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A non-animal origin tree shrew sperm in vitro capacitation liquid and application thereof

A technology of animal origin and capacitation, applied in the field of animal reproductive biology, can solve the problems that have not been reported, and achieve the effects of reasonable formula, eliminating the risk of introducing pathogenic factors, and easy quality control

Inactive Publication Date: 2019-01-11
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now, more and more people of insight believe that the tree shrew is the most promising new type of experimental animal to replace the precious primate, and the research on its sperm capacitation is conducive to the development of in vitro fertilization and artificial reproduction technology of this animal. However, there has been no report on the research on tree shrew sperm capacitation, and the development of non-animal-derived component capacitation solution has not been reported because it is extremely challenging.

Method used

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  • A non-animal origin tree shrew sperm in vitro capacitation liquid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] A method for in vitro capacitation of tree shrew sperm using non-animal-derived in vitro capacitation liquid, the specific steps are:

[0023] 1) Preparation of semen washing liquid and capacitation liquid: prepare tree shrew semen washing liquid and capacitation liquid respectively, and adjust the pH to 7.3-7.4; the formula of the semen washing liquid is NaCl 0.6975g / 100ml, KCl 0.0356g / 100ml, MgSO 4 0.0143g / 100ml, KH 2 PO 4 0.0161g / 100ml, Glucose 0.0901g / 100ml, pyruvic acid 0.0011g / 100ml, lactic acid 185μl, CaCl 2 2H 2 O 0.0294g / 100ml, NaHCO 3 0.0168g / 100ml, cyclodextrin 0.1000g / 100ml, Hepes (Na salt) 0.1302g / 100ml, HepesAcid form 0.1192g / 100ml; the capacitation solution formula is NaCl 0.6662g / 100ml, KCl 0.0236g / 100ml, MgSO 4 0.0143g / 100ml, KH 2 PO 4 0.0161g / 100ml, Glucose 0.0901g / 100ml, pyruvic acid 0.0011g / 100ml, lactic acid 185μl, CaCl 2 2H 2 O0.0294g / 100ml, NaHCO 3 0.2100g / 100ml, cyclodextrin 0.1000g / 100ml.

[0024] 2) Sperm collection and countin...

Embodiment 2

[0031]A method for in vitro capacitation of tree shrew sperm using non-animal-derived in vitro capacitation liquid, the specific steps are:

[0032] 1) Preparation of semen washing liquid and capacitation liquid: prepare tree shrew semen washing liquid and capacitation liquid respectively, and adjust the pH to 7.3-7.4; the formula of the semen washing liquid is NaCl 0.6975g / 100ml, KCl 0.0356g / 100ml, MgSO 4 0.0143g / 100ml, KH 2 PO 4 0.0161g / 100ml, Glucose 0.0901g / 100ml, pyruvic acid 0.0011g / 100ml, lactic acid 185μl, CaCl 2 2H 2 O 0.0294g / 100ml, NaHCO 3 0.0168g / 100ml, cyclodextrin 0.1000g / 100ml, Hepes (Na salt) 0.1302g / 100ml, HepesAcid form 0.1192g / 100ml; the capacitation solution formula is NaCl 0.6662g / 100ml, KCl 0.0236g / 100ml, MgSO 4 0.0143g / 100ml, KH 2 PO 4 0.0161g / 100ml, Glucose 0.0901g / 100ml, pyruvic acid 0.0011g / 100ml, lactic acid 185μl, CaCl 2 2H 2 O0.00441g / 100ml, NaHCO 3 0.252g / 100ml, cyclodextrin 0.0655g / 100ml.

[0033] 2) Sperm collection and counting...

Embodiment 3

[0040] A method for in vitro capacitation of tree shrew sperm using non-animal-derived in vitro capacitation liquid, the specific steps are:

[0041] 1) Preparation of semen washing liquid and capacitation liquid: prepare tree shrew semen washing liquid and capacitation liquid respectively, and adjust the pH to 7.3-7.4; the formula of the semen washing liquid is NaCl 0.6975g / 100ml, KCl 0.0356g / 100ml, MgSO 4 0.0143g / 100ml, KH 2 PO 4 0.0161g / 100ml, Glucose 0.0901g / 100ml, pyruvic acid 0.0011g / 100ml, lactic acid 185μl, CaCl 2 2H 2 O 0.0294g / 100ml, NaHCO 3 0.0168g / 100ml, cyclodextrin 0.1000g / 100ml, Hepes (Na salt) 0.1302g / 100ml, HepesAcid form 0.1192g / 100ml; the capacitation solution formula is NaCl 0.6662g / 100ml, KCl 0.0236g / 100ml, MgSO 4 0.0143g / 100ml, KH 2 PO 4 0.0161g / 100ml, Glucose 0.0901g / 100ml, pyruvic acid 0.0011g / 100ml, lactic acid 185μl, CaCl 2 2H 2 O0.0441g / 100ml, NaHCO 3 0.084g / 100ml, cyclodextrin 3.0831g / 100ml.

[0042] 2) Sperm collection and counting...

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Abstract

The invention relates to a non-animal origin tree shrew sperm in vitro capacitation liquid and application thereof, belonging to the technical field of animal reproductive biology. The specific stepsare as follows: preparing tree shrew ishing semen and capacitation liquid; separating and removing the epididymis cauda of tree shrews; placing the epididymis cauda in centrifugal tubes containing ished semen and incubating for 10 min to allow sperm to swim out. transferring the sperm suspension to the sterilized centrifuge tube, adding the semen ishing liquid, gently mixing, and centrifuging; discarding supernatant, adding semen ishing liquid, and centrifuging; discardingthe supernatant and resuspending the sperm in the capacitation solution to make the sperm concentration 10* 106 sperm / mL.5 ml of that semen is divided into incubation tubes and incubating in a CO2 incubator. A method for in vitro capacitation of tree shrew spermatozoa adopting animal-free component capacitation liquid avoids that risk of introduce pathogenic factors into in vitro capacitation or in vitro fertilization system, and is easy to carry out quality control on the capacitation liquid.

Description

technical field [0001] The invention belongs to the technical field of animal reproductive biology, and in particular relates to a non-animal source tree shrew sperm in vitro capacitation solution and application thereof. Background technique [0002] Although human and mammalian sperm have the ability to move when they are just ejaculated or taken out from the tail of the epididymis, they cannot immediately combine with egg cells. They need to stay in the female reproductive tract for a period of time and undergo a series of physiological and biochemical changes before they have the ability to fertilize egg cells. This phenomenon is called "sperm capacitation". A large number of studies have shown that sperm capacitation is a physiological stage that all mammalian sperm must go through before fertilization. [0003] Early studies on sperm capacitation used the method of in vivo capacitation, that is, taking sperm from the reproductive tract of mated female animals for fert...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/12C12N2500/14C12N2501/90C12N2501/999
Inventor 李亚辉杨明华李雪宋文菲解道豪吴文静
Owner YUNNAN AGRICULTURAL UNIVERSITY
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