Method, pretreatment solution and kit for in vitro maturation of sheep oocytes

An oocyte and pretreatment solution technology, applied in the field of embryo engineering, can solve the problems of unsatisfactory experimental results, unfavorable late embryo development, oocyte toxicity, etc., and achieves good application prospects and improves the effect of in vitro development ability.

Inactive Publication Date: 2016-08-17
三亚雪古丽现代生态农业综合开发有限公司
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Problems solved by technology

Even so, the results of such experiments are not ideal.
The reason is that the meiosis inhibitors used in these reports are all chemical inhibitors (such as 3-isobutyl-1-methylxanthine (IBMX) and forskolin (FSK)), although in ovo The process of mother cell maturation can effectively inhibit meiosis, but it has a toxic effect on oocytes to some extent, which is not conducive to the later development of embryos

Method used

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  • Method, pretreatment solution and kit for in vitro maturation of sheep oocytes
  • Method, pretreatment solution and kit for in vitro maturation of sheep oocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 pretreatment liquid

[0043] Step 1. extracting follicular fluid from 1-3mm follicles on the isolated sheep ovary;

[0044] Step 2. Centrifuge the extracted follicle fluid at 14,000 rpm for 10 minutes, extract the supernatant, and filter it with a filter membrane with a pore size of 0.45 μm;

[0045] Step 3. The resulting filtrate, namely SFF, was added to TCM199 containing BAS at a final concentration of 3 mg / ml at a final concentration of 10% (v / v) to prepare the Pre-IVM solution.

Embodiment 2

[0046] Example 2 In Vitro Maturation of Sheep Oocytes

[0047] Step 1. Oocyte collection: Use a 10ml syringe to extract follicles with a diameter of 4-6mm from the surface of the isolated sheep ovary. Put the extracted oocytes into a 100mm-diameter petri dish, and select grade A (uniform cytoplasm, surrounded by multi-layered cumulus granulosa cells) and grade B (homogeneous cytoplasm, less than 3 layers of eggs) under a stereomicroscope. Cumulus granulosa cells wrapped or partially exposed) cumulus-oocyte complexes (COCs) were used for in vitro maturation culture.

[0048] Step 2. Oocyte pretreatment: Wash the selected A and B grade COCs twice with Pre-IVM solution, and then put them in CO 2 Culture in the Pre-IVM solution in the incubator for more than 2 hours, and the culture condition is 5% CO 2 The air temperature is 38.6°C, the humidity is saturated, and the incubation time is 4 hours.

[0049] Step 3. Oocyte maturation culture: IVM solution (TCM199+10ug / ml FSH+1ug / ml...

Embodiment 3

[0050] Example 3 Verification of Sheep Oocyte Maturation Rate

[0051] experimental method :

[0052] 1. Culture of Mature Sheep Oocytes

[0053] Two types of in vitro matured sheep oocytes were prepared using conventional methods and methods provided by the present invention respectively.

[0054] The preparation method of class I mature oocytes is a conventional maturation method, and the steps are as follows:

[0055] 1) with embodiment 2 step 1

[0056] 2) IVM solution (TCM199+10ug / ml FSH+1ug / ml LH+1ug / ml E2+10%FBS) CO 2 Equilibrate in the incubator for more than 2 hours, and carry out in vitro maturation culture in the medium. The COCs collected in the previous step are placed in the IVM solution for 24 hours. The culture condition is 5% CO 2 air, temperature 38.6°C, saturated humidity.

[0057] The preparation method of type II mature oocytes is the same as Steps 1-3 of Example 2.

[0058] 2. In vitro fertilization

[0059] Semen: SOF + 2% estrous sheep serum. ...

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Abstract

The invention discloses a method for in vitro maturation of sheep oocytes, a pretreatment solution and a kit. The invention relates to a pretreatment solution for in vitro maturation of sheep oocytes, which is characterized in that it contains sheep small follicle fluid and conventional culture solution components for in vitro maturation of sheep oocytes. The invention also discloses a method for in vitro maturation of sheep oocytes, which is characterized in that a pretreatment step is added on the basis of the conventional in vitro maturation method. At the same time, the present invention also verifies that the cleavage rate and blastocyst rate of fertilized eggs obtained after in vitro fertilization of oocytes cultured by the maturation method of the present invention are significantly higher than those of oocytes cultured by conventional maturation methods. The pretreatment solution and the in vitro maturation culture method provided by the invention can effectively promote the in vitro maturation of sheep oocytes, and have the characteristics of no toxicity to the oocytes, less restriction, good effect and the like.

Description

technical field [0001] The invention relates to the field of embryo engineering, in particular to a method for in vitro maturation of sheep oocytes, a pretreatment solution and a kit. Background technique [0002] In livestock reproduction, ovary-derived oocytes are one of the main sources for producing animal embryos. Therefore, in vitro maturation of oocytes is an important platform technology for animal breeding, cloning, and transgenic animal production. [0003] In 2003, Wang Xuguang compared the in vitro maturation effects of Xinjiang sheep oocytes under different FSH and LH concentrations in different seasons, and found that the appropriate culture time for in vitro maturation of sheep oocytes was 24-26 hours. At the same time, different EH and LH concentrations were explored. The effect of electrical activation scheme on in vitro maturation of oocytes, found that EH+A23187+CHX is the most suitable activation scheme for sheep oocytes, with a cleavage rate of 69.8% and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075
Inventor 张家新赵丽霞陈秀亮
Owner 三亚雪古丽现代生态农业综合开发有限公司
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