Anti-freezing liquid for improving survival rate of sperms of Chinese prawns and cryopreservation method of anti-freezing liquid

Abstract

The invention discloses an anti-freezing solution for improving the survival rate of sperms of Chinese prawns and a cryopreservation method of the anti-freezing solution, and belongs to the technical field of low-temperature biology and reproductive science. The invention provides an anti-freezing solution for cryopreservation of sperms of Chinese prawns, which comprises a basic solution and an anti-freezing agent, and the anti-freezing agent is dimethyl sulfoxide with the volume fraction of 10% or glycerol with the volume fraction of 10%; the basic liquid contains 23.69 g of sodium chloride, 1.1 g of potassium chloride, 14.8 g of magnesium chloride hexahydrate, 4.3 g of sodium sulfate, 0.23 g of sodium bicarbonate, 1.12 g of calcium chloride and distilled water with the constant volume of 1000 mL. According to the cryopreservation method provided by the invention, 10% dimethyl sulfoxide or 10% glycerol is adopted as an antifreeze agent, the sperm density is controlled to be 1 * 10 < 7 > / mL, and step-by-step cooling is carried out, so that the survival rate of unfrozen sperms reaches 90% or above. The long-term preservation effect of the Chinese prawn sperms is remarkably improved, and the method is suitable for germplasm resource protection and efficient breeding.

Description

Technical Field

[0001] The invention relates to the technical field of low-temperature biology and reproduction, in particular to an antifreeze solution for improving the survival rate of Chinese shrimp sperm and a freezing preservation method thereof. Background Art

[0002] Sperm cryopreservation technology is a hot topic in cryobiology and reproductive science. Cryopreservation involves completely inhibiting sperm cell metabolism and halting energy consumption at ultra-low temperatures, enabling long-term sperm storage. Upon use, the sperm is revived by thawing the frozen cells, restoring their fertilizing capacity. Research suggests that sperm cryopreservation can extend the in vitro storage time of sperm and significantly increase the utilization rate of high-quality male animals.

[0003] The Chinese shrimp (Fenneropenaeus chinensis) is an arthropod of the family Penaeidae, order Decapoda. Also known as giant prawn, prawn, or prawn, it has a long, laterally compressed body and a thin, smooth, and transparent carapace. The frontal margin of the cephalothorax is a prominent central feature, with a slightly raised base. The dorsal margin of the horn is covered with 7-9 teeth, and the ventral margin has 3-5 teeth. Except for the first cephalothorax segment and the telson, all other segments possess a pair of appendages. The shrimp possess five pairs of legs, the first three pairs being chelate-like and the last two pairs being claw-like. The telson ends in a pointed, spinous tip with a longitudinal groove on the dorsal surface. Females are bluish-green, males are brownish-yellow, and females are larger than males. Chinese shrimp are a major export aquatic product, with tender, delicious meat and a rich nutritional profile, including protein, fat, various vitamins, and essential trace elements. They can be eaten fresh or dried. They also have medicinal value, being effective in the treatment of skin ulcers and neurasthenia. Chinese shrimp farming has become one of the most representative sectors of marine aquaculture. Most shrimp are annual, and the male and female gonads mature asynchronously. Research on hybrid breeding, germplasm selection, gynogenesis, and species conservation all involve sperm cryopreservation. Long-term sperm storage ensures the long-term stability of genetic resources, reduces time and space constraints, and significantly improves the feasibility of selective breeding by ensuring sperm supply without seasonal restrictions or the high cost of rearing parent stock.

