Development of an in vitro culture system to support the growth of immature oocytes collected from early antral follicles

In vitro embryo production currently relies on oocytes from large antral follicles, leaving a significant proportion of ovarian follicles unused. This has led to a growing interest in developing technologies for in vitro maturation of oocytes from earlier follicular stages, such as early antral follicles (EAFs). EAFs are abundant and near the fully grown stage, making them a promising source. Various culture systems have been created for EAFs in different species, but challenges remain, including limited embryo production and species-specific differences. To address these issues, a Long In Vitro Culture (LIVC) system has recently been developed, demonstrating notable enhancements in embryonic production, particularly in cattle. This study aimed to test LIVC technology in sheep to enhance mature oocyte production and fertility preservation. We assessed the effects of seasonality and culture conditions on oocyte morphology, viability, and meiotic competence. Initial findings showed LIVC's effectiveness in sheep, especially during the breeding season, in isolating higher-quality cumulus-oocyte complexes (COCs) from EAFs. In a subsequent study, we compared single versus group cultures of COCs to determine the best conditions for promoting meiotic competence. Group culture proved more effective, enhancing global transcription activity and meiosis resumption. The final study focused on comparing the LIVC system's efficiency in lambs and adult sheep. Although some parameters were comparable, COCs from lambs showed lower developmental competence than those from adult sheep, highlighting age-specific variations in oocyte quality and culture response. In conclusion, our studies confirmed the LIVC system's feasibility in sheep and provided insights for expanding fertilizable gamete sources and optimizing culture techniques. Further research is necessary to understand the underlying mechanisms and refine protocols for broader application in reproductive biotechnologies.