Ghrelin-containing sheep embryo in-vitro culture solution and culture method thereof

A technology for in vitro culture and sheep embryos, which is applied in the field of in vitro culture of sheep embryos and in vitro culture fluid for sheep embryos, can solve the problems of low early embryo development rate and cannot meet the needs of in vitro embryo early development, and achieve low development rate and convenient Promote the application and promote the effect of follicle growth

Inactive Publication Date: 2012-09-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to solve the problem that the in vitro culture medium of sheep embryos in the prior art cannot meet the needs of the early development of in vitro embryos, resulting in a relatively low early embryo development rate, and provides a ghrelin-containing product that has the function of improving the early development rate of in vitro embryos. Sheep embryo in vitro culture medium and its culture method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: prepare the sheep embryo in vitro culture fluid containing ghrelin

[0020] Accurately weigh 60 mg of ghrelin (Sigma Company), dissolve it with 20 mL of TCM199 culture medium, then add 15 mg of glutamine, 800 mg of bovine serum albumin, 10,000 IU of penicillin, and 10,000 μg of streptomycin to the culture medium. Gently stir evenly, then add TCM199 culture medium to 100mL, stir again and mix well. After being sterilized by suction with a 0.22 μM microfiltration membrane, store at 4°C for later use.

Embodiment 2

[0021] Embodiment 2: The ghrelin-containing culture medium is used for in vitro culture of goat embryos

[0022] Goat oocytes were matured in TCM199 medium containing 10 μg / mL of FSH, 10 μg / mL of LH, 100 IU / mL of penicillin, 100 μg / mL of streptomycin and 10% serum by volume, and then carried out IVF. The early embryos after fertilization were further cultured for 7 days in the TCM199 culture medium without ghrelin (control group) and the in vitro culture medium of sheep embryos containing ghrelin prepared in Example 1, respectively. On the second day of culturing, the early embryo division rate in the control group was 64.9%, and the early embryo division rate in the ghrelin-containing sheep embryo in vitro culture medium prepared in Example 1 was 78.3%, which was 13.4% higher than that of the control group. On the 7th day of cultivation, the early embryo blastocyst stage development rate in the control group was 24.0%, and the early embryo blastocyst stage development rate i...

Embodiment 3

[0023] Embodiment 3: The ghrelin-containing culture fluid is used for in vitro culture of sheep embryos

[0024] Sheep oocytes were matured in TCM199 medium containing 10 μg / mL of FSH, 10 μg / mL of LH, 100 IU / mL of penicillin, 100 μg / mL of streptomycin and 10% serum by volume, and then carried out IVF. The early embryos after fertilization were further cultured for 7 days in the TCM199 culture medium without ghrelin (control group) and the in vitro culture medium of sheep embryos containing ghrelin prepared in Example 1, respectively. On the second day of cultivation, the early embryo division rate in the control group was 62.7%, and the early embryo division rate in the ghrelin-containing sheep embryo in vitro culture medium prepared in Example 1 was 76.4%, which was 13.7% higher than that in the control group. On the 7th day of culturing, the early embryo blastocyst stage development rate in the control group was 25.3%, and the early embryo blastocyst stage development rate ...

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Abstract

The invention provides a ghrelin-containing sheep embryo in-vitro culture solution. The sheep embryo in-vitro culture solution takes a TCM199 culture solution as a matrix; and the matrix also contains 400-800mg / mL ghrelin, 0.15mg / mL glutamine, 100IU / mL penicillin, 100mug / mL streptomycin and 5-10mg / mL bovine serum albumin. In the embryo in-vitro culture solution provided by the invention, ghrelin is used as the endogenous ligand of a growth hormone secretagogue receptor, and has the functions of regulating the secretion of reproductive hormone, promoting the growth of ovarian follicle and ovulation, adjusting the ovarian function, participating in the early developmental regulation of embryo and the like. By applying ghrelin to the sheep embryo in-vitro culture, the ectogenesis of the embryo can be effectively promoted, thus the problem of low ectogenesis rate of the sheep early embryo at present is solved. The sheep embryo in-vitro culture solution provided by the invention has relatively low economic cost, and is convenient to popularize and use in production.

Description

(1) Technical field [0001] The invention relates to a ghrelin-containing sheep embryo in vitro culture solution and a method for using the culture solution to cultivate sheep embryos in vitro, belonging to the technical field of livestock embryo engineering. (2) Background technology [0002] With the development of modern reproductive biotechnology and the gradual improvement of embryo in vitro production technology, sheep embryo in vitro production technology (in vitro fertilization or cloning) has become an important way to speed up the breeding of breeds and the expansion of excellent populations. Although living offspring have been obtained through in vitro embryo production technology, in vitro embryos obtained by in vitro fertilization or cloning generally have developmental blockage during the culture process, that is, embryos stop developing and degenerate after reaching a certain stage, resulting in in vitro embryos. The development rate of blastocysts is much lowe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
Inventor 王争光白惠琴白骊骅俞颂东
Owner ZHEJIANG UNIV
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