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Culture medium used for bovine in vitro fertilization and method for improving bovine in vitro fertilization

A technology of culture medium and bovine body, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve the problems of limited efficiency of in vitro fertilization

Active Publication Date: 2021-03-12
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

[0015] However, the efficiency of bovine frozen semen for in vitro fertilization with oocytes remains limited

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: the culture method of bovine in vitro fertilization embryo (ex vivo collection)

[0059] 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.

[0060] 1. Reagents

[0061] In the specific test of the present invention, if not otherwise specified, the relevant reagents used are described in detail as follows:

[0062] 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-ser...

Embodiment 1A

[0100] Embodiment 1A: the culture method of bovine in vitro fertilization embryo (ex vivo collection)

[0101] 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.

Embodiment 1B

[0102] Embodiment 1B: the culture method of bovine in vitro fertilization embryo (in vitro collection)

[0103] 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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PUM

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Abstract

The invention relates to a culture fluid used for bovine in vitro fertilization and a method for improving bovine in vitro fertilization. The method for bovine in vitro fertilization comprises the following steps: washing the mature cumulus-oocyte complex in the fertilization medium, then transferring it to the fertilization medium, putting it into an incubator, and putting it into an incubator for subsequent use; tube, thawed in a water bath at 37°C, inject the semen into the centrifuge tube containing the semen preparation culture medium, centrifuge and discard the supernatant; add the semen preparation culture medium to the above centrifuge tube, resuspend the sperm pellet, and take an appropriate sperm suspension for spermatogenesis Counting; add the calculated volume of sperm suspension into the fertilization culture drop filled with oocytes, and put the culture plate into the incubator to incubate the sperm and eggs together, and complete the in vitro fertilization operation, ready for in vitro culture of embryos. The involved fertilization culture fluid and semen preparation culture fluid contain ingredients such as sodium pyruvate and heparin. The method of the invention can significantly improve the efficiency of bovine in vitro fertilization.

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. Application in fertilization. The bovine in vitro fertilization method of the invention has excellent technical effect. 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. Because it is inseparable from embryo transfer technology (ET), it is also referred to as IVF-ET. In biology, animals obtained by transplanting in vitro fertilized embryos into mothers are calle...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075C12N5/076A61D19/02
CPCA61D19/02C12N5/0609C12N5/061C12N2500/12C12N2500/14C12N2500/20C12N2500/24C12N2500/32C12N2500/33C12N2500/34C12N2500/35C12N2500/36C12N2500/38C12N2500/40C12N2501/105C12N2501/11C12N2501/30C12N2501/31C12N2501/39C12N2501/392C12N2501/999
Inventor 王娜王小武郝少强赵明礼韩勇权郭春明许晓椿
Owner 天津力牧生物科技有限公司
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