241 TIME POINTS FOR IN VITRO MATURATION OF LION (PANTHERA LEO) OOCYTES

Abstract

The objective of the study was to establish a time point for successful in vitro maturation of lion (Panthera leo) oocytes using a model developed for the domestic cat (Gomez et al. 2003 Theriogenology 60, 239–251). As part of a game reserve management program, one adult free-ranging lioness (6–7 years old) and her 3 sub-adult cubs (18 months old) were chemically immobilized with 500 mg of a combination of tiletamine and zolazepam (Zolatil 100^®; Virbac SA, Carras, France) by remote injection (Dan-inject^®; Dan-Inject ApS, Borkop, Denmark) and within 20 min euthanized using 8 g sodium pentobarbitone (Euthapent^® KruVet, SA). Ovaries collected from the adult and 2 sub-adult females were transported to a laboratory in a flask containing warm (37°C) sterile saline. Within 2 h of collection, all visible follicles were aspirated using a 21G needle attached to a 5-mL syringe. To increase the number of recovered oocytes, ovaries were minced using a scalpel blade in a 60-mm Petri dish containing warm search medium (HEPES-buffered TCM-199, 2.2 mM Ca lactate, 0.36 mM pyruvate, 2 mM glutamine, 1.12 mM cysteine, 0.3% w/v fatty acid-free BSA, and 50 µg mL^-1 gentamicin). A total of 33 and 54 oocytes were recovered from the adult and sub-adult females, respectively, and cultured in 35-mm Petri dishes containing 3-mL of maturation medium (sodium bicarbonate-buffered TCM-199, 1 IU mL^-1 hCG, 0.5 IU mL^-1 eCG, 2.2 mM Ca lactate, 0.36 mM pyruvate, 2 mM glutamine, 1.12 mM cysteine, 0.3% w/v fatty acid-free BSA, and 50 µg mL^-1 gentamicin). Petri dishes containing oocytes were enclosed in a sealed plastic bags, filled with a humidified gas mixture of 5% CO₂, 5% O₂, and 90% N₂, and incubated at 38.8°C. After 26, 32, or 38 h of incubation, groups of 29 oocytes were fixed in 3 : 1 ethanol : acetic acid solution and stored at 4°C for 48 h. Fixed oocytes were stained with 1% w/v orcein and visualized with phase-contrast microscopy. Oocytes in telophase I or metaphase II were classified as mature. Each ovary had an average of 22.5 ± 3.0 antral follicles, where 8.8 ± 2.0 were 2-3 mm, and 13.8 ± 1.5 were 1 mm in diameter. There were no CLs present. Out of 87 oocytes recovered, 24.0 ± 3.7% had a uniform cytoplasm and >3-4 layers of cumulus cells, 42.2 ± 6.0% had a uniform cytoplasm and 2 or less layers of cumulus cells, and 33.8 ± 9.7% had no cumulus cells attached. None of the oocytes were mature at 26 h, but at 32 h and 38 h, the percentage of matured oocytes significantly (P < 0.05) increased to 63.9 ± 13.9% and 80.4 ± 7.1%, respectively. These results indicate that the domestic cat system used herein can be successfully applied for in vitro maturation of lion oocytes. However, unlike oocytes from a domestic cat, lion oocytes required a culture period of 32 to 38 h to reach metaphase II. Further studies are required to confirm these findings and to test fertilization rates of such matured oocytes, and their ability for further development.