. To date no study has been conducted on the semaelnity qouf certain indigenous South African ram breeds (in this case the Damara, Nama aAqfurikaner, Pedi and Zulu) and the 2 potential of their gametes (sperm) to be prese rvDeude. to practical reasons, the semen collected from the different breeds was done whiteh at id of the electro-ejaculator, which is not the preferred method. It is generally accepthteadt the quality of semen obtained when using the electro-ejaculator is inferior to thatt aoinbed when using the artificial vagina (Greyling & Grobbelaar, 1983). The objectives of this study were thus to charaiscete irndigenous South African ram semen macroscopically (volume, pH and sperm concentra) tiaond microscopically (sperm cell viability and motility rate), determine a suitabsleto rage temperature (5°C vs. 15°C) and storage time for diluted ram semen prior to AI. oA ltso determine the effect of storage temperature and period on ram sperm motility anldo cvitey characteristics of semen diluted with an extender containing glycerol, prior to cpryreoservation and artificial insemination. It was further to determine the optimal glycerol insciolun level in a standard cryopreservation diluent for South African indigenous ram semen, aconmd pare programmable slow cooling rates with the use of liquid nitrogen vapour in cthryeopreservation of indigenous ram semen. 3 Chapter 2 Literature review Factors affecting cryopreservation and post thabwil vitiy of ram semen 2.1 Description of semen Semen is the liquid cellular suspension containsinpge rm cells and secretions from the accessory organs of the male reproductive tracet .f lTuhid portion of the ejaculate is known as seminal plasma (Hafez & Hafez, 2000). The medicicatlio dnary describes semen as the penile ejaculate; a thick, yellowish-white, viscous fluciodn taining sperm cells. 2.2 Production of semen 2.2.1 Site of production Sperm cells are produced in the seminiferous tusb oufle the testis through a process called spermatogenesis. After formation in the seminifes rotubules, the sperm cells are forced through the rete testis and vasa efferentia ineto e tphididymis, where they are stored while undergoing maturation changes that make the spaeprmab cle of fertilizatio n(Hafez & Hafez, 2000). 2.2.2 Hormones involved in the control of spermeanteosgi s The functions of the testes, are namely the proiodnu cotf sperm and androgens (testosterone), regulated by specific hormones. These hormonesc alrle d the gonadotropins, and are released into the bloodstream (endocrine hormobnye sth) e pituitary gland located in the base of the brain. The production of sperm cells and roagnedns by the testes cease without gonadotropin (interstitial cell stimulating hormo naend spermatogenesis stimulating hormone) support. Production and release of thoensea dgotropins by the pituitary are in turn controlled by other centres in the brain (hypotmhaulas), which also respond to environmental stimuli. The main gonadotropins maintaining andu rlaetging spermatogenesis are FSH (SSH) and LH (ICSH) (Evans & Maxwell, 1987). 4 2.2.2.1 Follicle stimulating hormone (F S H) The Sertoli cells of all mammals have FSH recep taonrsd are known to regulate the differentiation and transformation of germ cells s tpoermatozoa. However, there appear to be species and age differences in the way in which FreSgHulates spermatogenesis. FSH has a critical role in regulating spermiogenesis, the cpersos that controls the formation of normal mature sperm with fertilising ability (Moudgal & iSram, 1998). 2.2.2.2 Luteinizing hormone (LH) In the male, LH is known as interstitial cell stilmatuing hormone (ICSH) (Hafez & Hafez, 2000). It acts on the Leydig cells of the teste ss ttiomulate testosterone production. The testosterone in turn acts on the seminiferous etusb utol promote spermatogenesis (Evans & Maxwell, 1987). Fosteer t al. (1978) stated an increase in both volume and atyc toivfi the Leydig cells to be caused by the secretory patotef rLnH . 2.2.2.3 The male sex hormone, testosterone Testosterone is an anabolic androgenic steroidr roinccgu naturally in both males and females (secreted by the adrenal cortex and ovaries in l sqmualntities). It is the principal male sex hormone, which belongs to the class known as anednrso.g Testosterone is produced by the interstitial (Leydig) cells of the testis, and a lcotcsally to stimulate the development of sperm, and via the circulating blood to promote the secaoryn dmale characteristics. Testosterone levels in the body are controlled bny eagative feedback mechanism that involves the hypothalamus, the anterior pituitarlya ngd, and the testes. Briefly, the hypothalamus releases gonadotropin-releasing hoer m(Gon RH) that is transported to the anterior pituitary via the portal system (that libeestween the two areas of the brain). The anterior pituitary then releases follicle stimunlagt ihormone (FSH) and luteinizing hormone (LH), which target the testes. FSH induces the nsiefemroi us tubules to produce sperm and a feedback hormone called inhibin. LH on the othern dh apromotes the production of testosterone by the interstitial cells of the tse.s Itnehibin and testosterone initiate a feedback on the anterior pituitary to inhibit the product iofn FSH and LH and, on the hypothalamus to inhibit the production of GnRH. When inhibin ands ttoesterone levels drop GnRH, FSH, and LH production increases once again (Evans & Max,w 1e9l8l 7). 5 2.2.3 Spermatogenesis Spermatogenesis is the process whereby spermatcooznota ining half the number of chromosomes (haploid) are produced, compared tsoo tmheatic cells. This process takes place in the seminiferous tubules of the testis. The g ceermlls progress from the diploid to haploid state and then change shape to become fully deevde losperm cells. Spermatozoa are the matured male gamete in many sexually reproducingga noisrms. Thus, spermatogenesis is the male version of gametogenesis. In mammals it oc icnu trhse male testes and epididymis in a stepwise fashion and in humans it takes approxilmy a6t4e days. Spermatogenesis is highly dependent on optimal conditions (e.g. temperatfuorre t)h e process to occur efficiently, and is critical in reproduction. Spermatogenesis starts pautberty and usually continues uninterrupted until death. A slight decrease in esne mproduction is discerned with an increase in age. The entire process of spermatogenesis ec asnu bb-divided into several distinct stages, each corresponding to a particular type of cesll toarg e of maturation (Hafez & Hafez, 2000). The spermatogenic process in mammals is compose dt hroefe functionally and morphologically distinct phases: the spermatogo (npiraolliferative or mitotic), spermatocytary (meiotic) and spermiogenic (differentiation) pha, swehsich are under the control of specific regulatory mechanisms (Russetl la l., 1990; Sharpe, 1994). The meiotic and spermiiocg en phases are very similar in all mammals. Spermateosgies nis divided into three phases (Figure 2.1). The first being spermatocytogenesis, entga iali nseries of mitotic divisions during which spermatogonia form the primary spermatocytes. Tehceo nsd phase is meiosis, when the primary spermatocytes undergo reduction divisiornm fiong rounded spermatids with haploid nuclei. The third phase is spermiogen,e sai sphase during which spermatids undergo a metamorphosis, forming sperm cells. The entire epsros cwill be completed within 46 to 49 days in rams. Time estimates reported are shonr tethre i boar (36 to 40 days) and longer in bulls (56 to 63 days). As spermatogenesis progsre, sthse developing gametes migrate from the basement membrane of the seminiferous tubouwleasr dt the lumen and then, towards the rete testis (Beardeent al., 2004). 2.2.3.1 Spermatocytogenesis There are two types of cells located along the mbaesnet membrane of the seminiferous tubules. The first are the Sertoli cells, which larreger, less numerous and are somatic cells which play a supporting role during both spermattogceynesis and spermiogenesis. Second are the spermatogonia, small, rounded and more rnouums ecells which are the potential 6 gametes. After migrating to the embryonic testehse, pt rimordial germ cells undergo a number of mitotic divisions before forming the gocnytoes. Before puberty these gonocytes differentiate into A0 (stem cells), A1 (dormant) and A2 (dormant )spermatogonia, located along the basement membrane of the seminiferous tubTulhees . A2 spermatogonium will divide, forming either dormant (1A) spermatogonium or an active 3()A spermatogonium (Figure 2.1), starting a new generation of developing germ c eTlhlse. active spermatogonia will undergo four mitotic divisions in bulls and rams, eventuy aflol rming 16 primary spermatocytes. In rams, these mitotic divisions are completed by 1d5a yto 17 (Bester, 2006). 2.2.3.2 Meiosis Meiosis is a two-step process. Each primary speorcmyatet will undergo a first meiotic division to form two secondary spermatocytes. Wthitihs division, the chromosome number in the nucleus is reduced by half so that nuclei ien sthecondary spermatocytes contain an unpaired (n) or haploid number of chromosomes. Tstheisp requires approximately 15 days in the ram. Within a few hours after their formatioena,c h secondary spermatocyte will again divide, forming two spermatids. Thus, four spermdsa tfiorm from each primary spermatocyte, or 64 from each active (3A) spermatogonium, in bulls and rams. As th1e sApermatogonia divide by mitosis to form A2 spermatogonia, the potential yield of spermatid hsi gisher than is actually realized. Degeneration of the spermatoag odnuiring mitotic division could account for this loss in efficiency. The Sertoli cells th erenmove the degenerating germ cells by phagocytosis. Following a resting or dormant state of several kwse, ethe dormant (A1) spermatogonium will divide, forming A2 spermatogonia which will vdidi e, forming new active (A3) and new dormant (A1) spermatogonia. Even though A0 spergmoantioa (reserve stem cells) will occasionally divide, forming new A0 and A1 spermgaotnoia, the formation of dormant spermatogonia from A2 spermatogonia is the key epsrso cto maintaining the continuity of spermatogenesis and thereby not diminishing thpel ys uopf potential gametes within the testes (Bearden et al., 2004). 7 Figure 2.1 Spermatogenesinisd icating the sequence of events and time inv ionl vsepdermatogenesis in the r am Source:h ttp://nongae.gsnu.ac.kr/~cspark/teaching/chap6l .htm 2.2.3.3 Spermiogenesis During spermatogenesis thsep ermatids are attached tthoe Sertoli cells. Each sperma ttihden undergoes a metamorphosis, forming a spermatozDouornin. g this metamorphosis the nuclear material will compact ina certain are aof the cell, forming the head of the sperm, whhile t rest of the cell elongates, forming the tail. Tahcero some, ac ap around the head of the sp,e rm will then form from the Golgi apparatus of the spermatids. As the cytoplafrsomm the spermatid is cast off during formation of the t ai cl,ytoplasmic droplewt ill form on the neck of the sperm. The mitochondria from the spermatiildl fworm in a spiral around the upper one-sixth of the tail, forming them itochondrial sheath. Newly formed specrmel ls will then be released from thee Srtoli cell and forced out thorugh the lumen of the seminiferous tubules into the rete testis. Sperm ce allsre unique cells in that thehya ve no cytoplasm, and after maturation possess the ability to be progressivmeolyti le. The process ofp sermiogenesis is completed after 15 to 17 dsa yin rams (Bester, 2006). 8 2.3 Seminal plasma The seminal plasma is the extracellular fluid tphraotv ides the medium for sperm cells. It is a composite mixture of secretions that originate f rotmhe male accessory organs of reproduction (Gundogan, 2006). Seminal plasma cahs issu synthesized and secreted by the testes and accessory sex glands in males and ap lsaiygsn ificant role in the development of sperm motility and hence its freezing ability. Mamalmian seminal plasma is thus composed of secretions from several glands in the reprodveuc ttriact (Mann & Lutwak-Mann, 1976) that are mixed with the sperm at ejaculation and counteri bto the majority semen volume and components (Mouraet al., 2006). The seminal plasma is known to contaointe pinrs, enzymes, lipids, electrolytes, sugars and various othero frasc, twhich may play significant roles in the metabolic regulation of sperm cells. In ejaculasteedm en, fructose is a major saccharide contained in the seminal plasma of most farm ansi.m Tahl is fructose in the seminal plasma plays an important role in sperm metabolism, aned sthperm cells utilize it to produce adenosine triphosphate (ATP) (Maxwetl l al., 1999). Certain accessory sex gland proteins are also known to bind and be absorbed into threm s pcell membrane, affecting its functions and properties (Mentzet al., 1990; Desnoyers & Manjunath, 1992). It is knotwhna t the seminal plasma proteins coat and protect sperms cdeulrling ejaculation. Many studies have shown that the low content of seminal plasma pnrost eisi associated with poor semen quality (White et al., 1987; Ashworthe t al., 1994). The seminal plasma proteins are mainly composed of albumin and globulin, in addition toa sllm quantities of non-protein nitrogen, amino acids and peptides (Zededta a l., 1996). These compounds make up the amphoteric property of the seminal plasma proteins, while ltohwe protein content in seminal plasma reduces its buffering capacity and in turn the sne mqueality (Paze t al., 1992). Seminal lipids play significant roles in the memnber astructure of the sperm cell, sperm metabolism, sperm capacitation and fertilization thoef female gamete (Hafez, 1993). In addition, some researchers have reported that trieodnus cin sperm concentration and motility are associated with a decrease in seminal plaspmida c loi ntent (Kelsoe t al., 1997; Tahae t al., 2000). Seminal plasma has also been reported ton tamina isperm motility and viability in many species (Baaest al., 1983; Graham, 1994; Maxweeltl al., 1996) and increase the sperm resistance to cold shock injury (Bergetr al., 1985; Barriose t al., 2000), by providing specific components that stabilize the membranteh eo ff rozen-thawed sperm cells (Maxwell & Watson, 1996; Olleroe t al., 1997). Maxwel let al. (1999) evaluated the effects of resuspending ram sperm cells in 20% seminal plaspmosat -thawing and reported the 9 penetration of sperm cells through the cervical umsu tco be improved, and fertility after cervical insemination of ewes significantly increda.s Mortimer and Maxwell (2004) subsequently reported frozen-thawed sperm, resudsepde in artificial seminal plasma or ram seminal plasma to have improved motility and inscereda plasma membrane stability, compared to those resuspended in PBS. It was stuegdg tehsat this was due to the components of the medium. The detrimental effects of seminal plasma on spmeromti lity (Iwamoto et al., 1993; De Lamirandee t al., 1984), viability (Dott, 1979) and post thaw siuvarvl rate (Ritar & Salamon, 1982, Kawanoe t al., 2004) have also been reported. Seminal plasms ab ehean shown to suppress sperm capacitation and to decapacitavtieo upsrely capacitated sperm (Cross, 1993). 