A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree Doctor of Philosophy. Johannesburg 2017 / The translocation of rhinoceroses is extremely stressful to the animal and this may strongly affect the success of translocation. The objectives of this study in white rhinoceroses were to (i) validate a non-invasive assay using faecal metabolites to assess the stress response in the white rhinoceros, (ii) to assess the stress response in the white rhinoceros associated with capture, handling, transportation and confinement, to be able to predict at capture which animals will adapt to confinement, and (iii) to investigate the use of the acute phase proteins (APP), serum amyloid A (SAA) and haptoglobin (Hp) as indicators of stress associated with capture and confinement. To achieve these objectives blood and faecal samples were collected from each rhinoceros at capture, and whenever possible, during confinement. To assess the hypothalamic-pituitary-adrenocortical axis (HPA) response of white rhinoceroses an ACTH challenge was performed and plasma cortisol and faecal glucocorticoid metabolites (FGM) were measured with a commercially available 125I RIA kit and a 125I corticosterone RIA kit, respectively. Gastrointestinal transit time was estimated with the use of an inert marker. Results showed a 4-to 8-fold increase in plasma cortisol within 15 to 20 minutes and a 3-fold increase in FGM concentration 60 to 90 hours later. Although this FGM measurement is non-invasive, relevant and robust, the time required to complete the extraction and assay could take several days. This measurement can be advantageous to monitor the stress of animals in confinement but has no application where animals are captured and immediately translocated. Measurement of plasma cortisol and FGM was also used to assess stress in rhinoceroses following transportation and in confinement. The results show that rhinoceroses have variable individual responses to capture and confinement and although there was evidence of behavioural habituation, HPA activity showed that there was no physiological habituation. We could not establish any predictor of success of habituation in the boma environment. The APP results showed that Hp is more likely to be an indicator of metabolic stress; rather than physical and psychological stress; while SAA responds rapidly to physical and psychological stress in the rhinoceros. It was also found that plasma cortisol was positively associated with SAA concentration when the animal is transferred to the boma, and that SAA may be a potential plasma biomarker to identify animals which could be at risk of failing to habituate to confinement. / MT 2018

Small populations of animals are vulnerable to the consequences of breeding within a closed group – inbreeding depression and genetic drift lead to reductions in genetic variability, which in turn can give rise to the amplification of deleterious traits. Traditionally, managers attempt to minimise these effects by controlling the genetic structure via a manipulation of the paternal line, in the case of rhinos usually by translocation of breeding and sub-adult bulls. This strategy depends on having access to detailed pedigree data, and, in particular, some knowledge of the parentage of the offspring within the population. This information is particularly difficult to obtain in long-lived, free-ranging populations (particularly for rare and endangered species) where the identity of the father must be inferred from behavioural observations, and often the identity of the mother cannot be determined once juveniles have dispersed. In this thesis I present the results of a study to determine the parentage within a free-ranging, enclosed population of southern white rhinoceros (Ceratotherium simum simum) located on Ongava Game Reserve (OGR) in the north of Namibia. I used genetic techniques to obtain a genotype for each animal in the population, and then used detailed reserve management records from the period 1993-2009 to constrain the statistical process of parentage assignment. Using these different methods, I was able to assign both parents for 22 of 23 offspring with 80% confidence (16 of 23 at 95% confidence, mother only in 1 of 23), making this study the first to successfully complete a comprehensive parentage analysis in a free-ranging population of southern white rhinoceros. The key to the success of this study was a combination of accurate pedigree data and a complete set of genotype data. The parentage assignments allowed me to construct a complete lineage diagram for each of the founder matrilines, and further analyse the status and reproductive success of the population. OGR’s southern white rhinoceros population is expanding at close to 14%, well over the expected maximum growth rate for rhinoceros metapopulations (9%). The mean inter-calf interval is about 2.2 years and average age at first parturition is 6 years, indicating good fecundity. Conception is strongly seasonal, occurring mainly (89%) during the rainy season. The current management practice is to replace dominant bulls after their breeding tenure, and also to remove all young bulls from the population before they reach breeding age (with the aim to restrict potential inbreeding). Only one calf of seven in the F2 generation is inbred. My analysis indicates that, at least in the 2006 cohort of eight calves, founder females bred only with the founder male, while offspring females (F1 generation) bred only with introduced males. This suggests some form of mate selection leading to inbreeding avoidance within the population. Finally, there is some evidence that certain matrilines exhibit/experience different reproductive potential (daughters in one matriline exhibit longer inter-calving intervals and male bias in calf birth sex ratios). These details indicate that genetic data provide valuable information for management. When reviewing management decisions to date, I found that one of the founder bulls was the more successful in terms of calves sired (10 of 13). Management had, however, selected the other founder bull for removal by sale based on the assumption that he was behaviourally dominant and territorial and therefore likely to have been more successful at breeding. I also found that introduced bulls were breeding successfully before they appeared to have established territories. Thus these findings challenge the assumption that male white rhinoceros reproductive success is related to dominant, territorial behaviour. I conclude that in order to optimally manage small, free-ranging enclosed populations of southern white rhinoceros it is essential to have reliable and accurate pedigree data (this includes a methodology for identifying individual rhino), as well as genetic data for the entire population. I recommend that conservation management programmes for rhinoceros populations incorporate both genetic and demographic data. This will allow for the development of white rhinoceros population management strategies that attempt to optimize genetic diversity and population health, and benefit the establishment of new, robust populations. Translocations of animals are an important aspect of meta-population management of rhinoceros and data which provides accurate insight into the true mating system and reproductive success within a population allows for the correct selection of individuals for this process. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Zoology and Entomology / Unrestricted

