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Oocyte in-vitro maturation culture method and application thereof

A technology for in vitro maturation culture and oocyte, which is applied to the in vitro maturation culture of oocytes and its application fields, and can solve the problems of unstable in vitro culture effect of oocytes and the like

Active Publication Date: 2022-01-18
北京亿里生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The quality of in vitro cultured oocytes is one of the key prerequisites for determining the success or failure of embryonic bioengineering research and application, but the effect of in vitro cultured oocytes is still unstable

Method used

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  • Oocyte in-vitro maturation culture method and application thereof
  • Oocyte in-vitro maturation culture method and application thereof
  • Oocyte in-vitro maturation culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Collection of bull's eye aqueous humor and preparation of maturation culture medium.

[0040] Cows that are about to be eliminated from the cattle farm are treated with estrus at the same time. When their physiological cycle is in estrus, they are pulled to the slaughterhouse for slaughter. Before slaughter, the cows to be slaughtered are given electric shocks. With a 5ml sterile syringe, the needle is gently inserted into the eye chamber at a 45-degree angle facing the eyeball, and the aqueous humor is extracted until the eyeball collapses. Then the aqueous humor was immediately transferred to a sterile centrifuge tube, sealed and placed in an ice box, and brought back to the laboratory to prepare an aqueous humor maturation solution in an ultra-clean workbench.

[0041] The formula of aqueous humor mature solution is: aqueous humor (basic fluid)+0.01IU / mL FSH+0.1IU / mL LH+1mg / mLE 2 +0.2mM sodium pyruvate.

Embodiment 2

[0042] Example 2 Acquisition of bovine oocytes, in vitro maturation, preparation of in vitro fertilized embryos and somatic cell nuclear transfer embryos.

[0043] (1) Obtaining bovine oocytes.

[0044] The ovaries of adult cattle were collected from the slaughterhouse, placed in 30°C normal saline and sent to the laboratory within 4 hours, washed three times in 37°C PBS solution, and then the follicles with a diameter of 2-8mm were extracted with a needle with a diameter of 0.7mm. Pick cumulus-oocyte-complexes (COCs) with uniform shape and compact structure under a stereoscope.

[0045] (2) Maturation of oocytes.

[0046] The picked cumulus-oocyte-complexes were randomly divided into two groups for the following two treatments.

[0047] Aqueous humor treatment group: use the aqueous humor maturation solution (see Example 1) for maturation culture, specifically, put cumulus-oocyte-complexes at 50-60 pieces / well into the chamber containing the aqueous humor maturation solutio...

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Abstract

The invention provides an oocyte in-vitro maturation culture method and an application thereof. The method of the invention comprises the step of applying aqueous humor from a non-human animal to in-vitro maturation of oocytes of a non-human mammal. Experiments prove that the oocytes matured by the maturation culture method has remarkably higher developmental rate of subsequent in-vitro fertilized embryos and nuclear transfer embryos than a control group, indicating that the method can effectively improve the maturing quality of the in-vitro oocytes. Aqueous humor is used as a basic culture solution for in-vitro maturation culture of the oocytes and has natural advantages as follows: the aqueous humor is rich in nutritional ingredients to meet various nutritional requirements required by in-vitro maturation of the oocytes firstly; various natural growth factors contained in the aqueous humor play important roles in promoting in-vitro maturation of oocytes and later-stage in-vitro embryonic development secondly; and aqueous humor is directly used for culture, thereby avoiding tedious preparation process of culture solution and facilitating large-scale popularization and application of in-vitro embryo culture thirdly.

Description

technical field [0001] The invention specifically relates to a method for in vitro maturation and culture of oocytes and its application. Background technique [0002] The maturation of oocytes cultured in vitro is a very important technical link in animal artificial assisted reproductive technology. With the research and application of embryo engineering technologies such as in vitro fertilization and nuclear transfer, it is becoming more and more extensive. [0003] In in vitro maturation of oocytes, factors such as basal medium, animal serum, hormones, growth factors, etc. have an important impact on the effect of in vitro maturation of oocytes. A large number of experiments have proved that in the in vitro culture of animal oocytes, adding an appropriate amount of growth factors to the maturation solution of animal oocytes has an important effect on the maturation of oocytes and early embryonic development. Most of the added growth factors are substances contained in an...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/84C12N2501/10
Inventor 丁方荣王海平王剑
Owner 北京亿里生物科技发展有限公司
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