Effects Of Metformin On Schizothorax Prenanti Sperm At 4℃ Storage And Its Mechanism

Schizothorax prenanti is a unique cold-water fish in our country and an important characteristic economic fish in the upper reaches of the Yangtze River.However,this population is facing problems such as overfishing and habitat destruction,its wild resources have declined sharply,and the scale of fishing has continued to shrink.The development and utilization of fish sperm preservation technology can promote the rapid development of artificial breeding technology in production,and promote the recovery and market supply of wild populations of Schizothorax prenanti.The timeliness of fish sperm preservation limits its wide application.With the prolongation of the storage time,the metabolites produced by sperm lead to various physiological functions and structural disorders of sperm,affecting sperm motility.Appropriate addition of protective agent in the fresh-keeping process of milt can effectively reduce the damage to sperm during low-temperature storage and prolong the storage time of sperm.Clinically,metformin is used to restore the body’s utilization of glucose,thereby improving and regulating the body’s metabolic disorders,and then restoring the body’s metabolic homeostasis.The effect of metformin on cryopreserved spermatozoa(4 ℃)was studied.The effect of metformin on cryopreservation of spermatozoa was assessed by measuring sperm motility,ATP content,plasma membrane integrity,mitochondrial membrane potential,and fertilization capacity.This study investigated the effect of different concentrations(100,200,400,800 μmol/L)of metformin on the sperm quality of Schizothorax prenanti stored at 4 ℃ for 72 hours,and whether metformin affects the sperm quality during freezing by activating AMPK.The following main results were obtained:1.The effect of metformin at four different concentrations(100,200,400 and 800μmol/L)on the quality of cryopreserved Schizothorax prenanti sperm was studied.The results showed that with metformin(400 μmol/L)in semen extender was the ideal concentration to maintain normal physiological conditions such as sperm motility,ATP content,plasma membrane integrity and mitochondrial membrane potential,showing that it can prolong the time of Schizothorax prenanti sperm in vitro stored at 4 ℃(P<0.05).2.The study also found that sperm glucose uptake is concentrated in the neck of the sperm,where the mitochondria were located.Metformin activates the AMPK pathway to enhance glucose uptake by sperm,thereby maintaining stable ATP levels and improving sperm quality and fertilization capacity during in vitro cryopreservation(P<0.05).3.The positive effects of metformin on sperm quality,glucose uptake,and fertilization capacity were significantly inhibited by Compound C,an AMPK-specific inhibitor that blocks AMPK phosphorylation(P<0.05).In summary,AMPK played an important role in vitro sperm storage.Metformin promoted glucose uptake by activating AMPK,maintained ATP content,prolonged storage time,and improveed the quality of Schizothorax prenanti sperm,indicating that metformin has a promising application prospect in the practice of in vitro preservation of fish sperm.