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

Philosophiae Doctor - PhD / Introduction: Plant extracts and herbal preparations are often marketed as natural and safe alternatives to conventional medicines for the prevention and treatment of a variety of ailments, without proof of efficacy and safety. Cardiovascular, hematopoetic, hepatic and renal impairment resulting from the use of conventional drugs is widely acknowledged. However, there is less awareness of the potential toxicity of herbal preparations and other botanicals, many of which are widely perceived by the public as being effective and harmless, and are commonly used for self medication without supervision. In addition, potential interactions between herbal medicines and conventional drugs may compromise with patient management. In the safety evaluation of most substances, non human primates are preferred to rodent species for preclinical animal safety studies, because of their biological similarity to humans. They are regarded to be the best metabolic models for humans in a broad range of investigations. Additionally, a disadvantage of using small animal species in toxicological testing is that they require higher doses of drugs and more frequent administrations than in larger species. In light of these considerations, vervet monkeys are used here to investigate toxicity of a plant-derived triterpene, oleanolic acid. The focus is to determine effects of different concentrations of this triterpene on the cardiovascular, hematopoetic, hepatic and renal systems. Materials and methods: 12 male vervet monkeys used in this study were equally divided into four groups, i.e. three treatment groups (4, 10 and 25 mg/kg bodyweight), and one control group. Each individual in a treatment group received a specified concentration of oleanolic acid in food for 16 weeks. Monkeys in the control group received the vehicle (food) alone. Bodyweight, body temperature, respiratory rate, heart rate, systolic pressure, diastolic pressure, and mean arterial pressure were recorded from ketamine-anaethetized monkeys at baseline and every second week until week 16. In addition, blood samples were collected at baseline and every fourth week for clinical biochemistry indicators (serum electrolytes, enzymes, proteins, lipids, nitrogenous compounds, bilirubins and glucose) and hematological tests (red cell count and its indices, hemoglobin, haematocrit, white blood cell and differential count and platelet count). Results: No animal showed deviation from their normal behavioral patterns, food and water intake, was in poor health or died during and after completion of the study. The average bodyweights were not statistically significantly different between controls and the treated groups. The biphasic changes in the average body temperature of treated monkeys were similar to those seen in the control group during the first eight weeks of the study. No statistically significant differences were found in body temperature determinations between controls and the treated groups. Fluctuations observed in the respiratory rates of the treated monkeys were not statistically significantly different from that of the control group. Although not statistically significantly different from the controls, the systolic, diastolic and mean arterial pressures in the group treated with 25 mg/kg oleanolic acid were lower at week 16 compared to baseline, while those of the groups treated with 4 and 10 mg/kg oleanolic acid were relatively unchanged. Except for a reduction in systolic pressure of the control group, other blood pressure parameters were stable. Heart rates in the treated groups were not statistically significantly different from those in the controls. In all groups, except the control, high density lipoprotein concentrations were higher at week 16 compared to baseline. Fluctuations in low-density lipoprotein and total cholesterol concentrations were similar between controls and the treated groups. The triglycerides were lower at week 16 compared to baseline for all four groups. Upward trends from baseline to the end of the study were observed in creatine kinase concentrations of the controls and the groups that received 4 and 25 mg/kg. Concentrations of this enzyme were unchanged in the group that received 10 mg/kg oleanolic acid between baseline and the end of the study. No statistically significant differences were found with cholesterol, triglyceride and creatine kinase concentrations between treated groups and the controls. Serum concentrations of aspartate aminotransferase were unchanged in the controls and the groups treated with 4 and 10 mg/kg oleanolic acid, but changes in this parameter over time were statistically significantly different (P = 0.0452) from the controls in the group that received 25 mg/kg oleanolic acid. Despite wide fluctuations in the alanine aminotransferase concentrations in the groups that received 4 and 25 mg/kg oleanolic acid, no statistically significant differences were found with any of the treated groups compared to the controls. No statistically significantly different changes were seen in alkaline phosphatase activities between controls and the treated groups. Reductions in gamma-glutamyl transferase activities in the groups that received 4 and 25 mg/kg oleanolic acid were not statistically significantly different from concentrations of this enzyme in the controls. In addition, no statistically significant differences were evident between controls and the group that received 10 mg/kg oleanolic acid. There were no statistically significantly different changes in the total and conjugated bilirubin and glucose concentrations between controls and the treated groups. Fluctuations over time in the serum albumin and globulin concentrations were similar between treated groups and the controls, whereas total protein concentrations were relatively constant. Consequently, no statistically significant differences were found between controls and the treated groups. Wide fluctuations were observed in the creatinine concentrations of the groups that received 4 mg/kg oleanolic acid, while no such changes were encountered in the controls and the group that received 10 and 25 mg/kg oleanolic acid. Serum urea concentrations increased in all groups over time, except for the group that received 10 mg/kg oleanolic acid. Both urea and creatinine concentrations in the treated groups were not statistically significantly different from concentrations in the controls. Serum concentrations of sodium, chloride, potassium, calcium and magnesium and phosphate in the treated groups were not statistically significantly different from these electrolyte concentrations in the controls. Decline in red cell and hemoglobin concentrations of the controls and the group that received 25 mg/kg oleanolic acid were not statistically significantly different between these groups. In addition, no statistical significant differences were found in red cell and hemoglobin concentrations between controls and the groups that received 4 and 10 mg/kg oleanolic acid. Controls and the treated groups showed upward trends in haematocrit concentrations. Mean corpuscular volumes were statistically significantly increased; P = 0.0027 (4 mg/kg), P = 0.0010 (10 mg/kg), and P = 0.0022 (25 mg/kg), while mean corpuscular hemoglobin concentrations were statistically significantly reduced; P = 0.0017 (4 mg/kg), P = 0.0004 (10 mg/kg), P = 0.0002 (25 mg/kg) in the treated groups as compared to the controls. No statistically significant differences were evident in the concentrations of mean corpuscular hemoglobin between controls and the treated groups. White blood cell counts of the treated groups were not statistically significantly different from those of the controls throughout the study period. No statistically significant differences were found in the differential white cells and platelet counts between treated groups and the controls. Discussions: The results of this study showed that administration of oleanolic acid had no effects on the general wellbeing, bodyweights, body temperature, respiratory and heart rates, and blood pressure of vervet monkeys. A statistically significant increase in the aspartate aminotransferase activity of the group treated with 25 mg/kg oleanolic acid, together with the increase in the alanine aminotransferase levels during the same time period, might indicate oleanolic acid-induced hypersensitivity, and accordingly hepatocellular alteration. However, since serum concentrations of these enzymes returned to baseline levels, as well as the absence of variations over time in other parameters of the hepatic function, particularly alkaline phosphatase activity, it is likely that there was no underlying subacute liver disease. Serum renal function parameters also appeared to be within normal physiological limits. No pronounced changes were observed in the hematological parameters of monkeys that received oleanolic acid. Conclusion: This study's results, suggest that oleanolic acid does not produce cumulative liver enzyme alterations, and has no detrimental effects on the renal, hematopoetic and cardiovascular systems of vervet monkeys.