230 IN VITRO MATURATION OF OOCYTES FROM ENDANGERED DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA)

Abstract

In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing of COCs were performed in warmed TCM-199-HEPES with antibiotics and polyvinyl alcohol. Degenerated oocytes or those with expanded cumulus cells were removed. A total of 15 COCs were cultured in TCM-199 with 10% heat-treated fetal calf serum, 10 μg/mL ovine FSH/LH, 1 µg/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO₂/air with high humidity. After 24 h of culture, matured oocytes, as revealed by the presence of a polar body, were activated with 7% ethanol for 10 min and further incubation for 3 h. Meiotic progression and activation were evaluated by staining with Hoechst 33342 and propidium iodide (1 μg/mL each) and visualization under a fluorescence microscope. Results at the end of incubations showed that 4/15 oocytes were degenerated, 4/15 were arrested at the MI stage, and 7/15 (46.7%) progressed to the MII stage. One oocyte was found to be at the 2-cell stage but it could not be established whether this was the result of the activation method used. These results demonstrate that it is possible to recover viable oocytes several hours after death and rescue them for subsequent in vitro maturation and fertilization. More studies are needed to characterize suitable conditions for oocyte maturation, fertilization, and culture in the dorcas gazelle. This would, in turn, help in the effort to rescue biomaterials from wildlife for generating offspring.

This work was supported by the Spanish Ministry of Education and Science (REN 2003–01587) and Acciones Integradas (HI20030336).