O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid and culture method

A technology of carnitine hydrochloride and oocytes, which is applied in the field of in vitro maturation and culture of buffalo oocytes, and can solve the problem of no significant impact on subsequent blastocyst formation

Inactive Publication Date: 2016-06-22
GUANGXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adding L-carnitine to preantral follicles of mice and pigs can improve the subsequent development of eggs, but it has also been reported that adding L-carnitine during egg maturation can improve the division rate of embryos, but it has no effect on subsequent blastocysts. The formation of

Method used

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  • O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Preparation of buffalo oocyte in vitro maturation solution containing O-acetyl-L-carnitine hydrochloride: first prepare a storage solution of 50mMO-acetyl-L-carnitine hydrochloride, and use 200μl EP tubes 110 μl (without 10% ECS, 3% BFF, 0.5 μg / ml FSH, 5 μg / ml LH, 10 μg / ml Cysteine ​​but containing NaHCO 3 26.2mM, Hepes 5mM TCM-199 solution), dilute 20 times when used, that is, take 50 μl of the storage liquid and add it to 950 μl of buffalo oocyte maturation working solution in vitro.

[0018] The specific in vitro maturation solution formula is as follows

[0019] Table 1 The formula of the buffalo oocyte in vitro maturation solution containing O-acetyl-L-carnitine hydrochloride

[0020]

[0021] Culture method: Use a 10ml syringe with a 12-gauge needle to extract 2-6mm follicles from isolated buffalo ovaries, select oocytes with complete cumulus cell coating under a common stereo microscope, and wash them with egg washing liquid first 2 times, then wash 2 times ...

Embodiment 2

[0029] The in vitro maturation of buffalo oocytes and the identification of subsequent embryonic development capabilities are the same as in Example 1.

[0030] Count the total number of inner cell masses of the blastocysts formed on the 7th day after parthenogenetic activation. First, put the blastocysts in 10 mg / ml Hoechst33342 for 5 minutes in the dark, then wash them in PBS for 2 to 3 times, and then press them into slices. . Under an ordinary fluorescent microscope, observe at a field of view of 200 times, and count the bright spots that emit blue fluorescence under the field of view.

[0031] The results showed that the total number of inner cell masses of the obtained blastocysts was 85.64, significantly higher than that of the control group, 56.40, and 29.24 higher than that of the control group, indicating that the quality of the blastocysts obtained in Example 2 was higher than that of the control group. (See Table 3) The buffalo oocyte in vitro maturation solution ...

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Abstract

The invention discloses O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid. A corresponding culture method is further established by an inventor accordingly. Experiments indicate that O-acetyl-L-carnitine hydrochloride is applied to buffalo oocyte in-vitro maturation, compared with buffalo oocyte in-vitro maturation liquid to which no O-acetyl-L-carnitine hydrochloride is added, buffalo ovum in-vitro maturation quality and the development capacity of subsequent embryos can be remarkably improved, O-acetyl-L-carnitine hydrochloride is low in cost, and the O-acetyl-L-carnitine hydrochloride containing buffalo oocyte in-vitro maturation liquid can be applied and popularized in buffalo buffalo in-vitro production conveniently.

Description

technical field [0001] The invention belongs to the technical field of in vitro culture of oocytes, in particular to a buffalo oocyte in vitro maturation liquid containing O-acetyl-L-carnitine hydrochloride and a culture method. Background technique [0002] With the development of embryonic biotechnology, compared with eggs derived from the body, immature eggs can be obtained from isolated ovaries. After in vitro maturation and culture, a large number of mature eggs can be obtained for subsequent in vitro fertilization, somatic cell cloning, embryonic stem cells, etc. research and production applications. However, the current in vitro maturation efficiency of buffalo oocytes is low, which limits the research and application of buffalo embryo bioengineering. [0003] The process of egg maturation and subsequent embryo development to final embryo implantation requires high energy. The addition of carbohydrates in in vitro culture medium plays a key role in the complete matu...

Claims

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Application Information

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IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2501/999
Inventor 许惠艳卢克焕陆阳清卢晟盛杨小淦谢龙
Owner GUANGXI UNIV
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