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Buffalo embryo freezing method in combination with laser membrane rupture

A freezing method and membrane rupture technology, which is applied in the biological field, can solve the problems of embryo freezing stage, lack of uniform standards for freezing liquid and freezing procedures, low temperature sensitivity of embryos, low freezing efficiency, poor embryo quality, etc., so as to improve the survival of frozen embryos The effect of increasing the rate of implantation, increasing the rate of conception of transplantation, and reducing the possibility of ice crystal formation

Active Publication Date: 2015-10-21
皇氏赛尔生物科技(广西)有限公司
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Problems solved by technology

[0002] Buffalo embryos produced in vitro are sensitive to low temperature and have low freezing efficiency, mainly due to poor quality of embryos caused by improper in vitro culture conditions and excessive lipid droplets in embryos.
For ultra-low temperature freezing of buffalo embryos, vitrification is mainly used today, but there is no uniform standard for embryo freezing stages, freezing liquids and freezing procedures
Moreover, buffalo embryos are not processed before freezing, so the freezing efficiency is still low, especially for 8-9d blastocysts, the freezing efficiency is only 50-60%
In addition, after vitrification-thawing, the thickness of the embryo's zona pellucida will increase by an average of 2.8 μm, and the hatching rate of frozen embryos is only about 50%, resulting in a conception rate of only 4-12% after thawing embryo transfer

Method used

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  • Buffalo embryo freezing method in combination with laser membrane rupture
  • Buffalo embryo freezing method in combination with laser membrane rupture

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Embodiment 1

[0019] A buffalo embryo freezing method combined with laser membrane rupture, the method is to culture buffalo embryos produced in vitro to expanded blastocysts, use a laser membrane rupturer to punch holes in the blastocyst cavity, discharge blastocyst fluid, and then use freezing liquid to Vitrification. In this embodiment, the freezing liquid is composed of the following materials: a 20% ethylene glycol (EG) solution, a 20% dimethyl sulfoxide (DMSO) solution, and a 0.5M sucrose solution . Further, the buffalo embryo freezing method combined with laser membrane rupture also includes the following steps:

[0020] (1) Using the XYClone laser membrane rupture system, adjust the parameters to 90% pulse intensity, 800μs pulse width, make a 15-20μm zona pellucida incision on the zona pellucida opposite to the inner cell mass of the blastocyst, and break the blastocoel Blastocyst fluid flows out.

[0021] (2) Take a 0.25mL plastic straw and make a 30° oblique section with a blad...

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Abstract

The present invention provides a buffalo embryo freezing method in combination with laser membrane rupture. The method comprises: culturing an in vitro fertilized buffalo embryo to be an expanded blastocyst; using a laser membrane rupture instrument to drill a blastocyst cavity; discharging blastocyst fluid; and then using freezing liquid to perform vitrification freezing. According to the present invention, discharging the blastocyst fluid before freezing can make the cryoprotectant enter an embryo more quickly, reduce the possibility of ice crystal forming in a cell, and thereby avoiding embryo damage to a large extent, and improving a freezing survival rate of the embryo. On the other hand, due to partial breaking of a zona pellucida before freezing, so that after thawing cultivation, the zona pellucida is easier to be hatched in a revived blastocyst cavity, and thereby facilitating embryo implantation of the embryo after transplanting, and improving a transplant pregnancy rate.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, in particular to a method for freezing buffalo embryos combined with laser membrane rupture. 【Background technique】 [0002] Buffalo embryos produced in vitro are sensitive to low temperature and have low freezing efficiency, mainly due to poor quality of embryos and excessive lipid droplets in embryos due to improper in vitro culture conditions. For the ultra-low temperature freezing of buffalo embryos, the vitrification method is mainly used today, but there is no uniform standard for the embryo freezing stage, freezing liquid and freezing procedure. Moreover, the buffalo embryos are not processed before freezing, so the freezing efficiency is still low, especially for 8-9d blastocysts, the freezing efficiency is only 50-60%. In addition, after vitrification-thawing, the thickness of the embryo's zona pellucida will increase by an average of 2.8 μm, and the hatching rate of frozen embryos is onl...

Claims

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Application Information

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IPC IPC(8): A01N1/02
Inventor 杨春艳尚江华郑海英谷毅鹏黄芬香梁贤威黄锋陈明棠覃广胜
Owner 皇氏赛尔生物科技(广西)有限公司
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