Kaempferol combined with epigallocatechin-3-gallate enhances bovine oocyte in vitro maturation by attenuating oxidative stress and improving mitochondrial function

This study investigated whether combined treatment with kaempferol and epigallocatechin-3-gallate (EGCG) offers superior protection during the in vitro maturation (IVM) and subsequent embryonic development of bovine oocytes compared to treatment with either agent alone. The underlying mechanisms were also examined. Preliminary dose–response assays revealed 0.1 µM kaempferol and 20 µM EGCG as the optimal concentrations when used individually. Accordingly, bovine cumulus–oocyte complexes were allocated to the following four groups: control, kaempferol only (0.1 µM), EGCG only (20 µM), and kaempferol + EGCG co-administration (0.05 µM + 10 µM, respectively). The combination treatment yielded the most favorable oocyte maturation and subsequent developmental outcomes. The first polar body extrusion rate was significantly higher in the combination group than in the control and EGCG groups ( P < 0.05), but did not differ significantly from the kaempferol-only group. In addition, cleavage and blastocyst formation rates were significantly higher in the combination group than in both single-treatment groups ( P < 0.05). Meanwhile, oocytes subjected to the combined treatment exhibited markedly lower intracellular reactive oxygen species levels and oocyte apoptosis rates, along with significantly higher glutathione levels, ATP production, and mitochondrial membrane potential than both individual treatment groups and the control ( P < 0.05). Molecular analyses further revealed synergistic effects in the co-administration group, which demonstrated the greater upregulation of SOD3 and SIRT2 (antioxidant defense), DNM1 and MFN2 (mitochondrial dynamics), and BCL2 (anti-apoptosis). Meanwhile, the pro-apoptotic gene BAX was downregulated in the combination treatment group ( P < 0.05). Overall, the results revealed that the combined administration of kaempferol and EGCG synergistically mitigates oxidative stress and preserves mitochondrial activity in bovine oocytes during IVM. This approach may therefore help in enhancing oocyte quality during livestock reproduction.