| With the continuous development of social economy,China's livestock industry has embarked on the fast track of rapid development and gradually moved towards intensive and large-scale cultivation.At the same time,artificial insemination technology has been developed rapidly in practical production.At present,the artificial insemination technology is developing rapidly in the cattle and pig breeding industry,but has been stagnating in the sheep breeding industry.The main reason for this phenomenon is that the sheep sperm is vulnerable to oxidative damage in the preservation process and is difficult to preserve effectively.A large amount of reactive oxygen species(ROS)will be produced in semen preservation of sheep,especially in cryopreservation.A large amount of accumulated ROS will have a lipid peroxidation reaction with sperm membrane to destroy the integrity of sperm membrane,resulting in the imbalance of the antioxidant system of sperm and the decline of semen quality,thus failing to provide high-quality semen for the artificial insemination of sheep.Therefore,it is of great significance to reduce the excessive ROS produced during semen freezing and improve the quality of freeze-thawed semen for the rapid propagation of sheep breeding and the production efficiency of sheep breeding.Current research shows that metformin not only lowers blood sugar,but also increases the antioxidant capacity of cells through its antioxidant activity.However,the effect of metformin on the cryopreservation of sheep semen remains unclear..This study studied the effect of metformin on semen cryopreservation of the Tong sheep,a national protected breed of livestock and poultry.By measuring sperm motility,sperm plasma membrane integrity rate,acrosome integrity rate,ROS content and the activation of metformin to AMPK,the study on metformin's effect on the quality of semen after freeze-thawed was conducted to provide technical support for the application of metformin in semen cryopreservation.This experiment mainly obtains the following research results:1.Different concentrations of metformin(0.25,0.5,1.0,2.0,4.0 mmol/L)were added to the semen cryopreservation diluents of the Tong sheep,which had positive effects on semen after frozen-thawed.When the added amount of metformin in the semen cryopreservation diluent was 1.0 mmol /L,the sperm motility,acrosome integrity rate,head plasma membrane integrity rate,tail plasma membrane integrity rate after cryopreservation and thawing reached 53.56%,52.37%,51.65%,42.62%,which were significantly higher than the control group(P < 0.05).When the concentration of metformin was 0.5 mmol/L,there was no significant difference in sperm motility,acrosome integrity rate and plasma membrane integrity rate of the head after freezing and thawing compared with that of the 1.0 mmol/L metformin treatment group(P > 0.05),bue the plasma membrane integrity rate of the tail was significantly lower than that of the 1.0 mmol/L metformin treatment group and higher than that of the control group(P < 0.05).When the concentration of metformin was 4.0 mmol/L,all indexes of cryo-thawed semen were significantly lower than those of the treatment group with 1.0 mmol/L metformin,and significantly higher than those of the control group(P < 0.05).However,there was no significant difference between the acrosome integrity rate with the control group(P > 0.05).2.Adding different concentrations of metformin(0.25,0.5,1.0,2.0,4.0 mmol/L)to the semen cryopreservation diluent of the Tong sheep can increase the total antioxidant level after semen frozen-thawed.When the concentration of metformin was 1.0 mmol/L,the protective effect on oxidative damage of freeze-thawed semen was the best,at the moment the sperm total antioxidant capacity(T-AOC)reached 12.23 U/m L,catalase(CAT)was 14.03 U/m L,glutathione peroxidase(GSH-Px)was 68.25 U/m L,and superoxide dismutase(SOD)was 255 U/m L,all significantly higher than that of control group(P < 0.05).Meanwhile,the level of reactive oxygen species(ROS)and malondialdehyde(MDA)were 1981.27 and 5.1 nmol/m L,which were significantly lower than the control group(P < 0.05).However,when the added concentration of metformin was 0.5 mmol/L,there was no significant difference in the total antioxidant capacity(T-AOC),superoxide dismutase(SOD)activity,reactive oxygen species(ROS)and malondialdehyde(MDA)content compared with the 1.0 mmol/L metformin treatment group(P > 0.05).The activity of glutathione peroxidase(GSH-Px)and catalase(CAT)were significantly lower than that of the 1.0 mmol/L metformin treatment group,but both were better than the control group(P < 0.05).When the added amount of metformin was 4.0 mmol/L,there was no significant difference in the content of MDA and SOD compared with the control group,but the activity of GSH-Px and the ability of T-AOC were significantly higher than that of the control group,and the ROS level was significantly lower than that of the control group(P < 0.05).3.The addition of metformin to the semen cryopreservation diluent of the Tong sheep can affect the semen preservation quality by mediating AMPK.In this study,AMPK was distributed in the whole head of sperm,but mainly concentrated above the equatorial ring of the Tong sheep sperm,while phosphorylated AMPK was mainly located below the equatorial ring of sperm.Compared with the Compound C(AMPK inhibitor)group and the Compound C + Metformin group,metformin significantly increased sperm motility,mitochondrial membrane potential and ATP level in semen after freeze-thaw(P < 0.05).Compared with the control group,Compound C group and Compound C + Metformin group were significant negative effects on sperm motility,mitochondrial membrane potential and ATP level in sperm after freeze-thaw(P < 0.05).There was no significant difference in sperm motility,mitochondrial membrane potential and ATP level in sperm between the Compound C treatment group and the Compound C + Metformin treatment group after freeze-thaw.This study showed that metformin added to the semen cryopreservation diluent of the same sheep could reduce the oxidative damage of sperm during cryopreservation and improve the quality of semen after cryopreservation,and the optimal concentration of metformin was 1.0mmol /L. |
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