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Method for culturing and cryopreserving bovine in-vitro fertilization embryos

A technology of embryo culture and embryo culture solution, applied in biochemical equipment and methods, embryo cells, culture process, etc., can solve the problem of reduced embryo survival rate

Active Publication Date: 2021-06-18
天津力牧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] However, poor freezing and thawing procedures for embryos obtained after in vitro development of fertilized eggs may result in reduced embryo survival

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: the culture method of bovine in vitro fertilization embryo (ex vivo collection)

[0068] In the present embodiment, in step (1), the collected oocytes were placed in transport medium containing HEPES at 38.8° C. without carbon dioxide supply, and transported back to the laboratory within 24 hours.

[0069] 1. Reagents

[0070] In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

[0071]The transportation culture fluid containing HEPES used in this example contains: glycine 50.0mg / L, L-alanine 25.0mg / L, L-arginine hydrochloride 70.0mg / L, L-aspartic acid 30.0mg / L, L-cystine dihydrochloride 26.0mg / L, L-glutamic acid 75.0mg / L, L-glutamine 100.0mg / L, L-histidine hydrochloride monohydrate 21.88mg / L, L-hydroxyproline 10.0mg / L, L-isoleucine 40.0mg / L, L-leucine 60.0mg / L, L-lysine hydrochloride 70.0mg / L, L-methionine 15.0mg / L, L-phenylalanine 25.0mg / L, L-proline 40.0mg / L, L-seri...

Embodiment 1A

[0116] Embodiment 1A: the culture method of bovine in vitro fertilization embryo (ex vivo collection)

[0117] With reference to the method of Example 1, tests were carried out on three kinds of cattle, Holstein (dairy breed), Simmental (beef breed), and Chinese buffalo (serving breed), respectively. The results showed that the cleavage rate was 85-90%. Within the range, the morula rate was in the range of 62-67%, the blastocyst rate was in the range of 50-55%, the apoptosis rate was in the range of 4-6%, and the blastocyst hatching rate on the 9th day was 72-5%. Within the range of 77%; the maturation rates of oocytes in vitro maturation of Holstein, Simmental, and Chinese buffalo were 83.3%, 80.6%, and 75.6%, respectively.

[0118] In the tests carried out for four kinds of cattle in Example 1 and Example 1A, when the embryos were cultured in vitro, preserved and thawed in step (3), the freezing solution containing dextran 40 and acetylcysteine ​​and thawing were used. The ...

Embodiment 1B

[0122] Embodiment 1B: the culture method of bovine in vitro fertilization embryo (in vitro collection)

[0123] With reference to the method of Example 1, four kinds of cattle were tested respectively for Chinese yellow cattle (Nanyang cattle, service breed), Holstein cattle (dairy breed), Simmental cattle (beef breed), and Chinese water buffalo (service breed), However, the formulations of the fertilization culture medium and the semen preparation culture medium were respectively modified, and the remaining test fluids and operations remained unchanged. The formula of the fertilization medium was changed to: containing 112.0mM sodium chloride, 4.02mM potassium chloride, 2.25mM calcium chloride dihydrate, 0.52mM magnesium chloride hexahydrate, 0.83mM potassium dihydrogen phosphate, 37.0mM sodium bicarbonate, 1.25 mM sodium pyruvate, 10μg / ml heparin, 4mg / ml bovine serum albumin (BSA), 100U / ml penicillin, 100μg / ml streptomycin, 10umoL / L α-lipoic acid, 25μg / ml folic acid, 3mg / ml ...

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Abstract

The invention relates to a method for culturing and cryopreserving bovine in-vitro fertilization embryos. The method comprises the following steps of: washing a mature cumulus-oocyte complex in a fertilization culture solution, transferring the mature cumulus-oocyte complex into the fertilization culture solution, and putting the mature cumulus-oocyte complex into an incubator for later use; taking a frozen sperm tubule from liquid nitrogen, unfreezing, taking a sperm suspension, and counting sperms; adding the sperm suspension into fertilization culture solution drops containing oocytes to co-incubate the sperms and the oocytes, completing in-vitro fertilization operation, completely removing granular cells around the embryos by using an oocyte stripping needle, putting the embryos into an embryo culture solution for culturing, transferring available embryos into a freezing solution, and putting the embryos into liquid nitrogen for cryopreservation; and when the cryopreserved embryos need to be used, taking the tubule from the liquid nitrogen, transferring the tubule into an unfreezing solution, putting the embryos into small drops of the embryo culture solution for culturing, and completing the unfreezing process. The invention further relates to the used freezing solution and the used unfreezing solution. According to the method, the survival rate of the bovine in-vitro fertilization embryos after cryopreservation and unfreezing can be remarkably increased.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, and relates to a technology for agricultural-animal husbandry and veterinary reproduction, in particular to a method for bovine in vitro fertilization. In addition, the present invention also relates to the use of related working test solutions used in bovine in vitro fertilization. The application in fertilization, in particular, relates to a method of bovine in vitro fertilization, for example, relates to a method of freezing and thawing bovine in vitro fertilization blastocysts with excellent effects. The bovine in vitro fertilization method of the invention has excellent technical effects. Background technique [0002] In Vitro Fertilization (In Vitro Fertilization) or (external fertilization) refers to the technology in which mammalian sperm and eggs complete the fertilization process in an artificially controlled environment outside the body. It is referred to as IVF in English....

Claims

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Application Information

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IPC IPC(8): C12N5/073A01N1/02
CPCA01N1/021A01N1/0221C12N5/0604C12N2500/12C12N2500/14C12N2500/16C12N2500/30C12N2500/32C12N2500/38C12N2501/02C12N2501/11C12N2501/31C12N2501/90C12N2501/999
Inventor 王娜王小武韩勇权郝少强赵明礼郭春明许晓椿
Owner 天津力牧生物科技有限公司
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