Mitigating cryodamage in ram semen using lutein-loaded chitosan nanoparticles: Insights from antioxidant and apoptotic analyses.

This study aimed to investigate the impact of adding lutein-loaded chitosan nanoparticles (LU-CNPs) to a semen extender on the quality, membrane integrity, kinematic parameters, redox status, apoptosis-like changes, ultrastructural integrity, and fertility of cryopreserved ram sperm. Semen samples from seven fertile rams (12-14 months old) were collected using the artificial-vagina technique and cryopreserved in a Tris-based extender containing LU-CNPs at concentrations of 0, 50, 100, and 150 μg/mL. Supplementation with 100 μg/mL LU-CNPs significantly improved sperm progressive motility, viability, membrane integrity, and kinematic parameters. This concentration also enhanced total antioxidant capacity and the activities of superoxide dismutase and glutathione peroxidase, while reducing malondialdehyde, hydrogen peroxide, nitric oxide, and related enzymatic activities in the post-thaw cryopreservation diluent recovered after centrifugation of sperm samples, compared with the control group (p < 0.05). Additionally, LU-CNPs reduced apoptotic and necrotic sperm cells, upregulated the anti-apoptotic Bcl-2 gene, and downregulated pro-apoptotic genes. Electron microscopy further revealed that incorporating LU-CNPs into the freezing extender preserved acrosome integrity, plasma membrane integrity, and the overall ultrastructural organization of cryopreserved ram spermatozoa. Additionally, insemination with semen supplemented with 100 μg/mL LU-CNPs improved fertility outcomes, resulting in a 27% increase in ewe pregnancy rates compared with the control group. Overall, supplementation with 100 μg/mL LU-CNPs in the freezing extender improved post-thaw sperm quality and enhanced fertility potential.