Evaluation of free rosmarinic acid and rosmarinic acid-loaded chitosan nanoparticles in buffalo sperm cryopreservation: Impacts on sperm quality, functionality, and fertility potential.

The aim of this study was to assess the effects of incorporating either free rosmarinic acid (RA) or RA-loaded chitosan nanoparticles (RA-CHNPs) into a buffalo semen extender on various parameters, including semen quality, antioxidant capacity, expression of apoptotic genes, semen microbiota, ultra-structural alterations, and fertility outcomes of cryopreserved buffalo sperm. Semen samples were collected from five healthy, fertile Egyptian buffalo bulls (Bubalus bubalis), aged 5-6 years, using the artificial vagina technique. The ejaculates were cryopreserved in a Tris-based extender supplemented with 100 μg/mL of either RA or RA-CHNPs, alongside a control group without antioxidant supplementation. Supplementing the cryopreservation extender with 100 μg/mL RA-CHNPs significantly improved sperm viability, progressive motility, membrane integrity, and kinematic parameters. Additionally, it significantly enhanced the activity of major antioxidant enzymes and markedly reduced levels of hydrogen peroxide, malondialdehyde, nitric oxide, and semen microbiota counts compared to the control group (p < 0.05). Furthermore, RA-CHNPs treatment significantly modulated the expression of apoptosis-involved genes, with reduced expression of caspase-3 and Bax and increased Bcl-2 expression. Transmission electron microscopy confirmed that RA-CHNPs helped preserve the ultra-structural integrity of spermatozoa, particularly the acrosome and plasma membrane. Notably, pregnancy rates increased by 19 % in buffalo cows inseminated with RA-CHNPs-treated semen compared to those inseminated with supplemented semen (p = 0.0157). In conclusion, loading rosmarinic acid onto chitosan nanoparticles enhanced its stability, water solubility, and bioavailability, ultimately improving its effectiveness in the semen freezing extender. The addition of 100 μg of RA-CHNPs significantly improved post-thaw sperm quality.