Innovative nanotechnology approach using salicylic acid loaded chitosan nanoparticles to improve post-thaw sperm...

This study was designed to determine whether salicylic acid-chitosan nanoparticles (SA-CNPs) added to a semen freezing extender could enhance post-thaw sperm quality and functionality in Rahmani rams, with a particular focus on oxidative stress, apoptosis, microbial contamination, and ultrastructural preservation. Semen was collected from six healthy rams over eight weeks, pooled, and cryopreserved in extenders containing different concentrations of SA-CNPs (0, 5, 10, 20, and 40 µM). Post-thaw analyses demonstrated that SA-CNPs significantly improved progressive motility, viability, and membrane integrity compared with controls (p < 0.05). Quadratic regression analysis revealed a dose-dependent response, with predicted optimal improvement in sperm quality observed at 30 µM. Sperm kinematics showed highest values in samples treated with 20 µM SA-CNPs. Acrosome reaction analysis showed more live sperm with intact acrosomes and fewer live or dead sperm with damaged acrosomes, peaking at 20-40 µM. Antioxidant defense was improved, with higher total antioxidant capacity, increased nuclear factor erythroid 2-related factor 2 and its downstream enzymes (catalase, glutathione peroxidase 1, superoxide dismutase1), alongside reductions in oxidative stress markers, with the strongest effects observed at 20 µM. Apoptotic markers were favorably modulated, as evidenced by reduced caspase-3 levels, increased Bcl-2 expression, and a decreased apoptotic sperm percentage. These effects followed a quadratic dose-response pattern, peaking at 30 µM, while Bax expression declined progressively with increasing SA-CNPs concentration. Additionally, SA-CNPs reduced total bacterial and coliform counts and preserved sperm ultrastructure, including plasma membrane, acrosomal cap, and mitochondria. Collectively, these results indicate that SA-CNPs supplementation enhances post-thaw sperm function, strengthens antioxidant defenses, mitigates apoptosis, reduces microbial contamination, and protects ultrastructure, with 20-30 µM identified as the optimal concentration range for maximal cryoprotective and functional benefits.