2.4 Semen collection methods 2.4.1 Semen collection using the artificial vagina The artificial vagina as a means to collect semse ena isy to use and the semen collected is generally relatively clean and the ejaculate isi lsairm to the natural ejaculate (Salisbuerty a l., 1978). The artificial vagina (AV) briefly consisotsf a rigid cylinder of rubber or PVC and a thin walled rubber tube for the inner lining. A wear-ttight jacket is formed inside the cylinder by folding both ends of the thin walled rubber t uobve r the outer cylinder. The water jacket is filled with water, warm enough (45-55°C) to bgr inthe inside temperature of the artificial vagina to a few degrees Celsius (°C) above normodayl btemperature. The temperature of the water simulates the thermal, while the pressuret hien AV provides the mechanical stimulation of the vagina over the glans penis (oDvoann et al., 2001). At one end of the artificial vagina, a graduated, glass semen coiollne cttube is fitted. A female, preferably in oestrus, is placed in a neck clamp and the maalello iws ed to mount. When the male mounts, the penis is deflected into the AV, where the maejlea culates naturally. The major disadvantage of this method of semen collectiont hiast the rams have to be trained beforehand to utilize this method (Mathewets al., 2003).To avoid contamination of the semen sample and prevent the transmission of vaeln deirseeases from one ram to another, all rubber parts should be thoroughly cleaned and dri nwsieth water, then with alcohol, and finally with distilled water and allowed to dry. 10 2.4.2 Semen collection using an electro-ejaculator The electro-ejaculation (EE) method of semen cotiollenc was first used by Gunn (1936), in Australia. The technique was based on the prin coipf lsetimulating the spinal cord, between the 4th lumbar and the s1t sacral vertebrae by placing one electrode in ethcetu rm and the other in the back muscle. By passing a few 5 to 10 se crohnytdhmic electric stimuli through the electrodes, an ejaculation can be induced ande tmhe ns collected in a glass tube. The animals generally experienced no harmful effects, no lons sb oi dy condition, no real change in disposition, and no special disinclination to fuerrt happlication of the treatment. However, during the application of this electro-ejaculatimone thod, the electric current produces general strong contractions of all body muscles, and ah st laignd temporary motor inability of the hindquarters and hind limbs, at the end of thisa ttmreent. Later a bipolar rectal electrode in contact with the floor of the rectum was introdu ctoe dfacilitate the process (Salisbuerty a l., 1978). Cartere t al. (1990) described the EE as a two phase proceses .f irTsht entailed an emission phase, involving stimulation of the lum bsayrmpathetic nerves which form the hypogastric nerve and which supply the ampullae vaansda deferentia. The second is an ejaculatory phase involving the contraction of uthret hral muscles, which are serviced by the sacral parasympathetic nerves, forming the pelnvidc paudendal nerves. Electro-ejaculators are basically electrical generators, which deliavenr oscillating current which serves as a stimulus to the nerve controlling the emission aenjadc ulation of semen. Researchers have claimed that EE generally produce ejaculates waitrhg elr volumes, but a lower sperm concentration than that obtained using the AV. Bdena rand Fuquay. (1980) indicated that the total number of sperm cells obtained using EE mis pcaorable, and fertility levels also seem to be comparable to that of ejaculates collected frtohme same rams when using the AV. According to Matthewse t al. (2003), semen collected with the aid of an AVd purcoe a higher sperm concentration, but with a similar volume asnpde rm morphology, when compared to that of semen collected by an EE. Carette ra l. (1990) also compared EE and AV semen collection methods in rams and found the repeaittya boilf the volume of the ejaculate obtained, sperm concentration, total sperm numpbeerrc, entage of normal sperm, and wave motion were slightly higher when using the artiafilc viagina technique. The advantages of the EE are that no prior tra iinsi nngeeded for rams, more ejaculates can be collected within a short period of time, and semcean be collected from superior sires that are incapable of mounting, possibly as a resulitn joufr y or ageing (Sundararamaent al., 2007). 11 2.5 Semen evaluatio n 2.5.1 The importance of semen evaluation Semen quality and its relationship to fertility aarsepects of major concern in the animal production industry. The average ejaculate volumf rea mo semen is 1.1mL (Seremaetk al., 1999). Semen however needs to be evaluated uslignhgt am icroscope to estimate the sperm viability and the percentage motile (and progreeslsyi vmotile) sperm cells, prior to its use in AI (Rowe et al., 1993). 2.5.2 Subjective assessment of semen 2.5.2.1 Colour and volume of the ejaculate The first measurement of raw or fresh semen toc aintdei quality is its overall appearance. Raw (unaltered) semen appears as a thick whiti sshli gtohtly yellowish fluid. The colour of ram semen varies from milky- white to pale creamn yc oi lour (Bage t al., 2002). According to Hafez and Hafez (2000), there exists a correlatbioentw een the colour and the sperm concentration of the semen ejaculate. The visc osf ityhe semen sample is often a reflection of the number of sperm cells present. In practhicee stemen sample should be free of any odour, as this is indicative of an infection or thperesence of urine, which could be detrimental to the fertilizing ability of the sem seanmple. Other contaminations considered to be detrimental to the ejaculate can be detectethde i nc olour of the semen e.g. blood, urine, and faeces, which may cause the semen to be p ibnrko twonish in colour. White clumps or flakes in the ejaculate indicate pus and the prcees eonf an infection in the reproductive tract of the male. Hafez and Hafez (2000) further reported age of rathme and body condition, season of the year, skill of the technician involved and the fureqncy of collection to affect the ejaculate volume. The ejaculate volume generally ranges beentw 0e.5 and 2mL in mature rams, and 0.5 and 0.7mL in young rams. The ejaculate volume gweilnl erally decrease if a ram is collected three or more times per day, or for a lengthy pde orifo time. Gil et al. (2003) using the AV to collect semen from rams, regarded a volume of beentw 0e.75 and 2mL to be normal. 12 2.5.2.2 Sperm concentration Sperm concentration generally refers to the numobf esrp erm cells per millilitre of semen (Hafez, 1993) .Sperm concentration in the ejaculate serves aso of nthee criteria in semen characteristics, to qualify fertile males for brienegd purposes (Graffeer t al., 1988). The concentration of semen is essential to determinwe hmouch to dilute the semen, while providing adequate number of sperm cells in eacshe minination dose. The sperm concentration in the ejaculate is physically meeads uwr ith the aid of a haemocytometer or a spectrophotometer. The haemocytometer is compofs ae dm oicroscope slide with a precisely calibrated chambers generally used for counting b rloeodd cells (Evans & Maxwell, 1987 ). According to Hafez and Hafez (2000) there existcso rar elation between the colour of the semen sample and the concentration of the ejac. uTlhaete density of the semen sample is then a reflection of the number of spermatozoa presTenatb.l e 2.1 demonstrates how sperm concentration varies, based on the semen sampoluer c. ol Table 2.1 Subjective assessment of semen concioen turasting colour variation Semen score Ejaculate colour Number of sper9m/m(1L0) Mean Range 5 Thick creamy 5.0 4.5-6.0 4 Creamy 4.0 3.5-4.5 3 Thin creamy 3.0 2.5-3.5 2 Milky 2.0 1.0-2.5 1 Cloudy 0.7 0.3-1.0 0 Clear (watery) Insignificant Insignificant Source: Hafez and Hafez, 2000 2.5.2.3 Sperm motility Sperm motility is the simplest trait to evaluate tqhuality of a semen sample. Hafez and Hafez (2000) reported sperm motility assessmenitn vto lve the subjective microscopic estimation of the viability of the sperm cells atnhde quality of the sperm motility. Sperm motility in raw and extended semen at va risotuesps of the freezing process can be assessed microscopically by examining a uniformp dorfo semen on a slide with a coverslip, under a phase contrast microscope, fitted to a w satarmge at 37°C. This motility assessment is 13 generally made on the basis of an arbitrary scraolme f0 to 5 (0 = no motility, 1 = 20%, 2 = 40%, 3 = 60%, 4 = 80% and 5 = 100% motility) (Kazarast et al., 1997). Although it is important to look for progressively motile spermp e(rsm moving in a straight line), it may be just as relevant to evaluate the viability – toe drmetine if sperm are alive and motile (total motility or sperm being able to propel themselvoersw fard with a beating tail). For people evaluating semen for the first time, the proces ass osfessment seems difficult, inaccurate and not very repeatable. Although useful, these sperm motility evaluationres naot completely reliable or repeatable, because of the small number of sperm evaluated ,la tchke of objectivity and human bias (Grahame t al., 1980). 2.5.2.4 Sperm morphology The structure or morphology, of the sperm cell bheaes n studied using light and electron microscopy techniques. The sperm as such has befeinne d as a highly structured cell, streamlined to deliver DNA to the oocyte. Primarby noarmalities may occur during spermatogenesis in the testis, while secondary rambanlioties may occur during maturation in the epididymis and tertiary abnormalities resuoltm fr poor handling of the semen following ejaculation. Generally sperm abnormalities asseodc iawtith the head are classified as primary and those associated with the mid piece or speirlm a sta secondary. Abnormalities of the sperm head include twin, tapering or pyriform, rdo,u snhrunken, large, narrow, elongated and diminutive heads. Abnormalities of the neck on oththee r hand include broken necks and loose necks (Evans & Maxwell, 1987). The most common abnormalities of the sperm mid-ep iienc lude bent, broken, and short, enlarged or thickened, double, filiform, vestigmiaild -piece or abaxial attachment of the mid- piece to the head of the sperm cell. The princaipbanl ormalities of the tail include coiled, twin, broken, crooked, kinky, or truncated tailsa.l iSsbury et al. (1978) reported the ageing of the sperm cells to result in morphological chan geevse,n in semen kept under controlled temperatures. Periods of high ambient temperat utoreges,ther with high humidity may render a male infertile for up to 6 weeks and many abnol rmspaerm cells may appear in the ejaculates collected during the recovery periode. rTahm’s fertility is often questionable when 20% or more cells are abnormal in a semen sampill ee.t Gal. (2003) regarded semen with less than 10% abnormalities, to be normal for s.h eSepasonal variations may influence the 14 percentage of abnormal sperm, with the number noof rambalities being highest in spring, and declining as the natural breeding season advances. 2.5.3 Objective semen evaluation 2.5.3.1 Introduction Semen quality and its relationship to fertility aorfe major concern in animal production. The accurate evaluation of semen quality is thus of ousttm importance. Conventionally, the laboratory tests for standard semen evaluation oastt mAI centres use light microscopy to estimate sperm survival and the percentage of em osptilerm (Rowee t al., 1993). Although useful, these tests are not completely reliablere poer atable because of the small numbers of sperm eventually evaluated, lack of objectivityd, ahnuman bias (Grahamet al., 1980). More objectivity and repeatability in the assessmenstp oefr m motility can be achieved with the aid of the Computer Assisted Sperm Analysis (CASA) (iDs a&v Siemers, 1995) . 2.5.3.2 Semen evaluation with the aid of the coemr paustsisted sperm analyser (CASA) Computer Assisted Sperm Analysis (CASA) has beetrno dinuced in the laboratory as a routine method to improve the accuracy and repeiliatyta obf sperm quality data collection, to avoid technician error resulting from the subjeec teivvaluation of different technicians and to reduce the time spent in semen evaluation (eJta anle., 1996). The use of this Computer Assisted Sperm Analysloisw asl for the objective measurement of several sperm parameters, for example motility,c wh hoiffers a more reliable, unbiased and repeatable method of assessing sperm motility, thea nsubjective evaluation by the human eye (Colenbrandeert al., 2003). Several CASA systems are available comcimalelyr, and may differ in their mode of functioning and in their ilaitby to detect and measure the motility of sperm in different species. The majority of CASAs tseyms (e.g. the ISASTM by Proiser, the Hobson Sperm Tracker using Sound and Vision or CSETRMO system by Hamilton Thorne), record the path and type of movement of a grouspp oefr m in a wet preparation under a cover slip using a video camera. The signal receivedh bey c tamera is digitized and the information is processed by a computer which reconstructs einadcivhi dual sperm path trajectory for a certain number of frames. Subsequently, these s pterarmjectories are mathematically processed, permitting them to be defined in a nuicmale frorm (Quintero-Morenoe t al., 2003). The CASA system is able to determine a series mofe sne variables, including the number of 15 moving sperm, curvilinear velocity (VCL), linear lovecity (VSL), linear coefficient (LIN), straightness coefficient (STR), frequency of spehrema d displacement (BCF), etc. The kinematic parameters obtained from the CASA sysaterem t hus useful for research purposes, making the identification of sperm sub-populatiocnos- existing in an ejaculate possible (Quintero-Morenoe t al., 2003). 2.5.3.2.1 Advantages of using the computer as sipsetermd analysis (CASA) system The CASA provides an accurate evaluation of semaerna mpeters such as spermatozoa motility by avoiding errors that may arise as au rlte sof subjective evaluation of different technicians and reduces the time spent on semelnu aetivoan (Janee t al., 1996) . More objectivity and repeatability in assessing spermt ilmityo can be achieved by the Computer Assisted Sperm Analysis (CASA) (Davis & Siemers9, 51)9. The use of CASA offers a more reliable, unbiased and repeatable means of asgse ssspinerm motility, compared to examination by the human eye (Colenbranedte arl ., 2003). Individual spermatozoa can be analysed and video images of the sperm cells aprteu rcead and analysed by the software. 2.5.3.2.2 Disadvantages of using the computert eads sipserm analysis (CASA) The major problems with CASA are centered on thgeh hciost of the instruments, which suggests its use only in sophisticated laborat otrhieast perform a high number of routine semen evaluations (Verstegetn a l., 2002). The instrument settings are relativelbyj escutive and different CASA instruments use different matahteicmal algorithms (Rabinovitch, 2006). The degree of comparability of measurements acarlol sinss truments is not quite clear. There are problems encountered regarding the atcec ucroaunting of high and low sperm concentrations (Rabinovitch, 2006). The measuresm oebntained following sperm counting, include a statistical counting error, while CASAq ureires extensive training and cross validation regarding technician competencies (Veegresnt et al., 2002). The clinical significance of the kinematical variables is howre sveverely limited. The analyses are not standardized due to the different instrument sgestt iannd algorithms (Rabinovitch, 2006). 2.6 Effect of environmental factors on sperm prtoiodnu cand quality The fertilization rate generally depends on thei labvaility of a sufficient number of fertile sperm in the vicinity of the fertile ovum. In tu rtnh,e quality of these sperm depends on a number of biological and environmental factors. tCaienr factors, such as inadequate nutrition, 16 high ambient temperatures, and aging of the anihmaavle negative effects on the overall semen production. On the other hand, an extendeodto pehriod in small stock, frequent semen collection, and certain genetic factors c opuoldsitively stimulate sperm production (Flowers et al., 1997). A thorough knowledge of factors affectinspge rm quality and ultimately semen production is important in all cAeIn tres (Soderquiset al., 1996). 