Long in vitro culture of cumulus-oocyte complexes collected from early antral follicles in sheep: a promising strategy for producing competent oocytes

In vitro culture of cumulus-oocyte complexes (COCs) derived from early antral follicles (EAFs) has shown promising results in cattle (1,2). In this study, we investigated whether this technology can be transferred to sheep. Sheep ovaries were collected during the breeding (October–November) and nonbreeding season (March–May). COCs were isolated from EAFs (350-450 µm) and individually cultured for five days in TCM199 supplemented with 0.15μg/mL Zn sulfate, 10-4IU/mL FSH, 10ng/mL estradiol, 50ng/mL testosterone, 50ng/mL progesterone and 5μM Cilostamide (1). After long in vitro culture (LIVC), they were in vitro matured under standard conditions used for this species (Mara et al., 2014). The diameter of the oocytes collected from EAFs did not differ between the breeding and non-breeding seasons (109.4 ± 0.2 µm vs 108.8 ± 0.2 µm). After LIVC the diameter increased in both seasons (GLM, p<0.000), but oocytes grew significantly more in the breeding season (116.3 ± 0.2 µm) compared to the non-breeding season (114.9 ± 0.2 µm). However, they were significantly smaller than fully grown oocytes collected from antral follicles (133.6 ± 4.5 µm). Oocyte viability decreased significantly after LIVC only during the non-breeding season (from 76.5 ± 3.7% to 64.7 ± 5.1%; GLM, p<0.000). After LIVC, both groups showed a decrease in gap junction communication and a shift in chromatin configuration from the non-surrounded nucleolus (NSN) to surrounded nuclear envelope (SNE) (p<0.000). Global transcription activity exhibited a reduction in both groups after LIVC, with a greater decline observed during the breeding season (p<0.05). Oocytes collected from EAFs showed no meiotic competence before LIVC. After LIVC, oocyte meiotic competence significantly increased in both seasons (17.7 ± 4.4 and 10.3 ± 3.8% in the breeding compared to the non-breeding season; p<0.000). However, MII rates were lower compared to those obtained from COCs collected from antral follicles (p<0.000). In conclusion, our study suggests that the breeding season is associated with improved efficiency of the LIVC technology consequent to a higher quality of the COCs isolated from EAFs, which may have important implications for optimizing the LIVC system in mammals with seasonal reproduction.