Post-thaw quality, apoptosis, oxidative stress, microbiota, and in vivo fertility of buffalo bull semen in response to quercetin nano-emulsion or hesperidin-loaded chitosan nanoparticles supplementation.

This study evaluated the effects of quercetin nano-emulsion (QNE) and hesperidin-loaded chitosan nanoparticles (HLCN) supplementation on post-thaw semen quality, apoptotic markers, acrosomal integrity, oxidative stress, microbial load, and fertility of Egyptian buffalo bulls (Bubalus bubalis). Semen samples were collected via artificial vagina from seven fertile bulls (aged 5-6 years). The samples were cryopreserved using a Tris-based extender alone (control; CON) or supplemented with quercetin nano-emulsion at 5 µM (QNE5) or 10 µM (QNE10), or with hesperidin-loaded chitosan nanoparticles at 12.5 µM (HLCN12.5) or 25 µM (HLCN25). The results demonstrated that the QNE10, HLCN12.5, and HLCN25 achieved significantly higher post-thaw progressive motility, sperm viability, plasma membrane function, and acrosomal integrity compared to both the QNE5 and CON groups (P < 0.05). Notably, the HLCN25 group exhibited superior sperm kinematic parameters, which were significantly higher than those of all other treatment groups (P < 0.01). Total antioxidant capacity (TAC) and catalase (CAT) activity increased markedly in all nano-supplemented groups, peaking in the HLCN25 group (P < 0.05). Conversely, all treatment groups exhibited a significant reductions in oxidative stress markers (hydrogen peroxide and malondialdehyde) and total bacterial and coliform counts compared to the CON treatment (P < 0.05). Furthermore, HLCN25 significantly enhanced fertility, yielding a 40% increase in pregnancy rates compared with the CON treatment (P < 0.05). Molecular docking simulations demonstrated that both quercetin and hesperidin exhibit high binding affinities for the antioxidant proteins peroxiredoxin 3 (PRDX3) and cytochrome c. Notably, hesperidin displayed superior binding stability and lower free energy scores than quercetin, suggesting its enhanced therapeutic potential in mitigating oxidative stress. In conclusion, our findings suggest that QNE and HLCN are promising nano-antioxidants for the cryopreservation of buffalo semen. The 25 µM of HLCN dose, provided the most notable improvements in sperm quality and fertility outcomes, making it the most effective concentration for reducing cryodamage in buffalo spermatozoa.