Thesis (M.Sc. (Zoology)) --University of Limpopo, 2001. / A South African rehabilitation centre for illegally kept vervet monkeys required an evaluation of the genetic status of vervet monkeys , to determine whether animals from different geographical areas may be kept in the same enclosures and mixed during release back into the wild. Animals originating from three geographical regions (the former Transvaal, KwaZulu-Natal and the Eastern Cape) were studied using biochemical genetic and morphological approaches to address this question. The most prominent trend from allozyme data was derived from the locus PRT-2 (an unspecified serum protein), where each of the three populations could be characterized by the absence or presence of unique alleles. A significant deviation of genotypes from Hardy­ Weinberg equilibrium was found at the PGD-1 locus in all the populations studied. Nevertheless, statistical coefficients indicated little genetic divergence, with genetic distance values of 0.001-0.003, gene flow values of 4.300 - 16.310 and an overall fixation index value of 0.046. Average heterozygosity did not differ appreciably among populations (2.5-3.3%). The morphological study identified suitable traits, free from the influence of growth allometry, which can be used for inter-population comparisons. No significant morphological differences between conspecific populations were however found. It is concluded that vervet monkeys from the species' wider distribution range is relatively monotypic, but that monkeys from different geographical areas should not be unduly mixed, pending the results of finer grained molecular studies.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The present study describes molecular aspects of inherited congenital cataract in captive-bred Vervet monkeys. Congenital cataracts are lens opacities that are present at birth or soon after birth and include hereditary cataracts or cataracts caused by infectious agents. The MRC Primate Unit is housing a colony of captive-bred Vervet monkeys in which 7.5% is suffering from congenital cataract. However, the parents of the affected individuals were asymptomatic. Six families within the colony have been identified to be affected by two types of morphologies (Y-sutural and total cataract). Based on the evidence provided above, it was speculated that the colony was affected with autosomal recessive cataract. The main aim of this study was to facilitate a strategy for managing breeding programs by minimizing cataract occurrences in captive-bred Vervet monkeys. Integrated combination of clinical, molecular and bioinformatic strategies were used to identify and assess reciprocal candidate susceptibility genes for cataracts. The genes that are known to be responsible for most human congenital cataract cases were prioritized. The genes include Heat shock transcription factor 4 (HSF4), Crystalline Alpha A (CRYAA), glucosaminyl (N-acetyl) transferase 2 (GCNT2) and Lens intrinsic membrane protein 2 (LIM2). Twenty two subjects were selected based on their morphology (5 carriers, 5 controls and 12 cataracts). 2ml of blood was collected for Deoxyribonucleic acid (DNA) extraction. Coding exons and flanking regions were screened by polymerase chain reaction (PCR) amplification and sequenced. The CLC DNA workbench was used for results analysis. The screening of four genes revealed 20 sequence variants which were not present in the control individuals. Sequencing of HSF4 revealed three mutations: R116R, L245>L and P421>L in exon 5, 10 and 14, respectively. The coding exons for CRYAA showed two sequence variants: S134W and K166N in exon 3. Twelve mutations were identified in exon one of all three GCNT2 transcripts (A, B and C). These mutations include: G212G, H256>H, M258>V, N275>N, V16>I, Y122>F, S15>S, S24>N, S38>S, I118>I, D194>D and Y373>Y which was found in exon three of all transcripts. There were no mutations in LIM2, however, three single nucleotide polymorphisms (SNPs) were identified in exon 2 (P66>P) and 3 (I118>T and A127>T). The above mutations were conserved when aligned with other species. The sequence variations vary among the families and those individuals with the same or different cataract phenotype. Based on these findings, it can be concluded that the four candidate genes harbour mutations that are responsible for both phenotypes. The effect of these mutations in Vervet monkeys is not yet understood, however, their impact will be further investigated. For future studies, it will be of absolute importance to screen the entire family to verify that indeed cataract formation in this colony is inherited in an autosomal recessive manner.

