Impact of Antioxidant Supplementation on In Vitro Growth of Porcine Oocytes Derived from Early Antral Follicles

Introduction: Oxidative stress (OS), a key factor impairing oocyte developmental competence, poses a major challenge in in vitro growth (IVG) systems. While antioxidant supplementation can mitigate OS-driven deficits, combining agents with complementary mechanisms—such as melatonin (Mela) and astaxanthin (Asta)—may offer synergistic benefits. This study evaluates the effects of Mela and Asta, individually and in combination, on oocyte quality, gene expression, and epigenetic markers during IVG. Methods: Growing oocytes from early antral follicles (EAFs; 1–1.5 mm) were cultured for 4 days in a 3D agarose system. Initial experiments tested Mela (1, 10, 100 µM/mL) to assess survival rates and expression of BMP15, CAT, SOD1, and GPX4 via qPCR. Subsequently, the optimal Mela concentration was combined with Asta (0.05, 0.1, 0.25 µM/mL) to determine synergistic effects. Histone H3 methylation (Me-H3-K4) and global transcriptional activity (nascent mRNA accumulation) were analyzed to evaluate chromatin remodeling and transcriptional dynamics. Results: Mela supplementation significantly improved oocyte survival compared to controls, with 1 µM/mL Mela yielding optimal results. Combining 1 µM/mL Mela with 0.05 µM/mL Asta further enhanced survival rates and upregulated antioxidant genes (CAT, SOD1, GPX4) and BMP15. This combination also elevated Me-H3-K4 levels and nascent mRNA accumulation, indicating enhanced transcriptional activity and chromatin remodeling. These changes correlated with reduced ROS impact and improved oocyte quality post-IVG. Conclusion: Co-supplementation with Mela (1 µM/mL) and Asta (0.05 µM/mL) synergistically mitigates OS during IVG by boosting antioxidant gene expression, enhancing epigenetic modifications (Me-H3-K4), and promoting global transcription. This strategy significantly improves oocyte developmental competence, offering a promising approach to optimize IVG systems.