Silymarin as a functional antioxidant enhances oocyte competence and embryo development in Tharparkar cattle...

In vitro matured oocytes generally exhibit lower developmental competence than in vivo matured oocytes due to various culture-associated factors, the most significant being oxidative stress. This study evaluated the effects of silymarin supplementation during In Vitro Maturation (IVM) of Ovum pick-up (OPU) derived oocytes in indigenous Tharparkar cattle (Bos indicus). Cumulus-oocyte complexes (COCs) were matured in vitro in media with varying concentrations of silymarin (100, 200, or 300 μM) against a control. Post-IVM, the effect of silymarin on oocyte competence and blastocyst production at these concentrations was evaluated. Further, functional outcomes were validated through molecular analyses of genes related to oocyte competence (Growth differentiation factor 9 [GDF9] and Bone morphogenetic protein 15 [BMP15]), apoptosis (B-cell lymphoma 2 [BCL2] and BCL2-associated protein X [BAX]), and proliferation (Proliferating cell nuclear antigen PCNA). Supplementation with 200 μM silymarin significantly enhanced cumulus expansion (4.74 ± 0.03 mm²), nuclear maturation (90.01 ± 2.44%), and blastocyst formation (55.07 ± 2.1%), while markedly reducing intracellular reactive oxygen species (ROS) (11.51 ± 0.17 AFU). Evaluation of oxidative stress markers in the spent IVM medium showed increased total antioxidant capacity (TAC, 29.27 ± 0.78 U/mL), reduced nitric oxide (NO, 18.33 ± 0.45 μmol/L), and reduced malondialdehyde (MDA, 3.83 ± 0.12 μmol/L). Gene expression analysis confirmed upregulation of GDF9, BMP15, BCL2, and PCNA, and downregulation of BAX. Collectively, these findings demonstrate that silymarin supplementation at 200 μM effectively mitigates oxidative stress, enhances oocyte quality, and maximizes preimplantation embryo development, providing a practical and efficient strategy to improve in vitro reproductive outcomes in Tharparkar cattle.