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A method for cryopreservation of Chinese prawn sperm at ultra-low temperature

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 general effect and poor repeatability, and achieve the effect of reducing the formation of ice crystals, increasing viscosity, protecting ice crystals and osmotic pressure damage

Active Publication Date: 2022-04-12
山东省海洋科学研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

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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  • A method for cryopreservation of Chinese prawn sperm at ultra-low temperature
  • A method for cryopreservation of Chinese prawn sperm at ultra-low temperature
  • A method for cryopreservation of Chinese prawn sperm at ultra-low temperature

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Experimental program
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Effect test

Embodiment 1

[0021] To prepare ultra-low temperature cryoprotective solution with sterilized natural seawater as the base solution and different concentrations of dimethyl sulfoxide (DMSO), ethylene glycol (EG), and glycerol (Gly) as anti-freeze protection agents, the fresh prawns collected The semen and different cryogenic cryoprotectants were diluted with a volume ratio of 1:5 and then placed in a programmed cooling device that had been pre-cooled at 0°C, running at 0°C for equilibration for 20 minutes; -5°C / min to -80°C ; Equilibrate at -80°C for 5min; drop to -180°C at -20°C / min, and then put it into liquid nitrogen at -196°C for storage. Take out from the above-mentioned liquid nitrogen frozen semen, quickly put it into a 37°C water bath, shake until the semen dissolves, carry out quality inspection, and further determine the optimal type and concentration of osmotic cryoprotectant. The results are shown in Table 1.

[0022] Table 1 Screening of optimal penetrating cryoprotectants for...

Embodiment 2

[0026] Taking sterilized natural seawater as the base solution, 10% Gly as anti-freeze protection agent, the dilution volume ratio of semen and ultra-low temperature cryoprotective solution is 1:5, and the cooling procedure is the same as that in Example 1, except that different concentrations of trehalose and glucose are set respectively. , maltose, vitamin C, bovine serum albumin as the non-permeable anti-cryoprotectant in the experimental group were cryopreserved at ultra-low temperature, and the quality of the frozen sperm was tested to further determine the optimal type and concentration of the non-permeable anti-cryoprotectant. The results are shown in Table 2.

[0027]Table 2 Screening of optimal non-permeable cryoprotectants for cryopreservation of Penaeus chinensis spermatozoa

[0028]

[0029]

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

Embodiment 3

[0032] Taking sterilized natural seawater as the base liquid, 10% Gly as the anti-freeze protection agent, respectively using 0.25mol / L bovine serum albumin or trehalose as the impermeable anti-freeze protection agent, the cooling procedure is the same as that in Example 1, the difference is that the Five dilution volume ratios of 1:1, 1:3, 1:5, 1:7, and 1:9 were respectively set for the semen and ultra-low temperature cryoprotective solution of Penaeus chinensis in experimental groups 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 optimal dilution ratios for cryopreservation of Penaeus chinensis spermatozoa

[0034]

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

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Abstract

The invention relates to a method for cryopreservation of Chinese prawn sperm at ultra-low temperature, and belongs to the technical field of sperm preservation. Specifically, the collected Chinese prawn semen was diluted with ultra-low temperature cryoprotectant solution, then the temperature was lowered to -180°C, and then transferred to liquid nitrogen for storage. In the present invention, the seminal fluid of Penaeus chinensis diluted with ultra-low temperature cryoprotectant solution is cooled down to -180°C and stored in liquid nitrogen. Molecular combination, hydration occurs, and the crystallization process of weakened water increases the viscosity of the solution to reduce the formation of ice crystals. At the same time, it can maintain a certain molar concentration inside and outside the cell, reduce the concentration of electrolyte in the unfrozen solution inside and outside the cell, and make the cell Protects cells from solution damage and ice crystal damage.

Description

technical field [0001] The invention relates to the technical field of sperm preservation, in particular to an ultra-low temperature cryopreservation method for sperm of Penaeus chinensis. 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 farmed shrimp species in my country. The individual is large, the carapace is thin, smooth and transparent, and the shrimp meat is crispy and soft, which has a very high economic value. However, in recent years, due to the increasing demand year by year, the size of the wild population of Penaeus chinensis in China has begun to decline sharply year by year, and it is heavily dependent on artificial proliferation and release. Therefore, it is very necessary to carry out germplasm conservation of Penaeus chinensis in China, and then to carry out germplasm selection and estab...

Claims

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Application Information

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Patent Type & Authority Patents(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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