Study On Determination Of PCV-2 Infection And Elimination Methods Of In Vitro Produced Porcine Embryos

In vitro fertilization(IVF)and somatic cell nuclear transfer(SCNT)are important breeding techniques for livestock.High-quality MII oocytes produced from in vitro maturation(IVM)are required for the two techniques listed above.The ovaries used for IVM operations are primarily acquired from commercial abattoirs,and the pathogen status of slaughtered animals becomes an unavoidable issue.Our previous monitoring data showed that Porcine circovirus type 2(PCV-2)infection rate in slaughterhouse-derived ovary is 77.5%,which is one of the main pathogens in slaughterhouse-derived ovary.However,the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production(IVP)of embryos are unclear,and currently there are no relevant studies.Therefore,the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development.Polymerase chain reaction(PCR)was used to detect PCV-2 DNA in ovaries,follicular fluid(FF),oocytes,cumulus cells and IVP embryos.The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated.We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR.Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a.PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation.Moreover,virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers.Besides in IVF embryos were tested,we also tested the copy number of PCV-2 virus at various stages during the development of somatic cell nuclear transfer embryos.Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium.Viruses in oocytes,embryos and culture fluids can cause potential biosecurity risks for in vitro embryo production,embryo transfer and cloning animals,disease model animals and xenotransplantation animals,therefore,we added the antiviral drugs amantadine and selenomethionine to the in vitro culture medium,and evaluated the inhibitory effect of drug addition on PCV-2 replication during in vitro culture.First,we assess the concentration of amantadine and selenomethionine for PCV-2-negative embryos during in vitro embryo development.Results of subsequent drug addition experiments showed that the amantadine and selenomethionine addition can significantly decrease the PCV-2 virus copy numbers in embryo during embryo development.Further,the viral Cap protein expression level decreased obviously after amantadine and selenomethionine addition.In summary,For the first time,this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.It was found that follicular fluid will be a potential source of virus infection in oocytes during the maturation of oocytes.At the same time,during embryo development,the susceptibility of embryos to viruses increases with development,resulting in high levels of virus DNA in embryos in the blastocyst stage.Subsequent experiments with antiviral additions results imply that addition of amantadine and selenomethionine in embryo culture medium is a practical strategy to eliminate the PCV-2 in naturally infected oocytes and improve the biological safety of in vitro production(IVP).