Abstract: This study aimed to investigate the effects of AMPK activators metformin (Met) and acadesine (AICAR) on sheep semen cryopreservation. Firstly, Met and AICAR with different concentrations (0, 100, 200, 300, 400, 500 μmol·L^-1) were added into the frozen diluent. After freezing and thawing, the optimal concentration (400 μmol·L^-1 Met, 200 μmol·L^-1 AICAR) were selected based on sperm motility, motion performance and membrane integrity; Secondly, the collected semen was froze using different frozen diluents (control group:diluent; Met group:diluent + 400 μmol·L^-1 Met; AICAR group:diluent + 200 μmol·L^-1 AICAR). After thawing, the sperm AMPK protein expression, acrosomal enzyme activity, metabolic index, mitochondrial function and antioxidant enzyme activity were examined. The results showed that the addition of 400 μmol·L^-1 Met and 200 μmol·L^-1 AICAR could significantly improve sperm motility, motion performance and membrane integrity(P<0.05) after thawing. In 400 μmol·L^-1 Met group, the total sperm motility, acrosome integrity rate and plasma membrane integrity rate were 43.20%, 91% and 46%, respectively. Compared with the control group, the level of AMPK phosphorylation in the sperm of the Met and AICAR groups was significantly increased after thawing (P<0.05), the acrosome enzyme activity of sperm was significantly increased(P<0.05); the pyruvate level was significantly decreased (P<0.05), the lactate dehydrogenase activity, lactic acid content, and ATP content were significantly increased(P<0.05). Compared with the control group, the dilution of Met group and AICAR group was more conducive to maintain mitochondrial membrane potential (P<0.05), increase ATPase and antioxidant enzyme activity (P<0.05). The addition of appropriate concentrations of Met and AICAR could improve semen quality of cryopreservation in sheep.

Key words: sheep semen, cryopreservation, AMPK activator, semen quality