[0004] Ultra-low temperature cryopreservation (preservation in liquid nitrogen at -196°C), a core method for germplasm conservation, has established standardized protocols for mammals (such as cattle and pigs) and fish (such as salmon and tilapia). However, in crustaceans, particularly commercial shrimp (such as Penaeus vannamei and Penaeus japonicus), the application of this technology has lagged behind due to the unique sperm structure (absence of acrosomes and the presence of spinous processes) and low-temperature sensitivity. Research on sperm cryopreservation in Penaeus chinensis began in the 1990s. Prior studies on sperm cryopreservation in Penaeus chinensis include experiments by Ke Yafu et al. (1996), who achieved a sperm survival rate of 60% after freezing, and by Zou Yan et al. (2021), who achieved a sperm survival rate of over 85%. Therefore, further improving the sperm survival rate after freezing in Penaeus chinensis is of great value and significance for the aquaculture of Penaeus chinensis. Summary of the Invention

[0005] The purpose of the present invention is to provide an antifreeze solution and a cryopreservation method for improving the survival rate of Chinese shrimp sperm, so as to solve the problems existing in the above-mentioned prior art. The base liquid defined in the present invention can effectively reduce the sperm metabolism rate and reduce the toxic damage of metabolites to sperm; the antifreeze agent used in the present invention can not only play a good antifreeze protection role, but also will not cause sperm poisoning due to excessive antifreeze concentration, thereby reducing sperm cell damage; the present invention limits the final sperm density to 1×10 7 The present invention's cryopreservation method can achieve a 90% survival rate for Chinese shrimp sperm after thawing, and the long-term survival rate (3 months) remains stable at over 81%, a significant improvement over existing technologies.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: An antifreeze solution for cryopreservation of Chinese shrimp sperm, comprising a base liquid and an antifreeze agent, wherein the antifreeze agent is 10% by volume of dimethyl sulfoxide or 10% by volume of glycerol; the base liquid contains 23.69g of sodium chloride, 1.1g of potassium chloride, 14.8g of magnesium chloride hexahydrate, 4.3g of sodium sulfate, 0.23g of sodium bicarbonate, and 1.12g of calcium chloride, and is diluted to 1000mL of distilled water.

[0008] Technical solution 2: Application of the antifreeze solution in improving the survival rate of frozen-preserved sperm of Chinese shrimp.

[0009] Technical solution three: A Chinese shrimp sperm cryopreservation kit, comprising the antifreeze solution.

[0010] Technical Solution 4: A cryopreservation method for improving the survival rate of Chinese shrimp sperm, comprising the following steps: diluting and filtering the Chinese shrimp sperm with the base liquid, mixing the filtered Chinese shrimp sperm with antifreeze solution, equilibrating at 4°C, and allowing the sperm to settle on the surface of liquid nitrogen, and finally storing the sperm in liquid nitrogen.

[0011] Furthermore, the density of the diluted and filtered Chinese shrimp sperm is 1×10 7 pieces / mL.

[0012] Furthermore, the volume ratio of the filtered Chinese shrimp sperm to the antifreeze solution is 1:3.

[0013] Furthermore, the 4°C equilibration time is 30 minutes.

[0014] Furthermore, the residence time of the liquid nitrogen surface is 15 minutes.

[0015] The present invention discloses the following technical effects:

[0016] The present invention adopts sterilized artificial seawater as the base liquid; using the base liquid to appropriately reduce the sperm storage density can effectively reduce the sperm metabolic rate, reduce the toxic damage of metabolic products to sperm, and thus prolong the life of sperm. The base liquid in the method of the present invention is controllable and easy to prepare, and has a better sperm preservation effect than other base liquids; the present invention adopts a 10% volume fraction of dimethyl sulfoxide as the antifreeze; the antifreeze is to prevent sperm from being damaged by ice crystals and oxidation, which affects the sperm preservation effect and the survival rate and fertilization rate of sperm after thawing. The antifreeze used should be controlled within the concentration range accepted by Chinese shrimp sperm, maintaining good permeability while reducing toxicity. The present invention adopts 10% dimethyl sulfoxide, which can not only play a good antifreeze protection role, but also will not cause sperm poisoning due to excessive antifreeze concentration, reduce sperm cell damage, and has the highest survival rate compared with other antifreezes; the final sperm density of the present invention is 1×10 7 The density of the sperm is higher than 1000 mmol / mL. Too high a density increases the chance of sperm collisions, making sperm less likely to survive and increasing the rate of deformities. Furthermore, the present invention employs a cooling method that involves first fully equilibrating the sperm, then allowing it to remain on the liquid nitrogen surface for a period of time before finally being placed in an ultra-low temperature liquid nitrogen tank, thus preventing sperm from being damaged by crystallization. Equilibrium is the process of interaction between sperm and the cryoprotectant, allowing the protective agent to penetrate the sperm and exert its cryoprotective effect. This cryopreservation cooling procedure is simple and convenient.

