Swelling of the apical ridge and anterior acrosome of motile bovine sperm was observed using differential interference contrast optics. Transmission electron microscopy indicated that the acrosomal matrix was extended into complex folds and/or projections. Outer acrosomal and plasma membrane integrity was retained. Anterior acrosomal swelling (AS) was first observed after neat-semen had been slowly cooled to 4°C and stored for 1 day. With subsequent dilution (25 X 10⁶ sperm/ml) and incubation of sperm in a Tris fructose medium at 37°C, a majority of the motile sperm had AS. Of these unique conditions, storing sperm (1500 X 10⁶ /ml) in seminal plasma (SP) at 4°C was very conducive to AS. Replacing SP with egg yolk-citrate (EYC) inhibited AS. Using a Tris buffer with 54 mM fructose as an incubation medium eliminated the necessity of storing sperm in SP provided storage in EYC at 4°C was ≥3 days. AS occurred after storage at 37 or 4°C but not at 21°C. Storing cauda epididymal sperm at 4°C with and without 30.8 mM fructose or glucose in EYC or egg yolk-Tris (EYT) media demonstrated that AS formation required the presence of either sugar. Storing cauda sperm in EYT at 4°C with 0, 3.9, 7.7, 15.4, 30.8, and 61.7 mM fructose indicated that a minimum of 7.7 mM fructose was necessary for a strong AS response. Storage media pH was measured at the end of storage. Media pH was 6.7 with 0 mM fructose and had decreased during storage to 5.7-6.0 in media giving a strong AS response. Further experimentation with cauda sperm demonstrated that storage at reduced media pH would induce AS with or without the presence of fructose. Conversely, storage at normal pH's (6.6-6.8) inhibited AS even with fructose in the media. The proportion of motile sperm with AS was estimated from wet smears initially. Evaluation of acrosomal morphology from dry smears of vitally stained sperm was developed as a means of quantifying the proportion of viable sperm with AS. This technique also allowed closer scrutiny of the acrosomal morphology of viable sperm. The physiological importance of AS is unclear. It may be a unique form of sperm deterioration or a prelude to the true acrosome reaction since AS occurs without loss of sperm viability. / Doctor of Philosophy

The first experiment was designed to determine if the antigenicity of egg yolk diluter could be eliminated by the addition of specific antibodies. Two virgin heifers were immunized with 20% yolk - 2.1% citrate - 7% glycerol with penicillin and streptomycin. Non-immune serum and immune sera having titers of 1:640 and 1:2560 were collected and frozen. Serum antibody titers were determined by the passive hemagglutination test. Utilizing equivalent proportions test it was determined that 80 volumes of anti-egg-yolk-diluent sera titered 1:2560 was necessary to neutralize 1 volume of 20% egg yolk diluent. The second experiment examined the effect of immune sera against egg yolk diluter upon the viability of bovine spermatozoa in that diluter. Using a split-ejaculate technique, 7 ejaculates from 3 bulls were diluted in egg yolk-citrate diluter or skimmilk diluter, cooled, glycerolated and stored at S°C. On the first day after semen dilution, complement-fixed immune serum titered 1:2560, 1:640 and non-immune serum were added to aliquots of diluted semen (1:9 v/v). Aliquots of each diluter without serum served as controls. Each treatment was evaluated immediately after addition of serum and again after 48 hr storage at 5°C. Percent intact acrosomes, percent motility and percent agglutination were measured from unfixed smears at 0, 3, 6 and 9 hr of incubation at 37°C. Both immune and non-immune serum treatments were characterized by head to head agglutination. Within both diluters, there were no significant differences in motility or intact acrosomes due to immune and nonimmune sera. However, all serum treatments were significantly higher (P < .01) in motility and percent intact acrosomes than the non-serum controls. The overall percent intact acrosomes across all hours, days and diluters were 77.9, 81.1, 81.2 and 81.7 for control, non-immune serum, 1:640 serum and 1:2560 serum, respectively. Differences in the percent head to head agglutination were not due to anitbody titer of the serum. / Master of Science

