225 IN VITRO MATURATION OF LION OOCYTES

The potential for rescuing immature oocytes from the ovaries of females of rare species of felids that die or undergo ovariohysterectomy has been reported in only two studies (Johnston et al. 1991 Biol. Reprod. 45, 898-906; Jewgenow et al. 1997 J. Reprod. Fertil. Suppl. 51, 33-39), in which oocytes were maturated in complex media (Eagle's MEM, M199, respectively). The domestic cat is used as a research model for endangered species but there may be some differences that perturb the adaptation of in vitro maturation (IVM), fertilization, or culture methods of lion oocytes compared to cat oocytes. Therefore, in the present study we evaluated the in vitro response of lion oocytes to our system for in vitro maturation of domestic cat oocytes in a simple medium (Merlo et al. 2005 Theriogenology 63, 2032-2039). A 14-year-old lioness, referred to our clinic because of pyometra, underwent ovariohysterectomy. After premedication with acepromazine 0.1 mg/kg i.m. (Prequillan; ATI, Bologne, Italy) and ketamine 5 mg/kg i.m. (Ketavet 100; Intervet, Boxmeer, The Netherlands), anesthesia was induced with ketamine 0.05 mg/kg i.v. and diazepam 0.02 mg/kg i.v. (Diazepam; Intervet) and maintained with isoflurane (Forane; Abbott, Rome, Italy) after intubation. Ovaries were removed and stored in saline solution at room temperature until collection of oocytes (within 1 h). A total of 53 small follicles (2-4 mm), five corpora lutea and a 15-mm follicle were present on the ovaries. Oocytes were collected by aspirating visible follicles of each ovary with a 21-ga needle connected to a vacuum pump (K-MAR-5100; Cook Australia, Brisbane, Australia) at -75 mmHg; then CL were removed and the ovaries were minced with a scalpel blade in a 60-mm petri dish containing HEPES-SOF (H-SOF) for recovery of additional oocytes. A total of 45 oocytes were recovered, of which 19 were degenerate (42.2%); of the remaining 26, 12 were fully surrounded by cumulus cells, 9 had only corona radiata, and 5 were denuded. Degenerate oocytes were discarded and all other oocytes were washed twice in H-SOF and matured in a 35 mm petri dish containing SOFaaBSA 5 mg/mL plus 0.1 IU of porcine FSH-LH (Pluset; Laboratorios Calier, Barcelona, Spain), 25 �L/mL insulin-transferrin-selenium (ITS) (Sigma, Madrid, Spain), 1.2 mm l-cysteine (Sigma), and 25 ng/mL epidermal growth factor (EGF) (Sigma) for 24 h in a humidified atmosphere of 5% CO2 in air at 38.5�C. After maturation, cumulus cells were removed by pipetting oocytes into a 0.25% trypsin solution for 2 min. Then, denuded oocytes were washed once in H-SOF plus 10% FCS to inactive trypsin and twice in H-SOF before being stained with Hoechst 33342 (10 �g in 10 mL PBS) for 30 min at room temperature. After washing in PBS, oocytes were observed using fluorescence microscopy to determine maturation rate. Oocytes in telophase I or metaphase II were considered mature. Of 26 oocytes, 19 (73.1%) were mature and 7 (26.9%) were at the GV stage. These results demonstrated that lion oocytes can undergo successful IVM at a frequency that is similar to that of cat oocytes cultured in the same system (76.9%, P > 0.05). Furthermore, the maturation rate obtained in a simple medium was higher or similar to those previously reported (mean: 22.9% and 69.7% respectively, by Johnston et al. 1991 and Jewgenow et al. 1997).