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Animal-origin-free antifreezing solution and cryopreservation method thereof on seminal liquid of machin

An animal-derived, cryopreservation technology, applied in the field of animal reproduction, can solve the problems of inability to use in vitro fertilization or artificial insemination, and the survival rate is less than 40%, and achieve the effect of eliminating the risk of pathogenic factors.

Inactive Publication Date: 2015-04-22
YUNNAN AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0005] Animal-derived substances such as egg yolk and skim milk mainly play a role in resisting cold shock and improving the survival rate of frozen semen in the freezing of animal semen. The survival rate of sperm frozen with non-animal-derived antifreeze is usually far lower than 40%. , not for in vitro fertilization or artificial insemination
Previous studies have shown that mouse, goat, and human sperm can be successfully cryopreserved in chemically defined non-animal origin antifreeze, but similar techniques have not been reported for primate sperm freezing

Method used

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Embodiment

[0023] Weigh 0.2g Tris, 1.2g TES, 2g lactose, 2g glucose, 0.2g raffinose, 10000IU penicillin, 5mg streptomycin sulfate and dissolve them in 80mL Milli-Q ultrapure water in turn, mix well and add Milli-Q ultrapure water Dilute the water to 100mL, adjust the pH to 7.0-7.2 with 1N NaOH or 1N HCl, and make a diluent for cryopreservation. Add 10% glycerol by volume to the semen diluent to make 2× antifreeze solution for semen cryopreservation. At room temperature of 20-23°C, the semen of the cynomolgus monkey and the diluent were mixed in a 4mL test tube at a volume ratio of 1:9 and left for 1 hour. Then, add an equal volume of 2× antifreeze solution containing glycerol to the test tube at room temperature, add five times, add 1 / 5 of the volume each time, with an interval of 5 minutes between each time, the final concentration of glycerin is 5%, mix well Then equilibrate at room temperature for 30 minutes. Next, the obtained semen was sucked into a 0.25 mL frozen straw and sealed...

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Abstract

The invention discloses an animal-origin-free antifreezing solution and a cryopreservation method of the animal-origin-free antifreezing solution on seminal liquid of a machin. According to the antifreezing solution, 0.2 g of Tris 1.2 g of TES, 2 g of lactose, 2 g of glucose, 0.2 g of raffinose, 10,000 IU of penicillin G sodium salt and 5 mg of streptomycin sulphate are dissolved in 80 mL of Milli-Q ultrapure water in sequence, the Milli-Q ultrapure water is added after even mixing for achieving the constant volume of 100 mL, pH is adjusted to be 7.0 to 7.2 through 1 N of NaOH or 1 N of HCl, a diluent is prepared, glycerinum is added to the diluent, and the 2* antifreezing solution is prepared. The risk of introducing pathogenic factors in an external fertilization or artificial fertilization system is completely eradicated, a sperm low-temperature freezing injury mechanism and a freezing protection mechanism can be understood better, and quality control over the antifreezing solution can be carried out easily.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, in particular to a non-animal source antifreeze solution and a method for freezing and preserving the semen of cynomolgus monkeys. Background technique [0002] Although the sperm of many animals can be successfully cryopreserved, a considerable number of sperm lose their motility due to damage during the cryopreservation process. For most animals, only about 50% of the sperm survive even with the most advanced equipment and the best freezing procedures. The reason why the efficiency of sperm freezing is so low is mainly due to the lack of understanding of the mechanism of sperm freezing damage and the mechanism of action of some cryoprotective substances. [0003] In the cryopreservation of most animal sperm, in addition to glycerin, some animal-derived substances such as egg yolk, skimmed milk, milk, etc. are generally added to the antifreeze solution, and some even a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 李亚辉杨明华
Owner YUNNAN AGRICULTURAL UNIVERSITY
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