NL2028946B1

NL2028946B1 - Cryoprotective solution and ultra-low temperature cryopreservation method of stichopus japonicus sperm

Info

Publication number: NL2028946B1
Application number: NL2028946A
Authority: NL
Inventors: Zhang Libin; Yang Hongsheng; Liu Shilin; Xu Shuai; Sun Jingchun; Lin Chenggang; Sun Lina
Original assignee: Inst Oceanology Cas
Priority date: 2020-09-25
Filing date: 2021-08-10
Publication date: 2022-06-16
Also published as: CN112075415B; NL2028946A; CN112075415A

Abstract

The invention relates to the technical ﬁeld of ultra-low temperature cryopreservati on of stichopus japonicus germplasm, in particular to a cryoprotective solution, an activator 5 formula and an ultra-low temperature cryopreservation method of apostichopu japonicus sperm based on a program controlled freezer and CASA. The cryoprotective solution is composed of a diluent, an antifreeze, and an additive, wherein the diluent is natural seawater, the antifreeze is one or more of aprotic polar solvents, and the additive is glucose. The method of the present invention has a large amount of frozen storage (more 10 than 200 mL of fresh stichopus japonicus semen can be cryopreserved in one batch), can precisely control the cooling process, and has a good application effect after thawing. It can solve the problem of unsynchronized maturation of male and female sea cucumbers and a series of germplasm degradation of stichopus japonicus in actual production.

Description

-1- CRYOPROTECTIVE SOLUTION AND ULTRA-LOW TEMPERATURE

CRYOPRESERVATION METHOD OF STICHOPUS JAPONICUS SPERM TECHNICAL FIELD

[01] The invention relates to the technical field of ultra-low temperature cryopreservation of sea cucumber germplasm, in particular to a cryoprotective solution, an activator formula and an ultra-low temperature cryopreservation method of apostichopu japonicus sperm based on a program controlled freezer and CASA.

BACKGROUND ART

[02] Apostichopus japonicus, also known as stichopus japonicus, belongs to echinodermata, holothuroidea, aspidochirotida, stichopodidae, apostichopus, natural habitat in the northern part of the Western Pacific, including the Far East coast of Russia, the coasts of Japan and South Korea, and the Yellow Sea and Bohai Sea of China. It is the most common marine higher invertebrates. Stichopus japonicus is a nourishing product with the homology of medicine and food. Among the more than 20 edible sea cucumbers distributed in the seas of China, the nutritional quality and economic value are the highest. Its health and medical value has been widely recognized since ancient times. As the market demand for stichopus japonicus and the scale of the industry have expanded year by year, the over-harvesting of wild stichopus japonicus has become more and more serious, resulting in a sharp decline in the resource quantity and germplasm quality of wild stichopus japonicus. Stichopus japonicus have been assessed as endangered (EN) species by the Red List of Threatened Species of the World Conservation Union (IUCN Red List of Threatened Species or IUCN Red List); cultured sea cucumbers have also experienced a series of germplasm degradation problems such as frequent disease, low survival rate, and slow growth rate, and the frequent occurrence of stichopus japonicus diseases causes about 3 billion yuan in economic losses every year, causing heavy losses to the stichopus japonicus industry and becoming an important bottleneck restricting the healthy development of stichopus japonicus breeding. The offshore environment has an impact on the survival rate, quality, and yield of cultured sea cucumbers. Sea cucumber breeding itself has an impact on the marine environment, resulting in increasing

Do environmental pressure, and may even cause the seed ginseng to fail to lay eggs, sperm, or insufficient vitality of sperm and eggs, leading to the serious consequences of not being able to breed. For example, in the summer of 2018, the seashore was raging with high temperatures, and the continuous high temperature weather was earlier than in previous years and lasted longer. Cofferdam cultured sea cucumbers in Liaoning, Shandong and other places died under the influence of high temperature and high humidity, and economic and germplasm resources were severely lost. At present, researchers mostly use the method of hybridization between southern and northern sea cucumbers, which are geographically isolated, to improve the germplasm quality of stichopus japonicus. However, because the male and female sea cucumbers in the north and south do not mature at the same time, it is necessary to use methods such as ripening and ovulation promotion in the process of sea cucumber hybridization. Not only the operation is complicated and the cost is high, but there are also problems such as the failure to achieve hybridization due to the failure of ripening and ovulation promotion. Therefore, it is urgent to solve the problem of preservation of high-quality germplasm resources of stichopus japonicus.