2.6.1 Age of the ram Much attention in the past has been paid to thec te fof f age of the individual on semen production and the season of collection, regardthineg s perm morphology in bulls (Almquist & Amann, 1976; Almquist, 1982). The age of the bautl lsemen collection generally affects the volume of the ejaculate, the sperm concenntr,a taiond sperm motility. Several studies have suggested that an increase in age of the ims aalses ociated with a decline in certain semen parameters (Centola & Eberly, 1999). Agenin gro idents appears to cause certain histological changes in the testes, which in tuernsu rlts in the decline of sperm quality (Tanemurae t al., 1993; Centola & Eberly, 1999). The scrotal cmircfeurence and ejaculate volume normally increase with increasing ram agpe t,o u 5 years of age. These findings seem to indicate that the genital system of the ram urgnodes maturational changes during this period (Osinowoe t al., 1988; Toee t al., 2000). In men, semen volume, sperm concentr,a tion total sperm count, sperm motility, progressive mlitoy,t iand normal morphology have been found to decrease as age increases (Tanemetu raal. , 1993; Pasqualottoe t al., 2005). Similarly, quantitative analysis of sperm motilcithy aracteristics using CASA has indicated an age-related decline in linearity (LIN), straighnt eli velocity (VSL), and average path velocity (VAP) (Sloter et al., 2006). Shannon and Vishwanath (1995) and Garent earl . (1996) have reported the morphology of sperm, semen concentration, semen motility andv othluem e of the ejaculate to improve with an increase in the age of the bulls. This supptohret sf indings of Osinowoe t al. (1988) who reported mature rams to generally have higher elajatec uvolumes, sperm concentrations and total sperm per ejaculate than younger rams. Lardn g(f1o987) also found sperm output to increase with an increase in scrotal circumfere nGceen.erally, scrotal circumference can be used as an indicator of sperm production in shTeeope e(t al., 2000) 17 2.6.2 Season of the year Seasonality has been shown to affect semen quina lbituy lls, boars, bucks, stallions, and rams (Thongtip et al., 2008). Seasonal variation in the thyroid acyti vaint d seminal characteristics has also been observed in Iranian fat-tailed raZmasm (iri et al., 2005). It was specifically shown that the highest values for thyroid stimunlga tihormone (TSH), T4, free T4 index, testosterone, total sperm number, percentage n osrpmearlm, percentage live sperm, sperm concentration, semen volume and scrotal circumcfeer ewnere recorded from early summer to winter with the lowest values being detected a te tnhde of spring. It has also been suggested that the thyroid gland may be involved in seasotrnaanl sition of reproductive activity in the ram (Thongtipe t al., 2008). Low semen quality with a decreased sperm conceionntr aatnd motility and increased percentage abnormal sperm has also been foundy rioni dthectomized rams (Brookeest al., 1965). Most studies found evidence that seasono ollef cction significantly affects semen production (Graffere t al., 1988; Stalhammaert al., 1988). According to Schwaebt al. (1987) the highest volume of semen, sperm concentratinodn , naumber of sperm per ejaculate are produced during winter. Menendez-Buxadetr a l. (1984) also reported semen quality to be higher in winter. These results are contrary to ftihnedings of Fuentee t al. (1984), who obtained the lowest semen quality during wintera. sSoenal effects on semen quality are caused by several factors, such as ambient temupre roart humidity, day length, and available feed quality. Seasonal variations in total protceoin tent of the seminal plasma were found in rams, being higher in autumn than in summer andte wr i(nGundogan, 2006). 2.6.3 Daylight length (Photoperiod) Sexual behaviour in the ram can be influenced bnyy m factors, including season of the year, genetics, breed differences, hormonal effects, -wpoesatning management, ambient temperature and nutrition (Mickelseent al., 1982). Photoperiod is however the main environmental cue affecting sheep reproduction m(Cinheau et al., 1992). Variation in the sexual response of sheep breeds, to photoperiotidmicu lis appear to be affected by the latitudes where the animals are raised. Sheep oaantds gexhibit great seasonal variation in semen quality (Leboueeft al., 2000). Animals in the temperate zones are h igahffleycted by photoperiod, while those in the tropical zones alerses sensitive. D’Alessandro and Martemucci (2003) reported an improvement in therc epnetage of motile sperm to occur during decreasing photoperiod. 18 Reproductive responses to photoperiod are detedrm tion ea large extent by the degree of photo responsiveness, the nature of the photopice riosdignal, nutritional and social environment (Wildeus, 1995; Walkden-Brown & Res, ta1ll996). The nature of the photoperiodic signal is however important in detienrinmg the reproductive activity in seasonal breeders (Walkden- Brown & Restall, 19 P96h)o.toperiodic signals are translated into effects on the reproduction system by chaning etsh e pattern of secretion of melatonin from the pineal gland (Shelton, 1980; Wildeus, 1;9 W95alkden- Brown & Restall, 1996). This results in changes in the pulsatile releas eG noRf H, from the hypothalamus (Mori, 1992). In sheep and all mammals, the circulatinvge lsle of melatonin are generally low during the day and high at night. This profile of melanto nsiecretion is an endocrine signal, which relays the photoperiodic information to the reprcotdivue axis (Karsche t al., 1985). As short days are characterized by a longer duration of tmoneilna secretion compared to long days, attempts have been made to mimic the duration manpdli taude of the presence of melatonin in the blood. Continuous melatonin administration nvuiat rition, subcutaneous or intravaginal implants can stimulate an early onset of breedicntgiv iaty by mimicking the onset of short photoperiodic environments (Poultoent al., 1987). In a study conducted by Daveitd al. (2007), melatonin implants were found to producesh ao rter response than photoperiodic treatment as such, which was less repeatable. eArcactedl production of sperm by induction has been performed using different methods. Thnecsleu die the administration of Clomid® (Herbert et al., 2002), testosterone implants (Adamopouloetu sa l., 1990). However these methods implicate certain problems regarding an imheaal lth, embryo mortality, fertility, immunology and environmental contamination. As bdl oteostosterone levels and therefore the sexual activity are affected by photoperiod, ramees dn to be treated by other means that are less expensive and easier to apply. 2.6.4 Ambient temperature and testicular thermolraetgioun 2.6.4.1 Ambient temperature For normal semen production to occur the testese htoa vbe at a temperature several degrees below that of normal body temperature, otherwiser msp production may be affected. To provide the necessary thermoregulation for spergmeanteosis, the ram has large sweat glands in the skin of the scrotum, as well as a system uosf cles that raise or lower the testes nearer to the body for the purpose of temperature regounla. tBi lood flow to the testes also helps to 19 regulate temperature through a heat exchange miescmh.a Hneat is transferred from the testes to the blood and is then transported to other poaf rtthse body for dissipation. If the temperature in the testes cannot be kep t elnoowugh, as can happen in warm weather (e.g. ambient temperatures over 32°C for long pdesr io r short spells of very high temperatures (38 °C or more)), the production ormf naol, viable sperm will be affected. Fully developed stores of sperm are less affected thoasne tshperm still in the developing stages. High body temperatures produced in rams by high mseurm temperatures or with fever is generally a cause of poor quality semen. This aalsffoe cts semen formation or spermatogenesis and ultimately induces temporaeryili tsyt. These high temperatures can also affect mating, with subsequent reduced sexual iatyc.t iEv levated body temperature during periods of high ambient temperature leads to tuelsatri cdegeneration and reduction in the percentage of normal and fertile spermatozoa i ne jtahceulate (Maraei t al., 2008). 2.6.4.2 Testicular thermoregulation Several physiological mechanisms play a signifi craonlet in testicular thermoregulation. These include the regulation of blood flow, the tcroln of the testis position, relative to the body by scrotal musculature, sweating, countere-cnut rrheat exchange in the vascular cone, and overall radiation of heat from the scrotal ascuerf. The counter-current exchange of heat in the neck of the scrotum has been identified asp trhime ary mechanism of regulating the temperature in the testes. It has been shown hthea st ctrotum and testes have complimentary temperature gradients that contribute to testic tuhlearmoregulation (Kasteliect al., 1996). The testicular vascular cone is made up of a coxm vpelenous network that surrounds the highly coiled testicular artery. The counter-curt,r ehneat exchange within the vascular cone functions by allowing, the transfer of heat frome twharm blood flowing down the testicular artery towards the testis, to the cooler blood rrneintug from the testis through the testicular venous system (Cooekt al., 1994). Waites and Moule (1961) reported the cteoru-cnurrent exchange to only cool the testis if a temperaturraed igent exists between the venous and arterial blood. The extent of this heat exchangeen tdhepends solely on the magnitude of the temperature gradient. The vascular cone also palna yism portant role in the radiation of heat from the scrotum, as the scrotal skin overlying vthaescular cone is usually the warmest area on the scrotum (Acevedo, 2001) 20 2.6.5 The effect of nutrition on semen quality faenrtdil ity Nutrition has a direct and dramatic effect on cteuslatir size, which again has a corresponding effect on sperm production. Rams grazing on pas toufr efluctuating quality may have testes which double (or halve) in size during the year dtou ethe seasonal variation in quality of the pasture. Research has shown that an improvemennut triniti onal intake of both protein and energy during the two-month period prior to matimnga y increase the testicular size and subsequent sperm production by as much as 100%ri.t ioNnuatl changes also affect testicle size much more rapidly than is reflected in the lwiveight or general body condition. This highlights the importance of checking the rams'r oredpuctive soundness prior to the mating season. On the other hand, rams should not be eadll otow become over-fat (body condition score more than 4), as obese rams tend to bee lxeusas llsy active and are more prone to heat stress (Hafez, 1993). It is well documented thaetq audate nutritional management is crucial for successful mating in sheep flocks (Smith & Abkainmijo, 2000; Fernandeezt al., 2004). Carbohydrates, protein and nucleic acid metaboliasnmd their deficiency may impair spermatogenesis and libido in males, with resu ltlaonwter fertility rates, embryonic development and survival, post-partum recovery viaticetsi , milk production, later development and lower survival rates in the offnsgp r(iSmith & Akinbamijo, 2000). Vitamin A is essential for sperm production. Ramesfi cdient in Vitamin A often have soft testicles and produce poor quality semen. Wheres rhaamve spent six months or more without access to any green feed, supplements which co Vnittaaimn in A may be required (e.g. green hay, vitamin supplements). A number of studies have demonstrated that spergmenaetosis in rams is sensitive to an increase in protein intake. This effect has beelant erde to an increase in testicular size, due to an increase in the volume of the seminiferous epliuitmh and the diameter of the seminiferous tubules (Oldhame t al., 1978; Hotzele t al., 1998). The improvement of testicular efficiency with nutrition has also been reported by Oldheatm a l. (1978). It has been shown that rams maintained on a high plane of nutrition produce em soprerm than those raised on a low plane of nutrition. 21 Masters and Fels (1984) demonstrated testiculaer t osi zbe controlled by nutrition, even to the extent that well-fed rams in spring may have la rtgeesrtes, compared to poorly-fed rams in autumn. Nutrition appears to mediate its effecti nbcyr easing the frequency of pulses of LH and probably FSH (Lindsaeyt al., 1984; Boukhliqe t al., 1997; Hotzeel t al., 2003). However, the energy components of the diet, particularly ftahtety acids, appear to play a key role in reproductive responses following changes in nuotnr.it iFatty acids for example can stimulate the GnRH-dependent pathways that initiate changne ste si ticular function (Boukhliq & Martin, 1997; Blachee t al., 2002). 2.7 Factors affecting the viability of sperm after sne cmoellection 2.7.1 Temperature The most important physical condition that sperme eaxrtremely sensitive to is temperature. An excessive, fast decrease or increase in temupre racat uses sperm mortality (temperature shock). Such a change normally involves damaghee t op ltasma membrane of the sperm cell, which contain temperature sensitive, unsaturatettdy faacids. These lipids are sensitive to oxidization and excessive peroxidization disruphtse tcell membrane, rendering the cell incapable of fertilization (Bester, 2006). 2.7.2 Semen pH Stored semen following collection produce hydrogioenns , and as a result the pH decreases. Therefore, buffers are usually required to main staeimn en at acceptable pH levels. If extended semen is maintained at body or room temperaturee ,s ptherm will be metabolically active, secreting acids, increasing the pH and will sooen, difi not introduced into the female reproductive trac tL. atif et al. (2005) also reported that in an acidic pH envmiroent, the motility of sperm is affected, probably due to aa ncghe in the metabolic activity and a disturbance in the cellular respiration of the smp ecrell. 2.7.3 Osmotic pressure Semen and diluents must be isotonic as sperm mina itnhteair maximum metabolic activity when semen is diluted with an isotonic extendera. nSswon (1949) observed that bovine sperm are more sensitive to hypertonic solutions of smod icuitrate than to hypotonic solutions. It was suggested that, as a result of glycolytic moelitsamb, an increase in the osmotic pressure of semen occurred during storage. However, botho thoynpic and hypertonic extenders reduce 22 the metabolic activity and disrupt the membrane ginrity, which leads to clumping and finally death of the sperm (Lateift al., 2005). 2.7.4 Concentration of sperm per ejaculate A too concentrated semen sample decreases speramb omlice tactivity, due to an increase in potassium content of the sperm cell. Dilution dnooets n ormally change the metabolic activity of the sperm, but will increase its lifespan. Exscivees dilution (1 to 1000) on the other hand will depress the motility and metabolism. So wheenm esn is prepared for artificial insemination (AI), the semen is diluted in an exdteern and the number of sperm per AI dose is standardized. This reduces the direct advantagheig ohf sperm output in the ejaculate on the fertilizing potential. However, when a sample islu tdeid to standard sperm cell numbers, the seminal plasma is also diluted. The final dilutrioanti o of seminal plasma to the extender used in the AI straws generally depends on the spermc ecnotnration at semen collection (Karoliina, 2009) 2.7.5 Gas environment CO2 stimulates aerobic metabolism, if kept below 51 0to% .Too much oxygen however also decreases the sperm cell metabolism. The gaseovuirso nemnent under which semen is stored can thus influence the motility via a change inra incet llular ATP. The possible explanation for the CO2-based inhibition of sperm motility is that the sperence of CO2 in the semen storage environment leads to the depletion of intracell uAlaTrP. The CO2 reacts with water to generate carbonic acid, a reaction catalyzed bray cienltlular carbonic anhydrase. An increase in CO2 concentration in an aqueous environment, resu ltas idnecrease of the pH in that environment. The extent of this change in pH isu nac tfion of the magnitude of the C2 O increase, and the buffering capacity of the sonlu. t(ioBencice t al., 2000) 2.7.6 Light exposure Exposure to light can depress the sperm metabaotleic, mr otility, and fertilizing capacity that only occurs aerobically. It is recommended to n eevxeprose semen to direct sunlight as ultraviolet light can be lethal to sperm. That ihsy w it is so important to transport the semen following collection inside a box, to protect ito fmr direct sunlight (Bester, 2006). 