Five experiments were conducted to characterize a discontinuous bovine serum albumin (BSA) gradient and to determine its ability to isolate highly viable, morphologically normal, Y-chromosome-bearing bovine spermatozoa. The column consisted of a 500 ml separatory funnel in which 26 ml of extended semen was layered on top of a discontinuous gradient of 4% BSA (60 ml) over 10% BSA (60 ml). Results indicated that the greatest percentage of the applied semen was recovered from the bottom 20 ml of the gradient (fraction 6) when 1 x 10⁹ sperm cells were applied to the column. The column was able to exclude spermatozoa with midpiece and tail abnormalities but sperm cells with head abnormalities were similar in frequency in separated and unseparated semen. Semen isolated from fraction 6 was more viable and more resistant to damage by freezing and aging than semen from the top fractions of the column. The proportion of Y-chromosome-bearing spermatozoa isolated form fraction 6 did not differ from the proportion in unseparated semen. Cows inseminated with separated semen and unseparated semen gave birth to similar proportions of male and female calves. / Master of Science

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2007. / For spermatozoa to gain access to the oocyte for fertilization, lytic enzymes need to be released during the acrosome reaction. These enzymes, which are stored and transported within an organelle termed the acrosome, make it possible for spermatozoa to collectively penetrate the layers of cells and glycoproteins that surround and protect an oocyte. Acrosomes may thus be viewed as essential for fertilization and their shape, size and volume were examined morphometrically by utilizing automated morphometric analysis equipment. In addition to the acrosome being necessary for normal unassisted fertilization, spermatozoa also need the ability to migrate to the oocyte. Following zona pellucida binding, sperm tail thrust movement initiates zona penetration into the space created by the digestive action of the acrosomal enzymes. Therefore the motion characteristics of spermatozoa were also quantified in terms of kinematic properties. In the treatment of male sub fertility, assisted reproductive techniques are applied. In the application of such techniques, a motile sub-population of spermatozoa was obtained by employing a procedure (swim-up selection) that selects cells on the basis of their kinematic ability. This study presents an analysis of the morphometric and kinematic qualities of spermatozoa populations that are subjected to swim-up selection and investigates the relationship of these morphometrical and kinematic qualities. Computer-assisted semen analysis, swim-up selection and automated sperm morphology analysis tests were all used to evaluate spermatozoa populations. Results indicated that, irrespective of acrosome size, higher kinematic parameter measurements were observed post-swim-up. A significant inverse relationship between the population’s average acrosome size and a number of kinematic parameters was observed. Our results indicated that for a post-swim-up population of spermatozoa an increase in the average acrosome size was significantly related to a decrease in the kinematic parameters VAP, VCL and the VSL within the same population.

