The Beneficial Impact of Silymarin-Loaded Chitosan Nanoparticles on the Quality of Frozen-Thawed Buffalo Sperm: Antioxidant and Anti-Apoptotic Effects.

The objective of this study was to evaluate the impact of incorporating silymarin-loaded chitosan nanoparticles (SMCS-NPs) into semen extender on the semen quality, redox balance, enzymatic activities, apoptotic genes, and ultrastructural integrity of cryopreserved buffalo sperm. Semen samples were obtained using the artificial vagina technique from seven fertile bulls aged between 4 and 6 years. The collected semen was cryopreserved in a tris extender supplemented with varying levels of SMCS-NPs: 0 μg/mL (SMCS-NPs0), 50 μg/mL (SMCS-NPs50), 100 μg/mL (SMCS-NPs100), and 200 μg/mL (SMCS-NPs200). The inclusion of 100 μg of SMCS-NPs in the cryopreservation media significantly improved sperm progressive motility, viability, membrane integrity, and kinematic parameters. Additionally, this concentration improved key antioxidant enzyme activities, reduced malondialdehyde, hydrogen peroxide, and nitric oxide levels, and enhanced enzymatic activity in the seminal plasma compared to the control group (p < 0.05). The supplementation of SMCS-NPs notably boosted the expression of apoptotic genes like caspase 3, Bcl2, and Bax in sperm. Examination through electron microscopy revealed that enriching the cryopreservation medium with 100 μg or 200 μg of SMCS-NPs maintained the integrity of acrosome and the plasma membrane, and preserved the structural integrity of cryopreserved buffalo spermatozoa. The docking exploration revealed strong affinities for key proteins regulating apoptosis (caspase) and oxidative stress (catalase and glutathione peroxidase), emphasizing their therapeutic potential in reducing apoptosis and oxidative damage. In conclusion, supplementing the semen freezing extender with 100 μg of SMCS-NPs improved post-thaw sperm quality.