Effect of nanoliposomal gamma-oryzanol on cryosurvival, oxidative balance, and fertility of frozen–thawed ram spermatozoa

The purpose of this study was to examine how gamma-oryzanol (GO) and gamma-oryzanol nanoparticles (GO-N) affected the quality of ram sperm after freeze-thaw treatment. Epididymal sperm from Shall rams collected from a slaughterhouse were diluted and divided into seven groups: a control group (0 µM), three concentrations of GO (50, 100, and 200 µM), and three concentrations of GO-N (50, 100, and 200 µM). The results demonstrated that treatment with 100 µM GO-N significantly improved the total motility (70.45%), progressive motility (60.21%), and velocity parameters (VCL, VSL, and VAP) compared to the other groups (p < 0.05). Plasma membrane and acrosomal integrity increased to 75.76% and 77.35%, respectively, while the percentage of morphological abnormalities decreased to 16.67% (p < 0.05). In the same group, flow cytometry revealed a reduction in DNA fragmentation to 17.22%, an increase in mitochondrial membrane potential to 66.11%, a decrease in H₂O₂ concentration to 13.29%, and the highest sperm viability (76.76%). MDA levels dropped to 3.42 nmol/mL, while SOD and GPx activities increased to 138.16 and 74.69 U/mg protein, respectively. Fertility assessments also indicated that the use of 100 µM GO-N in the extender significantly improved the pregnancy rate, lambing rate, and prolificacy compared with the other treatments (p < 0.05). In conclusion, the addition of 100 µM GO-N to the cryopreservation extender appears to be an effective strategy for enhancing the quality and functionality of thawed ram sperm, and may be valuable in breeding programs, reproductive biotechnology, and assisted reproductive technologies.