[03] The currently published papers or patents on the cryopreservation method of stichopus japonicus sperm adopt the "two-step" method of cooling (controlling the height of the sample from the liquid nitrogen surface to control the cooling rate) and visual observation of sperm motility with a microscope. There are human subjective factors in these two modes of operation, and the accuracy of the results is greatly affected by the cooling device and the counting device. In the papers published by Shao et al. in 2006 (Shao MY, Zhang ZF, Yu L, et al. Cryopreservation of sea cucumber apostichopus japonicus (Selenka) sperm[J]. Aquaculture Research,2006,37(14):1450-1457) and papers published by Yuta Mizuno et al. in 2018 (Mizuno Y, Fujiwara A, Yamano K, et al. Motility and fertility of cryopreserved spermatozoa of the Japanese sea cucumber apostichopus japonicus[J]. Aquaculture Research, 2018; 1-10.), the sea cucumber sperm is diluted with preservation solution and then cryopreserved. The preservation solution formula used is artificial seawater (423.00mM NaCl, 9.00mM KCI, 9.27mM CaCl, 22.94mM MgCl:, 22.50mM MgSO:), 10mM Hepes buffer and 15%-20% dimethyl sulfolide. Hepes buffer is not only expensive but also produces certain biologically toxic hydrogen peroxide when exposed to visible light. The operation time of Shao et al. is up to one hour, and the

-3- loss of sperm motility is large. After thawing, the sperm life is only 1200s, which is difficult to apply to production; In the studies of Yuta Mizuno et al. and the patent filed in 2019 (Chang Yaqing, Zhan Yaoyao, Zhao Tanjun, etc., Sea cucumber sperm cryopreservation method [P].CN110326610A, 2019-10-15.), the survival rate of all processed sperm after thawing is less than 15%, and the sperm viability rate, especially the proportion of sperm capable of forward movement, is positively correlated with the fertilization rate and hatching rate. The higher the sperm motility after thawing, the better the long-term preservation of sperm and the actual production application. However, the process of the above patent documents is not clear enough, and it is greatly affected by the subjective factors of the operator, and at the same time, it is greatly affected by the freezing device. Using the methods in the above patent documents, the sperm of stichopus japonicus have no vitality after thawing.

SUMMARY

[04] The purpose of the present invention is to solve the above-mentioned problems in the prior art and to solve the problems of unsynchronized maturation of male and female sea cucumbers and a series of germplasm degradation of stichopus japonicus in actual production. Based on the program controlled freezer and CASA, the invention provides an ultra-low temperature cryopreservation solution formula, an activator formula and an ultra-low temperature cryopreservation method of apostichopus japonicus sperm.

[05] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is:

[06] A cryoprotective solution, which is composed of a diluent, an antifreeze, and an additive; wherein the diluent is natural seawater, the antifreeze is one or more of aprotic polar solvents, and the additive is glucose.

[07] The said is dimethyl sulfoxide (DMSO) and/or dimethylacetamide (DMA).

[08] The cryoprotective solution is natural seawater, dimethyl sulfoxide (DMSO) and glucose, wherein the respective volume-to-mass ratio is 85mL-90mL: 10mL-15mL:

1.98g.

[09] The cryoprotective solution is natural seawater and dimethylacetamide (DMA), and the volume ratio is 80mL:20mL.

