Method for fertilizing oocytes in vitro of young animals twice

Abstract

The invention provides a method for fertilizing oocytes in vitro of young stock twice, which comprises the following steps of: performing FSH super-ovulation processing on 45 to 100-day young female animals which are receptors and taking more than 100 oocytes from each young female animal for later use; mixing the oocytes in vitro matured for 25 to 26 hours and sperms to finish in-vitro fertilization of the first time; and after 5 to 6 hours, performing the in-vitro fertilization of the second time, transferring the fertilized oocytes into a culture plate for culture after 13 to 14 hours, and counting a clearage rate after 48 hours. The matured oocytes need treating with 0.1 percent hyaluronidase for 30 to 60 seconds, and then are added into a fertilization medium for incubation after cumulus cells are partially removed from the oocytes in a blowing and sucking way, wherein the concentration of the fertilization medium is 50 to 70 mu l per drop and is well balanced for 2 to 5 hours; semen is unfrozen with a water bath and is placed into a CO2 box, and after 20 to 25 minutes, supernatant fluid is extracted and centrifugated for 2 to 5 hours, and then the precipitated sperms are added into the fertilization medium for incubation according to the density of 2-4*106 sperms per ml; ova fertilized for 5 to 6 hours are taken out and are placed into the fertilization medium; the sperms are unfrozen by the similar method, are added into the fertilization medium, and after 13 to 14 hours, are washed and transferred into a well-balanced four-hole culture plate for culture, wherein each hole of the four-hole culture plate contains 500 mu l of the washed solution. The method has the advantages of simplicity, practicability and the capacity of making the fertility rate of young cows and ewes reach 80 to 92 percent while blastocyst rate reach 40 to 50 percent.

Description

technical field [0001] The invention relates to in vitro fertilization of young animals, in particular to an in vitro fertilization method for oocytes, which can increase the fertilization rate and embryo development rate by adopting effective two-time fertilization. Background technique [0002] In vitro fertilization refers to the process of completing the fertilization of sperm and eggs in an external environment. JIVET (juvenile invitro embryo transfer) is the early assisted reproductive technology of juvenile animals. It is an embryonic biotechnology system integrating the technology of inducing follicle development in juvenile animals, in vitro fertilization, embryo transfer and other technologies; its main technical links include superovulation of juvenile animals, egg Mother cell collection, in vitro maturation culture, in vitro fertilization, in vitro development of early embryos, embryo transfer, etc., in vitro fertilization is one of the key steps. The traditiona...

AI Technical Summary

Problems solved by technology

This shows that domestic research on the double fertilization of porcine oocytes in vitro has been reported in the literature, but there are fewer reports on the double in vitro fertilization of young animal oocytes

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