[0017] The present invention demonstrates that the method for preserving sperm has a high recovery rate after thawing, with a survival rate of up to 90%, a significant improvement over existing technologies. This method is simple to operate and low-cost, providing reliable technical support for the conservation of Chinese shrimp germplasm resources and large-scale breeding. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1

[0024] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0025] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution is prepared sterile artificial seawater, and the base solution is configured as follows: 23.69g sodium chloride, 1.1g potassium chloride, 14.8g magnesium chloride hexahydrate, 4.3g sodium sulfate, 0.23g sodium bicarbonate, 1.12g calcium chloride, mixed with 900mL distilled water, fixed to 1000mL, and then sterilized in an autoclave;

[0026] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:3 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% dimethyl sulfoxide by volume, which is prepared as follows: 10 mL of dimethyl sulfoxide is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and sterilized to obtain the antifreeze agent;

[0027] (3) Cryopreservation of Penaeus chinensis sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 15 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0028] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0029] Example 2

[0030] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0031] The difference from Example 1 is that the antifreeze agent is 10% glycerol. The preparation is as follows: 10 mL of glycerol is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and mixed, and sterilized.

[0032] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution is prepared sterile artificial seawater, and the base solution is configured as follows: 23.69g sodium chloride, 1.1g potassium chloride, 14.8g magnesium chloride hexahydrate, 4.3g sodium sulfate, 0.23g sodium bicarbonate, 1.12g calcium chloride, mixed with 900mL distilled water, fixed to 1000mL, and then sterilized in an autoclave;

[0033] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:3 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% glycerol, which is prepared as follows: add 10 mL of dimethyl sulfoxide to a beaker, add 90 mL of the base solution to dilute it, and make up to 100 mL. Shake well to mix, and sterilize to obtain the antifreeze agent.

[0034] (3) Cryopreservation of Penaeus chinensis sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 15 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0035] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0036] Example 3

[0037] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0038] The difference from Example 1 is that the base liquid is Ren's solution (Leagene brand purchased from Qingdao Qingkesaier Biotechnology Co., Ltd.).

[0039] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution was Ren's solution (Leagene brand purchased from Qingdao Qingkesaier Biotechnology Co., Ltd.);

[0040] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:3 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% dimethyl sulfoxide by volume, which is prepared as follows: 10 mL of dimethyl sulfoxide is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and sterilized to obtain the antifreeze agent;

[0041] (3) Cryopreservation of Penaeus chinensis sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 15 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0042] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0043] Example 4

[0044] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0045] The difference from Example 1 is that the base liquid is Hank's solution, which is configured as follows: 8.01 g sodium chloride, 0.4 g potassium chloride, 0.14 g calcium chloride dihydrate, 0.35 g sodium bicarbonate, 0.06 g potassium dihydrogen phosphate, 0.1 g magnesium chloride hexahydrate, 0.1 g magnesium sulfate heptahydrate, 0.06 g disodium hydrogen phosphate dihydrate, and 10 g glucose, and distilled water is added to make the volume to 500 mL. The remaining steps are the same as in Example 1.

[0046] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution was Hank's solution, which was prepared as follows: 8.01 g sodium chloride, 0.4 g potassium chloride, 0.14 g calcium chloride dihydrate, 0.35 g sodium bicarbonate, 0.06 g potassium dihydrogen phosphate, 0.1 g magnesium chloride hexahydrate, 0.1 g magnesium sulfate heptahydrate, 0.06 g sodium hydrogen phosphate dihydrate, and 10 g glucose. Distilled water was added to make the volume up to 500 mL.