White rhinos in the Umfolozi Game Reserve (UGR) are managed with a 'Sink Management Policy' which utilises natural dispersal patterns of white rhinos from an area of high density (the core), to areas of low density (vacuum zones). This study was initiated to determine how white rhinos utilised the vacuum zones, as the management staff of the UGR felt that white rhinos were not dispersing into them but utilising them only as areas of trophic resources. Significantly different white rhino densities were established in the Makhamisa and Masinda vacuum zones by removing white rhinos from Masinda. Changes in the density of white rhinos were monitored and the age and sex compositions determined. No significant changes in the density of white rhino groups were recorded during the study, however, the density of individuals in Makhamisa increased significantly in the dry season, while the density in Masinda did not change. The reason for this increase could not be determined, however, the most likely explanation was that rhinos moved toward the Makhamisa study area in search of surface water which was present in the White Umfolozi River. During the dry season the water level in the Umfolozi River was low so the white rhinos where able to cross into the study area. However, owing to the above average rainfall, resources were not limited in either study area, and most likely in most of the reserve. Thus, it could not be determined why white rhinos would need to enter the Makhamisa study area. Changes in grass height, grass colour, and the availability of water in pans and streams were monitored throughout the study. The grassland community compositions of the two study areas were found not to be significantly different. The total utilisation of the different grassland communities by white rhinos during the study were determined for both study areas. White rhinos in the two study areas utilised the grassland communities in a similar pattern throughout the study. During the wet and dry seasons, white rhinos primarily foraged in the short grassland community (Panicum coloratum & Themeda-Urachloa), their staple grassland community, and were not observed to switch and start foraging in the tall grassland community (Themeda & Themeda-Panicum). their reserve community. White rhinos in both study areas were able to forage in their stable grassland community throughout the dry season because of the above average rainfall experienced during the study The results of the study suggest that white rhinos outside the study areas did not utilise the vacuum zones for trophic resources. However, as the study was conducted in a year with above average rainfall, these findings may not represent how white rhinos utilise the vacuum zones in years with average or below average rainfall. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

