Graphene oxide and silymarin combination: A novel approach to improving post-cryopreservation quality of ram sperm.

Graphene oxide (GO) has been extensively studied for its diverse biomedical applications, including drug delivery, imaging, and tissue engineering. Silymarin, as a flavonoid complex derived from the milk thistle plant, has recently shown potential health benefits, particularly concerning reproductive health. This study aims to evaluate the effects of GO and silymarin supplementation, both individually and in combination, on the characteristics of frozen-thawed ram sperm. Semen samples were collected using standard artificial insemination (AI) techniques with an artificial vagina. The collected semen was evaluated and cryopreserved in a tris-based extender containing varying concentrations of silymarin and GO (0, 10, or 20 μg/mL) or their combination. Post-thaw assessments evaluated sperm motility, viability, morphological abnormalities, DNA integrity, membrane integrity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and total antioxidant capacity (TAC). Our findings revealed that the combination of 20 μg/mL silymarin and 20 μg/mL GO significantly enhanced total motility, viability, membrane integrity, and DNA integrity of sperm. Additionally, this treatment effectively reduced morphological abnormalities and MDA levels post-thawing. Notably, SOD and TAC activities were improved following the freeze-thaw compared to other treatment groups. In conclusion, the combination of silymarin and GO significantly improves the quality of frozen-thawed ram sperm by enhancing sperm parameters while reducing oxidative stress markers. The results suggest their potential as effective additives in cryopreservation protocols, providing a promising avenue for improving reproductive outcomes in rams and potentially other livestock species.