A low-temperature freezing storage tube and a low-temperature freezing storage method and application of grouper

Abstract

The present application belongs to the field of ultra-low temperature cryopreservation technology, and in particular, relates to a cryopreservation tube and a method and application of cryopreservation of grouper. The bottom of the cryopreservation tube of the present application is concave upward to form a hollow tube that exceeds the top of the tube, and the two ends of the hollow tube are connected. Since there is no blockage at both ends of the hollow tube, the airflow is smooth, and the hollow tube also increases the surface area of ​​the hollow cryopreservation tube, thereby accelerating the cooling / heating rate, improving the uniformity of cooling / heating, and improving the freezing effect. The sperm motility rate after thawing is high, thereby solving the technical problem of poor sperm freezing effect of cryopreservation tubes in the prior art.

Description

Technical Field

[0001] The present application relates to the field of ultra-low temperature cryopreservation technology, and in particular to a cryopreservation tube and a grouper cryopreservation method and application. Background Art

[0002] Ultra-low temperature cryopreservation is an effective method for sperm preservation in vitro. The preservation method mainly involves mixing sperm with diluent and protective solution, cooling through a series of procedures, and finally storing it in liquid nitrogen. Ultra-low temperature cryopreservation technology is not only conducive to the preservation and utilization of genetic resources, but also plays an important role in the aquaculture industry.

[0003] Grouper is a precious marine fish with delicious meat, rich nutrition, high economic value, important production application value, and is an important marine farmed fish in the south. However, it takes 4-5 years for male grouper to reach sexual maturity, and there is a problem of asynchronous sexual maturity between female and male broodstock, which is not conducive to the stable supply of seedlings and seriously restricts the development of grouper farming. Ultra-low temperature freezing preservation technology is an effective means to solve the asynchronous sexual maturity of female and male broodstock of grouper. The semen of sexually mature male grouper is collected and ultra-low temperature frozen for long-term storage, and can be used at any time according to production needs, overcoming the problems of insufficient male fish and asynchronous sexual maturity of males and females, and ensuring the supply of seedlings.

[0004] The cryopreservation containers used in ultra-low temperature cryopreservation technology are mainly wheat straws and cryotubes; wheat straws have better effects in sperm cryopreservation, but due to their complex structure and small capacity, it is difficult to freeze large amounts of semen; cryotubes have large volumes, poor cooling effects, and poor semen cryopreservation effects. Summary of the invention

[0005] In view of this, the present application provides a low-temperature freezing preservation tube and a grouper low-temperature freezing preservation method and application, which are used to solve the technical problem that the freezing effect of sperm in the cryopreservation tube in the prior art is poor.

[0006] The first aspect of the present application provides a low-temperature freezing storage tube, comprising a tube body and a tube cover;

[0007] The bottom of the tube body is concave upward to form a hollow tube that exceeds the top of the tube body;

[0008] The two ends of the hollow tube are connected;

[0009] The tube cover is provided with a tube cover through hole corresponding to the hollow tube;

[0010] The pipe cover and the pipe body are threadedly connected.

[0011] Preferably, the low-temperature freezing storage tube further comprises a rubber sealing ring;

[0012] The rubber sealing ring is located at the top of the tube body;

[0013] The outer diameter of the rubber sealing ring is the same as the outer diameter of the tube body.

[0014] It should be noted that the sealing property of the low-temperature freezing storage tube can be improved by the rubber sealing ring.

[0015] Preferably, the outer diameter of the tube body is 1 to 1.5 cm, the thickness of the tube body is 0.05 to 0.1 cm, and the height of the tube body is 3.5 to 4 cm;

[0016] The outer diameter of the hollow tube is 0.2-0.4 cm, and the height is 4.2-4.8 cm;

[0017] The diameter of the through hole of the tube cover is 0.2-0.4 cm.

[0018] The second aspect of the present application provides a method for ultra-low temperature freezing and preservation of grouper, using the above-mentioned hollow cryopreservation tube as a freezing container for ultra-low temperature freezing and preservation, comprising the following steps:

[0019] Step S1. mixing the grouper semen with a cryoprotectant, and then placing the mixture into the cryopreservation tube and sealing the tube;

[0020] Step S2. pre-freeze the low-temperature cryopreservation tube containing the grouper semen and the cryoprotectant above the liquid nitrogen surface;

[0021] Step S3. placing the pre-frozen cryopreservation tube containing the grouper semen and the cryoprotectant into liquid nitrogen for ultra-low temperature cryopreservation;

[0022] The cryoprotectant includes antifreeze and protection liquid.

[0023] Preferably, in step S1, the antifreeze agent comprises dimethyl sulfoxide;

[0024] The protective solution includes glucose and sodium chloride.

