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High levels of polyunsaturated fatty acids (PUFAs) in ram sperm make it susceptible to the cold condition during the cooling process, which decreases the reproductive potential of chilled semen. Supplementation of chilled sperm medium with suitable antioxidants is a valuable method to protect sperm fertility rate during the liquid-storage. The purpose of this study was to evaluate the effects of mitochondria-targeted (MitoQ and Mito-TEMPO) and untargeted (SOD and CoQ10) antioxidants on ram’s sperm quality and fertility potential during the cooling process. Semen samples were collected and diluted in the extender and divided into eight groups. The first group was control and the other groups were supplemented with 100 U/mL superoxide dismutase (SOD), 1 μM CoQ10 (Q10), 50 and 100 nM MitoQ (MQ50 and MQ100), 50 and 100 μM Mito-TEMPO (MT50 and MT100), 50 nM MitoQ+50 μM Mito-TEMPO (MQ/MT), respectively. Then, the samples were cooled at 5 ◦C up to 50 h and sperm total motility (TM), progressive motility (PM), viability, membrane integrity, lipid peroxidation (LPO), and mitochondria active potential (MAP) were assessed during 0, 25, and 50 h of cooling storage. Moreover, artificial insemination was performed using 25 h-chilled semen to evaluate sperm fertility potential. In the results, no significant differences (P > 0.05) were observed between groups at time 0. The highest (P ≤ 0.05) TM, PM, viability, membrane integrity, MAP, as well as the lowest (P ≤ 0.05) LPO were detected in the MQ/MT group compared to the other groups. The pregnancy rate, parturition rate, and lambing rate were higher (P ≤ 0.05) in MQ100, MT100, and MQ/MT compared to the control group. In conclusion, supplementation of the ram sperm cooling extender with a combination of 50 nM MitoQ and 50 μM Mito-TEMPO as the powerful mitochondria-targeted antioxidants is a useful strategy to preserve sperm quality during cooling storage.
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