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In-vitro embryo culture method

A culture method, technology of embryo culture solution, applied in the field of embryo culture in vitro, can solve the problems of reducing the effect of adding drugs and covering up the real effect of bovine embryo development, etc.

Active Publication Date: 2017-12-29
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, in actual experiments, we found that mineral oil can adsorb some drugs added to the culture medium, reduce the effect of added drugs, and cover up the real effect of added drugs on the development of bovine embryos

Method used

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  • In-vitro embryo culture method

Examples

Experimental program
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Effect test

Embodiment 1

[0024] The present invention utilizes the non-mineral oil-added culture system to carry out in vitro culture of bovine embryos, and the specific method is as follows:

[0025] 1. Preparations

[0026] Prepare embryo culture medium 3 hours before embryo culture, prepare a 4-well culture dish, 500 μl culture medium per well, put 50 embryos in each well, no need to cover mineral oil, put in 5% CO 2 , 5% O 2 and balance N 2 middle;

[0027] 2. Embryo culture operation in vitro

[0028] After the fertilized eggs are collected, put them into 600μl of hyaluronidase, precipitate for 30s, draw the supernatant to 100μl, shake on the shaker for 5min; add 1ml SOF-HEPES to the centrifuge tube, and move all the liquid to 1000mm After washing the embryos for 3 times in a plate (for the method of washing the embryos, refer to the content of the invention in the instruction manual), put them into the above-mentioned embryo culture plate, add 5% serum on the 3rd day of culture, and judge th...

experiment example 1

[0043] We used GSK-J4 (a cell-permeable prodrug that is an inhibitor of trimethylation demethylase at lysine 27 of histone 3) added to bovine embryo culture medium to examine its effect on embryo As a result, the adsorption of mineral oil to the added drug was found, and the specific embryonic development rate (%) results are as follows:

[0044] (1) The culture medium was not replaced during the culture period

[0045] In the experiment without mineral oil coverage and with mineral oil coverage, the ratios of the four stages of bovine embryo development to 2 cells, 4 cells, 8 cells and blastocyst are basically the same, indicating that whether adding mineral oil to cover the culture medium is important for bovine embryos. Developmental outcomes were not affected, thus demonstrating the feasibility of a bovine embryo culture system without mineral oil supplementation. The results are shown in Table 4.

[0046] Table 4 GSK-J4 was not added to the embryo culture medium

[004...

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Abstract

The invention discloses an in-vitro embryo culture method which specifically comprises the following steps: 1) placing an embryo culture medium in an atmosphere of CO2, O2 and balanced N2 in a culture dish before embryo culture; 2) placing a zygote into a hyaluronidase, allowing standing still, absorbing part of supernatant, abandoning the part of the supernatant and oscillating remaining liquid with an oscillator, so as to obtain mixed solution; 3) adding SOF-HEPES into the mixed solution, transferring the mixed solution to a plate, washing an embryo for several times, placing the washed embryo into the culture dish in step 1), adding 5% of serum into the culture dish on the third day of culture and waiting for blastocyst formation. In the in-vitro embryo culture method, mineral oil is mainly eliminated, and the influence of the covered mineral oil on adsorption of added medicines, so that the actual functions of the added medicines are reflected; meanwhile, a culture system without addition of the mineral oil can also achieve a desired effect of in-vitro embryo culture.

Description

technical field [0001] The invention belongs to the technical field of embryo culture, and in particular relates to a method for in vitro culture of embryos. Background technique [0002] In vitro culture of oocytes is the main channel for in vitro production of embryos, and the effect of culture is affected by various factors such as animal species, culture medium components, and operating proficiency. At present, the in vitro culture of animal oocytes is usually carried out by adding culture fluid to a four-well culture plate or a petri dish and covering the culture fluid with mineral oil. [0003] However, in actual experiments, we found that mineral oil can adsorb some drugs added in the culture medium, reduce the effect of added drugs, and cover up the real effect of added drugs on the development of bovine embryos. Contents of the invention [0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for cultu...

Claims

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Application Information

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IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2500/02C12N2501/73C12N2501/998
Inventor 丁威汤静陈军邢军刘晨
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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