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Deep-freezing preserving fluid of loach sperms and freezing recovery method and application thereof

A technology of ultra-low temperature freezing and cryopreservation, which is applied in the preservation, application, animal husbandry and other directions of human or animal body, can solve the problem of few male individuals of loach, and achieve the effect of solving the problem of few male individuals, high recovery rate and high efficiency of the method.

Inactive Publication Date: 2018-09-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can solve the problems of few male loach individuals and timely fertilization of sperm eggs in actual production, and provide convenience for large-scale production of loach seedlings and genetic breeding research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Ultra-low temperature cryopreservation of loach sperm:

[0027] (1) Preparation of semen: 6 male loach with well-developed selective glands and strong physique were treated with LHRH-A 2 +DOM mixed injection method for artificial induction of labor, after the effect time is reached, squeeze out the semen, collect the semen and mix with the freshly prepared pre-cooled preservation solution according to the volume ratio of 1:6;

[0028] (2) Subpackage: subpack the diluted semen into 0.5mL cryopreservation tubes, each containing 0.3mL;

[0029] (3) Programmed cooling: place the cryopreservation tubes in the programmed cooling box (1°C / min), and then move them together into a -80°C refrigerator for 4-6 hours;

[0030] (4) Ultra-low temperature storage: put the cryopreservation tube in liquid nitrogen for long-term storage;

[0031] Described cryopreservation solution is prepared by the following method:

[0032] 480mg NaCl, 680mgKCl, 22mg CaCl 2 , 12 mg MgSO 4 , 240mg ...

Embodiment 2

[0034] Resuscitation of loach sperm from ultra-low temperature cryopreservation and detection of sperm motility after resuscitation

[0035] (1) Resuscitate the cryopreserved material in Example 1 after being frozen for 1 month: put the cryopreservation tube in a water bath at 28°C, shake it gently to make the temperature uniform, take it out immediately when only a small amount of solid remains, and place it in the air Continue shaking to completely melt.

[0036] (2) Sperm motility was tested under a microscope. Take 45 μL of 0.6% NaCl solution on a glass slide, align the field of view under a magnification lens of 10×40, take 5 μL of semen and mix it in the droplet, and quickly observe the movement of sperm. Statistical sperm motility, that is, the percentage of activated sperm moving in a fast straight line to the total number of sperm detected; record the life span of sperm, that is, the experience from being activated to when more than 90% of the sperm lose their motili...

Embodiment 3

[0041] Application of loach spermatozoa after freezing and thawing in artificial reproduction:

[0042] (1) 6 female loach with well-developed selective glands and strong physique were treated with LHRH-A 2 +DOM mixed injection method for artificial induction of labor, after the effect time is reached, eggs are extruded;

[0043] (2) Select eggs with good quality and divide them into 6 parts after mixing. Take 3 of them and add the resuscitated semen (10,000-20,000 eggs / mL semen) dropwise, add an appropriate amount of 0.6% NaCl solution to mix and activate fertilization, and then add more 0.6% NaCl solution after 1 minute to wash away the ethylene glycol in the preservation solution. Alcohol, DMSO and excess sperm. The other 3 eggs were inseminated with fresh semen. All fertilized eggs were hatched at 24-26°C, water was changed regularly, and dead eggs were sucked out in time. The fertilization rate was counted in the middle stage of the gastrula, and the hatching rate was...

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Abstract

The invention belongs to the technical field of fish sperm cryopreservation, and particularly discloses deep-freezing preserving fluid of loach sperms and a freezing recovery method and application thereof. The method is utilized for carrying out deep-freezing (-196 DEG C) cryopreservation on sperms of loaches, and the motility rate of the resurgent sperms reaches 72.3%. Loach eggs are fertilizedwith the resurgent sperms, the fertility rate reaches 81.5%, the hatchability reaches 84.9%, and the spersms have no remarkable differences (P>0.05) with fresh sperms. The method is large in cryopreservation amount, good in effect and easy and convenient to implement and needs no expensive instrument and equipment, the problem that during actual production, the number of male loaches is small, andthe eggs cannot be fertilized in good time can be solved, and convenience is provided for large-scale production and genetic breeding studies of loach fry.

Description

technical field [0001] The invention belongs to the technical field of cryopreservation of fish sperm, and in particular relates to an ultra-low temperature cryopreservation solution of loach sperm, a cryopreservation method and application thereof. Background technique [0002] Loach (Misgurnus anguillicaudatus) is known as "water ginseng" for its rich nutritional value and unique nourishing and medicinal effects. It is favored by consumers in China, Japan and South Korea. It is a small economic fish emerging in my country. . With the development of the loach breeding industry, the demand for seed is increasing day by day, especially the high-quality seed of artificial propagation is in short supply, which cannot meet the market demand. For this reason, many scholars have carried out research on the artificial propagation of loach. However, it has been found in actual production that the male loach is smaller and the number is significantly less than that of the female loac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/021A01N1/0226
Inventor 周小云罗双双呼光富祝东梅
Owner HUAZHONG AGRI UNIV
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