Beneficial effects of carvacrol loaded phytosomes on enhancing cryotolerance of Buffalo semen following cryopreservation

Sperm cryopreservation technology underpins genetic advancement in animal breeding and ongoing research aims to minimize cryoinjuries. This study aimed to develop carvacrol-loaded phytosomes (CLNPs) to enhance the physicochemical properties of carvacrol in aqueous cryopreservation extenders. Semen samples from five buffalo bulls were collected, extended and cryopreserved with varying CLNPs concentrations (0, 2.5, 5, 10, and 20 µg/mL). The freshly prepared CLNPs exhibited an average particle size of 286.7 ± 11.27 nm, a polydispersity index of 0.189 ± 0.05, and a zeta potential of - 11.4 ± 0.26 mV. Supplementing the freezing media with CLNPs significantly enhanced sperm progressive motility, viability, and plasma membrane integrity after both equilibration (5 °C for 4 h) and thawing. Furthermore, sperm kinematic parameters were significantly higher in all CLNPs-treated groups (P < 0.05). Compared to the CLNPs-free extender, CLNPs supplementation significantly reduced the percentage of dead sperm with intact acrosomes and increased the percentage of live sperm with intact acrosomes (P < 0.001). Post-thaw oxidative stress markers, including H₂O₂ and MDA, were significantly lower in all CLNPs groups (P < 0.001). Notably, the addition of 10 or 20 µg/mL of CLNPs increased TAC and significantly decreased nitric oxide levels compared to the control. Mitochondrial membrane potential and viable sperm counts were significantly higher in the CLNPs-treated groups (P < 0.001). CLNPs also significantly decreased the total bacterial count, spore-forming bacteria, and coliform bacteria in the post-thawed semen microbiota (P < 0.001). Higher CLNPs concentrations (10 or 20 µg/mL) appeared to provide superior protection, as evidenced by a greater proportion of sperm cells displaying normal nuclear, plasma membrane, mitochondrial, and acrosomal morphology. The pregnancy rate in the 20 µg/mL CLNPs group (86%, n = 43/50) was higher than in the control group (72%, n = 36/50). Molecular docking analysis revealed binding energies of - 6.22, - 4.93, - 4.44, and - 5.36 kcal/mol for Cox7c, Hsp70, PrxIII, and ATP1B1, respectively. This study introduces a novel nanotechnology-based approach using CLNPs to enhance buffalo semen cryopreservation, potentially significantly advancing assisted reproductive technologies in buffalo.