[10] The natural seawater is natural seawater filtered with a 0.45um filter membrane;

4- dimethyl sulfoxide (DMSO) is dimethyl sulfoxide with a purity greater than or equal to

99.97%, dimethylacetamide (DMA) is dimethylacetamide with a purity greater than or equal to 99.97%, and both DMSO and DMA are aprotic high-polarity solvents.

[11] An ultra-low temperature cryopreservation method of stichopus japonicus sperm using the cryoprotective solution,

[12] (1) Semen acquisition: acquiring the sperm of stichopus japonicus in the breeding period and waiting for use;

[13] (2) Mixing semen with cryoprotective solution: mixing the fresh semen of stichopus japonicus and the cryoprotective solution in a container with a volume ratio of 1:5-1:7;

[14] (3) Program cooling: after mixing, placing it in a program controlled freezer, running the cooling program, the cooling program is to equilibrate at 0°C for 5 minutes, cool to -80°C at a cooling rate of 10-15°C/min, equilibrate at -80°C for 5 minutes, and cool to -180°C at a cooling rate of 20°C/min, and equilibrate for 5 minutes, and then taking out after 5 minutes of equilibration to realize ultra-low temperature cryopreservation of stichopus japonicus sperm.

[15] The sperm container after the cooling procedure is placed in liquid nitrogen for long-term storage.

[16] The cryopreserved stichopus japonicus germ is thawed in a 25°C water bath, shaken gently to make the temperature uniform, and when there is only a small amount of solids left, take it out immediately (about 120s), continue to shake it in the air to completely melt; pipette the frozen semen and mix it with the activator to make the total dilution rate of the original stichopus japonicas germ of 600 times, the activator 1s filtered natural seawater containing Swt% of fetal bovine serum.

[17] The fresh semen of stichopus japonicus in step (2) 1s to dilute the collected sperm by 600 times with an activator, detect the sperm and collect the sperm with activity greater than 90%, and wait for use.

[18] The advantages of the present invention:

[19] The cryopreservation method of the present invention is based on two instruments, a program controlled freezer (Kasu Micro-Digitcool) and CASA (Myron ML.-608JZ}, which can accurately control the cooling rate and quantitatively analyze sperm vitality, and can quickly and mass cryopreserve sperm (more than 200mL of fresh semen can be cryopreserved in one batch); the operation time is short (the

-5- operation is completed within 20 minutes after the sperm is dissected) to avoid the problem of large loss of sperm vitality; the preservation solution used is extremely low-cost, safe and non-toxic, and easy to prepare, and the effect is good; after the mixing of sperm activator and sperm, the sperm can be activated immediately to show high vitality, maintaining high vitality for more than 3 hours; after thawing and activation of frozen sperm, the sperm viability rate is as high as 60.07+5.88%, and the sperm rapid motility rate is as high as 43.39 £5.88%, the sperm curve motility speed is as high as 103.51+15.46um/s; it provides convenience for the protection of stichopus japonicus germplasm resources, the large-scale production of seedlings and genetic breeding research, and the data results are objective and credible, which is beneficial to the further detailed study of the ultra-low temperature cryopreservation method of stichopus japonicus sperm and its freeze-damage mechanism. The concept of using aprotic polar solvents in the cryoprotective solution is conducive to the development and selection of new antifreeze. In addition, the method of the present invention has a large amount of frozen storage (more than 200 mL of fresh stichopus japonicus semen can be cryopreserved in one batch), can precisely control the cooling process, and has a good application effect after thawing. It can solve the problem of unsynchronized maturation of male and female sea cucumbers and a series of germplasm degradation of stichopus japonicus in actual production.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[20] The specific embodiments of the present invention will be further described below in conjunction with examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[BI] The method of the present invention aims at the problems of low recovery rate and poor motility ability existing in the current ultra-low temperature cryopreservation of stichopus japonicus sperm. The present invention obtains two kinds of cryopreservation solutions suitable for ultra-low temperature cryopreservation of stichopus japonicus sperm and the cryopreservation resuscitation method (including one sperm vitality activation solution) by systematically studying the related factors of cryopreservation of stichopus japonicus sperm. The configuration method of preservation solution and activation solution is simple, convenient for storage, and has good effect; the cryopreservation method has large amount of frozen storage and short