[0047] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:3 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% dimethyl sulfoxide by volume, which is prepared as follows: 10 mL of dimethyl sulfoxide is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and sterilized to obtain the antifreeze agent;

[0048] (3) Cryopreservation of Penaeus chinensis sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 15 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0049] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0050] Example 5

[0051] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0052] The difference from Example 1 is that the volume ratio of sperm to antifreeze is 1:1, that is, sperm and antifreeze are placed in a 1.8 mL cryotube at a dilution volume ratio of 1:1.

[0053] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution is prepared sterile artificial seawater, and the base solution is configured as follows: 23.69g sodium chloride, 1.1g potassium chloride, 14.8g magnesium chloride hexahydrate, 4.3g sodium sulfate, 0.23g sodium bicarbonate, 1.12g calcium chloride, mixed with 900mL distilled water, fixed to 1000mL, and then sterilized in an autoclave;

[0054] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:1 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% dimethyl sulfoxide by volume, which is prepared as follows: 10 mL of dimethyl sulfoxide is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and sterilized to obtain the antifreeze agent;

[0055] (3) Cryopreservation of Penaeus chinensis sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 15 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0056] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0057] Example 6

[0058] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0059] The difference from Example 1 is that the volume ratio of sperm to antifreeze is 1:5, that is, sperm and antifreeze are placed in a 1.8 mL cryotube at a dilution volume ratio of 1:5.

[0060] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution is prepared sterile artificial seawater, and the base solution is configured as follows: 23.69g sodium chloride, 1.1g potassium chloride, 14.8g magnesium chloride hexahydrate, 4.3g sodium sulfate, 0.23g sodium bicarbonate, 1.12g calcium chloride, mixed with 900mL distilled water, fixed to 1000mL, and then sterilized in an autoclave;

[0061] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:5 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% dimethyl sulfoxide by volume, which is prepared as follows: 10 mL of dimethyl sulfoxide is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and sterilized to obtain the antifreeze agent;

[0062] (3) Cryopreservation of Penaeus chinensis sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 15 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0063] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0064] Example 7

[0065] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0066] The difference from Example 1 is that the cooling steps are: equilibration at 4° C. for 30 minutes, staying on the surface of liquid nitrogen for 5 minutes, and then immersing in liquid nitrogen.

[0067] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution is prepared sterile artificial seawater, and the base solution is configured as follows: 23.69g sodium chloride, 1.1g potassium chloride, 14.8g magnesium chloride hexahydrate, 4.3g sodium sulfate, 0.23g sodium bicarbonate, 1.12g calcium chloride, mixed with 900mL distilled water, fixed to 1000mL, and then sterilized in an autoclave;

[0068] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:3 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% dimethyl sulfoxide by volume, which is prepared as follows: 10 mL of dimethyl sulfoxide is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and sterilized to obtain the antifreeze agent;

[0069] (3) Cryopreservation of Chinese shrimp sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 5 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0070] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0071] Example 8

[0072] Antifreeze solution for improving sperm survival rate of Chinese shrimp and freezing preservation method thereof

[0073] The difference from Example 1 is that the cooling steps are: equilibration at 4° C. for 30 minutes, staying on the surface of liquid nitrogen for 30 minutes, and then immersing in liquid nitrogen.

[0074] (1) Dilution of Penaeus chinensis sperm: The collected Penaeus chinensis sperm (healthy male Penaeus chinensis with well-developed gonads) was diluted and filtered with an equal amount of base solution. The base solution is prepared sterile artificial seawater, and the base solution is configured as follows: 23.69g sodium chloride, 1.1g potassium chloride, 14.8g magnesium chloride hexahydrate, 4.3g sodium sulfate, 0.23g sodium bicarbonate, 1.12g calcium chloride, mixed with 900mL distilled water, fixed to 1000mL, and then sterilized in an autoclave;

