Method for improving in-vitro utilization rate of sheep oocyte

A technology for oocytes and sheep, which is applied in the field of improving the utilization efficiency of sheep oocytes in vitro, and can solve the problems of low maturation rate of negative oocytes

Inactive Publication Date: 2010-11-24
新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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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

Method used

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  • Method for improving in-vitro utilization rate of sheep oocyte
  • Method for improving in-vitro utilization rate of sheep oocyte

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Embodiment

[0026] Experimental operation method:

[0027] The sheep ovaries that were slaughtered within 30 minutes of being slaughtered were placed in physiological saline containing 1000IU / ml penicillin and 1000IU / ml streptomycin at a temperature of 30-35°C, and washed 3 times with physiological saline within 3 hours; The oocyte was extracted, a 10ml syringe with a 9-gauge needle was used to aspirate the follicles with a size of 6 mm on the surface of the ovary, the follicular fluid recovered in the syringe was poured into a 45mm dish, and the oocytes were picked up under a microscope and added with a concentration of 30 μMol. / L of BCB staining solution, stained in a 36°C water bath for 90 minutes; BCB+ and BCB- oocytes were separated under a microscope; BCB+ oocytes were directly washed twice with mature culture solution and cultured for 24 hours; - Oocytes were washed twice with a maturation medium containing 80 μMol / L of the PDE3 inhibitor milrinone for 8 hours, washed 3 times with...

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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 mature culture 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-methylaminopurine 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 phosphodiesterase inhibitor (PDE) milrinone, a method for inhibiting BCB negative oocytes with poor quality after BCB screening, which 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, they are classified 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 mammalian oocyte meiosis recovery is regulated by the second messenger cAMP, and PDE is a key enzyme regulating intracellular cAMP levels, so PDE can block the recovery of meiosis in oocytes cultured in vitro, It plays an important role in the regulation of oocyte maturation and division. [0003] In a previous study of a screening and culture method ...

Claims

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Application Information

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IPC IPC(8): C12N5/075
Inventor 黄俊成汪立芹赵云程刘明军林嘉鹏陈童
Owner 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心
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