A bovine embryo vitrification freezing tube thawing and direct transplantation method

A technology for vitrification and freezing of bovine embryos, which is used in medical science, preservation of human or animal bodies, and animal delivery, etc., can solve the problems of high production equipment requirements, unfavorable long-term storage, low freezing efficiency, etc. The tube is easy to operate and the effect of simple operation

Active Publication Date: 2016-08-24
CHINA AGRI UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0009] The object of the present invention is to provide a bovine embryo vitrification freezing tube thawing, to overcome the problems of low freezing efficiency, high technical difficulty, unclear identification, unfavorable long-term storage and high requirements for production equipment in the above-mentioned several methods, It provides a new way to improve the efficiency of bovine embryo cryopreservation and solve the industrialization of embryo transfer in animal husbandry production

Method used

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  • A bovine embryo vitrification freezing tube thawing and direct transplantation method
  • A bovine embryo vitrification freezing tube thawing and direct transplantation method
  • A bovine embryo vitrification freezing tube thawing and direct transplantation method

Examples

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Effect test

Embodiment 1

[0054] Example 1 Preparation of solution

[0055] 1. Preparation of EFS35 freezing liquid

[0056] Ficoll and sucrose were added to the mPBS solution (Whittingham, 1971) to make the final concentration of 300g / L and 0.5mol / L, respectively, to obtain the FS solution; add ethylene glycol to the FS solution, the volume ratio of the two was 65:35 , The EFS35 solution obtained is the freezing liquid. In the EFS35 freezing liquid, the final mass percentage of polysucrose is 18%, and the final concentration of sucrose is 0.3 mol / L.

[0057] 2. Preparation of thawing solution

[0058] The thawing solution is mPBS solution containing 10% (V / V) fetal bovine serum (FBS), that is, adding fetal bovine serum to the mPBS solution, the volume ratio of the two is 9:1.

[0059] 3. Preparation of equilibrium pretreatment liquid

[0060] Ethylene glycol (EG) and mPBS solution are mixed and prepared according to a volume ratio of 1:9.

Embodiment 2

[0061] Example 2 Method and results of thawing and direct transfer of bovine embryos

[0062] The freezing liquid, thawing liquid, and equilibrium pretreatment liquid in embodiment 1 are all applied to the corresponding method steps of this embodiment. See the installation diagram figure 1 .

[0063] (1) First add a section of thawing solution with a length of 7.5cm to the thin tube for freezing bovine embryos, and then put a section of 0.5cm of air;

[0064] (2) Then put in a section of EFS35 refrigerant with a length of 0.4cm, and then put a section of 0.5cm of air;

[0065] (3) Load a section of EFS35 freezing solution with a length of 0.6cm, and at 25℃, transfer the bovine embryos into the equilibrium pretreatment solution and equilibrate for 3 minutes, then quickly transfer it into the EFS35 freezing solution with a length of 0.6cm, and in this section of freezing solution Place 1 bovine embryo in the middle, and then fill in a section of 0.5cm air;

[0066] (4) Load a section o...

Embodiment 3

[0069] Example 3 The effect of different freezing liquids on bovine embryos

[0070] Use EFS30 (add ethylene glycol to the FS solution, the volume ratio of the two is 70:30), EFS35 and EFS40 (add ethylene glycol to the FS solution, the volume ratio of the two is 60:40) as the freezing liquid, according to The method of Example 2 cryopreserved bovine embryos.

[0071] The results showed that after using EFS30 as the freezing solution, the survival rate of bovine embryos after thawing was 90%, and the expansion rate of in vitro culture was 87% (26 / 30); while using EFS40 to cryopreserve bovine embryos, the survival rate after thawing was 94% , The expansion rate of in vitro culture is 90% (28 / 31). The cryopreservation effect of the above two freezing liquids of EFS30 and EFS40 is lower than that of EFS35. The reason is that the concentration of EG in EFS30 is low. When in equilibrium, EG cannot fully penetrate into the bovine embryo cells as an antifreeze protection agent, causing th...

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Abstract

The invention provides a bovine embryo vitrification tube swinging thawing and direct transplanting method and belongs to the technical field of embryo low-temperature biology. The method includes (1) freezing a bovine embryo and using a fine tube to first add a thawing liquid followed by air; (2) loading a freezing liquid followed by air; (3) loading a freezing liquid, placing the bovine embryo in the freezing liquid, and then loading air; (4) loading a thawing liquid, closing a tube opening, and performing freezing; (5) performing tube swinging thawing and direct transplanting, wherein the freezing liquid is an mPBS solution containing ethylene glycol (EG), polysucrose and sucrose, and the thawing liquid is an mPBS solution containing fetal bovine serum (FBS). According to the bovine embryo vitrification tube swinging thawing and direct transplanting method, the operation is simple and convenient, requirements for skills of operators are not high, the frozen bovine embryo can be directly transplanted after tube swinging thawing, the thawed embryo has a survival rate of 100% and an expansion rate of 94%, a pregnancy rate reaches 57% after transplantation, and the method is efficient, simple, easy to operate, and applicable to industrialized popularization.

Description

Technical field [0001] The invention relates to the technical field of animal embryo cryopreservation and embryo transfer, in particular to a method for thawing and direct transfer of a bovine embryo vitrified freezing tube. Background technique [0002] Since Rall et al. (1985) invented the embryo vitrification cryopreservation method, the freezing process can be performed at room temperature without the aid of a freezer, and the procedure has been greatly simplified. After improvement by many researchers, the current vitrification method of oocytes / embryos only takes about 1 minute to complete a procedure. In the process, many researchers creatively invented many frozen load-bearing objects and named them various methods. The following methods are commonly used today: Cryoloop, Open Pulled Straw (OPS), GMP, Microdrops, semi-thin tube Law (hemi-straw). However, there are still many problems in the application of the above methods. For example, the number of oocytes / embryos fr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02A61D19/04
Inventor 朱士恩傅祥伟周艳华张有文
Owner CHINA AGRI UNIV
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