Optimization And Production Application Of Cryopreservation Method For Transgenic Goat Semen

In the early stage,the research team used lentiviral transgenic technology to construct a lentiviral expression vector of the structural protein E2 of the classical swine fever virus and the structural protein VP1 of the foot-and-mouth disease virus,.packaged the virus particles,infected the embryo with the lentivirus,and prepared the transgenic goat for the genic engineering vaccine.Study the antigenic proteins that provide the virus.In this study,PCR technology was used to screen transgenic rams from transgenic embryo transfer progeny.At the same time,in order to rapidly expand the transgenic sheep population and make the transgenic goat semen long-term preservation,fully utilize and improve the utilization rate of the excellent transgenic rams,this study analyzes the semen quality of transgenic rams and compares the transgenic sheep and common ram semen quality,and by optimizing goat semen cryopreservation dilutions and freezing procedures,explore a more optimized cryopreservation protocol.The results obtained are as follows:1.After the transgenic embryo transfer recipient ewe is producing offspring,the ram is selected to collect blood,and the genomic DNA is extracted from the blood for PCR identification.The PCR product is recovered by gel and connected to the T vector for sequencing verification,the adult transgenic male semen genome is extracted verification again.The results showed that foreign genes were integrated into the genomes of three PCDH-BP5/11-OFMDV-VP1 transgenic sheep and three PCDH-BP5/11-CSFV-E2 transgenic sheep semen.2.The transgenic rams and common rams were not significantly different from the ejaculation mass,semen density,sperm motility,plasma membrane integrity,mitochondrial activity and lipid oxidation level,etc(P>0.05).It shows that the transgenic ram has good performance and can be used as a breeding ram for expansion.3.A more optimized formula of frozen semen dilution of goat semen,frozen diluted A solution: Tris 0.5mg/mL,glucose 52mg/mL,fructose 52mg/mL,trisodium citrate 3.5mg/mL,EDTA 0.7mg/mL,lycopene 1.0mg/mL,kelp polysaccharide 1.0mg/mL,sputum polysaccharide 2.0mg/mL,cholesterol-rich cyclodextrin(CLC)1.5mg/mL,gentamicin 0.3mg/mL,generally formulated 100 mL,15% egg yolk was added to the final volume.Freeze diluted B solution: Add 5% glycerol to the A solution.Compared with the ordinary diluted liquid phase,the optimized dilutions showed significant differences in sperm motility,plasma membrane integrity,mitochondrial activity and lipid oxidation level after cryopreservation(P<0.05),and semen after transgenic sheep and common rams were frozen.There was no difference in freezing efficiency(P>0.05).This study demonstrates that the transgenic male goat sperm genome contains exogenous genes,and uses a optimized cryopreservation scheme to preserve a large number of transgenic sheep semen,which lays a foundation for the subsequent expansion of the transgenic sheep population.The frozen semen prepared by the optimized freezing preservation method of goat semen can be used for artificial insemination,and the lambing rate is close to that of natural mating,indicating that the optimized freezing dilution solution is excellent and can be used in clinical practice.