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: In this study the author presents new data that will shed light on the fairly nebulous knowledge of intracellular pathways involved in the physiologically induced acrosome reaction and the subsequent events leading to fertilization. The zona pellucidainduced acrosome reaction, sperm-zona interaction as well as various sperm motion characteristics were investigated. The first part of the study focussed on the role of extracellular signal regulated kinase (ERK), a member of the family of mitogen activated protein kinases, during the zona pellucida-induced acrosome reaction and sperm-oocyte interaction. It was shown that the inhibition of ERK significantly reduced the zona pellucida-induced acrosome reaction as measured by fluorescent microscopy. This suggests that ERKs are directly or indirectly involved in the signal transduction pathway through which the human sperm acrosome reaction is induced by the zona pellucida. In an attempt to provide further proof that ERK was involved in human acrosome signalling hemizona assays were employed to test sperm-oocyte binding. From these sperm-oocytebinding experiments it was clear that the inhibition of ERK leads to increased binding. These results support the idea that the zona pellucida-induced acrosome reaction, and therefore the physiologically relevant exocytotic event, is regulated by an ERKmediated signal transduction process. In the second part of the study the significance of phosphatidylinositol 3-kinase (PI3K) in the process of human sperm motility, acrosome reaction and sperm-oocyte binding, was investigated by employing the specific PI3K, LY294002. PI3K inhibition increased the percentage motility and percentage progressive motility in asthenozoospermia patients. Under the present laboratory conditions PI3K inhibition furthermore did not influence the acrosome reaction, whilst it enhanced sperm-oocyte binding. These results therefore imply that PI3K negatively affect sperm motility and zona-binding. In the last part of the study the possible effects of intracellular cGMP accumulation via acute in vivo sildenafil citrate (ViagraTM) administration was investigated on seminal parameters, induction of the acrosome reaction, sperm-oocyte binding and sperm motility. As was expected no changes in the macro- and microscopically seminal parameters were caused by sildenafil citrate when compared to placebo. Furthermore the acrosome reaction was also not initiated or potentiated by sildenafil citrate at concentrations of 50mg orally. Sperm-oocyte binding, smooth path velocity, straight line velocity and the percentage rapid cells all increased after sildenafil citrate treatment. From these results it appear that there are various role players in the zona pellucidainduced acrosome reaction intracellular signalling system. A thorough understanding of such signal transduction systems and the crosstalk in-between will ultimately yield information regarding the nature of receptors to which these signal transduction pathways are coupled in human spermatozoa as well as the intracellular effectors that ultimately regulate sperm function. Moreover, an understanding of these regulatory pathways will be essential for the future development of clinical approaches designed to enhance or preclude fertilization. / AFRIKAANSE OPSOMMING: Die outeur lê in hierdie studie nuwe data voor ten einde meer lig te werp op die relatief vae veld van intrasellulêre seintransduksie paaie betrokke by die fisiologiesgeïnduseerde akrosoomreaksie en die daaropvolgende gebeure wat aanleiding gee tot bevrugting. Die zona pellucida-geïnduseerde akrosoomreaksie, sperm-zona interaksie sowel as spermmotiliteitseienskappe is ondersoek. Die eerste gedeelte van die studie fokus op die rol van ekstrasellulêreseingereguleerde- kinase (ERK), 'n lid van die familie van mitogeen-geaktiveerde proteïenkinases, tydens die zona pellucida-geïnduseerde akrosoomreaksie en sperm-oosiet interaksie. Daar word aangetoon dat die inhibisie van ERK die zona pellucida geïnduseerde akrosoomreaksie, soos gemeet met behulp van fluorosensie mikroskopie, betekenisvol verminder. Dit suggereer dat ERK direk of indirek betrokke is by die seintransduksie paaie waardeur die akrosoomreaksie van die menslike sperm deur die zona pellucida geïnduseer word. In 'n poging om onomwonde te bewys dat ERK betrokke is by menslike akrosoom-seintransduksie, is hemizona essais gebruik om sperm-oesiet binding te bepaal. Van hierdie sperm-oosiet bindingeksperimente is dit duidelik dat die inhibisie van ERK aanleiding gee tot verhoogde binding. Hierdie resultate ondersteun dus die idee dat die zona pellucidageïnduseerde akrosoomreaksie en dus die fisiologies relevante eksositotiese gebeurtenis gereguleer word deur 'n ERK-gemediëerde seintransduksie proses. In die tweede gedeelte van die studie is die belang van fosfatidielinositol 3-kinase (PI3K) in die proses van menslike spermmotiliteit, akrosoomreaksie en sperm-oesiet binding ondersoek deur van die spesifieke PI3K inhibitor LY294002, gebruik te maak. Pl3K-inhibisie het die persentasie motiliteit en progressiewe motiliteit by astenozoospermiese pasiënte verhoog. Onder hierdie laboratoriumtoestande het Pl3K-inhibisie nie die akrosoomreaksie beïnvloed nie, terwyl sperm-oosiet binding verhoog is. Hierdie resultate beteken dus dat PI3K spermmotiliteit en zona-binding negatief beïnvloed. In die laaste gedeelte van die studie is die effekte van intrasllulêre cGMP akkumulasie deur akute in vivo sildenafil sitraat (ViagraTM) toediening op seminale parameters, induksie van die akrosoomreaksie, sperm-oesiet binding en spermmotiliteit ondersoek. Soos verwag is geen veranderinge in die makro- en mikroskopiese seminale parameters deur sildenafil sitraat in vergelyking met plasebo veroorsaak nie. Verder is die akrosoomreaksies ook nie deur 50mg orale sildenafil sitraat geïnisieer of potensieer nie. Sperm-oosiet binding, geplaneerde snelheid, reguitlyn snelheid en persentasie vinnigbewegende selle was almal vehoog na sildenafil sitraat behandeling. Uit hierdie resultate blyk dit dat daar verskeie rolspelers in die zona pellucidageïnduseerde akrosoomreaksie is. 'n Deeglike insig van al hierdie seintransduksiepaaie en die onderlinge kruiskontak tussen mekaar sal uiteindelik die nodige inligting rakende die reseptore waaraan hierdie seintransduksie paaie gekoppel is, verskaf sowel as die intrasellulêre effektore wat uiteindelik spermfunksie beheer. Nog te meer sal die begrip van hierdie regulatoriese paaie verder noodsaaklik wees vir die toekomstige ontwikkeling van kliniese benaderings om bevrugting te bevorder of te voorkom.

Thesis (PhD.(Reproductive Biology)--Faculty of Health Sciences)-University of Pretoria, 2007.

