In vitro maturation of Southern white rhino (Ceratotherium simum simum) oocytes

Abstract

Currently there are only two northern white rhinoceros left in the world, both are females. The decline in numbers, to the point of extinction, can be attributed to poaching, habitat destruction and poor reproductive management. The Southern White Rhinoceros is the closest relative to the northern white rhinoceros and can therefore be used as a model for the research of assisted reproduction technology. For the Southern White Rhinoceros these techniques will also be critical to support breeding programmes in captive animals and to increase genetic diversity in current populations. Assisted reproduction technologies in rhinoceros include semen collection and freezing, artificial insemination, ovum pick-up, in vitro fertilisation and culture and embryo transfer. Since there are only two females left of the northern white rhinoceros, the production of an embryo from collected oocytes and frozen northern white rhinoceros semen is the only way to save the species.

The first step would be the successful and repeatable collection of oocytes from cows. In South Africa this step is already achieved by Dr Morne de la Rey. The next step would be the in vitro maturation of the oocytes to reach Metaphase II and produce visible polar bodies. A visible polar body is an indicator of successful maturation and that the oocyte is ready for fertilisation. After fertilisation the presumptive zygotes are cultured until reaching blastocyst stage. Once reaching blastocyst stage, the embryos would be suitable for transfer into a surrogate cow. The surrogate cow would need to be at the correct stage in her cycle to receive an embryo so that it may result in a pregnancy.

This study focuses on the in vitro maturation of Southern white rhinoceros oocytes. From collected data an analysis was performed to determine the influence of various factors on the maturation outcome of oocytes. In terms of the environment, factors like season and housing conditions were evaluated. The influence of age on number of follicles aspirated, number of oocytes collected and eventual maturation was considered. Some of the cows were stimulated with GnRH to achieve a higher number of follicles for aspiration and subsequently more oocytes. The influence of this stimulation on oocyte maturation was considered. The main goal of this study was to assess the influence of the media used for maturation on the maturation rate. The first media was formulated by the San Diego Zoo and the second adapted from this formula by Embryo Plus. The biggest difference was the base media. San Diego Zoo media uses TCM199 as a base media where the Embryo Plus media uses DMEM/F12. DMEM/F12 has a higher glucose content than TCM199. Another adaption to the San Diego Zoo media was the substitution of horse follicular fluid with lactic acid and MEM amino acids. Factors such as volume of media, number of oocytes cultured together and CO2 percentage during maturation were also evaluated.