Humic–zinc nanoparticles enhance cryopreserved bovine sperm quality: evidence from kinematic, oxidative and <i>in silico</i> analyses

Semen cryopreservation is a crucial tool in assisted reproduction. However, the process often leads to oxidative stress, which significantly harms sperm function and structural integrity. Humic acid (HA), a naturally occurring compound recognised for its diverse biological activities, shows considerable promise for improving cryopreservation outcomes. This study investigated the cryoprotective potential of humic acid nanoparticles (H-NPs) and zinc-blended humic nanoparticles (HZn-NPs) in bull spermatozoa to evaluate sperm quality, kinematics, apoptosis, acrosome integrity, ultrastructure, redox homeostasis and in silico analysis. Bull semen samples were cryopreserved in media supplemented with either 20 or 30 µg/mL of H-NPs or HZn-NPs, alongside a control group. The findings reveal that supplementing semen with either H-NPs or HZn-NPs significantly improved post-thaw sperm quality parameters compared to the control group (p < .05). Notably, HZn-NPs at a concentration of 20 µg/mL demonstrated the most substantial enhancement across key indicators, including motility, viability, acrosome integrity and various kinematic parameters. Both H-NPs and HZn-NPs significantly increased antioxidant capacity (TAC and SOD) and reduced oxidative stress markers (MDA, NF-κB, H2O2 and NO), concurrently decreasing apoptosis and necrosis in cryopreserved sperm (p < .05). Transmission electron microscopy provided further evidence of improved sperm ultrastructure in the treated samples. Molecular docking studies offered a mechanistic explanation for these observations, revealing potential binding interactions between HA and key proteins involved in redox homeostasis and apoptosis (GPX, BCL-2, caspase-3 and NDUFA4). These findings collectively demonstrate that HZn-NPs significantly enhance bull semen cryopreservation by improving antioxidant capacity, sperm quality and kinematics, while effectively mitigating oxidative stress and apoptosis.