Capturing and holding of white rhinoceros (Ceratotherium simum) for the reintroduction to new reserves or breeding in zoos often involves a risk of mortality. Non-invasive techniques to monitor the stress experienced by these animals may guide the selection of management techniques that reduce risks to animal well-being. The aim of the study was to evaluate the biological relevance of a developed technique to monitor stress hormone metabolites in faecal samples of wild-caught and captive-bred white rhinoceros. Faecal corticosterone concentrations were measured via radioimmunoassay (125I RIA), in seven white rhinoceros (3 males and 4 females), at three sites, before and after an adrenocorticotropic hormone (ACTH) challenge test and control saline injection. Administration of ACTH resulted in a significant increase in faecal corticosterone concentrations (up to 350% above pre-ACTH baseline) within 1-2 days of injection, returning to baseline 4 days post-injection. It was found that individual baseline corticosterone concentrations fluctuate naturally and vary between individual animals, suggesting that an adequate baseline period of faecal sampling is needed in order to accurately assess responses to ACTH stimulation. Furthermore, the technique proved sensitive enough to detect elevations in faecal corticosterone concentrations due to environmental stressors. Data of faecal corticoid concentrations were correlated with gastrointestinal transit (GIT) times before and after ACTH and saline treatment by using art glitter as a digestive marker. This showed that gut passage times correlated to the ACTH-induced time to peak. Overall the results confirm that measurements of faecal corticosterone metabolites with the validated 125I RIA is a useful diagnostic tool to monitor adrenocortical activity in white rhinoceros. This study can therefore provide a methodology for examining chronically heightened adrenal activity in these animals and consequently be used to inform management strategies that aim to improve the welfare of white rhinoceros in captivity. / Dissertation (MSc)--University of Pretoria, 2011. / Anatomy and Physiology / unrestricted

As part of the larger survey to map the geographical distribution of Babesia and Theileria parasites in the Southern African rhinoceros population, white rhinoceroses were sampled during routine immobilizations in the Kruger National Park. Polymerase Chain Reaction (PCR) and Reverse Line Blot (RLB) hybridization assays were used to screen for the presence of haemoprotozoa and complete blood counts were used to assess associated changes in clinical parameters. Of the 195 rhinoceroses sampled, 36.4% tested positive for the presence of Theileria bicornis, with no significant change in the haematological parameters measured. None of the rhinoceroses sampled tested positive for Babesia bicornis, the parasite linked to mortalities in black rhinoceroses. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Philosophiae Doctor - PhD / The survival of free-ranging (in situ) African elephant and Southern white rhinoceros populations are currently being challenged on a daily basis in Africa. Reproductive health is considered a vital component of species conservation. Conservation of the last mega land mammals may ultimately require intervention by breeding management or combined with assisted reproductive technologies (ART). There is a strong case for gathering baseline information, both physiological and biological, of any species, as opportunities arise. During this study a total number of 21 ejaculates collected over two seasons from 12 free-ranging African elephant bulls were characterised, as well as 10 ejaculates collected from 10 free-ranging Southern white rhinoceros bulls from two populations. Ejaculates were collected from adult bulls by means of electroejaculation under anaesthesia. Routine semen analysis was combined with Computer-aided sperm analysis (CASA), Computer-aided sperm morphology analysis (CASMA), Transmission electron microscopy (TEM) and Genomics as diagnostic tools. Additionally, sperm functionality within different media was investigated and sperm subpopulation classification according to the motion pattern displayed. The results presented is based on the evaluation and classification of ≈ 45 000 individual African elephant spermatozoa and ≈ 18 000 individual Southern white rhinoceros spermatozoa. The average elephant ejaculate contained a total number of 47 x 10⁹ spermatozoa (volume of 56 ± 38mL x concentration of 818 ± 750 x 10⁶/mL) that recorded a total motility of 81 ± 29% of which 62 ± 26% were progressively motile. CASA recorded velocities for curvilinear velocity (VCL 241 ± 58μm/s), straight-line velocity (VSL 173 ± 181μm/s) and average path velocity (VAP 201 ± 54μm/s), and kinematics at straightness of track (STR 86 ± 85%), linearity of track (LIN 67 ± 16%), amplitude of lateral head displacement (ALH 4 ± 0.75μm) and beat cross frequency (BCF 21 ± 3Hz). Structural analysis revealed 68 ± 11% of the spermatozoa were viable (intact plasma membrane) and 77 ± 11% maintained acrosome integrity. Ejaculates contained 55 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 6.83 ± 0.26μm and width 3.32 ± 0.18μm (total head area of 20.17 ± 1.96μm²) of which 38.95 ± 0.92% is covered by an acrosomal cap. The average rhinoceros ejaculate contained a total number of 1.1 x 10⁹ spermatozoa (volume of 24 ± 24mL x concentration of 83 ± 96 x 10⁶/mL) that recorded a total motility at 82 ± 8% of which 28 ± 23% were progressively motile. CASA recorded velocities for VCL (85 ± 29μm/s), VSL (44 ± 25μm/s) and VAP (69 ± 30μm/s, and kinematics at STR (63 ± 14%), LIN (51 ± 16%), ALH (2 ± 0.16μm) and BCF (16 ± 6Hz). Structural analysis revealed 73 ± 10% of the spermatozoa were viable (intact plasma membrane) and 76 ± 4% maintained acrosome integrity. Ejaculates contained 62 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 5.5 ± 0.17μm and width 2.9 ± 0.19μm (total head area of 14.8 ± 1.43μm²) of which 36.3 ± 0.59% is covered by an acrosomal cap. Based on a Boolean argument and CASA data exploration it was possible to derive elephant and rhinoceros CASA cut-off criteria to sort between activated and hyperactivated motile spermatozoa. For the genomic component of this study, the CatSper1 (Loxodonta africana) gene was identified,sequenced and verified in a free-ranging (natural) African elephant population. Multivariate analysis(MVA) was applied to examine the associations between the semen and sperm parameters and the traits they accounted for in this study. Our understanding of wildlife reproductive sciences can substantially progress as the analytical techniques applied and the combination thereof is expanded. This investigation presents a new set of comprehensive semen and sperm threshold values for future investigations.

