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Application of C-type natriuretic peptide to promotion on in vitro maturation of bovine oocyte

An oocyte and in vitro maturation culture technology is applied in the application field of C-type natriuretic peptide in promoting the in vitro maturation of bovine oocyte, which can solve problems such as uncertainty, achieve less toxic effect, and accelerate the breeding and propagation of fine varieties. Technical system, the effect of improving the ability of in vitro development

Active Publication Date: 2015-05-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the above is only a mechanism study. Whether CNP can inhibit the meiosis of livestock oocytes such as cattle and be used for in vitro fertilization and embryo production is still uncertain. There is no research report on this aspect so far.

Method used

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  • Application of C-type natriuretic peptide to promotion on in vitro maturation of bovine oocyte
  • Application of C-type natriuretic peptide to promotion on in vitro maturation of bovine oocyte

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Oocyte maturation culture

[0031] (1) Oocyte collection

[0032] Use a 10ml syringe containing egg extraction solution (TCM199+1%PVA+200uM IBMX+1% double antibody) to extract follicles with a diameter of 3-8mm from the surface of bovine ovaries obtained from the slaughterhouse. Put the egg-absorbing liquid after absorbing oocytes into a 100mL domestic petri dish, and select grade A (uniform cytoplasm with 3 or more layers of dense cumulus granulosa cells) and B grade (uniform cytoplasm with few layers) under a stereo microscope. In 3 layers of compact cumulus granulosa cells (wrapped or partially exposed) oocytes are used for in vitro pre-maturation culture.

[0033] (2) Oocyte pre-maturation treatment

[0034] The selected grade A and B cumulus-oocyte complexes (cumulus-oocyte complexes, COCs) were washed 3 times in egg washing solution (TCM199+1%PVA+200uM IBMX+1% double antibody), containing CNP The pre-maturation solution (TCM199 culture solution with 2...

Embodiment 2

[0042] Example 2 In vitro fertilization and in vitro embryo production

[0043] (1) In vitro fertilization

[0044]Using the petri dish micro-drop method, first wash the mature oocytes in the fertilization fluid (BO fluid + 10mM caffeine + 3mg / ml BSA) for 2-3 times, then put them into the balanced fertilization fluid (the amount of 15 oocytes per 50 μl fertilized fluid), oocytes cultured in pre-maturation fluid without CNP as a control; then the frozen semen was processed by the flotation method, and the sperm was washed in the semen fluid (BO fluid + 20ug / ml heparin sodium + 6mg / ml BSA) to float for 20-30min, then take 600-800μl of the supernatant and put it into a 1.5ml centrifuge tube for centrifugation (1500 rpm, 5min) for 2 times, remove the supernatant after centrifugation and add the final volume of washing semen to 250μl, take 50μl after treatment The semen was added to the fertilization fluid that had been put into the oocyte, and the final concentration of sperm wa...

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Abstract

A novel method for maturation of oocytes in vitro, comprising premature culturing in culture liquid of oocytes with C-type natriuretic peptide as meiosis inhibitor and culturing for maturation in maturation liquid. The method of present invention processes oocytes in vitro, and it can promote the maturation of cytoplasm and enhance the development ability. The method uses the ovary and oocyte resource of abattoir, and can increase the speed of breeding selection and propagation technology system, improve efficiency of breeding and level of technology.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of C-type natriuretic peptide in promoting bovine oocyte maturation in vitro. Background technique [0002] In livestock breeding, non-hormone-stimulated oocytes derived from ovaries are widely used to produce animal embryos after in vitro maturation. Therefore, in vitro oocyte maturation is an important platform technology for breeding, cloning and transgenic animal production. [0003] At present, the small number of high-quality breeding cattle, slow breeding speed and backward production performance are the key factors restricting the healthy and sustainable development of my country's cattle industry. The cell numbers of bovine somatic cell cloned embryos (NTEs) and in vitro fertilized embryos (IVFEs) are significantly lower than those of in vivo embryos, which reflects that the in vitro culture system of cattle is not ideal. The quality of bovine in vitro ferti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075C12N5/073
CPCC12N5/0609C12N2501/11C12N2501/31C12N2517/10
Inventor 田见晖贾振伟张家新安磊吴中红
Owner CHINA AGRI UNIV
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