-6- operation time, which is suitable for mass application. It provides convenience for the protection of stichopus japonicus germplasm resources, the large-scale production of seedlings and genetic breeding research, which is beneficial to the further detailed study of the ultra-low temperature cryopreservation method of stichopus japonicus sperm and its freeze-damage mechanism.

[22] Example 1

[23] a. Preparation of cryopreservation solution: according to the principle of current use and preparation, a fresh ultra-low temperature cryoprotective solution was prepared. The formula (1) of ultra-low temperature cryoprotective solution for stichopus japonicus sperm was as follows: 85mL of natural seawater filtered with a

0.45pm filter membrane, 15mL of dimethyl sulfoxide (DMSO) with a purity >99.97%, and 1.98g of glucose; formula (2) was as follows: 80mL of natural seawater filtered with a 0.45um filter membrane, and 20mL of dimethylacetamide (DMA) with a purity >99.97%. The prepared cryoprotective solutions were pre-cooled in a 4°C constant temperature refrigerator until use.

[24] b. Acquisition of semen: stichopus japonicus in the breeding period was taken, and the ginseng body after discharging a large amount of water in the body cavity (to avoid seawater contact with sperm to activate it) was dried. The sea cucumber was dissected from the abdomen using a sterile scalpel to prevent the gonads from being scratched. The complete gonads were picked out using a tweezer. The body fluid on the surface of gonads was adsorbed using an absorbent paper, then the gonads were transferred to a sterile culture dish, cut into pieces with scissors and filtered into a sterile 50ml centrifuge tube with a 300-mesh silk sieve, and placed in a constant temperature incubator at 4°C for use. At the same time, the vitality of fresh sperm was detected, and only sperm with vitality greater than 90% can be used for freezing experiments. The procedure of sperm vitality detection was that 1198ul of activator (filtered natural seawater containing Swt% of fetal bovine serum) and 2ul of fresh sperm were mixed, diluted by 600 times and activated, 10ul of the mixed solution was pipetted and added to the sperm counting plate, and placed in CASA to detect vitality.

[25] c. Mixing semen with cryopreservation solution: the fresh semen of stichopus japonicus and cryopretective solution were fully mixed according to a certain volume ratio and then dispensed into 2ml cryotubes. During the whole process, the cryotubes were stored in a cryotube box and placed in a thermal insulation box with ice. The

7- mixing volume ratio of semen and cryopreservation solution in cryoprotective solution formula (1) and (2) was 1:7. Taking a total volume of 480ul as an example, it contained 420ul of cryoprotection solution and 60ul of fresh semen of stichopus Japonicus.

[26] d. Program cooling: the cryotubes were placed into the program controlled freezer using a cryotube rack, then the cooling program was run, the cooling program was equilibrated at 0°C for 5 minutes, cooled to -80°C at a cooling rate of 10- 15°C/min, equilibrated at -80°C for 5 minutes, and cooled to -180°C at a cooling rate of 20°C/min, and equilibrated for 5 minutes, and then the cryotubes were taken out after 5 minutes of equilibration and put in liquid nitrogen (-196°C) for long-term storage.

