Effects Of Adding Mito-TEMPO In Diluents On Frozen Semen Quality Of Sheep

Artificial insemination is an important technical measure for animal genetic improvement.Semen preservation is the critical link in artificial insemination,and cryopreservation is the primary method of semen preservation.During cryopreservation,oxidative stress caused by excessive production of intracellular reactive oxygen species and the destruction of the antioxidant enzyme system are the main factors of cryopreservation damage.In this study,0 μM,20 μM,40 μM,and 60 μM were added to the frozen diluent of sheep semen.Frozen-thawed sperm motility,plasma membrane integrity,DNA integrity,mitochondrial activity,seminal plasma antioxidant related indicators,and seminal plasma mitochondrial enzyme activity,were evaluated for frozen semen quality and sperm protein expression related to energy metabolism,for the Mitochondrial targeted antioxidants(Mito-TEMPO)in sheep semen freezing diluent will provide a scientific basis for the application.The main results are as follows:1.After frozen thawed of each group,the curvilinear velocity(VCL)and straight-line velocity(VSL),average path velocity(VAP)were significantly higher than those of the control group(P < 0.05).40 μM group of sperm vitality,motility,DNA integrity rate were significantly higher than the control group(P < 0.05),and the other experimental group.Seminal plasma malondialdehyde(MDA)content and activity of lactate dehydrogenase(LDH),glutamic-oxaloacetic transaminase(GOT)activity and succinate dehydrogenase(SDH)activity was significantly lower than the control group,and the other experimental groups(P < 0.05);Plasma membrane integrity rate,mitochondrial activity,superoxide dismutase(SOD)activity,glutathione peroxidase(GSH-Px)activity and total-anti-oxidant capacity(T-AOC)of sperm in 20 μM and 40 μM groups were significantly higher than those in the control group and 60 μM group(P < 0.05);The catalase(CAT)activity in seminal plasma of each experimental groups was significantly higher than that of the control group(P < 0.05).2.Frozen semen from the control group and the 40 μM group were selected for Western Blotting to detect the expressions of sperm energy metabolism-related proteins Glut3 and Glut8 after thawing.The results showed that the expression of GLUT 3 and GLUT 8 in 40 μM thawed sperm was significantly increased as compared with the control group(P < 0.05).In conclusion,adding appropriate concentration(40 μM)of Mito-tempo to the diluent can improve the activity of antioxidant enzymes,protect the integrity of sperm plasma membrane and DNA,reduce the activity of mitochondria related enzymes,improve the activity of mitochondria,reduce the oxidative damage of sperm,and promote the sperm energy metabolism transporters GLUT 3 and GLUT 8 Therefore,the quality of frozen semen of sheep was improved.