动物实验,体外受精技术 z-bo 2020-10-14 447

　　(1) The collection of egg cells follows

　　13-14 hours after HCG injection, collect egg cells from Faraday tubes. This method is the same as that described in the second part of "Mouse Embryo Transfer". The collected egg stromal cell-oval complex was removed with 0.1% hyaluronidase to remove cumulus cells, and the cells were placed in TYH droplets.

　　(2) Collection and volume of sperm

　　Collect mouse sperm from the epidermis according to the method described in the first section of this chapter, or collect sperm using pseudo-vaginal and electrical stimulation methods. I will. The semen was centrifuged, washed 2-3 times with TYH solution, placed in TYH droplets, and incubated at 37° C., 5% CO 2 to enable the sperm to gain fertility. The fertile semen was transferred to TYH droplets containing oocytes, and the sperm concentration of the final solution reached 4×106/ml.

　　(3) In vitro fertilization In vitro fertilization is accomplished by incubating TYH droplets containing egg cells and fertilized sperm under 5% CO2 at 37°C for 6 to 8 hours. Remove the petri dish from the incubator and wash the treated egg stromal cells with TYH solution. At this time, the second pole and two pronuclei were observed in the fertilized egg cell.

　　(4) Culture of fertilized eggs The fertilized eggs are cultured in TγYH droplets at 37°C and 5% CO 2 for 24 to 28 hours. The microscope showed that the blastomere developed into 2-4 cells. Transfer these developed blastomeres to BMOC-3, TYH or Ham's F-10, wash 2-3 times, and then continue to incubate with the corresponding medium to form about 20 blastomeres.

　　(5) Embryo transfer According to the method described in Section 2 of this chapter, the embryos of in vitro fertilization are transferred into the Faropius tube of the pseudopregnant recipient female mouse.