[0025] Preferably, in step S1, the concentration of glucose in the protective solution is 0.3 mol / L, and the concentration of sodium chloride is 0.1 mol / L;

[0026] The volume ratio of dimethyl sulfoxide to the protective solution in the cryoprotectant is 1:10.

[0027] Preferably, in step S1, the volume ratio of the grouper semen to the cryoprotectant is 2:1.

[0028] Preferably, in step S2, the pre-freezing time is 10 minutes and the height is 3 to 7 cm.

[0029] Preferably, in S1, the total volume of the grouper semen and the cryoprotectant is 700 μL to 1800 μL.

[0030] The third aspect of the present application provides the application of the grouper ultra-low temperature freezing preservation method in grouper farming, and the application specifically comprises the steps of:

[0031] Step S4. taking out the cryopreservation tube containing the grouper semen and the cryoprotectant from the liquid nitrogen and thawing it;

[0032] Step S5: mixing the thawed grouper semen and grouper eggs in clean water and stirring until the grouper eggs are fertilized.

[0033] Preferably, in step S4, the thawing is performed by placing the hollow cryopreservation tube containing the grouper semen and the cryoprotectant in a 40° C. water bath for 2 minutes.

[0034] In summary, the present application provides a low-temperature freezing preservation tube and a method and application for ultra-low temperature freezing preservation of grouper. The low-temperature freezing preservation tube includes a tube body and a tube cover. The bottom of the tube body is concave upward to form a hollow tube that exceeds the top of the tube body and the two ends of the hollow tube are connected. The tube cover is provided with a through hole corresponding to the hollow tube. The tube cover and the tube body are threadedly connected. Since there is no blockage at both ends of the hollow tube, the airflow flows smoothly, and the hollow tube also increases the surface area of ​​the hollow freezing preservation tube, thereby accelerating the cooling / heating rate and improving the uniformity of cooling / heating. When freezing a large volume of semen, for example, when the freezing volume is greater than 900 μL, the hollow tube improves the smoothness of the airflow and the specific surface area, thereby preventing hot air from being accumulated in the hollow tubular structure and causing uneven cooling / heating, thereby improving the freezing effect. The sperm motility after thawing is high, which is used to solve the technical problem of poor sperm freezing effect of freezing tubes in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 A schematic diagram of a tube body, a tube cover and a rubber sealing ring in a low-temperature freezing storage tube provided in an embodiment of the present application;

[0037] Figure 2 A schematic cross-sectional view of a cryogenic storage tube provided in an embodiment of the present application;

[0038] Figure 3A comparison chart of the low-temperature freezing storage effects of different protective solutions provided in Example 3 of the present application;

[0039] Figure 4 A comparison chart of the low-temperature freezing storage effects of different antifreeze solutions provided in Example 3 of the present application;

[0040] Figure 5 A comparison chart of low-temperature freezing storage effects of different antifreeze agent dilution ratios provided in Example 3 of the present application;

[0041] Figure 6 A comparison chart of low-temperature freezing preservation effects of different cryoprotectant dilution ratios provided in Example 3 of the present application;

[0042] Figure 7 A comparison diagram of the cryopreservation effects of the cryopreservation tube provided in Example 3 of the present application at different heights from the liquid nitrogen surface;

[0043] Figure 8 A comparison chart of the low-temperature freezing storage effects of the low-temperature freezing storage tube described in Example 1 provided in the embodiments of the present application and a conventional freezing storage tube without a hollow tube. DETAILED DESCRIPTION

[0044] The present application provides a low-temperature freezing preservation tube and a method and application of ultra-low temperature freezing preservation of grouper, which are used to solve the technical problem that the freezing effect of sperm in the cryopreservation tube in the prior art is poor.

[0045] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0046] Example 1

[0047] This embodiment provides a low-temperature freezing storage tube, the structure of which is as follows Figure 1-2 As shown, the low-temperature freezing storage tube includes a tube body and a tube cover; the bottom of the tube body is concave upward to form a hollow tube that exceeds the top of the tube body; both ends of the hollow tube are connected, and the tube cover is provided with a tube cover through hole corresponding to the hollow tube, and the tube cover and the tube body are threadedly connected, and the threaded connection method is that the tube cover is provided with an external thread, and the top of the tube body is provided with a corresponding internal thread, so that the tube cover and the tube body are threadedly connected.

[0048] As a further improvement, the low-temperature freezing storage tube also includes a rubber sealing ring, which is located at the top of the tube body, and the outer diameter of the rubber sealing ring is the same as the outer diameter of the tube body, so that after the tube cover and the tube body are threadedly connected, the threaded connection position of the tube cover and the tube body can be sealed, thereby improving the sealing of the low-temperature freezing storage tube.

