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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 technology, applied in the biological field, can solve uncertain problems, achieve the effect of less toxic effect, improve breeding efficiency and technical level, and accelerate the breeding of improved varieties and the effect of multiplication technology system

Active Publication Date: 2013-01-30
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) twice, 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 was 1×10...

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Abstract

The invention relates to application of a C-type natriuretic peptide (CNP) to promotion on in vitro maturation of bovine oocyte. The invention provides a culture liquid for in vitro maturation of bovine oocyte; and a conventional culture liquid is used as a matrix containing C-type natriuretic peptide. According to the invention, CNP is used for in vitro inhibition on bovine oocyte meiosis and premature treatment to promote synchronization of oocyte nuclear maturation and cytoplast maturation, and improve ability of ectogenesis. The CNP can be promoted and applied as a novel meiosis inhibitor to animal husbandry, so as to accelerate fine breed breeding and an expanding propagation technique system. As a biological activity peptide substance, the CNP provided by the invention has toxic effect on oocyte less than that of a chemically synthesized meiosis inhibitor.

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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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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