23 2.8 Semen extenders or diluents Cryopreservation as a technique for long term sgteo roaf semen has many advantages, but the freezing and thawing processes induce detrimenffteacl tes in terms of sperm ultrastructural, biochemical and functional damage (Watson, 200e0s)u, lrting in a decrease of sperm motility, membrane integrity and fertilizing ability (Purd2y0, 06). The detrimental effects induced by cryopreservation may however be compensated four sbinyg higher sperm numbers in the insemination dose (Watson, 1995). Related to tvhaisri,o us techniques of processing and freezing of sperm have been developed over thes ,y etoa rreduce the cryogenic injury to sperm (Salamon &Maxwell, 1995). The purpose of a semen cryopreservation exten dgeern iesrally to supply the sperm cells with a source of energy, to protect the cells from termatpuere related damage, while maintaining a suitable environment for the sperm to survive termarpiloy. Diluents are used in the semen cryopreservation process as these media increaes ee jathculate volume without affecting semen quality and preserve the fertilizing capa ocfi ttyhe sperm for the longest period of time possible. Egg yolk is a general component of semcreyon preservation extenders used for domestic animals. This yolk has been shown to ha veb eneficial effect on sperm cryopreservation as a protector of the sperm pla smmeambrane and acrosome against temperature related injury – in association withe or tcomponents, because of the lipids that it contains (Purdy, 2006). The semen extender for sraem en cryopreservation should also contain buffers for controlling the pH (6.7 to 7,. 0a)ntibiotics to prevent bacterial growth and cryoprotectants to prevent the crystallization oaf tewr within the sperm cells. This will ultimately allow sperm cells to be cryopreservefde cetfively (Salamon & Maxwell, 2000). 2.8.1 Components of ram semen extenders Buffers are essential to control the pH between a 6n.d7 7.0. Sodium citrate, egg yolk and Tris buffers are commonly used for this purpose. Lipgiedns erally provide protection of the sperm membranes from temperature changes. Skim milk agngd y oelk are generally good sources of lipids. Nutrients are also essential to provider geyn efor sperm cell. Fructose and glucose are typically used in the extenders. Antibiotics arec luinded to prevent bacterial growth, while glycerol serves as a cryoprotective agent in thme esne freezing process. It prevents the crystallization of water within the sperm cells, icwhh ultimately allows the sperm cells to be frozen rapidly (Holt, 2000) 24 2.8.1.1 Example of a semen extender 1) Egg yolk- citrate (Fraction A) 20% (v/v) egg yolk 80% (v/v) of a 2.9% (weight/vol) sodium citrate dyedhrate 1000 IU Penicillin/mL 1000 µg Streptomycin/mL 2) Egg yolk- citrate + glycerol (Fraction B) 20% (v/v) Egg yolk 66% (v/v) of a 2.9% (weight/vol) sodium citrate dyedhrate 14% (v/v) glycerol- provides final concentration 7 o%f glycerol Glycerol is added after the semen has been cool e5d° Ct . This prevents morphological damage to sperm, compared with glycerol added oamt rtoemperature. The glycerol fraction is generally added in three equally timed steps v aonludmes. The final volume of the extender should contain equal quantities of Fraction A an. d B 2.8.2 Cryoprotective agents Cryoprotective agents or cryoprotectants are inecdlu din the cryopreservation medium to reduce the physical and chemical stresses derrivoemd cfooling, freezing and thawing on the sperm cells (Gaoe t al., 1997; Purdy, 2006). Cryoprotectants and theidr emso of action has been the subject of many reviews, with glycerol aDnMdSO being the most commonly used cryoprotective agents (Karow, 1981; Mazur, 1984o; cBkrbank, 1995). Fetal bovine serum (FBS) is also often used in mammalian cryopreseiornv ast olutions, but it is not a real cryoprotective agent. However dextrans, glycolasr, cshtes, sugars, and polyvinylpyrrolidone provide considerable cryoprotection in a variet yb ioflogic systems (Mazur, 1981). The cryoprotectants can be classified as penegtr aotri n on- penetrating agen ts. The penetrating cryoprotectants or intracellulayro cprotectants (glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol) have low moleacru wl eights, and induce membrane lipid and protein rearrangement, resulting in increasedm bmrane fluidity, greater dehydration at lower temperatures, reduced intracellular ice fotiromna, and an increased survival rate to 25 cryopreservation (Holt, 2000). Additionally, then peetrating cryoprotectants are solvents that normally dissolve sugars and salts in the cryoprvreastieon medium (Purdy, 2006). Non-penetrating cryoprotectants or extracellulayro pcrotectants (egg yolk, non-fat skimmed milk, trehalose, amino acids, dextrans, and suc) ronse the other hand have relatively high molecular weights, and do not cross the plasma mraenmeb and only act extracellularly (Aisen et al., 2000). Therefore, the non-penetrating cryoprtoatnetc may alter the plasma membrane, or act as a solute, lowering the freezing temperera touf the medium and decreasing the extracellular ice formation (Amman, 1999; Kunedtu a l., 2002). Cryoprotectants generally protect frozen sperm s ceblyl one or more of the following mechanisms: Suppressing the high salt concentrsa; tiroenducing cell shrinkage at a given temperature; reducing the fraction of the solutiforonz en at a given temperature and minimizing intracellular ice formation. Combinatiso nof cryoprotectants may also result in the additive or synergistic enhancement of cell visvuarl following cryopreservation (Brockbank, 1992; Brockbank & Smith, 1993). 2.9 Semen cryopreservation techniques Two methods are currently being used for gameteo pcrreyservation. These entail the slow freezing and vitrification techniques. The slowe fzreing method uses a low concentration of cryoprotectants, which may be associated with cchaelm toi xicity and osmotic shock to the semen sample. Vitrification as such is a rapidz firnege method that decreases the occurrences of cold shock, but is usually not performed as hteraantsfer in sperm cells is too slow to permit vitrification without the risks of solutioenf fects or ice crystal formation (Araevt al., 2002). Vitrification also does not require expenes ifvreezing equipment and the method (vitrification/warming) only takes a few seconds a(cIhenko, 2003). Classical vitrification generally requires a high proportion of permeabryleo pcrotectants in the medium (30–50% compared to 5–7% for slow freezing) and seems tdoe btreimental for the sperm cells, due to lethal osmotic effects and possible chemical atlitoenras. The failure to successfully vitrify sperm can be further explained by the extreme tsiveintys i of spermatozoa to high concentrations of cryoprotectants and the low oiscm totlerance of most mammalian sperm (Gao et al., 1995). The cryopreservation technique thus idnecslu temperature reduction, cellular dehydration, eventual freezing and subesnetq uthawing (Medeiroset al., 2002). The lowering from room temperature to 4°C reduces tehlelu lcar metabolic activity and increases 26 the life span of the sperm cells. Cryopreservatgioen erally stops all cellular activities, restarting its normal metabolic functions, aftearw thing (Mazur, 1984). In domestic animal species, fast cooling betwee na n3d0 0°C causes cell damage in certain sperm cells, the so called ‘‘cold shock’’, which disependent on the cooling rate and temperature interval (Gilmoreet al., 1998; Watson, 2000). The cooling or freezinge rmatust be slow enough to allow water to leave the cell so bsmy osis, preventing intracellular ice formation, which causes irreversible damage to stpher m cells (Fiser & Fairfull, 1986). Sperm cells are usually frozen at fast rates (1°5C–/6 0min), which gives rise to best post thawing results (Byrnet al., 2000; Anele t al., 2003). Semen cryopreservation induces the formation orfa cinetllular ice crystals, osmotic and chilling injury that gives rise to sperm damage . ec.ygtoplasmic fracture, effects on the cytoskeleton and genome related structures (Isakcoh, e2n003). The membrane permeability is increased after cooling, and may be a consequefn cinec roeased membrane leakiness, and specific protein channels. Calcium regulation ias ina gaffected by cooling and this has severe consequences on cellular function, including celal tdh. The uptake of calcium during the cooling process then influences capacitation chsa nagned fusion events between plasma membrane and acrosomal membrane. As known, them s mpeermbrane is a structure that undergoes vast reorganization during the capaocnit aptriocess. Cold shock reduces membrane permeability to water and solutes, while damaghineg atcrosomal membrane (Purdy, 2006). The main changes that occur during semen freezrien gm a inly ultrastructural, biochemical and functional. These impair the sperm transpodr t saunrvival in the female reproductive tract and reduce the resultant fertility in domestic aanli mspecies. The ultrastructural damage has been found to be greater in the ram than in buelrl msp. Greater damage has also been detected in the plasma and acrosomal membranes, mitochol nsdhreiaath and axoneme (Salamon & Maxwell, 2000). In frozen-thawed semen, the speromti lmity is generally better preserved than the morphological integrity. The plasma aned othuter acrosome membranes of the sperm cell are the most cryosensitive. Biochemcichaln ges have been detected, including the release of glutamic-oxaloacetic transaminase (G OloTs)s,es of lipoproteins and amino acids, a decrease in phosphatase activity, a decreaseo oisne lyl bound cholesterol protein, an increase in sodium and a decrease in the potascsoiunmte nt, inactivation of hyaluronidase and 27 acrosin enzyme, the loss of prostaglandins, theu crteiodn of ATP and ADP synthesis and a decrease in the acrosomal proteolytic activity a(Smaoln & Maxwell, 1995). The cryopreservation protocol as such causes sl eivnejuraries to the sperm cell by way of several factors e.g. the dramatic changes in teamtuprer, submission to osmotic and toxic stresses derived from exposure to molar conceonntrsa toi f cryoprotectants and finally the formation and dissolution of ice in the intraceallru land extracellular environment. These damaging effects of cooling and freezing on ther msp me embrane differ among domestic species and is influenced by several componentms,e lnya cholesterol/ phospholipids ratio, content of lipids in the bilayer, the degree of rhoycdarbon chain saturation and protein/ phospholipid ratio (Medeiroest al., 2002). Boar sperm is generally the most senes. itBivull, ram and stallion sperm are also very sensitive;l ew hdiog and cat sperm are somewhat sensitive; rabbit, human, and rooster sperm ar ele tahset sensitive to cold shock (Parks, 1997). Cryoprotectants are generally included in cryoprrveasteion medium to reduce physical and chemical stresses derived from cooling, freezindg tahnawing of sperm cells (Gaeot al., 1997; Purdy, 2006). These cryoprotectants, as moneendti previously, are classified as either penetrating or non-penetrating (See 2.8). Glyciesr ofrle quently used as a cryoprotectant for the freezing of ram semen. Glycerol or dimethylf osxuidl e (DMSO) can however induce osmotic stress and toxic effects on the sperm,t hbeu te xtent of the damage varies according to the species and depends on the concentratitohne ocfr yoprotectant in the extender solution (Purdy, 2006). Egg yolk is a normal component omf esne extenders, protecting the sperm cell against cold shock and supporting the cell memb rdaunreing freezing and thawing. The protective mechanisms are determined by the pholisppidhso (lecithin) and the low density lipoproteins (Medeirose t al., 2002; Purdy, 2006). Egg yolk thus acts on thlel mcembrane, having a greater effect on bull than ram sperm .t hFeo rfreezing of ram semen in ampoules, 3 to 6% egg yolk has been used, but for straws anlledt pfreeezing higher concentrations are required (15-17%), although the effect is depen doenn tthe extender composition (Salamon & Maxwell, 2000). It would seem as if the increased concentration se gogf yolk in the semen extender may reduce the glycerol levels. In the formulation oefm sen extenders, glycerol may be added initially or later in a separate fraction (glyceartoeld fraction), after semen refrigeration. In the first situation, the complete extender is addeedr asfet men collection (one step method). In the 28 second situation a fraction of the extender (with golyucerol) is added after semen collection, and the remaining portion (with glycerol) is addaeftde r refrigeration, prior to semen freezing (two step method) (Evans & Maxwell, 1987). Effecet ivcryoprotection after a short (5-10 seconds) contact with glycerol, has been demonesdt rfaotr bull, boar and ram semen (0-5 minutes) – which proves that the penetration ofc egrloyl into the cell is not essential for sperm protection. This still remains a controvel rsuiabject (Barbas & Mascarenhas, 2009). The freeze-thaw process increases the maturatio ns poefrm membranes and induces capacitated acrosome reaction in sperm. These imcaotdioifns may not affect the initial sperm motility, but can reduce the lifespan, the abitlioty i nteract with the female reproductive tract and ultimately affect the sperm fertility (Medei roest al., 2002). Cooled sperm have displayed, an increase in the intracellular free2+ ,C taypical of capacitated sperm following chlortetracycline staining. Cryopreservation of seenm also induces the formation of reactive oxygen species (ROS), which impair good fertiliozna ti(Alvarez & Storey, 1993; O’Flaherty et al., 1997). In ram and buck semen freezing methods, there arney msimilarities, e.g. in the type of extenders, cryoprotectants, and cooling rates zeudtil i (Salamon & Maxwell, 2000). The cryopreservation extenders used for goat and ramme ns egenerally include either egg yolk or non-fat dried skimmed milk. Sanchez-Partiedt aa l. (1992) showed that low concentrations (50 mM) of proline and glycine-betaine improved tphoest thaw motility of ram sperm. In rams and bucks, semen may be diluted using eitnhe r oor two step method. Normally, diluted semen is cooled to 4-5°C during a 1.5-3rhio pde, and thereafter aspirated into mini straws (0.25mL). Freezing may then be performedr olivqeuid nitrogen vapour or in a programmable biofreezer. In the first method, dfi llsetraws are arranged horizontally at a height of 4 to 5cm over the liquid nitrogen vapofourr a variable time (10-20 min), with good post thawing results (Byrneet al., 2000; Leboeuef t al., 2000). Programmable freezers are frequently used at AtIr ecse nwhen freezing large quantities of semen straws. The freezing rates vary accordinthge t ore search laboratories, so for example, the freezer may be at the following freezing ra fterosm: 4 to -5°C at 20°C/min, -5 to -110°C at 55°C/min and -110 to -140°C at 35°C/min, follodw bey the immersion of the straws into liquid nitrogen (Byrnee t al., 2000; Leboeuef t al., 2000). 