The cryoprotective effects of celastrol nanoemulsion on post-thawed attributes and fertilizing ability of cryopreserved buffalo semen

Abstract Celastrol (CL), a natural molecule isolated from Tripterygium wilfordii Hook. f., possesses notable therapeutic potential across a range of disease states. However, its impact on semen quality following cryopreservation has not been extensively investigated. Thus, this research comprehensively evaluated the cryoprotective effects of celastrol nanoemulsion (CNE) on buffalo semen by analyzing sperm quality, sperm kinematics, acrosome status, oxidative stress, mitochondrial membrane potential, ultrastructure, microbiota, and apoptotic-like changes. Semen samples (n = 45) were collected from five buffalo bulls and subjected to cryopreservation following a standard procedure. Prior to freezing, Tris-extenders were supplemented with celastrol nanoemulsion (CNE) at concentrations of 0 (CNE0), 25 (CNE25), 50 (CNE50), 100 (CNE100), and 200 (CNE200) µg/mL. The results showed that CNE supplementation (excluding 200 µg/mL) significantly improved sperm progressive motility, viability, and membrane integrity in post-thawed samples and after 2 h of incubation at 37 °C with 5% CO 2 . Specifically, sperm kinematic parameters were significantly improved by 50 and 100 µg/mL CNE compared to other treatments ( P < 0.05). Furthermore, a superior percentage of sperm with intact acrosomes was remarked in the 25, 50, and 100 µg/mL CNE groups than in the other groups ( P < 0.001). Total antioxidant capacity (TAC) was significantly higher in the CNE100 group compared to the CNE0 group ( P < 0.001), while no significant differences were found between CNE100 and the other treated groups ( P > 0.05). Oxidative stress markers (MDA, NO, and H 2 O 2 ) were significantly lower in CNE50 and CNE100 groups compared to all other treatments ( P < 0.001). Furthermore, the CNE 100 µg/mL group exhibited a higher percentage of viable sperm and a lower percentage of apoptotic sperm than other groups ( P < 0.05). The addition of CNE to the freezing extender significantly reduced the total bacterial count, spore-forming bacteria, and coliform bacteria count in post-thawed buffalo bull sperm ( P < 0.001). Transmission electron microscopy analysis revealed that CNE 100 µg/mL improved and sustained sperm ultrastructure. Notably, the CNE 100 µg/mL treatment also improved the pregnancy rate compared to the control group (82.0% vs. 68.0%). Collectively, the results indicate that CNE at concentrations of 50–100 µg/mL can be effectively used for improving sperm cryo-resistance. This is achieved through improvements in sperm quality and kinematic parameters, attenuation of oxidative stress and microbiota, enhancement of mitochondrial function, preservation of sperm ultrastructure, and improved fertilizing capacity. This study highlights the potential of celastrol nanoemulsion as a nanotechnology-based strategy for optimizing assisted reproductive technologies in livestock.