Optimization of In Vitro Maturation in Ovine Oocytes: A Comparative Study of Maturation Media

Abstract

Background: One of the most common techniques in assisted reproductive technologies (ART) is in vitro embryo production (IVEP), the success of which depends on the intrinsic quality of oocytes and the composition of the culture medium. Objectives: This study investigates the effects of gonadotropin hormones (such as eCG and hCG) along with the antioxidant quercetin on the maturation of ovine oocytes, as well as the impact of different media on the expression of Bcl2 and Bax genes in blastocysts. Methods: After washing the oocytes, cumulus-oocyte complexes (COCs) that exhibited three or more layers of cells and homogeneous cytoplasm were selected and matured in BO-IVM, TCM (TCM-199 + 10% FBS + 10% ovine follicular fluid + 5 mg/mL FSH + 1 mg/mL estradiol-17β + 0.81 mM sodium pyruvate + 50 mg/mL gentamicin sulfate), and TCM+ (TCM + 20 µg/mL eCG + 5 µg/mL hCG + 15 µg/mL quercetin) media for 24 hours at 38.5°C, 20% O2, and 5% CO2. After maturation, oocytes were fertilized with frozen ram semen, and presumed zygotes were cultured in BO-IVC under uniform conditions. Embryo development occurred at 38.5°C in a humid atmosphere with 5% CO2, 5% O2, and 90% N2. Results: The results indicated that the BO-IVM medium had a significantly higher mean percentage of zygotes, morulae, blastocysts, and hatched blastocysts compared to TCM+ and TCM (P < 0.05). Moreover, blastocyst formation and hatched blastocysts were significantly more frequent in TCM+ than in TCM (P < 0.05). However, the BO-IVM medium demonstrated a significant improvement compared to other maturation media. Gene expression analysis showed no significant difference in Bcl2 expression among the groups, while Bax expression was significantly higher in TCM (P < 0.05). Overall, the BO-IVM medium was identified as the best option for optimizing oocyte maturation and producing high-quality blastocysts in ovine, with TCM+ yielding better results than the TCM medium without hormonal and antioxidant compounds. Conclusions: The results of this study showed that the addition of the antioxidant quercetin and the growth factors eCG and hCG improved the performance of the TCM medium. However, further research is needed to enhance the performance of the TCM maturation medium compared to the commercial BO-IVM medium.