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Method for separating XY germ cell of mammal

A technology for mammals and sperm, applied in biochemical equipment and methods, microorganisms, tissue culture, etc., can solve problems affecting the purity of separated sperm, low efficiency of separated sperm, and poor resolution, so as to maintain sperm vitality and ensure sperm vitality , the effect of shortening the time interval

Inactive Publication Date: 2006-11-15
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the shortcomings of flow cytometry sperm separation technology are that the efficiency of sperm separation is still low and the cost is high. The sperm diluent contains yolk and other substances that affect the coloring of sperm, which is affected by the poor resolution of XY sperm after fluorescent staining. The purity of the separated sperm is compromised, and the vitality of the sperm is greatly lost after separation, which affects its fertilization ability

Method used

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  • Method for separating XY germ cell of mammal
  • Method for separating XY germ cell of mammal
  • Method for separating XY germ cell of mammal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

0.0050

[0035] The full name of HEPES is N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid;

[0036] Add deionized water to 200 ml;

[0037] Adjust the pH to 7.0 with 1 mol / L sodium hydroxide solution;

[0038] Sterilize by filtration through a 0.22 micron pore size filter.

[0039] (3) Separation of X sperm and Y sperm by flow cytometry at a constant temperature of 16°C, the components and contents of the sheath fluid and receiving fluid used for the separation are respectively:

[0040] Element

g / 1000ml

TRIS

citric acid

fructose

Sodium chloride

Penicillin

streptomycin

23.88

11.63

8.55

0.8

0.29

0.25

[0041] The full name of TRIS is trihydroxymethylaminomethane;

[0042] Add deionized water to 1000 ml;

[0043] Adjust the pH to 6.8 with 5 mol / L of hydrochloric acid;

[0044] 0.22 micron pore size membrane filtration.

[0045] Element

g / 100ml

TRIS

citr...

Embodiment 2

[0053] The collected fresh semen of Longlin goats was added to the mixed solution of antibiotics and stored in a water bath at 25°C at a constant temperature. Mycin was 200, 500, 800 and 800, respectively.

[0054]Dilute the original semen to 130 million / ml, add the fluorescent dye Hoechst33342 to 30 μg / ml, and incubate in a water bath at 33°C for 60 minutes. After the water bath, add the pigment FD&C#40 to a concentration of 0.003% by mass.

[0055] In semen diluent, the concentrations of sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium bicarbonate, magnesium chloride hexahydrate, sodium pyruvate, glucose, HEPES, bovine serum albumin, and gentamicin sulfate are expressed in grams / 200 Milliliters represent 1.1, 0.04, 0.005, 0.15, 0.01, 0.04, 0.15, 1.5, 0.1, 0.005, respectively, and sodium lactate is 0.7 ml / 200 ml. Add deionized water to 200 ml; adjust the pH to 7.2 with 1 mol / L sodium hydroxide solution; filter with a filter membrane with a pore size ...

Embodiment 3

[0061] The collected fresh semen of Changbai boars was added to antibiotic mixed solution and stored in a water bath at 27.5°C at a constant temperature. Micrograms / ml means 180, 400, 700, 700 respectively;

[0062] Dilute the semen to 100 million / ml, add fluorescent dye Hoechst33342 to 30 μg / ml, and incubate in a water bath at 37°C for 45 minutes. After the water bath, add the pigment FD&C#40 to a concentration of 0.0035% by mass.

[0063] In semen diluent, the concentrations of sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium bicarbonate, magnesium chloride hexahydrate, sodium pyruvate, glucose, HEPES, bovine serum albumin, and gentamicin sulfate are expressed in grams / 200 Milliliters represent 1.2, 0.05, 0.01, 0.18, 0.02, 0.05, 0.20, 2.0, 0.8, 0.01, respectively, and sodium lactate is 0.75 ml / 200 ml. Add deionized water to 200 ml; adjust the pH to 7.0 with 1 mol / L sodium hydroxide solution, and filter with a filter membrane with a pore size of 0.22...

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Abstract

A method for separating non-human mammalian XY sperm, based on the principle that the DNA content of mammalian X sperm is more than that of Y sperm, the sperm DNA is stained with fluorescent dye Hoechst33342 and then separated by flow cytometry. The present invention improves the accuracy of sperm separation and the vitality of sperm after separation while ensuring the efficiency of separating XY sperm by improving the preservation method before sperm separation, the diluent of sperm, the sheath fluid and receiving liquid of separated sperm, and is suitable for The large-scale commercial separation and production of XY sperm of non-human mammals such as cattle, buffalo, and goats can be combined with new animal breeding technologies such as artificial insemination and in vitro fertilization to greatly improve the efficiency of animal husbandry production.

Description

technical field [0001] The present invention relates to a method for separating X sperm and Y sperm of non-human mammals. Background technique [0002] In animal husbandry production, selectively breeding livestock and poultry offspring with predictable sex will greatly improve the speed and efficiency of animal husbandry production. Using X or Y sperm to inseminate the female animal can determine the sex of the offspring before fertilization, thereby significantly changing the sex ratio of the offspring and achieving the purpose of sex control. Taking dairy cows as an example, dairy farmers wish to increase or renew their dairy herd by breeding more heifers from their high-quality, high-yielding nucleus herd, which can be achieved through sex control. Similarly, beef cattle farmers want to breed more steers, because the growth rate and meat quality of cattle are related to sex, bulls grow faster than cows, and steer beef is more expensive. I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06
Inventor 卢克焕陆阳清张明
Owner GUANGXI UNIV
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