Orientador: Fabiana Ferreira de Souza / Resumo: Os objetivos deste estudo foram validar a sonda fluorescente (MitoStatus Red) para análise de potencial mitocondrial em espermatozoide de cães por citometria de fluxo, associada a análise de integridade de membrana plasmática e do acrossomo (experimento I). Além disso, foi conduzida análise proteômica (abordagem shotgun) do plasma seminal de cães, sua correlação com a congelabilidade do sêmen e capacidade de ligação das células espermáticas em membrana perivitelina da gema de ovo (TL) (experimento II). No experimento I foram utilizado 10 ejaculados, com motilidade espermática >75% e as concentrações de 20 nM, 50 nM, 100 nM e 200 nM da sonda MitoStatus Red foram comparadas na citometria de fluxo. Concluiu-se que 20 nM foi a concentração ideal, sem danos as estruturas celulares. No experimento II foram utilizados 10 animais, 2 ejaculados/animal (n = 20). Os ejaculados foram avaliados por cinética espermática, citometria de fluxo (potencial mitocondrial, integridade de membrana plasmática e de acrossomo) e TL. A análise proteômica do plasma seminal foi avaliada por espectrometria de massas. Os ejaculados foram divididos em 3 clusters (cluster Low, cluster Medium, cluster Hight) de acordo com os parâmetros espermáticos pré e pós-descongelação. Os grupos foram correlacionados com as proteínas encontradas no plasma seminal. Concluiu-se que existe relação entre presença ou ausência de proteínas do plasma seminal de acordo com a qualidade espermática. / Mestre

As the estimated market demand for the Agulhas sole Austroglossus pectoralis exceeds the annual catch from trawlers, this species is a potential aquaculture candidate. Broodstock conditioning and gamete preservation is part of research and development aiming at establishing a breeding protocol for a new aquaculture species. Based on a literature review of the morphology of pleuronectiform spermatozoa, this study was designed firstly, to contribute to the field of spermatozoan morphology by describing the ultrastructure of A. pectoralis spermatozoa. This was followed by an experiment to cryopreserve mature spermatozoa to provide baseline data for future studies on this and related species. The testis of A. pectoralis was a paired structure encased in a membrane, the tunica albuginea. The primary testis was located on the dorsal surface of the rib cage and the secondary testis on the ventral side. The testis was of an unrestricted spermatogonial type, based upon observations of spermatogonia along the entire length of the lobule. Mature spermatozoa of A. pectoralis had an acrosome-free ovoid head 1.68 ± 1.6μm in length and 1.7 ± 1.6μm in diameter, a short mid-piece of 0.5 ± 0.1μm in length, containing 7 irregularly shaped mitochondria forming a ring-like structure at the base of the nucleus. The flagellae were 47.4 ± 4.8μm in length, most with two plasma membrane lateral fin-like projections. However, some flagellae had either zero or three lateral fin projections. Cross-sections of the flagellae showed an axenome with a 9+2 microtubule configuration. The proximal and distal centriols were coaxal, situated deep within the nuclear fossa. The structure of A. pectoralis spermatozoa conformed to the type 1 ect-aquasperm, also found in externally fertilizing species. This type has been suggested to be the plesiomorphic form in Neopterigians. Finally, this study contributed to a cryopreservation protocol for A. pectoralis spermatozoa by testing the two cryoprotectants dimethyl sulphoxide (DMSO) and glycerol. Glycerol, at a concentration of 10%, offered better cryoprotection than DMSO. This was established using flow cytometry analysis of post-thaw nuclear membrane integrity after 64 days of storage in liquid nitrogen. The toxicity of DMSO to isolated cellular proteins may have resulted in DMSO-treated sperm having the highest percent (35.2% ± 3.2%) of non-viable cells compared with 23.0% ± 2.5% and 27.8% ± 3.4% for glycerol and the control, respectively. The presence of sucrose in the Modified Mounib Medium extender solution may explain why 45.5% ± 5% of the sperm cells were potentially viable in the control treatment. Initially, the white margined sole Dagatichthys marginatus (Soleidae) was selected as the most suitable candidate for flatfish aquaculture in South Africa. Thus, the aim of this study was to investigate the cryogenic potential and ultrastructure of D. marginatus spermatozoa. However, due to a skewed sex ratio, there were not enough males available to study this species. A skewed sex ratio is common amongst soleids, thus, the need to develop effective cryopreservation methods and to develop an understanding of sperm morphology so that the best time for cryopreservation can be chosen. In conclusion, this first description of spermatozan morphology of A. pectoralis contributed to our understanding of soleid sperm ultrastructure. In addition, a comparison of testis appearance between fish sampled just prior to spawning season and fish with mature sperm provided information on the spawning season of this species. The findings from the cryopreservation experiment suggested that glycerol was a feasible cryoprotectant for this species when sperm was prepared under field conditions.

