In Vitro Embryo Production And Cryopreservation Of Transgenic FMD VP1 Dairy Goats

In vitro embryo production and cryopreservation technology have important application value in the preservation of livestock germplasm resources.In recent years,with the continuous advancement of research on animal transgenic technology,a variety of new materials for genetically modified animal breeding continue to appear.Some genetically modified animals have entered the stage of safety evaluation,and the cultivation of new varieties of genetically modified animals has important application value in the field of innovation in the livestock breeding industry.At present,all genetically modified animal breeding materials are fed and managed under closed conditions,the establishment of a set of methods for the preservation and utilization of genetic material of genetically modified animals is of great significance to the promotion of genetically modified breeding research.Most of the epitopes of the foot-and-mouth disease virus are concentrated on VP1,as the main immunogenic protein to stimulate Th and B cells to produce immune response.The research team of our laboratory prepared the VP1 transgenic dairy goats in the early stage.In this study,PCR identification was performed on the offspring of transgenic dairy goats,screen out transgenic positive offspring and verify the integration of foreign genes in transgenic ram semen.Use transgenic ram semen for in vitro fertilization to obtain transgenic embryos,at the same time using Cryotop vitrification technology,the components of the vitrification cryoprotectant are selected and optimized by the efficiency of freezing,thawing and recovery of early blastocysts and the expansion rate of blastocysts after continued culture and development.And check the quality of embryos after freezing and thawing,compare the total number of blastocysts,the number of trophoblast cells(TE),the number of inner cell masses(ICM)and apoptosis cells with normal embryos.The conditions suitable for insemination of transgenic goat sperm and cryopreservation of embryos were determined.Used for the preservation and utilization of genetically modified goat embryo resources.The results obtained are as follows:1.14 transgenic offspring milk goats were collected from the jugular vein to extract the goat blood genome for PCR identification,among them,8 dairy goats were identified as dairy goats with foot-and-mouth disease VP1 gene(OFMDV-VP1),4 ewes,4 rams,simultaneous extraction of semen genomes of 4 rams for identification,foreign genes are also integrated in semen.2.Collect transgenic ram semen by electrical stimulation,using Percoll method to process semen for in vitro fertilization to obtain in vitro embryos,and perform PCR identification to determine the transgenic embryos.Continue to culture and use it for subsequent experiments for cryopreservation and embryo transfer.3.Determined to adopt Cryotop method with 40%EG+0.5mol/L L-proline as cryoprotectant,and can be used for cryopreservation of transgenic goat embryos.The recovery rate after freezing and thawing is 89.9%,and the blastocyst expansion rate is74.5%.Comparing the total number of blastocysts,TE,ICM and apoptotic cells of normal embryos and frozen-thawed embryos,there is no significant difference(P>0.05).Embryo transfer with embryos recovered after freezing,the pregnancy rate of ewes is 50%.It can be used in clinical applications to provide help for cryopreservation and production applications of transgenic dairy goat embryo resources.