[0075] (2) The Chinese shrimp sperm filtered in step (1) was diluted with antifreeze at a volume ratio of 1:3 and placed in a 1.8 mL cryotube to obtain semen. The sperm concentration of the semen was 1×10 7 The antifreeze agent is 10% dimethyl sulfoxide by volume, which is prepared as follows: 10 mL of dimethyl sulfoxide is added to a beaker, 90 mL of the base solution is added for dilution, the volume is fixed to 100 mL, the mixture is thoroughly shaken and sterilized to obtain the antifreeze agent;

[0076] (3) Cryopreservation of Chinese shrimp sperm: The semen prepared in step (2) was placed in a 4°C refrigerator for 30 minutes, frozen on the surface of liquid nitrogen for 30 minutes, and then placed in liquid nitrogen at -196°C for long-term storage;

[0077] (4) Thawing and recovery of sperm: Take out the cryopreservation tube containing semen that has been stored in liquid nitrogen for a long time in step (3) from the liquid nitrogen tank, quickly put it into a 39°C water bath to thaw for 3 minutes, and observe the sperm survival rate by microscopic examination after thawing.

[0078] Penaeus chinensis sperm were cryopreserved according to the method described in Examples 1-8. The sperm survival rate was observed under a microscope after thawing. The results are shown in Table 1.

[0079] Table 1 Sperm survival rate of Penaeus chinensis sperm after cryopreservation and thawing

[0080] Group Survival rate Example 1 90％ Example 2 86％ Example 3 72％ Example 4 65％ Example 5 78％82％ Example 6 82％78％ Example 7 73％ Example 8 66％

[0081] Example 9

[0082] Sperm from Penaeus chinensis (Penaeus chinensis) frozen in liquid nitrogen for one month, three months, and six months, as described in Example 1, were taken out and placed in a water bath at 39°C for 3 minutes. Sperm survival was then observed under a microscope. The results are shown in Table 2.

[0083] Table 2 Sperm survival rate of Penaeus chinensis sperm after thawing every three months after cryopreservation

[0084] Retention time (months) Sperm survival rate (%) Store for one month 84.67±6.12 Store for three months 81.00±2.83 Store for six months 58.00±9.19

[0085] In summary, the preservation method provided in Example 1 of the present invention can achieve a survival rate of up to 90% after thawing, and the long-term preservation (3 months) survival rate is stable at above 81%, which is a significant improvement over the prior art.

[0086] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An antifreeze solution for cryopreservation of Chinese shrimp sperm, characterized in that: The invention comprises a base liquid and an antifreeze agent, wherein the antifreeze agent is 10% by volume of dimethyl sulfoxide or 10% by volume of glycerol; the base liquid contains 23.69g of sodium chloride, 1.1g of potassium chloride, 14.8g of magnesium chloride hexahydrate, 4.3g of sodium sulfate, 0.23g of sodium bicarbonate, and 1.12g of calcium chloride, and the volume is fixed to 1000mL with distilled water.

2. Use of the antifreeze solution according to claim 1 in improving the survival rate of frozen-preserved sperm of Chinese shrimp.

3. A kit for cryopreservation of sperm of Chinese shrimp, characterized in that: The antifreeze solution according to claim 1 is included.

4. A cryopreservation method for improving the survival rate of Chinese shrimp sperm, characterized in that: The following steps are involved: The Chinese shrimp sperm and the base liquid of claim 1 are diluted and filtered, the filtered Chinese shrimp sperm are mixed with the antifreeze solution of claim 1, equilibrated at 4° C., allowed to stand on the surface of liquid nitrogen, and finally put into liquid nitrogen for storage.

5. The cryopreservation method according to claim 4, wherein The density of the diluted and filtered Chinese shrimp sperm is 1×10 7 pieces / mL.

6. The cryopreservation method according to claim 4, wherein The volume ratio of the filtered Chinese shrimp sperm to the antifreeze solution is 1:

3.

7. The cryopreservation method according to claim 4, wherein The 4°C equilibration time is 30 minutes.

8. The cryopreservation method according to claim 4, wherein The residence time on the liquid nitrogen surface is 15 minutes.