29 2.10 Thawing of cryopreserved semen Before the thawing of semen straws is attempteed ,l iqthuid nitrogen tank must be filled to avoid an increase in temperat.u Are liquid nitrogen container temperature above -°1C2 0has been shown to lead to irreversible damage to theerm s.p For the thawing of semen, the canisters containing the semen straws must bed r auips eto the neck of the nitrogen tank and then lowered to the bottom. These manipulations mcaauyse temperature fluctuations in the straws remaining in the canister (Neut ra l., 2006) . Thus during the freeze-thawing of semen, the wagrm pihnase is critical for the survival of the sperm as, well as the cooling phase (Feist earl ., 1987). During thawing, frozen semen will cross the critical temperature range between -1d5 - 6a0n°C. The thawing rate is dependent on whether the cooling rate has been sufficiently htiog hinduce intracellular freezing, or low enough to produce cell dehydration. In the firstt aince, fast thawing is required to prevent recrystallization of any intracellular ice preseinn t he sperm cell. Sperm thawed at a fast rate are also exposed for a short period of time toc tohnec entrated solute and cryoprotectant, and the restoration of the intracellular and extraclaerll uequilibrium is more rapid, than with slower thawing (Fiseer t al., 1987). Ram and buck semen is generally thaw e3d8 atot 42°C for 30s, but thawing at higher temperatures (60C-7),5 m° ay produce similar post-thaw sperm motility, acrosome integrity and fertility of thep esrm (Evans & Maxwell, 1987). 30 Chapter 3 Materials and method s 3.1 Study location This study was conducted at the Agricultural Recshe aCrentre (ARC)-Irene campus (25° 55’ S; 28° 12’ E), South Africa (S.A). The centre isc aloted in the Highveld region of South Africa, at an altitude of 1525m above sea level.e Tchlimatic conditions and ambient temperatures range from hot days to cool nightsu imn mer, to moderate winter days with cool nights. 3.2 Experimental animals Eight indigenous rams, between 2 and 4 years o fa angde weighing between 40 and 56kg were used in the trials. Hundred and twenty eig1h2t8 )( ejaculates were collected during the entire study. Semen was collected twice a weekr y(e Mveonday and Tuesday) from Damara, Namaqua Afrikaner, Pedi, and Zulu rams, using tlheec treo-ejaculator. The animals were maintained on natural grazing, supplemented wit0h t3o0 350g concentrate per day, with water being availablaed lib. Plate 3.1 Damara ram used as a semen donor 31 Plate 3.2 Namaqua Afrikaner ram used as a semeno rd on Plate 3.3 Pedi ram used as a semen donor 32 Plate 3.4 Zulu ram used as a semen donor 3.3 Preparation of Diluents Most of the chemicals were supplied by Sigma ex cfoerp tthe ultrapure water which was obtained from TRANSFARM, South Africa. Glycerol w apsurchased from Pal Chemicals. All semen extenders were prepared on the day b esfeomreen collection, and freshly laid eggs (egg yolk) were always used. The egg yolk- citreaxttee nder was derived from two different portions, the one without glycerol (diluent A) atnhde other containing glycerol (diluent B). Different glycerol inclusion levels were used ahned two step dilution procedure was always used. 3.3.1 Preparation of the egg yolk- citrate exte nder The composition of the fractions (A and B) of thaem r semen extenders used is set out in Table 3.1. Following the preparation, the semene nedxetrs were stored at 5°C, until utilized. The procedure for the preparation and compositfio tnh eo sperm washing medium is set out in Table 3.2. This solution was stored and used w i3th0in days, thereafter a new batch was prepared . 33 Table 3.1 Preparation of egg yolk extender (g/10)0 mL Fraction A Na.Citrate. 2H20: 1.856g Glucose: 1.0g Water: 80mL CPA: 0mL Egg yolk: 20mL pH: 7.0 Gentamycin Sulphate (optional): 0.1g Fraction B Na.Citrate. 2H20: 1.856g Glucose: 1.0g Water: 66mL CPA%: 14mL Egg yolk: 20mL pH: 7.0 Gentamycin Sulphate (optional): 0.1g 3.3.2 Protocol for preparing the sperm washingt isoonl u(BO-W) (Brackett & Oliphant, 1975) Table 3.2 Preparation of 10xBO stock solution fAe c(etifve for 30 day s) Component Molecular Wt. mM g/100mL NaCl 58.44 112.00 6.5453 KCl 74.56 4.02 0.2997 NaH2PO4.H2O 137.99 0.83 0.1145 MgCl2.6H2O 203.30 0.52 0.1057 CaCl2.2H2O* 147.02 2.25 0.3308 Glucose 181.16 13.90 2.5181 Antibiotics (penicillin) or 5mL or 10mL Streptomycin (0.02g/ mL) 50mg/mL 0.68 or 2.5mL 0.2% Phenol Red 4mL Addition of ultrapure water (Sabax) to a volume1 o0f0 mL 34 The BO-W solution was filtere udsing a craft suction unit (Rocket) and a 250mtLe rf ilsystem and prepared by firstly dissolving the componenetsp asrately in approximately 5mL of ultrapure water (Sabax). Thereafter water was a dtod ead volume of approximately 80mL, after which the pH was checked and adjusted to T7h.4is. sperm washing solution was then stored at 4°C, until utilize d. Table 3.3 Preparation of 1xBO working solution fBfe c(etive for 2 weeks) Component Molecular Wt. mM g/100mL NaHCO3 84.01 37.0 0.3108 Na-Pyruvate 110.04 1.25 0.0138 Take 10mL of BO stock (10mL) (solution A) Solution B was prepared as set out in Table 3.3te. rA afdding all the ingredients listed in Table 3.3, ultrapure water (Sabax) was added tol uam ve of 100mL. The pH was check ed and adjusted to 7.4 after adding caffeine and ftihltenre d. 3.3.3 Preparation of the sperm washing solution-W (B) O 80 mL of BO working solution B was taken to prep tahre sperm washing solution, to which 3.0mg/mL BSA (Fraction V, A-9418) was added and stohleution filtered. The remaining 20mL was used for preparing IVF maturation medium. 3.4 Semen collection and quality evaluation 3.4.1 Semen collection The semen of all individual rams was collected wtihthe aid of an electro-ejaculator (Ramsem, South Africa). Two ejaculates were coellde cpter week/ram at an interval of two days. The semen was collected directly into 15mbLe stu, and immediately placed in a thermo flask at 37°C. The collected ram semen was then sptroarted to the laboratory for microscopic sperm evaluation within an hour. Thwe sraemen samples were firstly evaluated macroscopically for the ejaculate volume, pH ande rmsp concentration .Spermatozoa parameters (motility and velocity) were microscoaplliyc evaluated using the computerized Sperm Class Analys®e r(CASA system). 35 Plate 3.5 Electro-ejaculator used for semen coiollne ct Plate 3.6 Thermo flask used for temporary semerna gseto after collection 3.4.2 Semen evaluation 3.4.2.1 Semen concentration Semen concentration (sperm/mL) was determined wthiteh aid of a spectrophotometer calibrated for ram semen (SpermaCue®, Minitüb, Gaenrym). Briefly, 20µl of undiluted raw 36 semen sample was pipetted into a microcuvette (HCeume oAB, Ängelholm, Sweden). This sample was then inserted into the spectrophotom teot ergive an automated sperm concentration reading in terms of the number orf msp/meL (X106). Plate 3.7 SpermaCue® used for the determinatiosnp oefr m concentration Plate 3.8 Semen pH meter used in this study 37 3.4.2.2 Semen pH The semen pH was measured using a microprocess/omr Vp/°HC meter fitted with a glass probe (Hanna HI 931401, Portugal). The probe wnasse rdi in ultrapure water and wiped dry with a paper towel before and after each semen lsea mepasurement. 3.4.2.3 Sperm motility evaluation using the CASsAte smy The Computer Assisted Sperm Analysis (CASA) systweams used to analyse the sperm motility with the aid of a Sperm Class Analy®z-eSrCA® (V.4.0.0.1 Animal/Veterinary Microptic S.L, Barcelona, Spain). The sperm swim t-eucphnique was used, where 10µl of the semen sample was diluted with 500µl of the Bracaknedt Oliphant (BO) medium, developed by Bracket and Oliphant (1975) and stored for 5 umteins in an MCO-20 AIC Sanyo C2 O incubator (Sanyo Electric Biomedical Co., Ltd, Jna)p, a t 37°C. Following the storage period, 5µl of this semen solution was pipetted onto a wparerm- ed bevel-edged, frosted-end microscope glass slide (Thermo Scientific Menzeäl-sGelr, Germany), gently covered with a microscope cover slip (Menzel-Gläser, Germany) eavnadl uated under X10 magnification with the SCA® microscope projecting an image on a monitor. Tehseu lrts were saved on a Microsoft excel sheet. The motility parameters ueavateld were expressed as the percentage progressively motile sperm (sperm with forward mmoveent), percentage non-progressively motile sperm and percentage static (immotile) sp).e Srmperm velocity parameters evaluated included the static, slow, medium, rapid, curvialinr e(VCL), straight-line (VSL), average path (VAP), linearity (LIN), straightness (STR) and woleb b(WOB) velocities. Table 3.4 The definitions of sperm motility destocrsip when using the CASA system Descriptors Abbreviation Unit Description Curvilinear velocity VCL µm/s Velocity of progression along the entire trajeyc tor Average path VAP µm/s Velocity of progression along the smoothed velocity trajectory Straight line velocity V S L µm/s Velocity of progression from first to last coordinates Beat cross frequencyB C F Hz Frequency that the sperm head crosses the smoothed trajectory Amplitude of lateral A LH µm Mean lateral sperm head displacement along the smoothed head displacement trajectory Linearity of track LIN % VSL/VCL×100 Straightness of trac k STR % VSL/VAP×100 Wobble WOB % VAP/VCL×100 Source: Holte t al. (2007) 38 Table 3.5 Sperm Class Analyser® V.4.0.0 settingesd utos analyse the ram sperm cell motility and velocity characteristics Parameter Setting Brightness 166 Chamber Cover slide Circular 50% of Linearity Connectivity 12 Contrast 450 Optics Ph- Number of images 50 Images per second 50 2 Particle area 15 - 70µm Progressivity 80% of STR Scale 10X Slow VAP of 0 - 30 µm/s Medium VAP of 30 - 80 µm/s Rapid VAP of 80 µm/s and above Velocity on the average path points 7 Plate 3.9 Incubator used for semen incubation ptori osrperm motility evaluation 39 Plate 3.10 Sperm Class Analyzer® used for spermil imtyo etvaluation 3.4.2.4 Sperm morphology and viability Sperm viability was determined with the aid of ano sien/nigrosin stain (pH=8.39; osmolarity=411), manufactured by the OnderstepoFoarct ulty of Veterinary Sciences’ pharmacy (60µl of eosin/nigrosin stain plus 6µsl eomf en). This staining method indicates the percentage live or dead sperm cells, while allow ain good evaluation of the morphology of the sperm cell (Bjoerndahetl al., 2004) . The sperm smears were prepa roend a clean, warmed glass slide to avoid temper asthuoreck and evaluated on the same day of semen collewctiitohn t he aid of a fluorescent microscope (Olympus BX 51TF) – using an oil immersion objeect i(vX100 magnification). A total of 100 sperm/slide were evaluated and counted for eacmha al npier collection, using a DBC.6 Model laboratory counter (Han Lien International Corpd) athne gross structural sperm abnormalities recorded (Hidalgoe t al., 2007). Under the microscope the live sperm fluoresced ew ahsit no stain was absorbed by the cell, while the dead sperm on the other hand fluoresceedd, ars the cells absorbed the stain (Beardene t al., 2004). The live spermatozoa were further catezgeodr iinto morphologically normal or abnormal cells. Abnormalities were receodr dusing two different sets of criteria. The first set of criteria used the location of tahben ormality i.e. head (e.g. bulb, small, 40 enlarged, looped, etc.), mid-piece and tail (ew.ge. llsing, looping, partial or totally lacking, etc.) as described by Łukaszeweict za l. (2008). Sperm abnormalities were further classified ase er itbheing primary or secondary, according to the degree of the lesion. These abnormalitierse wtheus set out, using the following criteria (Loskutoff & Crichton, 2001): The primary sperm abnormalities included the foilnlogw: Sperm head: microcephalic (small heads), macrocliecp (hlaarge/swollen heads), double heads and abnormal acromosomes. Mid-piece of the sperlml: scweollen, elongated and abaxial bodies. Tail of the sperm: double and short ta ils. The secondary sperm abnormalities included theo wfoinllg: Sperm head: detached, loose and damaged acrosoimd-ep.i eMce of the sperm cell: bent and containing protoplasmic droplets. Tail of the sp ecremll: bent, shoe-hook and protoplasmic droplets on the tail. Plate 3.11 Fluorescent microscope (BX 51TF) usre sdp feorm morphology and viability 41 Plate 3.12 Eosin/nigrositna sined ram sperm cells. iAs al ive and normal ram sperm; B is a dead sperm with a mid-piece abnormality and C is a de, andormal sperm. 3.5 Liquid storage of rame ms en Two experiments were conducted where a comparisaosn mwade betweesne men stored at two different temperatures (5 and 15°C) for var iosutosrage periods. In the first experiment only fraction A (without glycerol) of the egg yo-clkitrate diluent was used and in the second experiment both fractions A and B were used. Foer e txhperiment where only fraction A of the egg yolkc-itrate extender was used, semen samples were dd i1lu:1t . For the experiment where egg yolkc-itrate diluent containing 14%gl ycerol was used, the samples were first diluted with 1:1, v/vo f fraction A, and later diluted 2:1 v/v with fraction (Bw ith glycerol) – resulting in a final glycerol inclusion level of7 4%. . After semen evaluations, the ejaculates were divided into two aliquotsT.h e one aliquot beinkge pt at 5°C and the other at 15°C a i n Defy VT60 coole.r Sperm motility and velocity evaluations weprer formedf ollowing 3, 6, 9 and 24h of storage. 42 Plate 3.13 Walk-in refrigerator used during semteonra sge and processing Plate 3.14 Defy VT60 cooler used during liquid sne smtoerage 3.6 Semen cryopreservation After macroscopic semen evaluation in the laboyra, tsoermen samples were diluted 1:1 with the egg yolk citrate (EYC) fraction A (without gleyrcol, at a ratio of 1:1, v/v), at 37°C and maintained in a cold room (Recam international, thS oAufrica) at 5°C for 2h. Two hours 43 after the addition of the first extender (FractAio)n, fraction B (with glycerol) of the egg yolk citrate extender was added at a ratio of 2:1, (avn/vd) the semen samples were further cooled for another 2h. The cooled semen samples werel othaedne d into 0.25mL straws. The semen straws were put into the straw holder and froze na ipnrogrammable freezer (CBS freezer 2100 series, Custom Biogenic Systems, SA), usicnugs ato mized freezing curve as set out in Table 3.4. The straws were then plunged into ai dli qnuitrogen tank (-196ºC) for later sperm analysis (Hammadeeht al., 2001). Table 3.6 The freezing rates used to cool indigse noraum semen during cryopreservation From(°C) To(°C) Rate of cooling°C( /min) 5 -5 4 -5 -110 25 -110 -140 35 Ram semen was pooled because of the low ejacuolalutem ves obtained from the indigenous rams, to test the three different glycerol concaetniotnr s. The pooled semen sample was diluted 1:1 v/v with an egg yolk citrate fraction (Awithout glycerol) and was then divided into 4 aliquots. The semen samples were then minaeindt a in a cold room (Recam international, South Africa) at 5°C for 2h. Two hrso uafter the addition of the first extender, one group from the four aliquots was diluted witYhC E fraction A, which served as a control. The other three aliquots were diluted with EYC tfiroanc B containing 7, 10 and 14% GLY in a ratio of 2:1(v/v) resulting in the final glyce roinl clusion levels of 2.3, 3.3 or 4.7% respectively. The samples were further cooled nfort haer 2h, then loaded into 0.25mL semen straws and frozen, by placing the straws 5cm abthoev eli quid nitrogen (LN2) for 10 minutes, before being plunged into L2N and stored in LN2 storage tank (-196°C) for future evaluation. 44 Plate 3.15 The programmable freezer used for sefmreeenz ing Plate 3.16 Freezing of semen in liquid nitrogeno uvra p 45 Plate 3.17 Liquid nitrogen tanks used for semerna gseto 3.7 Thawing of semen for the post-thaw semen aensa lys Semen straws were thawed 7 days after cryopresioenrv bayt placing them in a water bath [Julabo P (Julabortechnik GMBH) West Germany] a°tC 3,7 for 30 seconds (Purdy, 2006). Both ends of the sealed straws were cut with a opfa sircissors and the semen poured into 15mL test tubes. 