Abstract
PURPOSE: This study aimed to evaluate the effectiveness of single versus group culture strategies for cumulus-oocyte complexes (COCs) derived from early antral follicles (EAFs), with the goal of optimizing culture conditions to increase oocyte availability for assisted reproductive technologies. METHODS: COCs isolated from EAFs (350-450 µm) from sheep ovaries were cultured in TCM199 medium supplemented with 0.15 µg/mL Zn(++) as zinc sulfate, 10(-4) IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 µM Cilostamide. After 5 days of long in vitro culture (LIVC), COCs underwent in vitro maturation. This study investigated the effects of single and group culture conditions on COCs, focusing on morphology (integrity of oocyte-granulosa cell complex), viability, oocyte diameter, chromatin configuration, and ultrastructure. Additional factors influencing developmental competence were assessed, including global transcriptional activity, gap junction communication, and meiotic competence. Intracellular reactive oxygen species (ROS) levels and mitochondrial activity were also measured. RESULTS: No significant differences were observed between groups in terms of morphology, viability, oocyte diameter, chromatin configuration, ROS levels, or mitochondrial activity. However, group culture resulted in ultrastructural changes, with a notable reduction in global transcriptional activity, an increase in active gap junctions, and a higher rate of meiosis resumption (p < 0.01). CONCLUSION: Overall, group culture of COCs derived from sheep EAFs promoted meiosis resumption, suggesting that this approach could improve in vitro culture techniques, increase the availability of mature gametes, and support fertility preservation programs.