Comparative cryoprotective and fertility-enhancing effects of astaxanthin-loaded chitosan nanoparticles and free astaxanthin in buffalo bull semen.

This study evaluated free astaxanthin (ASX) and astaxanthin-loaded chitosan nanoparticles (ASX-CNPs) as additives to semen freezing extenders for Egyptian buffalo bulls. Five healthy and fertile buffalo bulls were used, and forty ejaculates were collected over eight consecutive weeks. Ejaculates that met standard quality criteria were pooled to eliminate individual bull variation, diluted with a Tris-egg yolk extender, and allocated into three experimental groups: a control group without supplementation, a group supplemented with 127 μM free ASX, and a group supplemented with 127 μM ASX-CNPs. Semen samples were cryopreserved in liquid nitrogen for one month and subsequently evaluated after thawing. The results showed that supplementation with ASX-CNPs significantly improved post-thaw progressive motility, sperm viability, plasma membrane integrity, and acrosomal integrity compared with both the control and free ASX groups (p < 0.05), while sperm abnormalities were significantly reduced. Moreover, ASX-CNPs significantly improved sperm kinematic parameters. Antioxidant analysis revealed significant increases in total antioxidant capacity and the activities of catalase and glutathione peroxidase, accompanied by significant reductions in nitric oxide, malondialdehyde, and hydrogen peroxide levels. In addition, ASX-CNPs significantly decreased apoptotic and necrotic spermatozoa, downregulated the pro-apoptotic genes Bax and Caspase-3, and upregulated the anti-apoptotic gene BCL2. Ultrastructural observations confirmed preservation of sperm membrane, acrosomal, and mitochondrial integrity. Furthermore, ASX-CNPs reduced semen microbial load and improved fertility, increasing pregnancy rates by 24.28% compared with the control. In conclusion, ASX-CNPs represent a promising nano-antioxidant additive for buffalo semen cryopreservation, offering superior protection and enhanced fertility outcomes compared with free ASX.