The aim of this study was to define the ovarian follicular reserve of wild African elephants in terms of its type of small follicles (SF), its establishment and distribution throughout the ovaries, and the change in numbers of SF in the embryo and fetus as well as throughout prepubertal and adult life. The large elephant population in Zimbabwe provided the opportunity to collect ovaries from elephants culled for management reasons and hunted professionally. In total, gross morphological and histological studies were done on the gonadal ridges from 5 embryos (76–96 days post conception) and ovaries from 11 fetuses (4.8–22.2 months), 29 prepubertal females (2 months–10 years), 24 adult females (11–55 years) and 7 aged females (56–70 years). Specimens were fixed in 4% buffered formalin before a series of 25 ìm thick sections were cut and examined using stereological protocols to count SF numbers in each section and thereby calculate the follicle reserve of the whole ovary. Prior to counting SF numbers, their distribution throughout the ovary was studied and the repeatability of counts was validated. Numbers of SF were highest in mid-term fetuses, lower in fetuses during the second half of gestation, even lower in calves younger than 4½ years, whereas the numbers in calves aged 4½–9 years were significantly higher than those in younger calves, and similar to what they were in late-term fetuses. The numbers of SF were substantially and highly significantly lower in elephant 10–15 years in age compared to calves aged 4½9 years, suggesting a reduction around puberty. Thereafter the ovarian reserve fell steadily until depletion around the age of 70 years. During adult life the ovarian reserve was composed of early-primary (EP) and true-primary (TP) follicles. By 45 years of age only TP follicles remained although these enabled oestrous cyclical activity for many more years; of 7 sets of ovaries recovered from females aged 57–70 years, 6 showed evidence of cyclical activity or pregnancy within the preceding 6 years. The study shows that EP and TP form the follicular reserve from before birth until 45 years, with TP forming the reserve thereafter, which depletes in some old elephants and persists to maximum life span in others. / Thesis (PhD)--University of Pretoria, 2012. / Production Animal Studies / 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.