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A bovine oocyte maturation medium in vitro containing trehalose and its culture method

A technology for in vitro maturation culture and oocyte, which is applied in the field of in vitro maturation culture and in vitro fertilization of bovine oocytes, can solve problems such as trehalose that has not yet been seen, achieves easy popularization and application, low economic cost, and solves the problem of low in vitro development rate of embryos. Effect

Inactive Publication Date: 2015-08-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there has been no report on the use of trehalose in the field of livestock reproduction technology, especially for in vitro maturation of oocytes

Method used

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  • A bovine oocyte maturation medium in vitro containing trehalose and its culture method
  • A bovine oocyte maturation medium in vitro containing trehalose and its culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of culture medium for in vitro maturation of oocytes containing trehalose

[0025] Weigh 10 mg of daidzein, dissolve it in 1 mL of DMSO, prepare the mother liquor and store it in a refrigerator at 4°C. Accurately weigh 30 mg of trehalose, first dissolve it with 20 mL of M199 culture medium (Gibco Company), and then add 2.5 μL of the above-mentioned daidzein mother solution, 5 mL of calf serum, 5000 IU of penicillin, 5000 μg Streptomycin, stirred gently, then added M199 culture medium to a volume of 50mL, and stirred again. After filtration and disinfection with 0.22 μM microfiltration membrane (Millipore Corporation, Bedford.Mass.USA), store at 4°C until use.

Embodiment 2

[0026] Embodiment 2: Culture solution containing trehalose is used for in vitro maturation of dairy cow oocytes

[0027] Dairy cow oocytes were cultured in the control medium (M199 medium) without trehalose and daidzein, the M199 medium containing only 0.6 mg / mL trehalose, and the M199 medium containing only 0.5 μg / mL daidzein. and the bovine oocytes prepared in Example 1 containing trehalose and daidzein in the in vitro maturation culture medium for maturation, and then in vitro fertilization. The specific method is as follows:

[0028] A. Maturation culture: Bovine oocyte maturation medium in vitro forms a culture droplet with a volume of about 80-100 μL on a disposable plastic culture dish, preheats it in an environment of 38-39°C for 2 hours, and washes the bovine oocyte Then put into the preheated culture droplet, at 38~39℃, 5%CO 2 Cultivate in the environment for 24 hours;

[0029] B. In vitro fertilization: Use the fertilization medium to form fertilization droplets ...

Embodiment 3

[0036] Example 3: Culture solution containing trehalose is used for in vitro maturation of bovine oocytes

[0037] Cattle oocytes were matured and cultured in the control medium without trehalose and oocytes containing the trehalose prepared in Example 1, respectively, and then fertilized in vitro. The method was the same as in Example 2.

[0038] The results showed that on the second day of cultivation, the cleavage rate of oocytes in the control medium without trehalose after fertilization was 71.3%, and the oocytes in the in vitro maturation of oocytes containing trehalose prepared in Example 1 The division rate of the cells after fertilization was 81.7%, which was 10.4% higher than that of the control group. On the 8th day of culture, the blastocyst stage development rate after fertilization of oocytes in the control culture medium without trehalose was 21.3%, and the eggs in the in vitro maturation of oocytes containing trehalose prepared in Example 1 The blastocyst deve...

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Abstract

The invention provides bovine oocyte in-vitro mature culture solution containing mycose and a cultural method. The bovine oocyte in-vitro mature culture solution comprises 0.1-1.0mg / mL of mycose, 0.1-10mu g / mL of daidzein, 100IU / mL of penicillin, 100mu g / mL of streptomycin and 8% to 20% serum, wherein the solvent is regular nutrient solution which is one or more of M199, DMEM, F12 and MEM. The combination of mycose and daidzein is applied to bovine oocyte in-vitro mature culture, the cytoplasm maturing of oocyte can be remarkably promoted in comparison with solely using mycose or daidzein, and the problem of low embryo in-vitro developmental rate after the in-vitro mature oocyte is fertilized. The bovine oocyte in-vitro mature culture solution is low in economical cost and convenient to popularize and applied to production.

Description

(1) Technical field [0001] The invention relates to a bovine oocyte maturation culture solution in vitro containing trehalose, and a method for in vitro maturation culture and in vitro fertilization of bovine oocytes by using the bovine oocyte maturation culture fluid in vitro. (2) Background technology [0002] With the development of modern reproductive biotechnology and the gradual improvement of embryo in vitro production technology, the use of in vitro fertilization, somatic cell nuclear transfer and other technologies to produce in vitro embryos has shown broad application prospects. In vitro production of livestock embryos, especially somatic cell nuclear transfer, is an effective way to rapidly expand the number of excellent populations. Obtaining a sufficient number of mature oocytes is a prerequisite for in vitro production of embryos. Although the collection of oocytes from living animals has been initially applied in production, this method has disadvantages suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075C12N5/073
Inventor 王争光李正操俞颂东
Owner ZHEJIANG UNIV
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