10µL of semen was diluted withµ 5L0 0BO sperm washing solution and stored at 37°C for 5 minutes in an MCO-20 AIC Sa n5y%oCO2 incubator (Sanyo Electric Biomedical Co., Ltd, Japan). The semen samples wtherne evaluated for sperm motility and velocity parameters using the same method as wfo rs eramen. 46 Plate 3.18 Water bath used during thawing of thmee sne straws 3.8 Statistical analyses Data were analysed using the statistical programnS Gtaet®. Analysis of variance (ANOVA) was used to test for significant differences in esne mvolume, semen concentration, semen pH, sperm morphology, sperm motility and in the treanttms e(temperature, storage periods, glycerol, different glycerol levels and freezing tmhoeds). Treatment means were separated using the Fishers protected t-test least signicfieca dnifference (LSD), at the 5% level of significance (Snedecor & Cochran, 1980). 47 Chapter 4 Characterization of South African indigenous rame sne 4.1 Introduction Several adapted indigenous sheep breeds with sour pgeerni etic traits are available in South Africa. These include the Damara, Namaqua, Ped iZ aunlud sheep breeds, each with unique traits that make them adapted to South African ictioonnds. Therefore, for preservation of these valuable indigenous genetic resources, eits sise ntial to maintain the animals with certain unique production qualities, using the nteiqcuhes of semen cryopreservation and AI (Ehling, 2006). Most of the South African indigenso suheep breeds are currently under threat of extinction mainly due to crossbreeding pract,i caensd thus their genetic properties need to be preserved – in order to be used in genetic s ihmeperpovement programmes of the specie and genetic resource banking (Holt, 1997). Cryoeprrveastion is then one of the technologies which can be utilized to preserve these genetioc urrecses e.g. the oocytes, sperm, somatic cells and embryos (Bester, 2006). Generally, the technique of semen cryopreservaintivoonl ves temperature reduction, cellular dehydration, freezing and thawing processes (Moesd eeitr al., 2002). The lowering of the environmental temperature, from room temperatur0e° C(2) to 4°C generally reduces the cellular metabolic activity, and thus increases tlhife span of the sperm cells. Cryopreservation as such, stops the cellular atyc,t irvei starting its normal metabolic functions after thawing (Mazur, 1984). Sperm cryopreservation usually induces the formna otifo intracellular ice crystals, osmotic and chilling injury that may cause sperm cell daem, acgytoplasmic fracture, and even has effects on the cytoskeleton or the genome relatreudc tsures (Isachenko, 2003). The main changes that occur during the freezing of gamerte s maostly related to ultra-structural, biochemical and functional activities, which mayt imulately impair sperm transport and decrease the survival rate in the female reprovdeu cttriact, post thawing – thereby reducing fertilization rate. Ultra-structural sperm damagse g ienerally greater in the ram than in the bull and seems to be species related (Salamon &w Melal,x 2000). 48 Sperm preservation protocols differ between anismpaelc ies, due to their inherent abilities to accommodate variations in semen extenders usedh ei nc otoling and freezing processes (Barbas & Mascarenha s2,009). These differences between species regarding thseit isveityn of their sperm to cooling are then largely attreibdu tto the compositional variation in the sperm plasma membranes (Bailety a l., 2000). Differences in fatty acid composition and sterol levels of the cell membrane have also besesno caiated with the tolerance of sperm to cold shock and cryopreservation. Thus, the obse rvvaerdiation among species in sperm survival rate, after freezing and thawing, has b aetetrnibuted to these differences. A higher ratio of unsaturated/saturated membrane fatty ,a acindds lower levels of cholesterol in the bull and ram sperm cell membranes, compared to the h uamnda ndog, have been suggested to be a reason for the differences encountered in colodc ks hand cryopreservation tolerance recorded between these species (White, 1993). Thmearye then also be considerable differences between breeds and between individuaalel sm, regarding the ‘freezability’ of their semen (Hiemstraet al., 2005). A thorough knowledge of the sperm physiology fosrp ae cific species or even breed is thus essential to maximize post-thaw sperm survival asnudb sequent fertility (Purdy, 2006). Protocols for different species, including the rhaamv e been developed and tested over time, on various exotic breeds. There is however a noe esdt utdy and characterize the quality of indigenous (in this case, South African) ram sem aesn ,it will ultimately determine the fertility rate achieved. It is deemed necessaryc rtyoo preserve indigenous ram semen and to develop extenders that may optimise the sperm ucryvoivsal and guarantee their survival. Semen quality and its relationship with male feityrt ilare of utmost importance in animal production. Moreover, standard semen analyseso aurtein er ly implemented to determine the acceptability of processed semen for breeding psuersp.o In this study, the Computer Assisted Sperm Analysis (CASA) system has been used to atceclyu r measure the motility characteristics of the indigenous ram sperm ceTllhse. aim of this study was thus to characterise indigenous South African ram semenr omscaocpically (volume, pH and sperm concentration) and microscopically (sperm motirliatyte s). 4.2 Materials and Methods Semen was collected from rams of different Southri cAafn indigenous breeds (Damara, Namaqua Afrikaner, Pedi and Zulu), during May (eonf da utumn – i.e. during the natural breeding season), 2009. Two rams per breed werilea balvea and used due the scarcity of these 49 breeds of rams. The ages of the rams ranged be t2w eaennd 4 years and all animals were maintained on natural pastures, supplemented w ictho mamercial pelleted diet. Water was available ad libitum. A total of four ejaculates were collected fromc he aram. Semen was collected twice a week, at an interval of two dauyssin g the electro-ejaculator. Samples were collected in graduated test tubes and placed ihne arm to flask at a temperature of 37°C. Semen was then transported to the laboratory faolru eavtion within a period of 1h. The raw or fresh semen was macroscopically and microscopi ceavllayluated for sperm concentration, sperm cell motility rate, and semen pH. The speormnc ecntration was determined with the aid of a spectrophotometer (Spermacue®) and the semHe uns ipng a pH meter (Microprocessor pH/mV/°C Meter Hanna HI 931401). A Computer Assdis Steperm Analysis (CASA) system was used to evaluate the sperm cell motility ra tes. The sperm viability (percentage live/dead) was rdmeitneed using an eosin/nigrosin stain (60µl eosin/nigrosin and 6µl semen in a thin sm. eTahri)s staining method indicates the live or dead status of the sperm cells and also allo wgso oad evaluation of the sperm cell morphology (normal or abnormal) (Bjoerndaehtl al., 2003). The semen smears were prepared on a clean, warmed microscope slide,o tiod atevmperature shock to the sperm cells, and evaluated on the same day of collection whieth atid of a fluorescent microscope (BX 51TF), using an oil immersion objective (X100 mafigcnaition). A total of 100 sperm cells per slide were evaluated and recorded for each rame jpaecru late, with the aid of a cell counter, with the gross structural normal/abnormalities bge riencorded (Hidalgoet al., 2007). The live sperm cells fluoresced green, as these sperm d oa bnsotrb the stain, while the dead cells coloured red, as they absorbed the stain (Beaertd eanl. , 2004). The live sperm cells were further categorized as morphologically normal orn oarbmal. Abnormalities were recorded according to the location of the abnormality e.ega. dh (i.e. bulb, small, enlarged, looped, etc.), mid-piece and tail (i.e. swelling, looping, par tioarl totally lacking, etc.), as described by Lukaszewicze t al. (2008). All data were analysed using the statistical Getn®S tparogram. The analysis of variance (ANOVA) was used to test for significant differensc ebetween the treatments. Treatment means were compared using the Fishers protecteesdt tf-otr the least significant differences (LSD), at the 5% level of significance (SnedecoCr o&c hran, 1980). 50 4.3 Results and Discussion In Table 4.1, the mean live-weight, scrotal circeurmefnce, semen volume, pH and sperm concentration of the indigenous rams are set ohuet. wTeight and scrotal circumference of the rams ranged between 41.3 and 57.4kg and 28 andc m3 1r.e3spectively. There were no significant (P<0.05) differences between the ramiths wregards to scrotal circumferences. The volume of the indigenous ram ejaculates ranged f0ro.4m to 0.9mL. The volume recorded in this study was generally lower to that reportedG bily e t al. (2003), who reported an ejaculate volume of 0.75 to 2mL as being normal for rams, nw huesing the artificial vagina (AV) to collect semen from rams. Hafez and Hafez (2000o) raelsported the semen ejaculate volume in rams to range from 0.5 to 2mL in mature ramsd, faronm 0.5 to 0.7mL in young yearling rams. Furthermore, the sperm concentration reco irnd ethde current study ranged from 0.9 to 1.3x109sperm/mL, which was also lower, when compared thoe ro tstudies. According to Evans and Maxwell (1987) the sperm concentratio na no fadult ram ejaculate was found to vary from 3.5 to 6.0x190 sperm/mL. This was also supported by Hafez ande zH (a2f000). Gil et al. (2003) however considered a sperm concentratfi o2n.5 ox109 sperm/mL for rams to be normal and acceptable. The mean sperm cell conactieonnt rrecorded in the current study was however higher to the reported results by Foeutr iael . (2004), on intensively managed Dorper rams (731.6x106 sperm/mL), also using the electro-ejaculator aes mthethod of semen collection. This difference could thus generally abtetributed to the method of semen collection, breed of the rams or age of the induivailds. The semen pH recorded in this study ranged from 6.5 to 7.3. Greyling and Grobbelaar8 3(1) 9recorded a similar semen pH for Boer goats (ranging from 6.40 to 7.02). Semen p cHo nissidered to be normal when it ranges between 7.2 and 7.8 (Prins, 1999). It is generaacllcye pted that ejaculates obtained following the use of the electro-ejaculator, tend to induecme esn with a higher pH, due to excessive stimulation of the accessory sex glands with tahlekiar line secretions (Greyling & Grobbelaar, 1983). The reason for the significantly (P< 0.0o5w) el r semen pH in the Pedi rams is unclear. This could possibly be attributed to less acces gsolarnyd fluid being produced. In Table 4.2 Pearson correlations between the beoidgyhwt, scrotal circumference, semen volume, sperm concentration, semen pH and totarlm s pmeotility are set out. Body weight was positively correlated with total sperm moti l(irty = 0.228). However negative correlations were found to exist between body weight and sc rcoitraclumference (r = -0.003), semen volume (r = - 0.773), sperm concentration (r = 7- 30.), semen pH (r = - 0.783). Semen volume was positively correlated with sperm conrcaetniotn (r = 0.997) and pH (r = 0.566). 51 The scrotal circumference was positively correl awteitdh semen volume (r = 0.197), total sperm motility (r = 0.537) and sperm concentrat(iro n= 0.172). Sarder (2005) recorded the increases in semen volume and total sperm/ejac utola btee associated with an increase in scrotal circumference. Langford (1987), Devkoeta a l. (2008), Hassane t al. (2009) and Okere et al. (2011) also found scrotal circumference to bei tipvoesly correlated with semen volume and sperm concentration. Scrotal circumfceer ewnas found to be positively correlated with sperm motility, and these traits were closceolyrr elated with the fertility in bulls (Okere et al., 2011. The sperm concentration was positivelyr eclaotred with semen pH (r = 0.556). Negative correlations were also recorded in thuisd ys,t between total sperm motility and semen volume (r = - 0.562), sperm concentratio=n - ( 0r .613), semen pH (r = - 0.613). Table 4.1 Mean (±SD) semen volume, pH and spermc ecnotnration of different South African indigenous ram breeds Breed Body Scrotal Ejaculate Sperm Semen pH weight(kg) circumference(cm) volume concentration (mL) (109/ mL) Damara 41.3±0.8b 30.8±0.4a 0.4±0.1b 1.3±48.5a 7.3±0.3a Namaqua Afrikaner 47.4±0.5ab 29.8±0.9a 0.9±0.2a 1.2±30.5ab 7.3±0.3a Pedi 57.4±0.4a 31.3±0.8a 0.5±0.1b 0.9±84.2b 6.5±0.4b Zulu 51.6±0.2ab 28.0±0.7a 0.5±0.3b 0.9±177.2b 7.3±0.4a a,b Values with different superscripts within a columdinff er significantly (P< 0.05) In Table 4.3 the sperm morphology of the indigen oraums semen determined using the eosin/nigrosin stain, is set out. Sperm morpholoisg yg enerally considered to be a good predictor of successful fertilizing capacity (Lukzaeswicz, 1988). The proportion of live sperm in the current study ranged between 32 and 64.3h%e. NTamaqua Afrikaner ram (32%) demonstrated a significantly lower proportion ovfe l isperm cells, compared to the Damara (58.8%), Pedi (59.3%) or Zulu (64.3%) rams. Thes orena for the lower % live sperm recorded in the Namaqua rams is not clear. It is speculathteadt the percentage live sperm in the raw ejaculates should have been high – due to the s ebmeienng collected in the breeding season. The Namaqua Afrikaner rams then resulted in theh ehsigt occurrence of abnormal sperm. These morphological abnormalities of the sperms cheallve been generally associated with a decrease in fertility rate of the rams (Mitheat t al., 2001). In the present trial the sperm abnormalities ranged between 5.2% and 8.2%, whriec hr eagarded as acceptable values for a high fertility rate. The proportion of abnormal srmpe cells recorded in this study were generally lower than those cited by Peerte azl . (1997) (9.4%) and Taheat al. (2000) (14.2%). This may indicate the semen collection and stai npinrogcedures used in this trial, to be acceptable. 