Method for improving in-vitro utilization rate of sheep oocyte

Abstract

The invention provides a method for improving the in-vitro utilization rate of sheep oocyte, which comprises the following steps of: washing poor quality BCB-oocyte obtained by chromosome segregation for 2 to 3 times by using mature culture solution which contains 50 to 100mu Mol / L PDE3 inhibitor milrinone and culturing for 6 to 9 hours, and washing the BCB-oocyte for 2 to 3 times by using matureculture solution which does not contain the milrinone and culturing for 24 hours; removing granular cells around the oocyte, transferring the oocyte to cleaning solution, picking the oocyte and removing a first polar body, and counting the removal rate of the polar body, namely maturation rate; and activating the oocyte from which the first polar body is removed for 4 to 5 minutes by using 5mu Mol / L ionomycin and washing the oocyte for 1 to 3 times by using the cleaning solution, activating the oocyte for 2 to 3 hours by using 2mMol / L 6-methyl aminopurine which is balanced in a CO2 box 2 to 3 hours ago, washing the oocyte for 3 or 4 times by using the culture solution which is balanced in the CO2 box 2 hours ago, culturing the oocyte in the culture solution for 48 hours, counting cleavage rate, and counting blastocyst rate after 168 hours. In addition, the BCB-oocyte obtained by the chromosome segregation is washed by the mature culture solution for 2 to 3 times and cultured for 24 hours to be directly utilized. The method has great practical value for the in-vitro utilization of the sheep oocyte.

Description

technical field [0001] The invention relates to a phosphodiesterase inhibitor (PDE) milrinone, a method for inhibiting BCB negative oocytes with poor quality after BCB screening, and can significantly improve the in vitro maturation rate of sheep oocytes. Background technique [0002] PDEs are a class of isoenzymes that can hydrolyze intracellular cyclic nucleotide second messengers. In mammals, according to PDE kinetics, substrate specificity, distribution in tissue cells, inhibitors and agonists, and amino acid sequences It is divided into 11 categories, PDE3 is one of them. Many studies have shown that the resumption of meiosis in mammalian oocytes is regulated by the second messenger cAMP, and PDE is a key enzyme that regulates the level of cAMP in cells, so PDE can block the resumption of meiosis in cultured oocytes in vitro, Plays an important role in the regulation of oocyte maturation and division. [0003] In a previous study of a method for screening and culturin...

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Problems solved by technology

[0003] In a previous study of a method for screening and culturing sheep oocytes in vitro (patent application number: 200910113567.9), it was found that the maturation rate of negative oocytes after BCB screening was significantly lower than that of positive oocytes. Therefore, how to Improving the quality of negative oocytes has become an urgent problem to be solved

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