Effects Of AMPK Activator On Cryopreservation And Semen Quality In Goats

The semen preservation technology can make semen transported and used across regions and time,which greatly improves the utilization rate of improved breeds of male and female livestock,and also has great significance in saving the cost of breeding and improving the breeds of livestock and poultry.At present,semen preservation technology is mainly divided into three types:room temperature storage,cryopreservation and cryopreservation.The research of goat semen preservation mainly focuses on cryopreservation.In the process of cryopreservation of goat semen,sperm energy metabolism plays an important role,which is very important to maintain sperm vitality and integrity.AMPK is a eukaryotic energy receptor,which regulates energy metabolism.Metformin,as an activator of AMPK,can activate AMPK and make it phosphorylated to participate in energy metabolism.Therefore,this experiment first studied the effect of metformin on cryopreservation of semen in vitro,then added appropriate amount of metformin to goat diet,and through feeding experiment,explored the effect of metformin on goat semen quality and its molecular mechanism.It provides some guidance theory for the application of metformin in animal husbandry.Experiment one:Effect of AMPK activator on cryopreservation in goat semenThe purpose of this experiment was to investigate the effect of AMPK activator on cryopreservation of goat semen.The semen of adult rams was collected and diluted with three kinds of semen diluents.After being treated at 4℃and cryopreserved,the sperm viability,plasma membrane integrity and acrosome integrity were detected,and the best semen diluents were screened for the next stage of the experiment.AMPK activator metformin(Met)and inhibitor Compound C were added to diluted semen to explore the effect of AMPK on goat sperm preservation in vitro.The experiment was divided into three groups:control group,CC group(adding 100 μM Compound C)and Met group(adding 200 μM Met).Sperm viability,plasma membrane integrity rate and acrosome integrity rate were measured after cryopreservation and cryopreservation at 4℃.The results showed that:1)after cryopreservation at 4 h,the sperm viability,plasma membrane integrity rate and acrosome integrity rate of diluted 3 semen were the highest(P<0.05),which showed that the effect of diluent 3 was the best,so the diluent 3 was screened out for subsequent experiments.2)Compared with the control group,the plasma membrane integrity rate and acrosome integrity rate in CC group were significantly lower(P<0.05).The plasma membrane integrity rate and acrosome integrity rate in Met group were significantly higher than those in control group and C C group(P<0.05).The results showed that AMPK activator could improve the cryopreservation quality of goat semen.Experiment 2:Effects of ration formulation added different levels of metformin in goat performance and reproductive performanceThe purpose of this experiment was to investigate the effects of AMPK activator on goat performance and reproductive performance.Twelve adult rams with similar body condition(34.13+4.21 kg)were randomly divided into three groups:control group(basal diet,C,n=4),150 mg/kg metformin supplementation group(basal diet+150 mg/kg Met,Met-150 mg/kg,n=4),300 mg/kg metformin supplementation group(basal diet+300 mg/kg Met,Met-300 mg/kg,n=4).Feeding trial period was 3 weeks.The inspection period was 8 weeks.The results showed that:1)With the increase of metformin dosage,the average daily gain decreased,but there was no significant difference between the two groups(P>0.05);compared with the control group,with the increase of metformin dosage,insulin and auxin decreased significantly(P<0.05),and estrogen level increased significantly(P<0.05).2)Compared with the control group,sperm viability,plasma membrane integrity and acrosome integrity increased significantly with the increase of metformin addition(P<0.05),but sperm viability was not significantly different in Met-150 mg/kg group(P>0.05).Therefore,adding AMPK activator metformin to the diet can improve the semen quality in goats.Experiment 3:Effects of Metformin on antioxidant activity and gene expression in autophagic signaling pathway in goat testisThe aim of this study was to investigate the effects of dietary AMPK activator on the expression of related genes in testicular AMPK and autophagic signaling pathway in goats.The expression of antioxidant markers and related genes,AMPK pathway related gene proteins and autophagy pathway related gene proteins in testicular tissues of goats were detected by ELISA,immunohistochemistry,qPCR and Western blot.The results showed that:1)Dietary metformin significantly decreased the contents of ROS and MDA(P<0.05),significantly increased the activities of antioxidant enzymes CAT,GSH-px,SOD and the expression of mRNA(P<0.05),and the highest level was at 300 mg/kg metformin.2)In testicular tissue,p-AMPK protein is mainly expressed in spermatogenic cells at all levels of seminiferous tubule,and in sperm,p-AMPK is mainly located in equatorial ring and tail of sperm.3)Dietary metformin significantly increased the expression of AMPK and p-AMPK in testicular tissue(P<0.05).The expression of Beclin-1 and LC3 genes and proteins increased significantly with the increase of metformin(P<0.05).