Supplementation of Thymoquinone Nanoparticles to Semen Extender Boosts Cryotolerance and Fertilizing Ability of Buffalo Bull Spermatozoa

SIMPLE SUMMARY: Semen cryopreservation is a reliable technique used in artificial reproduction technology to preserve and transmit buffaloes’ superior genetic qualities. Buffalo sperm cells are highly susceptible to cryopreservation due to the abundant polyunsaturated fatty acids in their plasma membrane. The use of nanoparticles in freezing extenders is one of the most recent techniques for reducing the detrimental effects of cryopreservation on the post-thawed quality of buffalo spermatozoa. In this study, thymoquinone nanoparticles (TQNPs) were used for the first time as an effective method to enhance the sperm quality and fertilizing ability of frozen–thawed buffalo sperm. The addition of optimal levels of TQNPs (25 to 50 µg/mL) improved post-thawed sperm parameters and sperm kinematics, as well as maintained the morphology of sperm by supporting the total antioxidant capacity and reducing membrane damage, apoptotic sperm, and oxidative stress-related markers. These data can provide a novel nanotechnology-based strategy for enhancing the cryopreservation efficiency of buffalo sperm. ABSTRACT: Thymoquinone nanoparticles (TQNPs) are broadly utilized in numerous pharmaceutical applications. In the present study, we tested the effects of TQNP supplementation on sperm quality and kinematics, acrosome exocytosis, oxidative biomarkers, apoptosis-like and morphological changes of frozen–thawed buffalo sperm, as well as the fertilizing capacity. Semen was collected from buffalo bulls, diluted (1:10; semen/extender), and divided into five aliquots comprising various concentrations of TQNP 0 (CON), 12.5 (TQNP12.5), 25 (TQNP25), 37.5 (TQNP37.5), and 50 (TQNP50) µg/mL, and then cryopreserved and stored in liquid nitrogen (−196 °C). The results revealed that TQNPs (25 to 50 µg/mL) provided the most optimal results in terms of membrane integrity (p < 0.001) and progressive motility (p < 0.01). In contrast, TQNP50 resulted in a greater post-thawed sperm viability (p = 0.02) compared with other groups. The addition of TQNPs to the extender had no discernible effects on sperm morphology measures. Sperm kinematic motion was significantly improved in the TQNP50 group compared to the control group (p < 0.01). TQNPs effectively reduced the content of H(2)O(2) and MDA levels and improved the total antioxidant capacity of post-thawed extended semen (p < 0.01). The addition of TQNP significantly increased the number of intact acrosomes (p < 0.0001) and decreased the number of exocytosed acrosomes (p < 0.0001). A significant reduction in apoptosis-like changes was observed in TQNP groups. The non-return rates of buffalo cows inseminated with TQNP50-treated spermatozoa were higher than those in the control group (p < 0.05; 88% vs. 72%). These findings suggested that the freezing extender supplemented with TQNPs could effectively enhance the cryotolerance and fertility of buffalo sperm.