The oxidative/antioxidant status and cryotolerance of donkey sperm are influenced by the combination of permeable and non-permeable cryoprotectants.

During cryopreservation, donkey semen is highly vulnerable to oxidative damage, impacting fertility. Although removing seminal plasma (SP) is standard practice for freezing, it eliminates the main antioxidants protecting against reactive oxygen species (ROS). This study aimed to evaluate the oxidative/antioxidant status and cryotolerance of donkey sperm cryopreserved with different permeable and non-permeable cryoprotectants. Thirty semen samples from ten donkeys were evaluated across eight treatments combining 5% DMF, 200 mM sucrose, 1% BSA, and 10% homologous SP, as follows: T1: DMF; T2: DMF/SP; T3: SUC/BSA; T4: SUC/BSA/SP; T5: DMF/SUC/BSA; T6: DMF/SUC/BSA/SP; T7: DMF/BSA; and T8: DMF/BSA/SP. Semen was frozen using a conventional freezing protocol. Post-thaw oxidative/antioxidant markers were evaluated, including total antioxidant capacity (TAC), ROS, the superoxide anion (O2-) and the hydroxyl radical (OH-). Sperm cryotolerance was assessed by motility, mitochondrial membrane potential (ΔΨM), and viability of frozen-thawed semen. For statistical analysis, mixed models were fitted and means were compared by the Tukey's test. The results showed that T6 and T8 showed higher total motility. Treatments containing SUC/BSA/SP exhibited higher TAC. Treatments T4, T7 and T8 showed higher ROS production, while T7 produced the highest O2-. Treatments T7 and T8 had the highest proportion of cells with high-ΔΨM; however, they exhibited the lowest sperm viability (p < 0.05). It is concluded that the use of different combinations of permeable and non-permeable cryoprotectants during semen freezing, including DMF, BSA, SUC, and SP, influences the oxidative/antioxidant status, as well as the cryotolerance of donkey sperm.