Process for frozen-preserving Chinese sturgeon semen under super-low temperature

Abstract

Technology for bulk preserving spermatozoa of Chinese sturgeon through freezing at ultralow temperature comprises collecting and detecting fresh spermatozoa, diluting, balancing, adding antifreeze, subpackaging, program cooling, and preserving in liquid nitrogen container of -196deg.C. Each bulk can freeze 100 mL fresh spermatozoa. when the first bulk spermatozoa is in the procedure cooling step, after 5 minute, starting diluting the second bulk spermatozoa, and repeating above steps until all spermatozoa is preserved 8-12 h before its effective time. The diluent is Tris-HCl 20-30 millimole, sucrose 30-60 mol, potassium chloride 2-10 millimole, and distilled water 1000mL, and regulating pH to 8-8.5 with HCl. The antifreeze is methanol. The invention can efficiently carry out bulk preservation of Chinese sturgeon spermatozoa through freezing at ultralow temperature and long term ultralow temperature freezing preservation, and provide enough spermatozoa resource for artificial propagation of Chinese sturgeon.

Description

technical field

[0001] The invention relates to a method for ultra-low temperature preservation of fish sperm, in particular to a method for ultra-low temperature preservation of Chinese sturgeon sperm. Background technique:

[0002] With the development of fish seed artificial breeding and genetic breeding research, fish sperm cryopreservation technology has attracted people's attention. The first successful cryopreservation of fish sperm was in 1968. White eel essence, obtained a fertilization rate of 80-89%. In recent years, ultra-low temperature cryopreservation of fish sperm has been gradually extended to Cyprinidae, Perch, Catrididae, Salmonidae, and salmon and trout.

[0003] The Chinese sturgeon is one of the rare fish species and is a first-class protected animal in my country. It is a large freshwater fish unique to my country and is mainly distributed in the Yangtze River system and the Pearl River system. In recent years, due to the intensification of water pol...

Examples

Embodiment 1

[0028] Embodiment 1: Take the freezing 500 milliliters of Chinese sturgeon fresh essence as an example below to illustrate the specific operation process:

[0029] 1. Squeeze the abdomen of the mature male Chinese sturgeon and collect more than 500ml of fresh semen. During the collection process, the sperm should be absolutely avoided from contacting with water. The collected fresh semen should be put into the sealed container after oxygenation according to the amount of 100ml per bag. Put 5 bags in total in plastic bags, and then store them in a refrigerator with a temperature of 4°C;

[0030] 2. Under the microscope of 10×10 times, the computer-aided detection of fresh semen has an average liveness of 85%, the average speed of the curve movement is over 160 microns / second, and the average speed of linear motion is over 90 microns / second, reaching a ratio of Standard ready to freeze;

[0031] 3. Preparation of Chinese sturgeon sperm dilution: 2000 ml dilution preparation met...

Embodiment 2

[0038] Embodiment 2: Take 1000 milliliters of frozen Chinese sturgeon as an example below, and 4000 milliliters of diluent is needed, and its configuration is as follows:

[0039] 1 mole of Tris-HCl 20ml, 1 mole of sucrose 30ml, 1 mole of potassium chloride 2ml, distilled water to 1000ml, HCl to adjust the pH value to 8.5, mix 4 times, a total of 4000ml was prepared, store in the refrigerator at 4°C for later use ;

[0040] Other operation methods are the same as the above-mentioned embodiment 1.

technical field

[0001] The invention relates to a method for ultra-low temperature preservation of fish sperm, in particular to a method for ultra-low temperature preservation of Chinese sturgeon sperm. Background technique:

[0002] With the development of fish seed artificial breeding and genetic breeding research, fish sperm cryopreservation technology has attracted people's attention. The first successful cryopreservation of fish sperm was in 1968. White eel essence, obtained a fertilization rate of 80-89%. In recent years, ultra-low temperature cryopreservation of fish sperm has been gradually extended to Cyprinidae, Perch, Catrididae, Salmonidae, and salmon and trout.

[0003] The Chinese sturgeon is one of the rare fish species and is a first-class protected animal in my country. It is a large freshwater fish unique to my country and is mainly distributed in the Yangtze River system and the Pearl River system. In recent years, due to the intensification of water pol...

Examples

Embodiment 1

[0028] Embodiment 1: Take the freezing 500 milliliters of Chinese sturgeon fresh essence as an example below to illustrate the specific operation process:

[0029] 1. Squeeze the abdomen of the mature male Chinese sturgeon and collect more than 500ml of fresh semen. During the collection process, the sperm should be absolutely avoided from contacting with water. The collected fresh semen should be put into the sealed container after oxygenation according to the amount of 100ml per bag. Put 5 bags in total in plastic bags, and then store them in a refrigerator with a temperature of 4°C;

[0030] 2. Under the microscope of 10×10 times, the computer-aided detection of fresh semen has an average liveness of 85%, the average speed of the curve movement is over 160 microns / second, and the average speed of linear motion is over 90 microns / second, reaching a ratio of Standard ready to freeze;

[0031] 3. Preparation of Chinese sturgeon sperm dilution: 2000 ml dilution preparation met...

Embodiment 2

[0038] Embodiment 2: Take 1000 milliliters of frozen Chinese sturgeon as an example below, and 4000 milliliters of diluent is needed, and its configuration is as follows:

[0039] 1 mole of Tris-HCl 20ml, 1 mole of sucrose 30ml, 1 mole of potassium chloride 2ml, distilled water to 1000ml, HCl to adjust the pH value to 8.5, mix 4 times, a total of 4000ml was prepared, store in the refrigerator at 4°C for later use ;

[0040] Other operation methods are the same as the above-mentioned embodiment 1.

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