Magister Scientiae (Medical Bioscience) - MSc(MBS) / The incidence of male infertility is increasing, with up to 50% of infertile males having “unexplained” (idiopathic) infertility. Newly developed molecular techniques have great value in detecting subtle causes of male infertility, as compared to idiopathic infertility which may be explained by standardizing and optimizing sperm functional and structural tests in non-human primate (NHP) sperm. The aim of the study was to evaluate sperm functionality utilizing the sperm of two NHP species, i.e.1) the rhesus monkey (Macaca mulatta) and 2) the vervet monkey (Chlorocebus aethiops), and further evaluate the effect of physiological media (including commonly used, and newly formulated sperm wash and sperm capacitating media) on NHP sperm functionality. Sperm functionality was evaluated by investigating the following sperm functions i.e.: sperm motility, vitality, acrosome reaction (AR), hyperactivation, and mitochondrial membrane potential (MMP). Sperm functional tests included computer-aided semen analysis (CASA), motility analysis, BrightVit staining for sperm vitality, flourescenin isothiocyanate (FITC)- conjugated peanut agglutinin (PNA) staining for sperm acrosome integrity, induction of hyperactivation by stimulants (sperm preparation media containing capacitating ingredients), and mitochondrial inhibitor (Oligomycin-A) for testing MMP. All functional and structural tests were investigated in both species, except for acrosome integrity, mitochondrial inhibition and functional tests compared over time that could not be successfully completed and investigated in the rhesus species. Motility analysis tests proved that within the vervet species, the use of different physiological media results in statistically significant differences in motility and kinematic parameters over a 1 hour time period. Hyperactivation tests proved that capacitating physiological media produced significantly higher percentages hyperactivation when compared to sperm wash media within the vervet species over a 1 hour time period. Furthermore, within both NHP species, sperm structural analysis (vitality and acrosome integrity) results showed that no significant differences are present when making use of different physiological media over a period of 1 hour incubation. The incubation of vervet sperm with different concentrations of mitochondrial inhibitor, Oligomycin-A (0 μM, 5 μM, and 25 μM), resulted in motility inhibition over a 1 hour incubation period. By the evaluation of these tests it was found that the use of different sperm wash [Human tubal fluid (HTF), Ham‟s F-10® and HD Sperm Wash Plus (HDSWP)] and sperm capacitation media [Human tubal fluid with added caffeine (HTFC) and HD Sperm Capacitating Plus (HDSCP)] resulted in significantly different results within sperm functional tests as compared to sperm structural tests. The study indicates that the composition of media, varying from simple to more complex, used for semen preparation plays an important role in determining NHP sperm functionality. Based on these findings further investigation in larger NHP sample groups and human sperm are required to evaluate the role of certain ingredients in the development of more cost-effective media producing satisfactory results in terms of sperm functionality for artificial reproductive technologies (ART).

Philosophiae Doctor - PhD / The vervet monkey, Chlorocebus aethiops, an Old World monkey, has been often used in biomedical research programs (toxicological studies and fertility) because of the inaccessibility of relevant human tissues. Data from nonhuman primates have been a vital component of advances in areas such as infertility, contraception, and other reproductive processes because of the phylogenetic closeness of the primates to humans. The aims and objectives of the study were 1) to describe the gross morphology, histology and ultrastructure of the male reproductive system, 2) to describe and compare the processes of spermatogenesis and spermiogenesis of the vervet monkey to humans and other nonhuman primates, and 3) to evaluate the vervet monkey as a possible experimental model for future human reproductive studies. Twenty-nine adult male vervet monkeys, aged between 5 and 11 years, were used. Gross morphological features of different organs of the reproductive tract were recorded. Light and electron microscopic techniques, and methacrylate sections were used on selected tissues of the reproductive tract. The results showed that the vervet monkey has a male reproductive system similar to many non-human primates studied and man. The epididymis was distinctively subdivided into the caput, corpus, and the caudal regions. No significant differences were observed on the epithelial height of these three regions. Four cell types, apical, principal, and basal cells, and the intraepithelial lymphocytes were observed. The basal cell distribution showed significant differences among three regions of the epididymis (P ≤ 0.01). There were numerous phagocytic vesicles found in three regions of the epididymis. The Sertoli cells showed perforated sleeve-like processes which encased elongated and mature spermatids ready for spermiation. The nuclei of the Sertoli cells were found to be multilobed (4 to 5) compared to the less lobular nuclei of the human Sertoli cells (2 to 3). The Leydig cells showed typical features of steroidogenic cells with abundant smooth endoplasmic reticulum, numerous large mitochondria, and few rough endoplasmic reticulum. It was concluded that the gross morphology and structure of the reproductive tract of the vervet monkey has many similarities to humans and other mammals. Secondly, the organization of spermatogenesis is similar to that found in humans, and is commonly known as a helical arrangement. The results further suggest that the vervet monkey could be regarded as suitable model for human male reproductive studies