Nanophytosomal delivery of resveratrol as an effective strategy for enhancing frozen–thawed bovine sperm quality

The aim of this study was to assess the effects of supplementing semen extender with free resveratrol (FR) or resveratrol-loaded nano-phytosomes (RPNPs) on the quality, antioxidant capacity, enzymatic activities, and apoptotic genes of cryopreserved bovine sperm. Semen samples were collected from five proven fertility Holstein cattle bulls aged 4-6 years using the artificial vagina method. Pooled semen was cryopreserved in a Tris-based extender supplemented with free resveratrol (FR) at a concentration of 90 μg/mL and RPNPs at concentrations of 30 μg/mL (RPNPs30), 60 μg/mL (RPNPs60), and 90 μg/mL (RPNPs90). Cryopreserved semen without any additives served as the control. Supplementing the freezing medium with free resveratrol (FR) or RPNPs at 60 or 90 μg/mL significantly enhanced sperm progressive motility, viability, membrane integrity, and kinematic parameters (p < 0.05). Additionally, RPNPs60 and RPNPs90 treated samples exhibited superior antioxidant activities (TAC, GPX, and SOD), reduced lipid peroxidation, and improved enzymatic activities in seminal plasma compared to the control group (p < 0.05). The supplementation of FR or RPNPs significantly improved the transcription of apoptotic genes such as caspase 3, Bcl2, and Bax in sperm. Electron microscopy observations revealed that fortifying the freezing media with 60 or 90 μg of RPNPs maintained the integrities of the acrosome and plasma membrane, and preserved the ultrastructure integrity of cryopreserved bovine spermatozoa. The docking analysis revealed that the binding affinity with key sperm function biomarkers, such as CAT, SOD, GPX, Caspase, Bax, and BCL2, exhibited binding energies of -8.5, -7.9, -7.5, -10.4, -9.0, and -9.8 kcal/mol, respectively. The addition of 60-90 μg RPNPs to the bovine freezing extender improved post-thawed sperm quality.