Multifaceted evaluation of cysteamine-loaded nanoliposomes in buffalo sperm cryopreservation: Impacts on motility, ultrastructural integrity, oxidative balance, apoptotic gene expression, and fertility.

This study aimed to evaluate the impact of incorporating cysteamine-loaded nanoliposomes (CY-NPs) into a semen extender on semen quality, sperm kinematics, redox status, apoptosis-like changes, ultrastructure, and fertility outcomes. Semen was collected from seven healthy, fertile bulls (4-6 years old) using the artificial vagina technique, and a total of 42 ejaculates were obtained over six weeks. Each sample was analyzed in three replicates. Pooled ejaculates were cryopreserved in a Tris-based extender supplemented with 0, 25, 50, or 100 μg/mL of CY-NPs. Polynomial regression analysis showed that progressive motility, membrane functionality, and viability peaked at 60-75 μg/mL. CY-NPs supplementation enhanced sperm kinematics, increased total antioxidant capacity, and elevated activities of key antioxidant enzymes, including catalase, superoxide dismutase, and glutathione peroxidase, while significantly reducing malondialdehyde, nuclear factor kappa B, and nitric oxide levels in seminal plasma (p < 0.05). The percentage of apoptotic sperm cells decreased significantly, accompanied by upregulation of the anti-apoptotic Bcl-2 gene and downregulation of pro-apoptotic genes Bax and caspase 3. Electron microscopy confirmed that CY-NPs protect sperm ultrastructure, including the plasma membrane, acrosome, and mitochondria. Notably, supplementation with 50 μg/mL CY-NPs improved frozen-thawed semen fertility, increasing buffalo cow pregnancy rates by 23.33 % compared to controls (p = 0.0001). Overall, adding CY-NPs to the freezing extender enhanced semen quality, velocity, antioxidant status, and enzymatic balance, reduced apoptosis, preserved structural integrity, and improved fertility of cryopreserved buffalo sperm.