[27] e. Thawing: the cryotubes in step d were taken out from the liquid nitrogen, placed in a 25°C water bath to thaw, shaken gently to make the temperature uniform, and taken out immediately (about 1208) when there was only a small amount of solids left, and shaken in the air until completely melt;

[28] f. Activation and sperm vitality analysis: the frozen semen was pipetted into the activator to dilute the fresh semen of stichopus japonicus up to 600 times, so as to maintain 70-120 sperm in each field of CASA, such as when the mixing volume ratio of semen and cryopreservation liquid is 1:7, it needs to be diluted 75 times again. For example, 740pl of activation solution and 10p of thawed frozen semen were mixed evenly; 10ul of activated frozen semen was pipetted, and added to the sperm counting plate, and the sperm vitality was analyzed using CASA.

[29] The statistical results show that the sperm viability rate of stichopus japonicus sperm preserved in formula (1) after thawing and activation is 60.07+5.88%, the rapid motility rate is as high as 43.39+5.88%, and the sperm curve motility speed is as high as 103.51+15.46um/s; The sperm viability rate of stichopus japonicus preserved in formula (2) after thawing and activation is 45.11+5.22%, the sperm rapid motility rate is as high as 26.68+1.34%, and the sperm curve motility speed is as high as

136.544+15.98 m/s.

[30] Comparative example 1

[31] a. Preparation of cryopreservation solution: according to the principle of current use and preparation, a fresh ultra-low temperature cryoprotective solution was prepared. In this example, the formula of ultra-low temperature cryopreservation

8- solution of stichopus japonicus sperm was as follows: 85mL of natural seawater filtered with a 0.45um filter membrane, and 15mL of dimethyl sulfoxide (DMSO) with a purity >99.97%. The prepared cryoprotective solution was pre-cooled in a 4°C constant temperature refrigerator until use.

[32] b. Acquisition of semen: same as Example 1

[33] c. Mixing semen with cryopreservation solution: the fresh semen of stichopus Japonicus and cryopretective solution were fully mixed according to a certain volume ratio of 1:7 and then dispensed into 2ml cryotubes. During the whole process, the cryotubes were stored in a cryotube box and placed in a thermal insulation box with I0 ice.

[34] d. Program cooling: same as Example 1

[35] e. Thawing: same as Example 1

[36] f. Activation and sperm vitality analysis: same as Example 1

[37] The statistical results show that the sperm viability rate of stichopus japonicus sperm in this example after thawing and activation is 46.29+7.55%, the rapid motility rate is as high as 284+2.44%, and the sperm curve motility speed is as high as

102.37+6.62um/s.

[38] Comparative example 2

[39] a. Preparation of cryopreservation solution: according to the principle of current use and preparation, a fresh ultra-low temperature cryoprotective solution was prepared. In this example, the formula of ultra-low temperature cryopreservation solution of stichopus japonicus sperm was as follows: 80mL of natural seawater filtered with a 0.45um filter membrane, and 20mL of propylene glycol (PG) with a purity >99.97%. The prepared cryoprotective solution was pre-cooled in a 4°C constant temperature refrigerator until use.

[40] b. Acquisition of semen: same as Example 1

[41] c. Mixing semen with cryopreservation solution: same as Example 1

[42] d. Program cooling: the cryotubes were placed into the program controlled freezer using a cryotube rack, then the cooling program was run, the cooling program was equilibrated at 0°C for 5 minutes, cooled to -80°C at a cooling rate of 15°C/min, equilibrated at -80°C for 5 minutes, and cooled to -180°C at a cooling rate of 20°C/min, and equilibrated for 5 minutes, and then the cryotubes were taken out and put in liquid nitrogen (-196°C) for long-term storage.

9-

[43] e. Thawing: same as Example 1

[44] f. Activation and sperm vitality analysis: same as Example 1

[45] The statistical results show that the sperm viability rate of stichopus japonicus sperm in this example after thawing and activation is 7.84+0.61%, the rapid motility rate is as high as 4.79+0.96%, and the sperm curve motility speed is as high as

48.45+12.94um/s.