[0049] As for the size of the low-temperature freezing preservation tube, the outer diameter of the low-temperature freezing preservation tube body can be 1-1.5 cm, the thickness of the tube body can be 0.05-0.1 cm, the height of the tube body can be 3.5-4 cm, the outer diameter of the hollow tube can be 0.2-0.4 cm, the height can be 4.2-4.8 cm, the diameter of the through hole in the tube cover can be 0.2-0.4 cm, and the outer diameter of the hollow tube is the same as the diameter of the through hole in the tube cover. As the preferred size of the low-temperature freezing preservation tube, the outer diameter of the low-temperature freezing preservation tube body is 1.25 cm, the thickness of the tube body is 0.08 cm, the height of the tube body is 4 cm, the outer diameter of the hollow tube is 0.33 cm, the height can be 4.6 cm, and the diameter of the through hole in the tube cover is 0.33 cm. The low-temperature freezing preservation tube of this preferred size can store 1.8 mL of semen, ensuring a large semen capacity, and can be adapted to common freezing boxes on the market.

[0050] Example 2

[0051] This embodiment provides a method for cryopreservation of grouper using the cryopreservation tube described in Example 1, the method comprising the steps of collecting grouper semen, loading the cryopreservation tube with a cryoprotectant, pre-freezing, and ultra-low temperature freezing.

[0052] The steps of collecting grouper semen include anesthetizing sexually mature male grouper with eugenol, wiping off seawater on the surface of the fish body, lightly pressing the abdomen to squeeze out semen, removing the initially squeezed semen, and collecting clean semen in a test tube.

[0053] The step of filling the cryoprotectant low-temperature freezing storage tube comprises mixing the cryoprotectant with the grouper semen in the test tube and filling the mixture into the low-temperature freezing storage tube described in Example 1.

[0054] The pre-freezing step includes placing a low-temperature freezing storage tube containing grouper semen and a cryoprotectant at a certain height above the liquid nitrogen surface for 10 minutes for pre-freezing.

[0055] The ultra-low temperature cryopreservation step comprises putting the pre-frozen low temperature cryopreservation tube containing the grouper semen and the cryoprotectant into liquid nitrogen for storage.

[0056] The cryoprotectant is a mixture of a protective liquid and an antifreeze agent, which can minimize the damage to grouper semen during low-temperature freezing and storage. The protective liquid can be any one of TS19, sucrose, glucose, 0.2mol / L glucose + 0.1mol / L sodium chloride, 0.25mol / L glucose + 0.1mol / L sodium chloride, 0.3mol / L glucose + 0.1mol / L sodium chloride, 0.35mol / L glucose + 0.1mol / L sodium chloride, and 0.4mol / L glucose + 0.1mol / L sodium chloride.

[0057] The antifreeze agent includes any one of dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycine (GLY), dimethylformamide (DMF), dimethylacetamide (DMA), and methanol (ME).

[0058] The volume ratio of the protective liquid to the antifreeze agent in the cryoprotectant can be selected to be 100:5-25.

[0059] The volume ratio of cryoprotectant to grouper semen can be selected from 1:2 to 19:1.

[0060] During the pre-freezing process, the height of the low-temperature freezing storage tube containing grouper semen and cryoprotectant above the liquid nitrogen surface can be selected to be no greater than 9 cm.

[0061] Example 3

[0062] In view of the differences in physiological characteristics of different fish, the cryoprotectants of other fish are not ideal for preserving sperm of grouper. In this example, the cryoprotectant in the low-temperature freezing preservation method for grouper provided in Example 2 is improved.

[0063] The improvements include improvements in the protective solution in the cryoprotectant, improvements in the antifreeze agent in the cryoprotectant, improvements in the dilution ratio of the antifreeze agent in the cryoprotectant, and improvements in the dilution ratio of the cryoprotectant.

[0064] The improvement test of the protective solution in the cryoprotectant includes adding 10% dimethyl sulfoxide to any one of TS19, sucrose, glucose, 0.2mol / L glucose + 0.1mol / L sodium chloride, 0.25mol / L glucose + 0.1mol / L sodium chloride, 0.3mol / L glucose + 0.1mol / L sodium chloride, 0.35mol / L glucose + 0.1mol / L sodium chloride, and 0.4mol / L glucose + 0.1mol / L sodium chloride, respectively, and then mixing them in the low-temperature freezing storage tube described in Example 1. The dilution ratio of grouper semen to the protective agent is 1:1. The freezing conditions are to fumigate on a floating plate 7 cm above the liquid nitrogen surface for 10 min, then put it into liquid nitrogen for storage, and then put it in a 40°C water bath for 2 minutes. The sperm motility in the thawed grouper semen is analyzed, and the results are as follows Figure 3 As shown, the results showed that 0.3mol / L glucose + 0.1mol / L sodium chloride was the best grouper sperm protection solution.

