Effects Of Glucose Levels And TLR7/8 Ligands On Sperm Motility And Acrosomal Status During Boar Sperm Preservation

In recent years,the scale of intensive pig breeding has gradually increased,and artificial insemination（AI）technology has been widely used.AI can save cost and improve efficiency,and greatly improve the utilization rate of genetic resources of high-quality breeding pigs.In production,the method of liquid storage of boar sperm at 17℃ is generally used to preserve and transport boar semen,and the maintenance of boar sperm motility and fertilization ability is the focus of sperm preservation in vitro.In addition,in the process of sperm preservation in vitro,according to the biological characteristics of sperm,applying treatment to control the sex of progeny individuals can effectively improve the production efficiency.This study attempted to explore the effects of different levels of glucose on the preservation of porcine sperm,and to explore the internal molecular mechanism.Based on this,to explore the separation of X and Y sperm to provide guidance for gender control.The research content is divided into two parts.The first part explores the effects of different glucose concentrations on the storage of boar sperm at room temperature and its molecular mechanism,so as to extend the preservation time of sperm on the premise of ensuring sperm motility and acrosomal integrity.The second part,using the molecular characteristics of X and Y sperm differences,optimize the glucose level,to explore a rapid and simple method to separate X and Y sperm,in order to achieve the purpose of rapid separation of boar X and Y sperm.1.Lysine acetylation participates in boar sperm motility and acrosome status regulation under different glucose conditionsFor the study of boar sperm in vitro preservation process glucose concentration effects on sperm motility and acrosome status,respectively in boar semen diluent adding high concentration（153.0 m M）glucose and glucose,low concentration（30.6 m M）in physiological temperature（37℃）and room temperature preservation（17℃）to preserve,under the condition of the glucose concentration of sperm viability,mitochondrial activity and acrosome integrity.The results showed that the sperm motility,mitochondrial membrane potential and acrosomal integrity of low glucose group were significantly higher than those of high glucose group at physiological temperature and normal temperature.In addition,the total lysine acetylation level of sperm protein under different glucose levels was also determined.It was found that the acetylation level of sperm protein in the high glucose group was higher than that in the low glucose group,and the acetylated substrate acetyl-Co A in the high glucose group was significantly higher than that in the low glucose group.In addition,different concentrations of lysine acetyltransferase KAT6A inhibitors were added to the diluted solution.It was found that sperm motility and acrosomal integrity were significantly increased in the high glucose group.These results indicate that glucose concentration in diluent can affect sperm motility and acrosomal integrity,and lysine acetylation induced by high glucose concentration and Ca²⁺are involved in the regulation of sperm motility and acrosomal integrity.These results provide theoretical guidance for the design of glucose levels and the improvement of preservation methods for boar semen in vitro.2.Isolation of X and Y boar sperm based on TLR7/8 specific ligand and glucose levelStudies have shown that specifically expressed Toll-like receptor 7/8（TLR7/8）in mouse and bovine X spermatozoa and the addition of TLR7/8 ligand resiquimod（R848）in semen can specifically bind to X spermatozoa and reduce its viability.In order to explore whether this method can also produce the same effect on boar sperm,so as to establish a separation method for X and Y boar sperm,this experiment added R848 to boar sperm stored at room temperature,and further optimized the conditions to explore the influence of R848 on the motility of boar sperm.The results showed that R848 supplementation decreased sperm motility and phosphorylation of Glycogen synthase kinase-3α（GSK3α）.Real time RT RCR was used to detect the abundance ratio of Y-sperm specific gene SRY and X-sperm specific gene PLP.No significant difference was found in the proportion of X and Y sperm in R848treated sperm.In view of the previous study that glucose concentration can affect sperm motility,we further optimized the glucose concentration in the diluent,and found that when2 m M glucose was added,R848 increased the ratio of SRY/PLP in high motility sperm,that is,R848 could reduce the ratio of X/Y sperm in high motility sperm to a certain extent under the condition of 2 m M glucose.These results provide guidance for the separation of X and Y sperm and sex control.In conclusion,high glucose concentration leads to a decrease in sperm motility during in vitro storage,accompanied by an increase in protein lysine acetylation.Inhibition of sperm protein lysine acetylation can improve sperm motility and delay acrosomal reaction,thus prolonging the preservation time.The addition of TLR7/8 specific ligand R848 reduced the motility of porcine sperm,and with the decrease of GSK3αphosphorylation level,the proportion of X sperm in highly motile sperm was reduced to a certain extent when glucose concentration was set at 2 m M.