[46] Comparative example 3

[47] a. Preparation of cryopreservation solution: according to the principle of current use and preparation, a fresh ultra-low temperature cryoprotective solution was prepared. In this example, the formula of ultra-low temperature cryopreservation solution of stichopus japonicus sperm was as follows: 85mL of natural seawater filtered with a 0.45um filter membrane, and 15mL of dimethyl sulfoxide (DMSO) with a purity >99.97%. The prepared cryoprotective solution was pre-cooled in a 4°C constant temperature refrigerator until use.

[48] b. Acquisition of semen: same as Example 1

[49] c. Mixing semen with cryopreservation solution: same as Example 1

[50] d. Program cooling: the cryotubes were placed into the program controlled freezer using a cryotube rack, then the cooling program was run, the cooling program was equilibrated at 0°C for 5 minutes, cooled to -80°C at a cooling rate of 25°C/min, equilibrated at -80°C for 5 minutes, and cooled to -180°C at a cooling rate of 20°C/min, and equilibrated for 5 minutes, and then the cryotubes were taken out and put in liquid nitrogen (-196°C) for long-term storage.

[51] e. Thawing: same as Example |

[52] f. Activation and sperm motility analysis: same as Example 1

[53] The statistical results show that the sperm viability rate of stichopus japonicus sperm in this example after thawing and activation is 12.97+0.46%, the rapid motility rate is as high as 7.83+0.53%, and the sperm curve motility speed is as high as

80.28+9.70um/s. [S4] Comparative example 4

[55] a. Preparation of cryopreservation solution: according to the principle of current use and preparation, a fresh ultra-low temperature cryoprotective solution was prepared. In this example, the formula of ultra-low temperature cryopreservation solution of stichopus japonicus sperm was as follows: 85mL of natural seawater

-10- filtered with a 0.45um filter membrane, and 15mL of dimethyl sulfoxide (DMSO) with a purity 299.97%. The prepared cryoprotective solution was pre-cooled in a 4°C constant temperature refrigerator until use.

[56] b. Acquisition of semen: same as Example 1

[57] c. Mixing semen with cryopreservation solution: the fresh semen of stichopus japonicus and cryopretective solution were fully mixed according to a volume ratio of 1:19 and then dispensed into 2ml cryotubes. During the whole process, the cryotubes were stored in a cryotube box and placed in a thermal insulation box with ice. Taking a total volume of 480ul as an example, it contained 456 of cryoprotection solution and 24ul of fresh semen of stichopus japonicus.

[58] d. Program cooling: same as Example 1

[59] e. Thawing: same as Example 1

[60] f. Activation and sperm vitality analysis: the frozen semen was pipetted to the activator to dilute the fresh semen of stichopus japonicus up to 600 times, so as to maintain 70-120 sperm in each field of CASA, such as when the mixing volume ratio of semen and cryopreservation liquid is 1:19, it needs to be diluted 30 times again. For example, 290ul of activation solution and 10ul of thawed frozen semen were mixed evenly; 10u of activated frozen semen was pipetted, and added to the sperm counting plate, and the sperm vitality was analyzed using CASA.

[61] The statistical results show that the sperm viability rate of stichopus japonicus sperm in this example after thawing and activation is 13.18+0.74%, the rapid motility rate is as high as 9.24+0.52%, and the sperm curve motility speed is as high as

101.3442. 15um/s.

It can be seen from the above examples that performing ultra-low temperature cryopreservation treatment on the apostichopus japonicus sperm using the specific freezing solution of the present invention under specific conditions, the sperm viability after activation can reach 60.07+5.88%, the sperm rapid motility rate can reach

43.39+5.88%, and the sperm curve motility speed can reach 103.51£15.46um/s, and the aprotic high-polarity solvent in the freezing solution can quickly penetrate into the cell, combine with water and electrolyte, produce a certain molar concentration in the cell, and reduce the concentration of the electrolyte solution of the unfrozen solution inside and outside the cell, lowers the freezing point and reduces the formation of ice crystals. At the same time, it avoids excessive exudation of intracellular water that

