In vitro culture of cumulus oocyte-complexes from early antral follicles in prepubertal and adult ewes

The long-term in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) derived from early antral follicles (EAFs) has shown significant potential in producing meiotically competent oocytes in adult ewes. However, the effectiveness of this approach in prepubertal ewes remains unexplored. This study aimed to evaluate the efficiency of the LIVC system in prepubertal ewes, addressing the challenge of limited EAF availability in adults. Both adult and prepubertal ewe ovaries were used to isolate COCs from EAFs (350–450 µm), which were then cultured in TCM199 supplemented with 0.15 μg/mL Zn ++ , 10 -4 IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 μM cilostamide. After five days of LIVC, a group of COCs underwent in vitro maturation, while others were evaluated for LIVC effectiveness, including morphology, oocyte diameter, chromatin configuration, gap junction communication, meiotic competence, intracellular reactive oxygen species (ROS) levels, mitochondrial activity, and distribution. Results showed a significant increase in oocyte diameter (116.3 vs. 113.4 µm, p < 0.01), higher COC morphological integrity (61.8% vs. 42.6%), and active gap junction communication in adults compared to prepubertal ewes. Moreover, prepubertal ewes exhibited a higher mitochondrial activity, along with spontaneous meiosis resumption (26.7% vs. 0%) after LIVC and a lower rate of meiosis resumption (21.0% vs. 59.4%) following in vitro maturation. No significant differences in ROS levels or mitochondrial distribution were observed between groups. In conclusion, COCs isolated from EAFs of prepubertal ewes showed restricted development during LIVC, highlighting significant age-related difference s in optimizing fertility preservation techniques for prepubertal animals. Lay Summary Fertility preservation in adult livestock can be achieved by culturing growing oocytes from early antral follicles, a stage characterized by a small fluid-filled cavity. Still, its effectiveness in prepubertal animals remains uncertain. This study compared the quality and growth of oocytes from adult and prepubertal sheep. After five days in culture, oocytes from adult sheep showed better growth, structure, and cell-to-cell communication. In contrast, those from prepubertal sheep grew less but showed higher energy activity and early signs of maturation. These findings reveal clear age-related differences and suggest that current methods may need adjustments for younger animals. This work supports the development of better fertility preservation strategies for immature livestock.