177 IN VITRO MATURATION AND FERTILIZATION OF OVARIAN OOCYTES OF FREE-RANGING GEMSBOK (ORYX GAZELLA)

Abstract

The gemsbok is a large antelope native to arid regions of southern Africa. Listed by the International Union for Conservation of Nature as a species of least concern, the gemsbok is an excellent model for the development of assisted reproductive techniques for the closely related but critically endangered scimitar-horned oryx (Oryx dammah) and addax (Addax nasomaculatus). Gemsbok were introduced to the White Sands Missile Range by the New Mexico Department of Game and Fish to preserve the habitat by providing big game hunting, which ensures the support and lobby of hunters. Gonads were collected from hunted gemsbok and transported in PBS to the laboratory, where gametes were harvested. Testes were stored at 4°C in PBS until sperm were needed for IVF (18 to 28 h). Ovaries were sliced to release follicular oocytes, which were placed in maturation medium (TCM-199 with FCS, pyruvate, gentamicin and LH, FSH and oestradiol) for 20- to 22-h in vitro maturation culture at 38.8°C in 5% CO₂ in air. Oocytes were then washed [Tyrode lactate (TL)-HEPES with BSA, pyruvate and gentamicin] and placed in groups of 5 to 10 in 50-μL drops of IVF-TL medium supplemented with pyruvate, gentamicin and BSA. Sperm were allowed to swim out of sliced epididymides into room temperature IVF-TL medium and then equilibrated for 30 min at 38.8°C. Approximately 2 × 10³ motile sperm were added to each oocyte drop and incubated under oil for 21 to 23 h at 38.8°C in 5% CO₂ in air. Domestic cattle oocytes were matured in maturation medium at 39°C during shipment to the field site, where they were washed and transferred to IVF medium as described for gemsbok oocytes. Approximately 1.7 × 10³ motile gemsbok sperm were added to each drop and oocytes were incubated as described for gemsbok oocytes. At the end of IVF culture, oocytes of both species were stripped of granulosa cells and placed in embryo medium (SOF supplemented with BSA, essential and nonessential amino acids, pyruvate and gentamicin; all media formulations from Applied Reproductive Technologies, Madison, WI, USA) at 38.8°C in 5% CO₂ in air. Embryo culture medium was refreshed after 48 h. Embryos were removed from culture after 6 days, examined for cleavage and fixed in PBS:formalin for staining and further analysis. Although gemsbok sperm were capable of fertilizing domestic cow oocytes at the same rate (38.3%) as gemsbok oocytes (37.1%), the antelope oocytes cleaved at a higher rate (29% vs cattle at 15.3%). These results indicate that chilled epididymal gemsbok sperm is capable of fertilizing gemsbok and domestic cattle oocytes and that protocols designed for in vitro maturation and fertilization of cattle oocytes may be successfully used in the field to produce gemsbok embryos (Table 1).

Table 1.  Fertilization of in vitro-matured gemsbok and cattle oocytes by chilled epididymal gemsbok sperm

The authors thank the New Mexico Department of Game and Fish, Troylyn Zimmerly, Dana Powers and the Biology Department of New Mexico Institute of Mining and Technology.