MSCAGR (Animal Science) / Department of Animal science / The quality of cryopreserved Boer goat semen may be influenced by the source and concentration of energy supplements in the extender. The aim of the study was to improve the protocols for cryopreservation of Boer goat spermatozoa using different disaccharides concentrations as supplements in tris- egg yolk extender. Two experiments were carried out to investigate the effect of (a) addition of three disaccharides (maltose, sucrose and trehalose) and (b) disaccharides combination (maltose and trehalose) at different concentrations using tris-egg yolk extender. For experiment 1: the study was replicated six times and was conducted in a 3 x 2 x 2 factorial arrangement with three different sugars (sugars: maltose, sucrose and trehalose), two sugar concentrations (0.12g and 0.22g) and two evaluation times (0 hours before cryopreservation and 120 hours after cryopreservation). For experiment 2: the study was replicated six times and was conducted in a 2 x 2 factorial arrangement with two sugar concentrations (0.12g and 0.22g) and two evaluation times (0 hours before cryopreservation and 120 hours after cryopreservation). Semen ejaculates were collected at 7.00-9.00 am from three Boer goats twice per week. After collection, the semen samples were pooled and diluted with tris-egg yolk extender at the ratio of 1:7 (semen to extender). Sperm quality (progressive motility, non- progressive motility, kinetic motions, viability (live/dead) and morphology) were analyzed using computer aided sperm analyzer (CASA). For experiment 1: sucrose 0.12g had higher progressive motility (PM %) when compared to maltose, and trehalose at 0h but reduced after cryopreservation. Sucrose 0.12 showed high percentage of kinetic motions (straightness and average path velocity) when compared to other sugars at 0 hours. More morphological defects M (CH) were revealed by maltose 0.12 at 0 hours. Sugar type (ST) and evaluation time (ET) showed no significant difference (P>0.05) in progressive motility (PM %), sperm kinetic motion, sperm viability and morphology. For experiment 2: mixed/combined 0.12g (maltose and trehalose) revealed more progressive motility (PM %) at 0h and reduced after cryopreservation. Table 6 and 9: of experiment 1 and 2 showed an interaction caused by sugar concentration level and evaluation time (L X ET) on the percentage of cut head M (CH%) and coiled M(C%) morphological abnormalities. In conclusion addition of maltose 0.12g to the extender showed almost similar results with that of trehalose at 0h and 120h. Therefore addition of maltose and trehalose to the extender might improve the quality of Boer goat spermatozoa prior and post cryopreservation. / NRF

The effects of miticides on the reproductive physiology of queens and drones were examined. The first study examined the effects of Apistan (fluvalinate), Check Mite+ (coumaphos), and Apilife VAR (74% thymol) on sperm production and viability in drones. Drones from colonies treated with each miticide were collected at sexual maturity. Sperm production was determined by counting the number of sperm in the seminal vesicles. Sperm for viability assays was analyzed by dual fluorescent staining. Apilife VAR and coumaphos significantly lowered (P<0.0001) sperm production and coumaphos treatments caused a significant decrease (P<0.0001) in the sperm viability. The effects of miticides on queens was examined by treating queen-rearing colonies and examining the number and viability of sperm in the spermathecae of newly mated queens. Queens from each treatment group were collected after mating and the spermathecae were removed and analyzed. Colonies treated with coumaphos failed to provide viable queens and were excluded. Apilife VAR was found to significantly decrease (P<0.0016) sperm viability. No significant differences in sperm numbers were found between treatments. The effect of miticides on sperm viability over time was also examined. Drones were reared as described, but the spermatozoa were collected as pooled samples from groups of drones. The pooled samples from each treatment were subdivided and analyzed periods of up to 6 weeks. Random samples were taken from each treatment (n = 6 pools) over a period of 6 weeks. The exposure of drones to coumaphos during development significantly reduced sperm viability for all 6 weeks, and caused a large decline in week 6. The potential impacts of these results on queen performance and failure are discussed. / Master of Science in Life Sciences