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.

Captive-breeding has been identified as an integral part of the conservation of threatened species. The black rhinoceros (Diceros bicornis) as a species is currently listed by the IUCN as critically endangered (CR), while the white rhinoceros’s (Ceratotherium simum) current status is near-threatened (NT). Three African rhinoceros subspecies currently occur in captive populations in regional population management programmes, namely the South-central black rhinoceros (Diceros bicornis minor), the Eastern black rhinoceros (Diceros bicornis michaeli) and the Southern white rhinoceros (Ceratotherium simum simum). Concerns have been raised, however, that these populations are not self-sustainable. This study aims to analyze the demographic parameters that contribute to the growth rates of the global captive populations of African rhinoceros. The study sets out to determine if these global captive populations are currently self-sustaining, which demographic factors have the most influence on the population growth rates, and whether or not 50- and 100- year targets set for the captive African rhinoceros populations are attainable. Demographic data from 1 January 2010 until 31 December 2010 were analysed through population census, life table and age structure analyses. Two additional concerns, namely poor reproductive performance of the female F1 generation and male-biased birth sex ratios, were also assessed. The results indicate that the captive populations of D. b. minor and C. s. simum are not self-sustainable, with population growth rates (λ) of 0.98 and 0.99, respectively. Diceros bicornis michaeli is the only subspecies with a growing population, with a population growth rate of 1.02. Sensitivity analyses conclude that fecundity rates, and not mortality rates, are the limiting factor to population growth in all three subspecies. While lifetime reproductive success values for D. b. minor and C. s. simum captive-born females are far lower than those of the founder generation, several factors need further investigation to determine the true cause of this. Birth sex ratio analysis shows no significant difference from parity for both black rhinoceros subspecies, however, quadratic logit regression conducted on the white rhinoceros data indicated a statistically significant male-bias. In all three subspecies, no significant link was found between maternal age and the sex of the offspring. Overall, the results of this study show that the 50 year and 100 year targets set at the GCAP workshop in 1992 are achievable. However, D. b. minor will only be able to achieve the target population sizes with an increase in fecundity rate of around 170% or alternatively, additional supplementation from the wild. Recommendations for the future include a global study of breeding husbandry at an institutional level, and the formalisation of a Global Species Management plan for D. b. minor. / Dissertation (MSc)--University of Pretoria, 2014. / I would like to acknowledge several organisations and people that have supported me throughout this MSc. The National Research Foundation (NRF), which provided me with the funding for my studies. My employer, The National Zoological Gardens of South Africa, a research facility of the NRF, and particularly the CEO, Dr Clifford Nxomani and my manager, Dr Abeda Dawood, who gave me support, encouragement and most importantly, time off to concentrate on this thesis. A special heartfelt thanks goes to my supervisors, Prof. Chris Chimimba, who never gave up on me, even though this study took a little bit longer than it was supposed to, and Dr Ed Stam, whose dedication, patience and endless comments (and good cappuccinos) helped shape this thesis. Lastly, my husband David, who always encourages me to do better and never allows me to become complacent; thank you, my love, for always pushing me to achieve. / Zoology and Entomology / MSc / Unrestricted