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Ultralow-temperature cryopreservation method for fenneropenaeus chinensis sperms

A technology of ultra-low temperature freezing and preservation method, which is applied in the field of ultra-low temperature cryopreservation of Chinese prawn sperm, which can solve the problems of poor repeatability and general effect, and achieve the effects of increasing viscosity, reducing the formation of ice crystals, and protecting ice crystals and osmotic pressure damage

Active Publication Date: 2021-10-01
山东省海洋科学研究院
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0004] Based on the above content, the present invention provides a method for ultra-low temperature cryopreservation of Chinese prawn sperm. Aiming at the problems of poor reproducibility and general effect in the existing studies on the cryopreservation of Chinese prawn sperm, a method for ultra-low temperature cryopreservation of Chinese prawn sperm is carried out. Experiments on the components and proportions of cryoprotective fluids and precise cooling procedures have established an efficient, stable and reproducible cryopreservation technology, enabling the survival rate of frozen semen to reach more than 85%.

Method used

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  • Ultralow-temperature cryopreservation method for fenneropenaeus chinensis sperms
  • Ultralow-temperature cryopreservation method for fenneropenaeus chinensis sperms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Prepare the ultra-low temperature cryoprotectant solution with sterilized natural seawater as the base solution and different concentrations of dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol (Gly) as the antifreeze protection agent, and the collected fresh Penaeus chinensis Semen and different ultra-low temperature cryoprotectants were diluted at a dilution volume ratio of 1:5, and then placed in a programmed cooling device that had been pre-cooled at 0°C, and operated at 0°C for 20 minutes; -5°C / min to -80°C ; Balance at -80°C for 5 minutes; -20°C / min to -180°C cooling program, and then put into -196°C liquid nitrogen for storage. The semen frozen in liquid nitrogen was taken out, quickly placed in a 37°C water bath, shaken until the semen melted, and the quality was tested to further determine the optimal type and concentration of the osmotic antifreeze protectant. The results are shown in Table 1.

[0022] Table 1 Screening of cryoprotectants with optim...

Embodiment 2

[0026] Sterilized natural seawater is used as the base liquid, 10% Gly is used as the antifreeze protection agent, and the dilution volume ratio of semen and ultra-low temperature cryoprotectant solution is 1:5. The cooling procedure is the same as in Example 1, the difference is that different concentrations of trehalose and glucose , maltose, vitamin C, and bovine serum albumin were used as non-permeable cryoprotectants for ultra-low temperature cryopreservation, and the quality of frozen sperm was tested to further determine the optimal category and concentration of non-permeable cryoprotectants. The results are shown in Table 2.

[0027]Table 2 Screening of the optimal non-permeable cryoprotectant for ultra-low temperature cryopreservation of Chinese penaeus prawn sperm

[0028]

[0029]

[0030] The above experimental results show that the survival rate of sperm in the experimental group with 0.25 mol / L trehalose and 0.25 mol / L bovine serum albumin was significantly...

Embodiment 3

[0032] Sterilized natural seawater is used as the base liquid, 10% Gly is used as the antifreeze protective agent, and 0.25mol / L bovine serum albumin or trehalose is used as the non-permeable antifreeze protective agent respectively. The cooling procedure is the same as in Example 1, except that the Chinese prawn semen and ultra-low temperature cryoprotective solution were set up in five dilution volume ratio experimental groups of 1:1, 1:3, 1:5, 1:7, and 1:9 for ultra-low temperature cryopreservation. The quality of the frozen sperm is tested to further determine the optimal dilution ratio. The results are shown in Table 3.

[0033] Table 3 Screening of the optimal dilution ratio for cryopreservation of Chinese prawn sperm

[0034]

[0035] The above experimental results show that the sperm survival rate is the highest when the dilution ratio is 1:5.

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Abstract

The invention relates to an ultralow-temperature cryopreservation method for fenneropenaeus chinensis sperms, and belongs to the technical field of sperm preservation. The method specifically comprises the following steps: diluting collected fenneropenaeus chinensis semen with an ultralow-temperature freezing protection solution, performing programmed cooling to-180 DEG C, and transferring into liquid nitrogen for preservation. According to the method, the fenneropenaeus chinensis semen diluted by the ultralow-temperature freezing protection liquid is subjected to programmed cooling to-180 DEG C and then is transferred into liquid nitrogen for preservation, the sperm stops metabolic activity in an ultralow-temperature environment so as to prolong the service life. The ultralow-temperature freezing protection liquid is combined with water molecules in the solution to initiate the hydration action. The crystallization process of water is weakened to increase the viscosity of the solution so as to reduce the formation of ice crystals. Meanwhile, a certain molar concentration can be maintained inside and outside cells to reduce the concentration of electrolyte in an unfrozen solution inside and outside the cells, so that the cells are prevented from being damaged by solutes, and the cells are protected from being damaged by the solution and the ice crystals.

Description

technical field [0001] The invention relates to the technical field of sperm preservation, in particular to a method for ultra-low temperature freezing and preservation of Chinese penaeus prawn sperm. Background technique [0002] Chinese prawn (Fenneropenaeus chinensis), also known as Chinese prawn, formerly known as Chinese prawn, belongs to Arthropoda, Crustacea, Decapoda, Penaeidae, Penaeus, It is one of the main cultured shrimp species in my country. The individual is larger, the carapace is thin, smooth and transparent, the shrimp meat is crispy and soft, and it has extremely high economic value. However, in recent years, due to the increasing demand year by year, the wild population of Penaeus prawns in China has begun to decrease sharply year by year, relying heavily on artificial breeding and release. Therefore, it is very necessary to carry out germplasm preservation of Penaeus prawn, and then carry out germplasm optimization and establish a high-quality species b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/021A01N1/0221A01N1/0226A01N1/0252
Inventor 邹琰宋爱环刘洪军刘童王英俊吴莹莹
Owner 山东省海洋科学研究院
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