>Magister Scientiae - MSc / Molecular genetics: strategies to indentify congenital cataract genes in captive-bred Vervet monkeys Zandisiwe Emilia Magwebu MSc thesis, Department of Medical Biosciences, University of the Western Cape The present study describes molecular aspects of inherited congenital cataract in captive-bred Vervet monkeys. Congenital cataracts are lens opacities that are present at birth or soon after birth and include hereditary cataracts or cataracts caused by infectious agents. The MRC Primate Unit is housing a colony of captive-bred Vervet monkeys in which 7.5% is suffering from congenital cataract. However, the parents of the affected individuals were asymptomatic. Six families within the colony have been identified to be affected by two types of morphologies (Ysutural and total cataract). Based on the evidence provided above, it was speculated that the colony was affected with autosomal recessive cataract. The main aim of this study was to facilitate a strategy for managing breeding programs by minimizing cataract occurrences in captive-bred Vervet monkeys. Integrated combination of clinical, molecular and bioinformatic strategies were used to identify and assess reciprocal candidate susceptibility genes for cataracts. The genes that are known to be responsible for most human congenital cataract cases were prioritized. The genes include Heat shock transcription factor 4 (HSF4), Crystalline Alpha A (CRYAA), glucosaminyl (N-acetyl) transferase 2 (GCNT2) and Lens intrinsic membrane protein 2 (LIM2). Twenty two subjects were selected based on their morphology (5 carriers, 5 controls and 12 cataracts). 2ml of blood was collected for Deoxyribonucleic acid (DNA) extraction. Coding exons and flanking regions were screened by polymerase chain reaction (PCR) amplification and sequenced. The CLC DNA workbench was used for results analysis. The screening of four genes revealed 20 sequence variants which were not present in the control individuals. Sequencing of HSF4 revealed three mutations: R116R, L245>L and P421>L in exon 5, 10 and 14, respectively. The coding exons for CRYAA showed two sequence variants: S134W and K166N in exon 3. Twelve mutations were identified in exon one of all three GCNT2 transcripts (A, B and C). These mutations include: G212G, H256>H, M258>V, N275>N, V16>I, Y122>F, S15>S, S24>N, S38>S, I118>I, D194>D and Y373>Y which was found in exon three of all transcripts. There were no mutations in LIM2, however, three single nucleotide polymorphisms (SNPs) were identified in exon 2 (P66>P) and 3 (I118>T and A127>T). The above mutations were conserved when aligned with other species. The sequence variations vary among the families and those individuals with the same or different cataract phenotype. Based on these findings, it can be concluded that the four candidate genes harbour mutations that are responsible for both phenotypes. The effect of these mutations in Vervet monkeys is not yet understood, however, their impact will be further investigated. For future studies, it will be of absolute importance to screen the entire family to verify that indeed cataract formation in this colony is inherited in an autosomal recessive manner.

Vervet Monkeys (Chlorocebus aethiops) are versatile primates of the suborder HAPLORHINI, family CERCOPITHECIDAE, subfamily CERCOPITHECINAE, and genus Chlorocebus (Skinner & Smithers, 1990). They are a widely distributed species that adapt easily to a variety of environments, occurring throughout the Northern and Southern Savanna, from Senegal to Sudan and south to the tip of Southern Africa (Estes, 1992). According to Estes (1992), vervets are opportunistic omnivores. being predominantly vegetarians that live on wild fruits, flowers, leaves, buds, seeds, pods, sap, roots and tubers. Occasionally they will feed on invertebrates (grubs, termites, grasshoppers) and vertebrates (bird and reptile eggs and chicks) (Skinner & Smithers, 1990). Not much ecological research has been done on vervets outside the tropics to date, and it was thus considered necessary to determine how vervets cope with the effects of temperate area seasonality. The aim of this study was to describe the habitat structure of a vervet monkey troop's territory and then to investigate the effects of seasonality on differences in their diet (both overall and with respect to sex differences). activity patterns and habitat utilisation. / Agriculture Animal Health and Human Ecology / M.Tech. (Nature Conservation)

Vervet Monkeys (Chlorocebus aethiops) are versatile primates of the suborder HAPLORHINI, family CERCOPITHECIDAE, subfamily CERCOPITHECINAE, and genus Chlorocebus (Skinner & Smithers, 1990). They are a widely distributed species that adapt easily to a variety of environments, occurring throughout the Northern and Southern Savanna, from Senegal to Sudan and south to the tip of Southern Africa (Estes, 1992). According to Estes (1992), vervets are opportunistic omnivores. being predominantly vegetarians that live on wild fruits, flowers, leaves, buds, seeds, pods, sap, roots and tubers. Occasionally they will feed on invertebrates (grubs, termites, grasshoppers) and vertebrates (bird and reptile eggs and chicks) (Skinner & Smithers, 1990). Not much ecological research has been done on vervets outside the tropics to date, and it was thus considered necessary to determine how vervets cope with the effects of temperate area seasonality. The aim of this study was to describe the habitat structure of a vervet monkey troop's territory and then to investigate the effects of seasonality on differences in their diet (both overall and with respect to sex differences). activity patterns and habitat utilisation. / Agriculture Animal Health and Human Ecology / M.Tech. (Nature Conservation)

