Effect Of Spermatozoa Treated With R848 On Sex Ratio Of Mouse Embryos

Sex control technology is very important to animal husbandry,but the development of sex control technology in China is relatively slow.The purpose of this experiment is to explore a timesaving,rapid,efficient and novel X/Y sperm separation control technique,and obtain the male and female offspring with skewed sex ratio.In this study,a feasible spermatostratification model was established by adding TLR7/8 ligand R848.Different concentrations of R848(0,0.3,1,3 μmol/L)were added to the model to incubate mouse sperm for 60 min.After obtaining semen from the upper(UP)and lower(Down)layers respectively,It is used for In vitro fertilization(IVF)and sperm motility,motion parameters and sperm plasma membrane integrity detection,to evaluate sperm fertilization ability and embryo development potential after fertilization,and finally use PCR method of SRY fragment to identify the sex of early embryos.The test results show that:(1)In the stratification model of X/Y spermatozoa,it was found that UP/Down spermatozoa obtained by 0.3 μmol/L R848 were significantly reduced in IVF compared with the corresponding0 μmol/L UP/Down spermatozoa(P<0.01).The fertilization ability of the 0.3 μmol/L UP/Down group before and after cleaning was compared.It was found that the fertilization rate of the unwashed Down group was significantly increased after cleaning(P<0.01),and the blastocyst rate of the UP group was significantly increased after cleaning(P<0.01).(2)With the increase of R848 concentration,the fertilization ability of sperm in the UP/Down group was decreased to varying degrees,and the higher the supplemental concentration,the lower the fertilization rate.Fertility rates in 0.3 μmol/L R848 UP/Down groups were significantly higher than those in 1 μmol/L UP/Down and 3 μmol/L UP/Down groups(P<0.01).The sperm motility and motility of incubated with different concentrations of R848 were significantly lower than those of 0 μmol/L group(P<0.01),and the higher the concentration was,the greater the effect on motility and motility was.With the increase of R848 concentration,the mean path Velocity(VAP)and Curvilinear Velocity(VCL)of sperm are reduced to varying degrees.The plasma membrane integrity of spermatozoa in the UP/Down group supplemented with 0.3 μmol/L R848 and 1 μmol/L R848 was significantly lower than that in the UP/Down group.The complete damage rate and half damage rate of plasma membrane were higher than those of 0.3 μmol/L and 0 μmol/L groups(P<0.01).However,there was no significant difference between 0.3 μmol/L group and 0 μmol/L group.(3)The sex of blastocysts obtained from IVF of semen in each concentration group was determined by Polymerase chain reaction(PCR).The percentage of XY embryos in 0.3 μmol/L UP group(63.6%)and the percentage of XY embryos in 1 μmol/L UP group(67.1%)were both higher than the corresponding XX ratio,and significantly higher than that in 0 μmol/L UP group(P<0.05).Meanwhile,the percentage of XX embryos in 0.3 μmol/L UP group(36.4%)and 1 μmol/L UP group(32.9%)were significantly lower than that in 0 μmol/L UP group(P<0.05).The percentage of XY embryos obtained in 0.3 μmol/L Down group(41.2%)and 3 μmol/L Down group(40.0%)were significantly lower than that in 0 μmol/L Down group.Meanwhile,the percentage of XX embryos in 0.3 μmol/L Down group(58.8%)and 3 μmol/L Down group(60.0%)were significantly higher than that in 0μmol/L Down group(P<0.05).The results showed that the sperm stratification model of mice incubated with R848 could effectively separate the X/Y sperm population and obtain the offspring with skewed sex ratio.Meanwhile,the separation rate was faster,the operation procedure was simple and the cost was lower;Under this model,add 0.3 μmol/L In the stratified semen obtained by R848,X/Y sperm was the best,and the sex ratio of early embryos was higher,which laid a foundation for the preparation and production of sex control semen of large domestic animals in China.