The main aim of this study was to classify and describe the plant communities in the home range of white rhinoceros in order to understand the animal seasonal foraging ecology. To provide a detailed habitat description, forty sample plots were stratified randomly. A TWINSPAN classification, refined by Braun-Blanquet procedures, was carried out on the Viewpoint section of the reserve. Ten plant communities, grouped into five major community types, were identified. The veld condition and ecological carrying capacity in these communities were measured, stating an abundance of food on long grass while the short grass equal the number of short grass feeders. A number of behavioural aspects such as activity data, home range utilization and dietary usage were examined. Statistical methods such as the Spearman rank-order correlation, Wilcoxon sign test and Student T-test were used on rhino data. The difference in usage of the home range during wet and dry seasons was insignificant. / Environmental Sciences / M.Sc. (Environmental Management)

The main aim of this study was to classify and describe the plant communities in the home range of white rhinoceros in order to understand the animal seasonal foraging ecology. To provide a detailed habitat description, forty sample plots were stratified randomly. A TWINSPAN classification, refined by Braun-Blanquet procedures, was carried out on the Viewpoint section of the reserve. Ten plant communities, grouped into five major community types, were identified. The veld condition and ecological carrying capacity in these communities were measured, stating an abundance of food on long grass while the short grass equal the number of short grass feeders. A number of behavioural aspects such as activity data, home range utilization and dietary usage were examined. Statistical methods such as the Spearman rank-order correlation, Wilcoxon sign test and Student T-test were used on rhino data. The difference in usage of the home range during wet and dry seasons was insignificant. / Environmental Sciences / M.Sc. (Environmental Management)

As the dry season progresses across southern Africa, the availability and quality of food declines for large herbivores. Female white rhinos compensate for these declines by expanding and/or shifting their home ranges. These changes may be to incorporate habitat types that contain high quality food or quite simply more food. To determine the factors that drive these seasonal changes in home ranges, I focused on dry season changes in the availability and quality of grass in habitats utilised by white rhinos in the Ithala Game Reserve, South Africa. I expected that if food quality was the main driver, white rhinos would follow optimal foraging principles and incorporate habitat types with the highest nutritional quality into their dry season home ranges. Alternatively, due to their large body size (>1000 kg) and thus ability to survive on low quality food, they may rather incorporate habitat types with high food availability. In contrast to previous studies, I found that during the dry season female white rhinos did not increase the size of their home ranges, but rather shifted their home range boundaries. This resulted in individuals increasing the amount of Bushveld and decreasing the amount of Wooded Grasslands within their dry season home ranges. When I explored the different factors that could explain these patterns, I found that changes in the crude protein content of grass was the key factor driving the incorporation and exclusion of habitat types in the home ranges. During the dry season, white rhinos incorporated the habitat that had the smallest seasonal reduction in crude protein content, while excluding the one with the largest decrease in crude protein. As a result, my results suggest that the search for high quality best explains the seasonal home range shifts of female white rhinos in the Ithala Game Reserve. / Thesis (M.Sc.Ecology)-University of KwaZulu-Natal, Pietermaritzburg, 2013.