52 Table 4.2 Pearson correlations between bodywesicgrhott,a l circumference, semen volume, sperm conatcieont,r semen pH and total sperm motility in South African indigesn roaums Variables Body weigh t Scrotal circumferenc e Semen volum e Sperm concentratio n Semen pH Body weigh t — Scrotal circumferenc e -0.003 — Semen volum e -0.773 0.197 — Sperm concentratio n -0.730 0.172 0.997 — Semen pH -0.783 -0.606 0.566 0.556 — Total sperm motility 0.228 0.537 -0.562 -0.613 -0.613 53 Table 4.3 Sperm morphology evaluation of raw sefmroemn South African Indigenous rams of different breeds Breed %_Sperm cell s %_Abnormal sperm Live Dead Head Midpiece Tail Damara 58.8±4.8a 36.0 ± 6.0b 1.5 ± 0.7b 1.5 ± 1.3a 2.2 ± 0.9a Namaqua Afrikaner 32.0±9.9b 59.0 ± 10.6a 4.5 ± 0.8a 2.5 ± 0.6a 2.2 ± 0.9a Pedi 59.3±4.5a 35.2 ± 5.3b 1.2 ± 0.5b 1.5 ± 1.0a 2.7 ± 0.9a Zulu 64.3±6.8a 29.5 ± 7.8b 1.2 ± 0.5b 2.0 ± 0.8a 3.0 ± 0.8a a,b values with different superscripts within a columdifnfe r significantly (P< 0.05 ) Table 4.4 Mean (±SD) sperm motility and velocittye sra of South African indigenous ram breeds, as recorded by CASA Characteristics Breed Damara Namaqua Afrikaner Pedi Zulu Total motility (%) 69.6±16.a5 37.1±19.9a 74.9±11.0a 56.0±22.6a Progressive motility 36.4±15.1b 17.4±14.7c 52.7±13.3a 32.6±15.4b (%) Non-progressive 32.2±13.7a 19.7±12.9a 22.2±19.3a 23.4±17.5a motility (%) Rapid (%) 59.75±13.a6 23.4±17.8b 60.1±19.5a 40.4±19.1ab Medium (%) 4.9±33.a0 5.0±2.0a 3.9±4.4a 7.0±3.0a Static (%) 30.3±16.a4 62.9±19.9a 25.1±11.0a 44.0±22.6a Slow (%) 5.1±3.1a 8.8±4.8a 11.0±18.6a 8.6±11.0a VCL(µm/s) 213.3±39.a3 143.4±20.3b 201.7±63.3a 193.0±47.0a VSL(µm/s) 128.5±39.a7 94.0±24.8a 143.7±58.1a 123.4±52.6a VAP(µm/s) 177.8±30.a3 117.8±21.3a 164.5±62.1a 148.0±49.5a LIN (%) 59.2±9.8a 65.1±11.0a 69.0±10.1a 61.8±12.2a STR (%) 71.5±16.a0 79.4±12.1a 86.5±6.7a 81.5±8.1a WOB (%) 83.8±6.6a 81.8±3.4a 79.7±10.2a 75.3±7.5a VCL = curvilinear velocity, VSL = straight-line voecl ity, VAP = average path velocity, LIN = linear,i tSy TR = straightness, WOB = wobble, ALH = amplitude otfe lra l head displacement, BCF = beat cross frequ e n c y a,b Values with different superscripts within a rowf edrif significantly (P< 0.05) In Table 4.4 the sperm motility and sperm velocraittye s of the indigenous rams, as measured by the CASA system, are set out. In this analysisp trhoep ortion of total motile sperm ranged from 37.1 to 74.9%. Ram as such had no effect on thael mtoot tile sperm, although the Namaqua 54 tended to record lower sperm motility rates thaen Dthamara or Pedi. The Pedi (52.7%) recorded a significantly (P<0.05) higher proportion of preosgsr ive motile sperm cells, compared to the Damara (36.4%), Namaqua Afrikaner (17.4%) and thuelu Z(32.6%) rams. The total sperm motility and progressive sperm motility in the Naqmua (Table 4.4) was significantly (P<0.05) lower, than the other breeds, when using the CASysAte sm for sperm evaluation. This lower motility could possibly be related to the lower cpeenrtage of live sperm as set out in Table 4.3. Furthermore the breeds did not differ in the petracgeen of non-progressive motile, static, medium motile or slow motile sperm. Breed also recorde de fnfeoct on the straight line sperm velocity, average path velocity (VAP), linearity (LIN), stgrahitness (STR) and proportion of wobbling (WOB) sperm cells, as evaluated by the CASA sys tem. 4.4 Conclusions In this study, the volume of the indigenous ram cueljates ranged from 0.4 to 0.9mL. Furthermore, the sperm concentration recorded rda nfrgoem 0.9 to 1.3x190sperm/mL, which is lower when compared to other studies. The semerne pcoHr ded ranged from 6.5 to 7.3. The Pedi ram semen recorded the highest total motile (74 .a9n%d) most progressive (52.7%) sperm cells, compared to the other breeds. The Namaqua ramlste rde sinu lowest total motile (37.1%) and progressive (17.4%) sperm cells, compared to otbhrere ds, as measured by CASA. The relatively small standard deviation in the semenlu mvoe is indicative of a satisfactory and repeatable semen collection technique being uslethdo, uagh EE is not the most acceptable technique used. Body weight was positively correedla wt ith total sperm motility. However negative correlations were found between body wt eiagnhd scrotal circumference, semen volume, sperm concentration, semen pH. Semen vo lwuamse positively correlated with sperm concentration and semen pH. Scrotal circumferencaes wpositively correlated with semen volume, total sperm motility and sperm concentrna taiond these traits are closely correlated with fertility especially in bulls. The sperm concenitorant was positively correlated with semen pH. Negative correlations were also recorded in thuisd ys,t between total sperm motility and semen volume, sperm concentration, semen pH. This study thus gave an overall knowledge regar dthineg characterization of the indigenous ram semen and semen quality in the breeds. The ushee o Cf At SA system offered a more reliable, 55 unbiased and repeatable means of microscopicaslleys asisng sperm motility traits, compared to traditional visual assessment. The low percentafg es poerm abnormalities demonstrated the technique used to collect and evaluate sperm abanliotirems to be acceptable for obtaining normal, viable sperm . 56 Chapter 5 Effect of storage temperature on the viabilityi louft edd ram semen stored for different periods of time 5.1 Introduction The three South African indigenous sheep breedsm (aDr a, Namaqua Afrikaner and Zulu) from which semen was collected, are adapted to the haarrids hSouth African conditions. They are generally more tolerant to ticks and resistant etrot acin diseases, while they are also said to be highly fertile and the dams are said to have ane lelexnct mothering ability, being very protective and able to defend their young against smaller aptorersd (Ramsaye t al., 2001). The characterization of these indigenous breeds hawvee vheor been neglected in the past, especially regarding their production potential although theayv e been crossbred with other exotic breeds – with no records regarding their production perfonrmceas. The need has however long been recognized to preserve the genetics of these b rfeoer dfusture use. Semen storage (long or short term) is a method of preserving the genetic poatel notfi these indigenous breeds. As the cryopreservation and thawing process of sne imndeuce serious damage to the sperm cells especially in rams, this may result in impairinge thsubsequent fertility rate following AI (Maxwell & Watson, 1996; Soderquiestt al., 1997). The use of raw, diluted and cooled semen shortly following semen collection may be an alatetirvne method to the cryopreservation of semen for use in AI programs. Compared to raw h(f)r essemen, cooled ram semen however exhibits a decrease in sperm motility and sperm pmh olrogical integrity over time – accompanied with a decline in the survival ratet hoef sperm in the female reproductive tract, with a reduction in fertilizing ability and increeads embryonic losses. The injury to the sperm cells are usually less pronounced in diluted anidlle cdh semen, compared to frozen/thawed ram semen (Maxwell & Salamon, 199 3I)r.respective of the semen diluent used, the dinlu triaote, temperature, or conditions of storage, the quaolfit yth e sperm deteriorates as the duration of storage increases (O’Haerat al., 2010). It is however important to know when dtheete rioration in sperm quality occurs, and therefore there ise ead n to study the effect of semen storage on sperm survival rate for different time intervalsh.e T temperature at which semen is stored plays a 57 critical role in acceptable sperm motility ratecso rreded. Temporary storage of ram semen at 5°C or 15°C would be better in terms of fertilizatioant er, following the transportation of semen, while also being more affordable to small scalem fearrs. The main method of long term semen storage (cryopreservation) is generally in liquiidtr ongen at a temperature of -196°C. This temperature then lowers the metabolic rate of tpheer ms, and contributes to enhanced sperm survival (Vishwanath & Shannon, 2000). Vishwananthd aShannon (2000) also reported the storage of semen at room temperature to be su pteor iothre storage at a temperature of 5°C, provided the medium with which sperm is dilutedh,i binits those pathways that are detrimental to their survival at higher temperatures. So for exlaem Gprasae t al. (2004) successfully used 15°C as the storage temperature for liquid ram semenfo, reb eartificial insemination being implemented . It is critical to inseminate at an optimal time idnugr the oestrous period, relative to ovulation, to achieve an acceptable fertility rate. It is thesno a vlital to preserve and store the semen used for AI under optimal environmental conditions. The rmecmoended maximum storage period for raw ram semen has been set as short as 6 to 12h. Dinegp eonnd the time required for the transportation of semen from the AI stations to ftahrems, a short-term storage time will make it possible to inseminate the ewes at an optimal dtiumrein g oestrus (Paulenezt al., 2002). The processing and storage has been shown to per odmeostabilisation of the cell membrane, hampering capacitation and acrosome integrity oef sthperm (Watson, 1981; Guillaent al., 1997). Semen extenders are thus generally addseedm toe n to supply the sperm with a source of energy, protect the cells from temperature relaintjeudry , and maintain a suitable environment for the sperm to survive temporarily (Purdy, 2006). The aim of this study was to determine an acceep tastbolrage temperature (5°C vs. 15°C) and different storage times for raw ram semen, prio Ar It.o 5.2 Materials and Methods Semen was collected during the winter (June, 20 f0ro9m), 6 healthy mature rams of different South African indigenous breeds i.e. the Damaram, aNqaua Afrikaner and the Zulu breed (2 58 rams per breed being used). The ages of the ranmgse dra between 2 and 4 years and all animals were maintained on natural pastures, supplemenittehd a w commercial pelleted diet. Water was availablea d libitum. Semen was collected using an electro-ejacularotomr fall the rams, twice a week for a period of 2 weeks, with 4 ejaculatesn gb eciollected in total from each ram. Semen was collected directly into graduated test tubehsic, hw were then placed into a thermo flask, with the water being maintained at a temperature of .3 A7°llC collected semen was then transported to the laboratory for macroscopic and microscopic ueavtaiol n within 1h of collection. The fresh undiluted semen was evaluated for sperm conceonntr,a stiemen pH, and sperm motility (refer to Chapter 3 for more details). The sperm concentnr atwioas determined with the aid of a spectrophotometer (Spermacue®), the semen pH wheit ha itd of a pH meter and the Computer Assisted Sperm Analysis (CASA) system used for mureinags the sperm motility attributes. After the initial evaluation, all semen samples were epdo oalnd diluted equally in an egg yolk citrate extender in the ratio of 1:1(v/v). The pooled se mseanmple was then divided into two; one sample being stored at 5ºC, and the other at 1f5o°rC p,e riods of 3, 6, 9, and 24h respectively. Sperm characteristics were then recorded for enatcehrv ial of storage. Data were analysed using the statistical prograemn,S Gtat®. The analysis of variance (ANOVA) was used to test for significant differences betnw ethee treatments. Treatment means were compared using Fishers protected t-test least fisciagnti difference (LSD), at the 5% level of significance (Snedecor & Cochran, 1980). 5.3 Results and Discussion Raw or fresh ram semen generally has a shorte f elirfteilspan outside the bodiyn (vitro) (Morrier et al., 2002). However, a decreased metabolic rate c eoxutlednd the lifespan of the sperm cells. Subsequently a low storage temperature generatlelyn desx the fertile lifespan of the sperm – by reducing the metabolic rate. Sperm cells from hf rejsaculates are generally more fertile for a few hours after collection and their metabolic sra atere high at higher temperatures. However, as the metabolic rate increases, the life span ofs ptheer m cells decreases (Hafez and Hafez, 2000). At normal body temperature (37°C) the sperm cellyl osnurvives for a few hours, because of this increased cellular metabolism. It t hiserefore imperative to lower and stabilize thei reonnvmental temperature in which the semen is to be storedr,e bthye decreasing the metabolic rate of the 59 sperm cells and thus increasing its longevity (sBkroin et al., 2000). The ejaculates from the three indigenous rams were pooled to eliminate the indduiavil seminal differences and compare the effect of two storage temperatures (5°C and 15°ICn) . Table 5.1 the sperm motility characteristics of the diluted semen of the inndoigues rams stored at 5°C or 15°C, as measured by the CASA system, are set out. Storage period (3h) After 3h of semen storage, no effect of tempera (t5u°rCe or 15°C) was recorded regarding all the sperm cell motility characteristics evaluated. Veasl ufor the different sperm characteristics of semen stored at a temperature of 15°C were geyn esruapllerior to those for semen stored at 5°C – except for the proportion of static (immotile) smpe crells (although these differences were not significant). Semen stored at 15°C also exhibite sdi ganificantly higher proportion of motile sperm (9.6%) at the medium rate of motility, comepda tro semen stored at 5°C (5.3%). Storage period (6h) After a 6h period of semen storage, the proporotifo nto tal motile sperm, progressively motile, non-progressively motile and rapid motile spermls c reelcorded were generally higher in semen stored at 15°C, compared to that at 5°C. Simil atrhlye, proportion of immotile sperm cells was higher in semen stored at 5°C. No temperature te wffeacs recorded regarding the proportion of sperm cells for the medium or slow motile, the V CVLS,L, VAP, LIN, STR, WOB, ALH and BCF characteristics. For 15°C, all values recorfdoer dt he sperm characteristics (CASA) tended to be higher than at 5°C. It was evident that haell pt arameters measured were decreasing with time and were not as metabolically active as foilnlogw a 3h interval. Storage period (9h) After 9h of semen storage at 5°C (15.1%), a sicganniftily lower progressive motility rate was recorded compared to 15°C (22.3%) group. Althouog hs ingnificant differences were recorded, the percentage total motile, non-progressive m,o rtailepid motile, medium motile, slow motile, STR, WOB and ALH sperm were higher when stored 5a°tC 1. Semen stored at 5°C recorded higher values for the immotile sperm, VCL, VSL, V,A PLIN and the BCF characteristics. 60 Following this period of time, irrespective of thseto rage temperature, it was evident that the sperm motility characteristics had drastically deeacsred. Storage period (24h) After 24h of semen storage, the proportion of t omtaoltile, progressive motile, non-progressive motile and rapid motile sperm cells were signifitclya(nP<0.05) higher at 15°C, compared to 5°C. The proportion of static (immotile) sperm cells w saigsnificantly (P<0.05) lower at 15°C. No significant differences were recorded for the protipoon of medium motile, slow motile, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF characterisst.ic In general values recorded tended to be higher for the semen that had been store1d5 °aCt , compared to 5°C. This is because the sperm cells kept at 5°C are believed to lose thmeoirt ility, while maintaining their viability (Appell & Evans, 1977). The proportion of total mileo tsperm recorded after 24h of storage at 15°C was higher (61.2%), compared to that at 3h.4 (%51), 6h (50.1%) and 9h (50.6%). The percentage rapid motile sperm recorded followingh 2o4f semen storage at 15°C was higher (48.6%) than following 3h (30.7%), 6h (37.0%) anhd (934.3%) of storage. The trend followed by the sperm motility rates over time was not csotnesnit, as a linear decrease in semen quality is generally expected with time. The cooling of semen to 4°C has been shown to haanv aed verse effect on the sperm motility rates. In a study conducted by Appell and Evans7 7()1,9 where semen samples stored at 3 temperatures ( 4°C, 20°C or 37°C) were compareedr,m s pviability followed a motility pattern very close to that in the semen samples kept amt rtoeomperature (20°C) and body temperature (37°C ). However at 4°C the sperm viability was lw perleserved, despite the loss in motility of the sperm. At 20°C and 37°C all static sperm weraed d, however this was not the case at 4°C. Semen stored at 15°C recorded a value for the mtootatille sperm of 51.4%, following a 3h storage period. This however increased to 61.2%er a2f4t h. This increase in the total sperm motility may be attributed to a high proportion iomf motile sperm that possibly regained their motility after a long time exposure to 15°C. Thee roavll percentage of total motile sperm however decreased from 39% (at 3h storage) to 2a7t% 2 4(h storage) in semen stored at 5°C. This decrease in recorded sperm motility of the esne mstored at 5°C after 24h period may be 61 attributed to the immobilisation of sperm cells t thgaenerally occurs at lower storage temperatures. Generally semen stored at 15°C ereds uinlt better preservation of the sperm motility characteristics, when compared to semeonr esdt at 5°C. Langford and Fiser (1980) reported the storage of ram semen in skim milk5 a°Ct 1 to be more satisfactory in terms of sperm survival, than at 4°C – which is in agreement wthiteh current study. The results recorded in this study however differ with the observations of O’Ha aetr al. (2010), who found the storage of semen at 5°C to be superior to 15°C in terms ohf bsopterm motility and viability of the semen stored for a period of 72h. Pauleentz a l. (2002) also reported the motility of sperm sto aret d5°C to be superior to storage at 15°C. The differeneceo rrded in the current study may be ascribed to the semen diluent use Md.orton et al. (2009) clarified the variation in the effectivesnse of the semen diluents in preserving the longevity and nptoiatel fertilising capacity of sperm - during liquid storage, using different constituents. Twhiass in a comparative study using reconstituted skim milk and CUE (Cornell University Extender) forar m semen storage at 4 or 15°C. Interactions between the type of diluent, dilutiroante and the temperature of storage have been reported by Maxwell and Salamon (1993). 