200OVARIAN STIMULATION, LAPAROSCOPIC OOCYTE RETRIEVAL, IVF AND BLASTOCYST PRODUCTION USING SEQUENTIAL MEDIA IN THE AFRICAN LION (PANTHERA LEO)

Abstract

The objective of this study was to test the efficacy of Gardner’s Sequential Medium on in vitro embryo production in lions. Three young (3–4yr) nulliparous lionesses were treated with a total of 50 IU purified porcine FSH (Sioux Biochemical, Sioux Center, IA) in a decreasing regimen over 4 days. At 80h, they were chemically immobilized with a combination of tiletamine and zolazepam (Zoletil; Virbac) and a single deslorelin implant (Ovuplant, Peptech Animal Health Pty, Ltd., North Ryda, New South Wales, Australia) was placed (s.c.) in the lateral thorax over the scapula to stimulate final oocyte maturation. Approximately 24h later, the lionesses were again chemically immobilized (Zoletil); ovaries were viewed, structures recorded and oocytes retrieved laparoscopically (Table 1). Just prior to oocyte retrieval, four lions were chemically immobilized. Semen was collected by rectal probe electrostimulation and diluted 1:10 in five media (TL HEPES, modified Tyrodes+6mgmL−1 BSA+10% FCS, HEPES-Hams F10+10% FCS, Biladyl and Tyrodes+5% lion serum) and incubated at room temperature and ambient atmosphere for 6h before evaluation and use in IVF. Since there were no differences in sperm motility in any of these media (>90% after 6h), spermatozoa from each male were pooled from all treatments for IVF. A total of 6, 10 and 3 grade A oocytes (73%) from Lionesses #1, 2 and 3, respectively, were inseminated with approximately 1×106 lion sperm/mL in 0.5-mL wells containing G1 Sequential Medium and cultured for 16h in humidified 6% CO2 at 38°C. Then, presumptive zygotes were washed and cultured in 50-μL microdrops of G1 under oil. After 72h, embryos were washed and cultured in 50-μL microdrops of G2 under oil. At 110h post-insemination, 10 had cleaved (53%) and, of these, 5 (50%) developed to the morula stage and 3 (30%) to the blastocyst stage. In conclusion, the present study has shown that lion oocytes can be successfully fertilized in vitro. Furthermore, the in vitro-produced embryos can be cultured to the blastocyst stage in G1/G2 Sequential Media. Table 1 Ovarian structures observed and oocytes collected from three lionesses