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Method for bovine in-vitro fertilized embryo culture and used culture solution

A technology of embryo culture medium and culture medium, which is applied in the direction of embryonic cells, culture process, tissue culture, etc., can solve the problems of limited wide application, low efficiency, high production cost of in vitro fertilized embryos, etc.

Active Publication Date: 2017-08-11
天津力牧生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although in vitro fertilization technology can be successfully applied to many mammals, the high cost and low efficiency of in vitro fertilized embryo production due to the low blastocyst rate of in vitro fertilization limit the wide application of this technology in the practice of rapid breeding of cattle

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Embodiment 1: the culture method of bovine in vitro fertilization embryo

[0146] 1. Reagents

[0147] Mature medium:Contains 0.01IU / ml FSH, 10IU / ml LH, 1μg / ml estradiol, 100ng / ml IGF, 50ng / ml EGF, 100U / ml penicillin, 100μg / ml streptomycin, 10% fetal bovine serum TCM-199 culture medium.

[0148] Wash semen: Containing 112.0mM NaCl, 4.02mM KCl, 2.25mM CaCl2 2H2O, 0.52mM MgCl2 6H2O, 0.83mM KH2PO3, 37.0mM NaHCO3, 1.25mM sodium pyruvate, 10μg / ml heparin, 4mg / ml of bovine serum albumin (Bovine serum albumin, BSA), 10 mM caffeine, 100 U / ml of penicillin, and 100 μg / ml of streptomycin in water.

[0149] Semen received: Containing 112.0mM NaCl, 4.02mM KCl, 2.25mM CaCl2 2H2O, 0.52mM MgCl2 6H2O, 0.83mM KH2PO3, 37.0mM NaHCO3, 1.25mM sodium pyruvate, 10μg / ml heparin, 4mg / ml of BSA, 100 U / ml of penicillin, and 100 μg / ml of streptomycin in water.

[0150] Pre-embryo culture medium: Contains 109.5mM NaCl, 3.1mM KCl, 26.2mM NaHCO3, 0.8mM MgCl2 6H2O, 1.19mM KH2PO3, 0.4m...

Embodiment 2

[0201] Embodiment 2: the culture method of bovine in vitro fertilization embryo

[0202] With reference to Example 1, the only difference is that the composition of the embryonic late stage culture solution is changed to:

[0203] 109mM NaCl,

[0204] 3.1mM KCl,

[0205] 26.0mM NaHCO3,

[0206] 0.5mM MgCl2 6H2O,

[0207] 1.3mM KH2PO3,

[0208] 0.4mM sodium pyruvate,

[0209] 1.5mM glucose,

[0210] 5mM Calcium Galactonate,

[0211] 10v / v% fetal bovine serum,

[0212] 1mM L-Glutamine,

[0213] 2v / v% essential amino acids,

[0214] 1v / v% non-essential amino acids,

[0215] 3mM glutathione, and

[0216] Water as dosing solvent.

[0217] The test was carried out as in Example 1, and all the indicators of the results were basically the same as those of the 3mM glutathione group in Example 1. For example, in this example, the morula rate was 0.633, the blastocyst rate was 0.508, and the apoptosis rate was 0.050.

Embodiment 3

[0218] Embodiment 3: the culture method of bovine in vitro fertilization embryo

[0219] With reference to Example 1, the only difference is that the composition of the embryonic late stage culture solution is changed to:

[0220] 110mM NaCl,

[0221] 2.9mM KCl,

[0222] 26.5mM NaHCO3,

[0223] 1.0mM MgCl2 6H2O,

[0224] 1.0mM KH2PO3,

[0225] 0.4mM sodium pyruvate,

[0226] 1.5mM glucose,

[0227] 5mM Calcium Galactonate,

[0228] 10v / v% fetal bovine serum,

[0229] 1mM L-Glutamine,

[0230] 2v / v% essential amino acids,

[0231] 1v / v% non-essential amino acids,

[0232] 3mM glutathione, and

[0233] Water as dosing solvent.

[0234] The test was carried out as in Example 1, and all the indicators of the results were basically the same as those of the 3mM glutathione group in Example 1. For example, in this example, the morula rate was 0.636, the blastocyst rate was 0.521, and the apoptosis rate was 0.050.

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Abstract

The invention relates to a method for bovine in-vitro fertilized embryo culture and a used culture solution. Particularly, on one hand, the invention relates to a method for culturing a bovine in-vitro fertilized embryo. The method comprises the following steps of putting the bovine in-vitro fertilized embryo into a bovine in-vitro fertilized embryo culture solution and carrying out embryo in-vitro culture under the conditions that the temperature is 38.5 DEG C, the content of CO2 is 0.5% and the humidity is 100%. The invention further relates to a bovine in-vitro fertilized embryo culture solution. The culture solution comprises NaCl, KCl, NaHCO3, MgCl2, KH2PO3, sodium pyruvate, glucose, calcium galactonate, fetal calf serum, L-glutamine, an essential amino-acid, a nonessential amino acid, glutathione and water. The method and the used culture solution have excellent technical effects in the specification, for example, the method has a higher blastocyst hatching rate when the bovine in-vitro fertilized embryo is cultured.

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 the application of bovine IVF embryo culture solution in the cultivation of bovine IVF embryos. Further, the present invention also relates to a method for culturing bovine IVF embryos using the bovine IVF embryo culture solution. 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 called test-tube animals. This technology was successfully developed in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0603C12N2500/12C12N2500/16C12N2500/30C12N2500/32C12N2500/34C12N2501/999
Inventor 许晓椿王小武郭晶赵明礼
Owner 天津力牧生物科技有限公司
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