5.4 Conclusions Semen stored at 15°C recorded higher sperm mo, tciloitmy pared to semen stored at 5°C, over the entire time period. It would thus seem, accordoin gth te results in this trial that ram semen diluted and stored at 15°C for a period of up to 24h wasre m soatisfactory in retaining sperm motility. However, the fertilizing capacity of the sperm l shtial s to be evaluated. It can also be seen as practical means of storing semen (15°C) for thel icaaptpion of artificial insemination in a short- term semen storage programme, incorporated wit hS AemI. en stored at 15°C also resulted in the better preservation of sperm characteristics aosr drecd by CASA, compared to the 5°C storage group. CASA can be seen as a very reliable tesatr dreingg the sperm characteristics. More studies need to be conducted to investigate the longer teefrfemct (more than 24h) of semen storage at different temperatures on sperm viability and tehlea trionship to actual fertilizing capacity. The ultimate test regarding sperm viability would howere vbe in the fertilizing ability of the sperm cell – an aspect not investigated in this study. 62 Table 5.1 The mean (±SE) sperm motility characteicrsis of pooled diluted South African indigenous s reamen stored at 5°C or 15°C following evaluation using the CASA sys tem Pooled semen characteristics Temperatures at the different storage periods 3h 6h 9h 24h 5°C 15°C 5°C 15°C 5°C 15°C 5°C 15°C Total Motility (%) 39.0±8.5abc 51.4±8.1ab 24.4±5.7c 50.1±9.6ab 31.3±8.8bc 50.6±10.0ab 27.0±7.1c 61.2±10.0a Progressive Motility (%) 20.3±5a.5b 30.4±6.8a 14.2±3.4c 24.8±5.4ab 15.1±4.3c 22.3±6.4ab 13.6±3.8c 28.8±5.6a Non- progressive Motility (%) 18.7 ± 3b.c9d 21.1±4.1abcd 10.2 ± 2.5d 24.9 ± 5.2abc 16.2 ± 5.0bcd 28.3 ± 5.0ab 13.4 ± 3.5cd 32.4 ± 5.3a Rapid (%) 25.9±6.b5c 30.7±6.2bc 17.7±4.3c 37.0±7.7ab 21.3±6.3bc 34.3±7.5abc 18.3±5.1c 48.6±9.3a Medium (%) 5.3±1.b4 9.6±2.5a 3.1±1.1b 5.0±1.2b 3.2±1.2b 6.1±1.5ab 2.6±0.8b 4.7±0.9b Static (%) 61.0±8.a5bc 48.5±8.1bc 75.6±5.7a 49.9±9.6bc 68.7±8.8ab 49.4±10.0bc 73.0±7.1a 38.8±10.0c Slow (%) 7.8±1.6ab 11.2±3.8a 3.6±1.0b 8.1±1.8ab 6.8±2.2ab 10.2±1.7a 6.2±1.8ab 7.9±1.4ab VCL (µm/s) 180.1±16.a4 184.3±16.4a 168.5±24.3a 186.5±10.9a 164.0±25.4a 163.4±13.2a 165.5±25.3a 203.6±17.4a VSL (µm/s) 112.3±14.a9b 130.8±14.7a 113.7±16.8ab 115.3±9.7ab 101.8±20.3ab 88.0±11.4b 100.9±17.5ab 122.6±14.3ab VAP (µm/s) 145.2±16.a1 153.2±14.7a 150.4±23.2a 148.1±8.7a 130.8±26.1a 121.4±13.8a 131.5±21.9a 163.2±16.6a LIN (%) 59.4±5.0ab 70.8±4.3a 59.8±7.6ab 62.04±4.2ab 54.6±7.4b 53.3±4.8b 53.5±7.3b 59.2±3.4ab STR (%) 75.1±4.a3 84.7±3.3a 70.4±8.9a 77.1±2.6a 69.5±8.8a 73.1±4.5a 68.0±8.7a 74.6±2.3a WOB (%) 78.4±4.0a 83.1±2.5a 75.6±9.6a 79.9±2.9a 69.9±9.5a 72.8±4.4a 69.6±9.1a 79.0±2.6a ALH(µm) 3.3±0.5a 3.5±0.2a 2.9±0.4a 3.3±0.1a 2.9±0.4a 3.2±0.2a 3.3±0.2a 3.6±0.2a BCF(Hz) 13.6±1.b9 14.8±0.7ab 14.2±2.3ab 16.4±0.8ab 14.8±2.0ab 18.0±1.2a 15.2±0.7ab 17.5±0.9ab VCL = curvilinear velocity, VSL = straight-line voecl ity, VAP = average path velocity, LIN = linear,i tSyTR = straightness WOB = wobble ALH = amplit ude of lateral head displacement and BCF = beat croressq uef ncy. a,b Values with different superscripts within a row fedrif significantly (P< 0.05) 63 Chapter 6 The effect of temperature and different storage sti,m on sperm motility of ram semen diluted with an extender containing glycerol 6.1 Introduction When utilising males with outstanding traits in oan tcrolled breeding program, it is necessary to store the semen for a period of time. This storcaogueld be in liquid form at lower temperatures or by cryopreservation of semen. As long term creyoseprvation and thawing of ram semen induces serious structural damage to the sperm a ncedl limpairs fertility, raw diluted and cooled semen is generally considered as an alternativfreo ztoe n semen, when used for AI within a short period after semen collection (Maxwell & Watson,9 61;9 Soderquiset t al., 1997). Compared to raw or fresh semen, diluted cooled ram semen ise hveorw also exposed to a decrease in sperm motility and morphological integrity, accompaniedit hw a subsequent decline in sperm survival rate in the female reproductive tract, a reducitnio fne rtility and even increased embryonic losses (Maxwell & Salamon, 1993). Regarding fertilization as such, it is essentia iln tsoeminate, in this case the ewe at an optimal time during the oestrous period, to achieve accbelep tfaertility results. It is also important to preserve and store ram semen under optimal consd,i tiofor maximal AI success. The recommended maximum storage period of raw seme nb eheans said to be as short as 6 to12h (Paulenze t al., 2002). Depending on the time needed to tran sspeomrten from the AI station to the farm, such a short storage time often makedsi ffiitc ult to inseminate the ewes at the prescribed time during oestrus. The length of thaetu rnal oestrous period in sheep has been reported to vary between 24 and 36h, with ovula gtieonerally occurring some 24 to 30h after the onset of oestrus. Timing of insemination shoulds thbue 12 to18h after the onset of oestrus (Hunter et al., 1980). Semen extenders are generally addede t os etmh en to supply the sperm cells with a source of energy, protect the celolsm f rtemperature related injury and maintain a suitable environment for the sperm to survive termarpiloy (Purdy, 2006). Cryoprotectants in the case of semen freezing are then added as comp oton ethnets semen extenders to protect the sperm from these temperature related injuries. In thiaisl tgrlycerol was included, as a cryoprotectant as 64 it is currently the most extensively used agenmt ianm malian semen cryopreservation. Glycerol as such is a penetrating cryoprotectant that ca museemsbrane lipid and protein rearrangement, which results in increased membrane fluidity anrdm peeability for ions and an increase in ATP consumption. Glycerol thus generally causes gr eastpeerrm cell dehydration at lower temperatures and an increased ability for spermls ctoe lsurvive cryopreservation (Holt, 2000). Certain artificial insemination centres even incel ugdlycerol in the semen extender for the conservation of raw ram semen at higher tempersa t(uHreackett & Wolynetz, 1982). In liquid semen diluents, glycerol has been shown to redhuec ed et cline in fertility associated with the aging of sperm (Shannon, 1964). Purdy (2006) anldsoic ai ted the presence of glycerol to induce osmotic damage to sperm, while Morrieet r al. (2002) only recommended glycerol for use in semen cryopreservation. The aim of this study was thus to determine thec et fof f temperature and different storage times, on sperm motility of ram semen diluted with an enxdter, containing glycerol. 6.2 Materials and Methods Semen was collected during the winter (June, 2o0u0t9s,i de the natural breeding season) from 6 healthy mature rams of different indigenous brei.eed.s t he Damara, Namaqua Afrikaner and the Zulu breed, with 2 rams per breed being used. Tghees aof the rams ranged from 2 to 4 years and all animals were maintained on natural pastures s aunpdplemented with a commercial diet. Water was availablea d libitum. Semen was collected twice weekly for a period2 owf eeks and 4 ejaculates were collected in total from each ramith, wthe aid of an electro-ejaculator. Semen was collected directly into graduated test tubes, w hwicehre then placed into a thermo flask, containing water at a temperature of 37°C. All eccotlel d semen was transported to the laboratory for evaluation within 1h of collection. The raw, duilnuted ejaculate was evaluated for sperm concentration, semen pH, and sperm motility (forr em odetails, see Chapter 3). The sperm concentration of each ejaculate was determined wthiteh aid of a spectrophotometer (Spermacue®), the semen pH with the aid of a pHe mr aent d the Computer Assisted Sperm Analysis (CASA) system used for measuring the sp meromtility. After initial evaluation, all semen samples were pooled to eliminate individaumal dr ifferences and diluted equally with an egg yolk citrate extender, containing 14% glyceirno lt he ratio of 1:1 (v/v), making a final 65 glycerol concentration of 7%. The pooled semen slea mwpas then divided into two, one sample being stored at 5ºC and the other at 15°C, fora sgteo rperiods of 3, 6, 9, and 24h. Sperm motility characteristics were then recorded at each int eorfv saelmen storage. All data were analysed using the statistical prmog, raGenStat®. The analysis of variance (ANOVA) was used to test for significant differensc beetween the treatments. Treatment means were compared using Fishers protected t-test feo rle tahst significant difference (LSD), at the 5% level of significance (Snedecor & Cochran, 1980). 6.3 Results and Discussion Raw extended ram semen generally has a very sehrotirlte flifespan and the reduced metabolic rate of sperm at a lower storage temperature s heoxutlednd the storage life of the semen (Maxwell & Salamon, 1993). Thus low temperaturensd t eto extend the fertile life of sperm by decreasing the cell metabolism (Morrieet r al., 2002). The sperm cells of fresh ejaculates are generally fertile for a few hours at a high rate m oeftabolism. A high temperature will increase the metabolic rate, and subsequently decreasei feth es plan of a sperm cell. At normal body temperature (37°C) the sperm cell lives for a feowu rhs only, due to this increased cellular metabolism (Brinskoe t al., 2000). The semen of the three South Africang ienndoi us breeds of rams were diluted in an egg yolk citrate diluenotn, tcaining 14% glycerol and stored at 5°C and 15°C, were compared in this study. In Table 6.1 the sperm motility characteristicst hoef diluted indigenous ram semen exposed to glycerol as a cryoprotectant and stored at the sttwoora ge temperatures for the different periods of time, as measured by the CASA system, are st.e t ou Storage period (3h) In the current trial, the effect of storage temptuerrea (5°C vs. 15°C) was recorded with the aid of the CASA system following 3h of storage – regard VinAgP (average path velocity) and V SL (straight-line velocity). Sperm stored at 15°C rredceod significantly (P<0.05) higher values for the VAP and VSL, when compared to that of semen stoart e5d° C. The percentage of total motile sperm, progressive motile, non-progressive mortailep,id motile, slow motile, VCL (curvilinear 66 velocity), LIN (linearity), STR (straightness), WO (Bwobble) ,ALH (amplitude of lateral head displacement), and the BCF (beat cross frequenchya)r acteristics were recorded to be numerically higher at an storage temperature ofC 1, 5c°ompared to semen stored at 5°C. The semen stored at 15°C resulted in numerically (ntaotti sstically) lower static (immotile) sperm percentage and a medium motile sperm percentagne t hthaat of semen stored at 5°C (Table 6.1). Storage period (6h) Following 6h of semen storage, no temperature te (ff5e°cC vs. 15°C) was recorded regarding all the sperm motility and velocity characteristics.l uVeas for the percentage of total motile sperm and percentage static sperm were similar for beomthe sn storage temperatures (5°C and 15°C). Storage period (9h) Following 9h of semen storage, no temperature te (f5fe°cC vs. 15°C) was recorded for the semen characteristics measured. The percentage of tootatil em sperm, progressive motile sperm and non-progressive motile sperm, rapid motile spermed, imum motile, slow motile, the VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF characteristics we eornce again only numerically higher for semen stored at 15°C, than at 5°C, althoughs et hdeifferences were not significant. The percentage of immotile sperm was higher in semoerne ds tat 5°C than at 15°C. Storage period (24h) A significant effect of storage temperature for s thtei mperature was only recorded for the percentage wobbling sperm, where semen stored °aCt 1re5corded a significantly (P<0.05) higher WOB than semen stored at 5°C. Semen stot r1e5d° Ca recorded 48.3% total motile sperm after 3h of storage but this increased to 50.4%lo wfoinl g 24h of storage. Similar percentages of total motile sperm were recorded after 3h and 2440h% ( and 40.8%, respectively) in the semen stored at 5°C (Table 6.1). This may be due to tehdeu cred metabolism induced by the low temperature of 5°C. The results of the presenty s wtuedre thus similar for the two temperatures. However it was found that most of the seminal cchtaerraistics (sperm motility characteristics) were higher in semen stored at 15°C, comparedo tsoe t hstored at 5°C, although these differences were not significant. 67 Table 6.1 The mean (±SE) sperm motility characteicrsis of indigenous ram semen diluted with gly csetrorle, d at two temperatures for different periods of time as mreads buy the CASA system Characteristics Temperatures at different storage periods 3h 6h 9h 24h 5°C 15°C 5°C 15°C 5°C 15°C 5°C 15°C Total Motility (%) 40.0 ± 7.4a 48.3 ± 9.0a 46.9 ± 8.3a 46.7 ± 8.8a 31.8 ± 6.4a 47.1 ± 7.9a 40.8 ± 7.7a 50.4 ± 10.6a Progressive Motility (%) 26.1 ± 5a. 2 32.9 ± 6.4a 25.6 ± 4.9a 29.5 ± 6.3a 22.1 ± 4.4a 34.9 ± 6.2a 21.7 ± 4.9a 33.9 ± 7.7a Non-progressive Motility (%) 13.9 ± 2a.b9c 15.4 ± 2.9abc 21.3 ± 4.0a 17.3 ± 3.4ab 9.7 ± 2.6c 12.2 ± 2.6bc 19.1 ± 3.8ab 16.4 ± 3.3abc Rapid (%) 28.7 ± 5.a8 35.7 ± 6.8a 33.3 ± 6.1a 33.3 ± 7.6a 25.2 ± 5.6a 37.8 ± 7.1a 31.6 ± 6.1a 40.3 ± 8.9a Medium (%) 5.9 ± 1.a3b 5.8 ± 1.7ab 6.7 ± 1.6a 5.5 ± 1.4ab 2.9 ± 0.9b 3.8 ± 1.1ab 3.4 ± 0.6b 4.1 ± 1.1ab Static (%) 60.0 ± 7.a4 51.8 ± 9.0a 53.1 ± 8.3a 53.3 ± 8.8a 68.2 ± 6.5a 53.0 ± 7.9a 59.2 ± 7.7a 49.7 ± 10.6a Slow (%) 5.4 ± 1.a5 6.8 ± 1.5a 6.9 ± 1.8a 7.9 ± 1.3a 3.7 ± 0.9a 5.5 ± 1.1a 5.9 ± 1.2a 6.0 ± 1.3a VCL (µm/s) 157.5 ± 9.a2 169.5 ± 6.6a 164.2 ± 9.8a 147.5 ± 12.6a 165.8 ± 11.4a 180.3 ± 8.8a 175.4 ± 8.3a 163.8 ± 9.0a VSL (µm/s) 102.3 ± 8.b4 125.5 ± 6.4a 101.9 ± 6.9b 96.6 ± 9.0b 110.1 ± 8.0ab 126.4 ± 6.9a 92.7 ± 6.6b 103.1 ± 9.6b VAP (µm/s) 121.6 ± 6.b9 140.6 ± 6.2a 120.8 ± 6.9b 111.0 ± 10b 126.8 ± 7.8ab 141.1 ± 7.0a 118.1 ± 5.7b 125.7 ± 7.2ab LIN (%) 65.2 ± 4.7ab 74.2 ± 3.2a 63.4 ± 3.8b 69.5 ± 4.5ab 66.5 ± 1.9ab 70.2 ± 1.9ab 53.2 ± 4.1c 62.3 ± 5.2bc STR (%) 84.0 ± 4.a5b 88.9 ± 1.8a 84.2 ± 1.9ab 83.8 ± 2.5ab 86.3 ± 1.8ab 89.4 ± 1.2a 77.9 ± 3.1b 79.9 ± 5.4b WOB (%) 77.7 ± 2.7ab 83.0 ± 2.2a 74.7 ± 3.0bc 77.0 ± 2.1ab 77.1 ± 1.9ab 78.5 ± 1.7ab 68.4 ± 3.7c 77.1 ± 2.4ab ALH(µm) 2.6 ± 0.3a 2.8 ± 0.1a 3.1 ± 0.2a 2.7 ± 0.2a 3.0 ± 0.2a 3.1 ± 0.1a 3.2 ± 0.4a 2.7 ± 0.3a BCF(Hz) 20.3 ± 2.a1 21.9 ± 1.2a 23.8 ± 1.0a 23.1 ± 1.2a 23.5 ± 1.1a 25.0 ± 1.0a 23.0 ± 2.3a 22.1± 2.3a VCL = curvilinear velocity, VSL = straight-line voecl ity, VAP = average path velocity, LIN = linear,i tSyTR = straightness WOB = wobble ALH = amplit ude of lateral head displacement and BCF = beat croressq uef ncy. a,b Values with different superscripts within a row fedrif significantly (P< 0.05 ) 68 Storage temperature and period had no significPan