The Protective Role of Salicylic Acid‐Conjugated Chitosan Nanoparticles Against Cryodamage in Buffalo Semen

Abstract

This study explored the development of salicylic acid-conjugated chitosan nanoparticles (SACNs) and their effectiveness in enhancing the cryopreservation of buffalo sperm. The assessment focused on post-thaw sperm quality and kinematics, microbiota profiles, oxidative stress markers, acrosome integrity, apoptosis, and docking analysis. Semen was collected from eight fertile buffalo bulls via an artificial vagina. Samples were diluted with a Tris-based extender and supplemented with SACNs at concentrations of 0 μM (SACNs0), 5 μM (SACNs5), 10 μM (SACNs10), 20 μM (SACNs20), and 40 μM (SACNs40). Following a standard freezing procedure, sperm quality and kinematic parameters significantly improved in the SACNs10, SACNs20, and SACNs40 groups, with the greatest improvement in the SACNs40 group (p < 0.05). Acrosome integrity was significantly improved in all SACNs-treated semen samples compared with the SACNs0 treatment (p < 0.05). Additionally, total antioxidant capacity and catalase activity were improved, while hydrogen peroxide and MDA levels were noticeably diminished in all SACNs-treated groups (p < 0.05). Although all SACNs concentrations markedly reduced apoptotic markers, caspase-3 activity, and reactive oxygen species, it showed the lowest levels in the SACNs40 group (p < 0.05). Salicylic acid exhibited strong docking interactions with key regulators of mitochondrial function and sperm motion, including UCP1, HSP70, cytochrome c, and the tubulin-colchicine complex, suggesting a multi-target mechanism of action. Furthermore, supplementation with SACNs at 10, 20, and 40 μM significantly declined coliform and total bacterial counts compared with the SACNs0 treatment (p < 0.05). These findings show that SACNs, acting as nano-antioxidant additives, enhance the cryoresistance of buffalo sperm, suggesting their potential to improve assisted reproductive technologies.