Rehabilitation-release is a form of species reintroduction where sick, injured, or rescued animals are rehabilitated before release back into the wild. Published research on rehabilitation releases of rehabilitant non-human primates is limited, and released troop mortality rates are generally high or difficult to determine. The objective of this study was to add to the limited scientific literature on primate rehabilitation and release by investigating factors affecting survival rates and welfare of a rehabilitant troop of vervet monkeys (Chlorocebus pygerythrus rufoviridis) released in Malawi in 2016, using pre-existing datasets from the Lilongwe Wildlife Trust. I hypothesized that 1) higher social rank, more complete forest strata use, close proximity to troop members, and frequent predator vigilance would be associated with greater survival, and 2) rank stability/ group cohesion will be strong post-release, activity budgets will show low levels of stress-related behaviors, and behavioral diversity will increase post-release, suggesting welfare improvements. The Lilongwe Wildlife Trust troop had a survival rate of 36%, which is comparable to other vervet releases. Using a combination of linear modeling, survival analysis, and preliminary social network analysis, I found that being a juvenile, being more highly ranked, and being in close proximity to others was significantly associated with lower risk of death – but these results were not consistent and should be considered with caution. Contrary to predictions, forest strata use did not differ greatly across individuals despite differences in survival. Interestingly, the troop’s mean hourly count of predator vigilance decreased post-release, but this did not influence individual survival. In support of my predictions, the troop’s dominance hierarchy appeared stable post-release, group cohesion was strong, and activity budgets showed low levels of stress-related behaviors. However, mean behavioral diversity across individuals decreased post-release, contrary to predictions. These findings suggest that vervet dominance hierarchy, age, and social proximity may influence post-release survival with higher ranking individuals, juveniles, and highly socially connected individuals more likely to survive. Juveniles may be more ecologically adaptable than adults and so better able to survive in a new habitat. Lower ranked individuals, as well as those with low social connectedness, may be more disconnected from the troop while traveling or foraging, placing them at a higher risk of predation but more research is needed to confirm this. Decreased behavioral diversity post-release may have been caused by an increase in foraging and troop movement and generalized behavior categorization may have limited the accuracy of behavioral diversity measurements. Future studies that wish to use behavioral diversity to assess welfare should use highly specific ethograms to capture unique behaviors. Release troops may also benefit from pre-release feeding regimes, such as platform feeders, that encourage more complete canopy use as well as more time at the release site prior to the start of the rainy season. Predator-awareness training is highly recommended to strengthen anti-predator behaviors, especially if the troop has any wild individuals. Finally, the Lilongwe Wildlife Trust’s extensive pre- and post-release monitoring provides vital insight into the troop’s social dynamics, behavioral repertories, and overall survival. Other rehabilitation centers should follow this strategy, since all newly monitored and reported releases will add valuable information to the development of the vervet monkey rehabilitation and release program.

Thesis (Master of Environmental Health)--Cape Peninsula University of Technology, 2020 / Background: The development of metabolic disease is accompanied by changes in gut microbiota phenotype, including a decrease of beneficial bacteria and increase of pernicious bacteria of the gastrointestinal tract. A Western (high-fat and high-sugar) diet, sedentary lifestyle and altered gut microbiota diversity have been associated with an increased risk of developing metabolic diseases such as type 2 diabetes and its associated risk factor, obesity. Many researchers have studied the link between the gut microbiota and diet. Hence our in vitro study is aimed at investigating the potential prebiotic effect of an aspalathin-rich unfermented rooibos extract, Afriplex GRT™ and aspalathin on the faecal bacterial diversity of vervet monkeys fed Western diet. Methodology: A total of six vervet monkeys (Chlorocebus pygerythrus) were selected from monkeys fed either a maize based normal diet (standard diet group; n=3) or a high fat diet (Western diet group; n=3) for more than 5-years. Faecal samples were collected from the animals in both groups at the Primate Unit and Delft Animal Centre (PUDAC) between 7 – 9 AM. Faecal samples from the two groups were divided into culture-independent baseline samples (before culture) and culture-dependent samples (after anaerobic culture). The culture-dependent samples were cultured under anaerobic conditions at 37°C for 10 hours, with or without Afriplex GRT™ extract or aspalathin. Bacterial genomic DNA (gDNA) was extracted from all samples using the NucleoSpin® DNA Stool extraction kit. Purified gDNA was sent for metagenomic sequencing for 16S rRNA gene analysis of microbial diversity using an Ion Torrent Next-generation Sequencing platform. Results: Results indicated that the Western diet affects the abundance of several bacterial species. Afriplex GRT™ and aspalathin significantly enhanced the relative abundance of health promoting butyrate-producing bacteria such as Faecalibacterium prausnitzii in both standard and Western diet groups (p= 0.02 and p=0.04, respectively). A similar trend was observed in other beneficial bacteria such as Eubacterium spp., Sutterella spp., and Dorea longicatena. Conclusion: Based on the data observed, it can be suggested that Afriplex GRT™ has a beneficial prebiotic effect on gut microbiota diversity and gut health.

