Bovine embryo vitrification freezing tube swinging thawing and direct transplanting 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: 2015-04-08
CHINA AGRI UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

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

Method used

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  • Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method
  • Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method
  • Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method

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Experimental program
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Effect test

Embodiment 1

[0054] The preparation of embodiment 1 solution

[0055] 1. Preparation of EFS35 freezing liquid

[0056] Add polysucrose and sucrose to mPBS solution (Whittingham, 1971) so that the final concentrations are 300g / L and 0.5mol / L respectively to obtain FS solution; add ethylene glycol to FS solution, the volume ratio of the two is 65:35 , the EFS35 solution is obtained as the freezing liquid. In the EFS35 freezing solution, 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 an mPBS solution containing 10% (V / V) fetal bovine serum (FBS), that is, adding fetal bovine serum to the mPBS solution, and the volume ratio of the two is 9:1.

[0059] 3. Preparation of equilibrium pretreatment solution

[0060] Prepared by mixing ethylene glycol (EG) and mPBS solution at a volume ratio of 1:9.

Embodiment 2

[0061] Example 2 Bovine Embryo Frozen Tube Thawing and Direct Transplantation Method and Results

[0062] The freezing solution, thawing solution, and equilibrium pretreatment solution in Example 1 are all applied to the corresponding method steps in this example. For a schematic diagram of the installation, see figure 1 .

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

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

[0065] (3) Fill in a section of EFS35 freezing solution with a length of 0.6 cm. At 25 ° C, transfer the bovine embryos into the equilibrium pretreatment solution for 3 minutes and then quickly transfer them into the EFS35 freezing solution with a length of 0.6 cm. 1 bovine embryo was placed in it, and then a section of 0.5cm air was inserted;

[0066] (4) Fill in a ...

Embodiment 3

[0069] Example 3 Effects of Different Freezing Liquids on Bovine Embryos

[0070] 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] It was found that after using EFS30 as the freezing liquid, 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 was 90% (28 / 31). The cryopreservation effects of the above-mentioned EFS30 and EFS40 freezing liquids are lower than those of EFS35. The reason is that the concentration of EG in EFS30 is low. When in equilibrium, EG, as an antifreeze protectant, cannot fully penetrate into the interior of bovine embryonic cells, ca...

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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 cryopreservation and embryo transplantation of animal embryos, in particular to a bovine embryo vitrification freezing tube thawing and direct transplantation method. Background technique [0002] Since Rall et al. (1985) invented the embryo vitrification cryopreservation method, the cryopreservation 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 oocyte / embryo vitrification method only takes about 1 minute to complete a procedure. During this process, many researchers creatively invented many frozen carrying objects, which were named in various ways. The following methods are commonly used today: Nylon Ring Vitrification (Cryoloop), Open Pulled Straw (OPS, Open Pulled Straw), Fine Glass Tube (GMP), Microdrops, Semi-thin Tube Law (hemi-straw). However, there are still many problems in th...

Claims

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Application Information

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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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