Individually and group culture of sheep oocytes derived from early antral follicles

BACKGROUND-AIM To overcome the limited number of fully grown oocytes for in vitro embryo production (IVP) several techniques have been developed to exploit preantral follicles in humans and animals. The main challenge is to support follicular growth in vitro and reduce the degeneration rates. Recent works in cattle have demonstrated that long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) derived from early antral follicles support oocyte growth and the acquisition of meiotic competence (Barros, et al, 2020. Journal of Visualized Experiments, 161, p.e61625). METHODS To assess whether this protocol can be applied in the sheep, in this study, isolated COCs from sheep early antral follicles (350-400 μm) were retrieved by rupturing the follicle wall with a 21-gauge needle. Subsequently, they were washed and cultured as a single (control) or group (3 COCs) in a well of 96-well plate containing TCM199 supplemented with 0.15 μg/mL Zn sulfate, 0.0001 IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone and 1.7 μg/ mL Cilostamide. After five days long in vitro culture (LIVC), the healthy COCs were selected and subjected to in vitro maturation. RESULTS Results indicated that COCs cultured in groups (n=81) had lower degeneration rates compared with those individually cultured (n=88; 48.1 vs. 65.9%, respectively; Chi-squared test p<0.05). Also, group culture improved the ability of COCs to resume meiosis and reach the metaphase II stage compared to the individual culture (20.6% vs. 13.3%, respectively), although this difference is not statistically significant. Therefore, there may be a signaling pathway that enhances the integrity and growth of COCs in the group culture method. CONCLUSIONS In conclusion, we demonstrated that the LIVC in the sheep could support a gradual transition of the oocyte from immature to mature stage, especially in the group culture method. This technology can increase the source of fertilizable gametes in the preservation programs and gives a prospective approach to livestock selection programs or conservation of endangered species.