High temperatures are a significant biological stressor for mammals, which they may adapt to through behavioral changes, physiological plasticity, and via genetic adaptation. Savanna monkeys (genus Chlorocebus) have a wide climatic range in Africa south of the Sahara, making them a good model species for understanding adaptations to heat stress in primates. Savanna monkeys have been observed to behaviorally mitigate high temperatures, and genetic signs of selection in response to climate have also been found (specifically in relation to cold). In this study, I investigate whether there is genetic variation and evidence for selection related to function in a heat shock protein gene (HSPA1L) in 73 wild savanna monkeys ranging from equatorial Africa to the southern coast of South Africa. Given the important role of heat shock proteins in buffering heat stress, I hypothesized that genetic variation would be associated with maximum summer temperatures, as those are most likely to be warm enough to induce a heat shock response. I found 45 single nucleotide polymorphisms (SNPs) outside of Hardy-Weinberg Equilibrium, and 10 SNPs with significant integrated haplotype scores, only one of which was in a protein coding region (17:40210341; piHS = 2.20). Using phylogenetic least squares modeling I found that maximum temperature of the warmest month was strongly but not significantly associated with the frequency of a derived allele nested within a regulatory region for HSPA1L (17:40207386; piHS = 2.57; b = 0.044, p = 0.061) presumably experiencing selection. I discuss implications of these results for heat tolerance in primates and resilience to climate change.

Glycine-N-acyltransferase (GLYAT, EC 2.3.1.13) has been characterised in a number of species including: humans, chimpanzees, rhesus monkeys and bovines. The characterisation of GLYAT from various species contributes to a better understanding of the diversity of the enzyme which in turn might help improve the current understanding of detoxification in mammals. The GLYAT enzyme of both the chacma baboon and vervet monkey has not been characterised. In this project, tissue samples were obtained from a chacma baboon (Papio ursinus) and a vervet monkey (Chlorocebus pygerythrus) to determine the nucleic acid sequence that encodes GLYAT in these two species to broaden our current understanding on the diversity of GLYAT in primates. A liver of a chacma baboon was used to extract total RNA. Complementary DNA (cDNA) was synthesised using an oligo (dT) primer. An open reading frame (ORF) encoding GLYAT of the chacma baboon was amplified with a PCR (polymerase chain reaction) using primers designed from a human GLYAT transcript. The PCR product containing an ORF encoding GLYAT of the chacma baboon was cloned, sequenced and expressed. The recombinant GLYAT of the chacma baboon expressed well in bacteria, but was insoluble and did not have enzyme activity. A crude cytoplasmic extract was prepared from the liver of a chacma baboon. The objective was to compare enzyme activity between the native and recombinant GLYAT. The prepared liver extract from the chacma baboon was assayed for enzyme activity and compared to the activity in a liver extract from bovine, previously prepared by Ms M Snyders. Both the chacma baboon and bovine liver extracts had GLYAT enzyme activity. To obtain sequence information on vervet monkey GLYAT, leukocytes were isolated from blood obtained from a living vervet monkey. A human GLYAT gene sequence was used as a reference DNA sequence in the design of PCR primers that were used to amplify the exons of GLYAT of the vervet monkey. All six GLYAT exons were individually amplified and PCR products were sequenced. The sequences were combined to reconstruct an ORF encoding GLYAT of the vervet monkey. The ORFs coding the GLYAT of both chacma baboon and vervet monkey were found to be 888 bp long (excluding stop codon) and encoded a protein of 296 amino acids. A fragment of 1256 bp of the chacma baboon GLYAT transcript was sequenced. The two GLYAT ORF sequences were translated to amino acid sequences and aligned to that of GLYAT of primates obtained from the Ensembl sequence database. The GLYAT amino acid sequences of the chacma baboon, vervet monkey and rhesus monkey formed a related group, distinct from other primates. The chacma baboon and vervet monkey sequences were 99 % identical to the rhesus monkey sequence and 92.6 % identical to the human sequence. There were 4 new variations introduced by GLYAT amino acid sequences from the chacma baboon and the vervet monkey. The vervet monkey introduced an isoleucine in place of a valine at position 32 and an arginine in place of a histidine or glutamine at position 224. The chacma baboon introduced a tyrosine in place of isoleucine at position 201 and an arginine in place of histidine or glutamine at position 240. The knowledge generated in this project will broaden the understanding of GLYAT diversity relating to GLYAT in primates. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011

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).