Mouse sperm freezing medium as well as preparation method and application thereof

A technology of cryopreservation and sperm, applied in the application, preservation of human or animal body, animal husbandry, etc., can solve the problem that the sperm protection ability of mice needs to be further improved, and achieve the protection of sperm fertilization ability, high in vitro fertilization rate, The effect of enhancing immune function and antioxidant function

Active Publication Date: 2019-08-02
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glutamine solution has the functions of enhancing immunity and anti-oxidation, and is often used as a traditional mouse sperm cryopreservation solution for inbred mice or genetically engineered mice, but its ability to protect mouse sperm needs to be further improved

Method used

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  • Mouse sperm freezing medium as well as preparation method and application thereof
  • Mouse sperm freezing medium as well as preparation method and application thereof

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Experimental program
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preparation example Construction

[0024] The present invention provides a kind of preparation method of described mouse sperm cryopreservation liquid, preferably comprises the following steps:

[0025] 1) Mix raffinose, skimmed milk powder and water, sterilize, and dispense into a tube of 990 μL each to obtain the base solution;

[0026] 2) Prepare the rosmarinic acid mother solution with a concentration of 100 mmol / L by using the base solution, and divide into one tube per 10 μL to obtain the rosmarinic acid mother solution.

[0027] In the present invention, the sterilization is preferably filter sterilization; the filter membrane preferably has a pore size of 0.22 μm. Prior to sterilization, the resulting mixture is preferably centrifuged in order to remove insoluble impurities. The rotational speed of the centrifugation is preferably 10000-14000 rpm, more preferably 12000 rpm, and the centrifugation time is preferably 10-30 min, more preferably 20-30 min.

[0028] In the present invention, the base liqui...

Embodiment 1

[0042] Preparation method of mouse sperm cryopreservation solution

[0043]Dissolve 18g of raffinose and 3g of skimmed milk powder in 100mL of double distilled water and mix. Centrifuge the resulting mixture at 12,000rpm for 30min. Take the supernatant and filter it with a 0.22μm filter to sterilize it. Pack in 990μL and mark it as liquid A. Store in a -20°C refrigerator; use Solution A to prepare a solution of rosmarinic acid at a concentration of 100mM, aliquot 10μL, label it as Solution B, and store in a -20°C refrigerator. Liquids A and B are mouse sperm cryopreservation liquids.

Embodiment 2～ Embodiment 5

[0045] Preparation method of working solution of mouse sperm cryopreservation solution

[0046] When in use, discard 10 μL of the solution A prepared in Example 1 and then mix it with 2 tubes of solution B to obtain a 20 mM rosmarinic acid freezing solution.

[0047] Discard 20 μL of solution A prepared in Example 1 and mix it with 3 tubes of solution B to obtain 30 mM rosmarinic acid freezing solution.

[0048] Discard 30 μL of solution A prepared in Example 1 and mix it with 4 tubes of solution B to obtain 40 mM rosmarinic acid freezing solution.

[0049] Discard 40 mL of the liquid A prepared in Example 1 and then mix it with 5 liquids B to obtain a 50 mM rosmarinic acid freezing solution.

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Abstract

The invention provides a mouse sperm freezing medium as well as a preparation method and an application thereof, and belongs to the technical field of in-vitro fertilization. The mouse sperm freezingmedium comprises rosmarinic acid mother liquor and a basic liquid which are subpackaged independently; the basic liquid is an aqueous solution containing raffinose with mass concentration being 16%-20% and skim milk powder with mass concentration being 2%-4%; a solvent of the rosmarinic acid mother liquor is the basic liquid. During using, a working solution containing rosmarinic acid with concentration of 20-50 mmol/L is prepared from the mouse sperm freezing medium by mixing. The mouse sperm freezing medium contains rosmarinic acid and is used for resisting too much active oxygen produced ina cryopreservation-recovery process, optimizing a life metabolism process of sperms and enhancing the immunologic function and antioxidant function and achieves the effect of protecting fertilizing capacity of the sperms, thereby increasing the in-vitro fertilization rate of the mouse sperms.

Description

technical field [0001] The invention belongs to the technical field of in vitro fertilization, and in particular relates to a mouse sperm cryopreservation solution and a preparation method and application thereof. Background technique [0002] Due to the needs of biomedical research, in the experimental process, it is often necessary to prepare or import genetically modified mice from abroad. However, due to the technical difficulty and high price of the prepared or imported mice, the number of mice obtained is limited, and they are used for scientific research. The experimental mice need to reach a certain statistically significant number. Due to the long cycle of natural reproduction, sperm cryopreservation and multiple in vitro fertilization methods can quickly obtain a sufficient number of mice for experiments; scientific research work When it's over, the mice are often euthanized, leaving the resources gone. At present, resources can be preserved by adopting the method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 马雪云刘腾飞章延嘉
Owner EAST CHINA NORMAL UNIV
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