| With the development of modern animal husbandry,efficient and accurate sex control technology has become an important technical means to improve the economic benefits of livestock production.Sex control of semen is the most important method to achieve sex control,which can not only bring huge economic benefits to breeding industry,but also speed up the breeding process and give full play to the genetic potential of superior males.Based on the differences between X and Y sperm,many methods have been reported for the separation of X and Y sperm,such as flow cytometry,Percoll and albumin gradient centrifugation,upstream and immunological methods.However,only flow cytometry has been proven to be the most stable and effective method to separate X and Y sperm based on the difference in DNA content between X and Y sperm and has achieved commercial application in dairy industry.However,the use of flow cytometry to separate X and Y sperm still has some problems,such as high economic cost and sperm damage in the separation process,which greatly limits its wide application in animal husbandry.In recent years,the demand for beef in China has increased rapidly,but the yield is obviously insufficient,and the number of viable cows is small,which seriously restricts the healthy development of beef cattle industry in China.Therefore,the beef cattle industry needs an economical,efficient and less harmful sperm sorting method,in order to rapidly increase the breeding of cows and promote the development of beef cattle industry.The immunological method,which isolates X and Y sperm based on their protein differences,is emerging as an attractive alternative to flow cytometry.Therefore,in this study,TLR7/8 immunofluorescence staining of testicular round sperm,epididymal sperm and mature sperm of Qinchuan cattle was conducted to determine the specificity of TLR7/8 in X sperm,and the effects of TLR7/8 excitant R848 on sperm motility were studied,and the purity of X and Y sperm after separation was evaluated.To establish the activated separation technology of TLR7/8 specific protein in beef sperm.The main results of this study are as follows:1.Testicular tissue,epididymis tissue and mature sperm of Qinchuan cattle were stained with TLR7 and TLR8 immunofluorescence,and sperm were labeled with PNA.The results showed that TLR7/8 was partially expressed in testicular round sperm and epididymal sperm,and TLR7-positive(TLR7~+)and TLR8-positive(TLR8~+)sperm accounted for 47.61%±2.21%and 47.57%±0.63%of testicular round sperm,respectively.TLR7~+sperm and TLR8~+sperm accounted for 45.87%±2.61%and 44.81%±2.26%of epididymal sperm,respectively.TLR7and TLR8 were located in the tail flagellum and middle part of the tail of mature sperm,respectively,and TLR7~+sperm and TLR8~+sperm accounted for 55.54%±1.65%and54.12%±4.20%of the total sperm,respectively.2.To investigate the effect of TLR7/8 agonist R848 on sperm motility,sperm were co-incubated with TLR7/8 agonist R848 at different concentrations(0,0.06,0.6,1,2,4μmol/L),and the proportion of floating sperm was analyzed.The results showed that the proportion of upper X sperm in R848 0.6,1,2 and 4μmol/L groups was significantly lower than that in the control group(P<0.05).Meanwhile,0.6μmol/L R848 was incubated with spermatozoa for 0,15,30,60 and 120 min),the number of upper-layer X sperm showed a time-dependent decline,that is,the number of upper-layer X sperm gradually decreased with the increase of time,and the number of upper-layer X sperm decreased by about 50%compared with the control group after incubating R848 sperm for 60 min.In addition,VAP,VSL and VCL of lower X sperm(lower sperm 1 m L)were significantly lower than those of upper Y sperm(upper sperm 1 m L)and control group(P<0.05),and the lower X sperm motility trajectory became shorter;Meanwhile,the motility of lower X sperm was significantly lower than that of upper Y sperm and control group(P<0.05),but there were no significant changes in plasma membrane integrity and acrosome integrity of upper and lower sperm compared with the control group(P>0.05),indicating that TLR7/8 agonist R848 inhibited the motility of lower X sperm,but had no significant effect on the quality of upper and lower Sperm.3.In order to investigate the effect of TLR7/8 agonist R848 on sperm motility,0.6μmol/L R848 was co-incubated with sperm for 1 h,and ATP and mitochondrial membrane potential of upper and lower sperm were detected.The results showed that the ATP and mitochondrial membrane potential of lower X sperm were significantly lower than those of upper Y sperm and control group(P<0.05).At the same time,sperm and R848 were incubated for 0,15,30,45 and 60 min,respectively.It was found that the ATP and mitochondrial membrane potential of R848 were significantly decreased when incubated with sperm for 30,45 and 60 min compared with the control group(P<0.05).In order to reveal the mechanism of R848 reducing SPERM ATP and mitochondrial membrane potential,Western blot analysis was performed on upper Y sperm and lower X sperm after incubating R848 with sperm for 1 h.The phosphorylation levels of NFκB and GSK3α/βin lower X sperm were significantly increased compared with that in upper Y sperm(P<0.05).In addition,the phosphorylation levels of NFκB and GSK3α/βwere significantly increased after R848 was incubated with sperm for 15 min and 30 min compared with the control group(P<0.05).TLR7 immunofluorescence staining was performed on X and Y sperm after sorting,and it was found that most X sperm were TLR7~+sperm,while only a few Y sperm were TLR7~+sperm.The purity of Y sperm was 88.6%,while that of X sperm was72.5%.In conclusion,TLR7 and TLR8 are partially expressed in testicular round sperm,epididymal sperm and mature sperm,and TLR7/8 agonist R848 inhibits glycolytic ATP production and decreases sperm mitochondrial membrane potential through GSK3α/βand NFκB phosphorylation pathways,thus reducing sperm motility and inhibiting sperm motility.But it has little effect on sperm quality.In addition,the purity of Y sperm and X sperm reached 88.6%and 72.5%respectively after sorting.Therefore,in this study,the activation separation technology of specific protein in beef cattle sperm was preliminarily established,which can provide theoretical reference for the development of new technology of bovine semen sex control.TLR7+sperm,while only a few Y sperm are TLR7+sperm.The purity of Y sperm was 88.6%,while that of X sperm was 72.5%. |
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