-11- causes cell shrinkage and damage to dissolution; After the fresh semen is mixed with the cryopreserved solution at the volume ratio of 1:5 to 1:7, it is cooled to -80°C at the optimal cooling rate (10-15°C/min), so that it could quickly cross the danger zone (0~ - 60 °C) while avoiding more ice crystal damage to sperm caused by too fast cooling rate, and then the apostichopus japonicus sperm can be stored for a long time (liquid nitrogen -196°C), and then activated by a specific activator to make its vitality reach the maximum instantaneously, and maintain high vitality for more than 3 hours, which is conducive to more efficient detection of sperm vitality and practical application; even if the same freezing solution is used but under different mixing ratios or different cooling rates or other types of antifreezes, due to unsuitable conditions, the sperm suffered more freezing damage in the process of ultra-low temperature freezing, and the activation after freezing cannot achieve the corresponding effect.

Thus, it can be seen that the solution provided by the embodiment of the present invention has unexpected technical effects.

Claims

S12 - Conclusions l. Cryoprotective solution, characterized in that the cryoprotective solution is composed of a diluent, an antifreeze and an additive, wherein the diluent is natural seawater, the antifreeze is one or more aprotic polar solvents and the additive is glucose.

Cryoprotective solution according to claim 1, characterized in that the aforementioned is dimethylsulfoxide (DMSO) and/or dimethylacetamide (DMA).

Cryoprotective solution according to claim 1, characterized in that the cryoprotective solution is natural seawater, dimethylsulfoxide (DMSO) and glucose, the respective volume-to-mass ratio being 85 ml - 90 ml : 10 ml - 15 ml : 1.98 gis .

The cryoprotective solution according to claim 1, characterized in that the cryoprotective solution is natural seawater and dimethylacetamide (DMA) and the volume ratio is 80 ml : 20 ml.

Cryoprotective solution according to any one of claims | -4, characterized in that the natural sea water is natural sea water filtered with a 0.45 µm filter membrane; dimethyl sulfoxide (DMSO) is dimethyl sulfoxide of greater than or equal to 99.97% purity, dimethylacetamide (DMA) is dimethylacetamide of greater than or equal to 99.97% purity, and both DMSO and DMA are high polarity aprotic solvents .

A method of ultra-low temperature cryopreservation of stichopus japonicus sperm using the cryoprotective solution according to claim 1, characterized by : (1) sperm acquisition: acquiring the sperm of stichopus japonicus in the culture period and waiting for use; (2) mixing sperm with a cryoprotective solution: mixing the fresh sperm of stichopus japonicus and the cryoprotective solution in a container

213 - with a volume ratio of 1:5 -1:7; (3) program cooling: after mixing, place in a program controlled freezer, run the cooling program where the cooling program should equilibrate for 5 minutes at 0°C, cool to -80°C at a cooling rate of — 15°C /min, to equilibrate at -80 °C for 5 minutes, and cool to -180 °C at a cooling rate of 20 °C/min, and to equilibrate for 5 minutes and then take it out after equilibration for 5 minutes to allow cryopreservation of stichopus japonicus sperm at ultra-low temperature. 7. A method according to claim 6, characterized in that the semen container is placed in liquid nitrogen after the cooling program for long-term storage.

A method according to claim 6, characterized by thawing the cryopreserved stichopus japonicus germ in a 25°C water bath, shaking gently to make the temperature uniform, and when only a small amount of solid remains , take out immediately (about 120 s), and shake in the air to melt completely; pipetting frozen sperm into the activator and mixing well to make the total dilution rate of 600 times the original stichopus japonica germ, and wherein the activator is filtered natural seawater containing 5% by weight fetal bovine serum.

A method according to claim 6, characterized in that the fresh sperm of stichopus japonicus is in step (2) of diluting the collected sperm 600 times with an activator, detecting the sperm and collecting the sperm having a vitality of more than 90%, and waiting for use.