Double staining method for bovine in-vitro fertilization blastocyst

Abstract

The invention discloses a double staining method for a bovine in-vitro fertilization blastocyst. The method comprises the steps that the bovine in-vitro fertilization blastocyst which is cultured for 7-9 days is processed with 0.5% pronase for 55 seconds, washed in a PBS for 2-3 times, stained in PI (1% TritonX-100 / PBS) with the concentration of 100 mug / ml for 20 seconds, washed in the PBS for 2-3 times, transferred into Hoechst33342 with the concentration of 45 mug / ml to be stained for 50 seconds, washed in the PBS for 2-3 times and then processed through preforming; the bovine in-vitro fertilization blastocyst is placed under an inverted fluorescence microscope after preforming is finished, observing and photographing are performed under ultraviolet light, cell nucleuses of an inner cell mass (ICM) are blue, and cell nucleuses of trophoderm cells are red. According to the double staining method, staining can be completed in 3 minutes, but at least 47 minutes are needed in previous reports; accordingly, the time needed by double staining of the blastocyst is greatly shortened, the efficiency is improved, and the bovine blastocyst quality can be quickly evaluated.

Description

technical field [0001] The invention belongs to the biological technical field of bovine in vitro fertilization, and relates to a method for evaluating the quality of bovine in vitro fertilized blastocysts, in particular to a simple and rapid double-staining method for bovine blastocysts. Background technique [0002] Since the successful establishment of the cattle IVP system, people have tried to improve the system from different angles, [0003] For example, changing the culture system, adding lipid metabolism regulators (lipidmetabolicregulators), adding cAMP regulators (Cyclicadenosinemonophosphate (cAMP) modulators), etc., but compared with in vivo embryos, the quality of bovine in vitro embryos is still poor, not only the number of blastocyst cells is lower Fewer and the transplant pregnancy rate is not high. This makes people realize the necessity of improving the quality of bovine embryos, so the inspection of embryo quality has gradually become one of the importan...

AI Technical Summary

Problems solved by technology

The current blastocyst double-staining method is established on the basis of Thous et al. using TritonX-100 to double-stain the blastocyst, but the biggest problem is that the procedure is cumbersome, and each step of staining takes a lot of time. The entire staining procedure takes at least 47 minutes. Above, so the staining time is long (Li Ruiqi, Sang Runzi. The effect of fructose on the early development of bovine embryos was tested by double staining of blastocysts[J]. China Journal of Animal Husbandry, 2010(9): 23-25; Li Rong, Liu Ying, Zhao Xingbo, et al. Application of serum-free medium IVD101 and G1 / G2 in bovine somatic cell nuclear transfer[J]. Journal of Animal Husbandry and Veterinary Medicine, 2008, 39(11): 1487-1492; ThouasGA, KorfiatisNA, FrenchAJ, etal .Simplifiedtechniquefordifferentialstainingofinnercellmassandtrophectodermcellsofmouseandbovineblastocysts[J].Reproductivebiomedicineonline,2001,3(1):25-29.)

[0004] Usually when evaluating the quality of blastocysts, due to the different ages and developmental stages of embryos, it is necessary to classify the embryos and dye them in multiple batches, so the dyeing takes a long time, the efficiency is low, and the effect of dyeing may be affected

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