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Cryopreservation is an effective method to spread qualified ram spermatozoa for reproductive goals in different farms, but cryopreservation's shocks reduce sperm quality. This study investigated the efficacy of the new mitochondria‐targeted antioxidant Mito‐TEMPO on post‐thawed quality of spermatozoa in sheep. Collected samples were divided into five groups and after dilution, received different doses of Mito‐TEMPO (0, 0.5, 5, 50 and 500 µM), and frozen. Thawed sperm motility parameters, malondialdehyde content, membrane functionality, abnormal morphology, mitochondria activity, acrosome integrity, DNA fragmentation, ROS concentration, viability and apoptotic‐like changes, were evaluated. According to the results, Mito‐TEMPO (5 and 50 μM) improved (p ≤ 0.05) motility parameters, average path velocity, membrane functionality, mitochondria activity and viability compared with the other groups. Moreover, apoptotic‐like changes, lipid peroxidation and ROS concentration were lower (p ≤ 0.05) in groups received 5 and 50 μM Mito‐TEMPO. Mito‐TEMPO showed no effect (p > 0.05) on sperm acrosome integrity, morphology and DNA fragmentation. In conclusion, Mito‐TEMPO as a targeted antioxidant could be an efficient cryo‐additive to enhance quality parameters of post‐thawed ram semen.

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