[0065] The improvement of the antifreeze in the cryoprotectant includes mixing 0.3 mol / L glucose + 0.1 mol / L sodium chloride protective solution with dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycine (GLY), N-formyl dimethylamine dimethylformamide (DMF), dimethylacetamide (DMA), methanol (ME), propylene glycol and dimethyl sulfoxide in a volume ratio of 1:1, propylene glycol and dimethyl sulfoxide in a volume ratio of 1:1, propylene glycol and dimethyl sulfoxide mixed antifreeze in a volume ratio of 1:1, ethylene glycol and dimethyl sulfoxide mixed antifreeze in a volume ratio of 1:1, glycine and dimethyl sulfoxide mixed antifreeze in a volume ratio of 1:1, and a volume ratio of Any one of a mixture of methanol and dimethyl sulfoxide in a volume ratio of 1:1, a mixture of propylene glycol, ethylene glycol and dimethyl sulfoxide in a volume ratio of 1:1:2, a mixture of propylene glycol, methanol and dimethyl sulfoxide in a volume ratio of 1:1:2, and a mixture of propylene glycol, methanol, dimethyl sulfoxide, glycine and ethylene glycol in a volume ratio of 1:1:1:1:1 is mixed and loaded into the low-temperature freezing storage tube described in Example 1, the dilution ratio of sperm to protective agent is 1:1, the freezing condition is to place it flat on a floating plate 7 cm above the liquid nitrogen surface and fumigate it for 10 minutes and store it in liquid nitrogen, the thawing condition is to bathe it in 40°C water for 2 minutes, and the sperm motility in the thawed grouper semen is analyzed, and the results are as follows Figure 4 As shown, the results showed that dimethyl sulfoxide was the best antifreeze agent for grouper semen.

[0066] The improvement of the dilution ratio of the antifreeze agent in the cryoprotectant includes mixing 5 parts by volume, 10 parts by volume, 15 parts by volume, 20 parts by volume and 25 parts by volume of dimethyl sulfoxide with 100 parts by volume of 0.3 mol / L glucose + 0.1 mol / L sodium chloride protective solution respectively and then loading them into the low-temperature freezing storage tube described in Example 1, the dilution ratio of sperm to protective agent is 1:1, the freezing condition is to fumigate on a floating plate 7 cm above the liquid nitrogen surface for 10 minutes, the thawing condition is to bathe in 40°C water for 2 minutes, and the sperm motility in the thawed grouper semen is analyzed, and the results are as follows Figure 5 As shown, the results showed that the volume ratio of dimethyl sulfoxide to protective solution was 1:10, that is, dimethyl sulfoxide with a volume fraction of 10% was the best antifreeze agent for grouper semen.

[0067] The improvement of the cryoprotectant dilution ratio includes mixing the cryoprotectant with grouper semen and then placing them into the low-temperature freezing storage tube described in Example 1, setting the freezing height from the liquid nitrogen surface to 7 cm, steaming time to 10 min, thawing conditions to 40°C water bath for 2 minutes, and the dilution ratio of grouper semen to cryoprotectant is 2:1, 1:1, 1:3, 1:5, 1:7, 1:9 or 1:19. The sperm motility in the thawed grouper semen is analyzed, and the results are as follows: Figure 6 As shown, the results showed that when the dilution ratio of the cryoprotectant was 50%, that is, when the ratio of grouper semen to the cryoprotectant was 2:1, the cryoprotectant had the best effect on the low-temperature preservation of grouper semen.

[0068] It should be noted that there are large physiological differences between freshwater fish and marine fish, and there are also physiological characteristics between marine fish. In addition, the physiological characteristics of hermaphrodites of grouper are very different from those of conventional marine fish, which makes the physiological differences between grouper semen and conventional fish huge. So far, there has been no research report on ultra-low temperature cryopreservation of grouper sperm. Moreover, since the physiological characteristics of sperm of different species of fish vary greatly, the effect of preserving grouper sperm using cryoprotectants and methods reported for other fish is not ideal. Example 3 of the present application has been improved through experiments to obtain the best antifreeze agent dimethyl sulfoxide and protective solution glucose and sodium chloride.

[0069] Example 4

[0070] This embodiment improves the freezing preservation method of the grouper low-temperature freezing preservation method provided in Example 3.

[0071] The invention comprises improving the height of the low-temperature freezing storage tube from the liquid nitrogen freezing in the pre-freezing step.

[0072] The improvement of the freezing height of the cryopreservation tube from the liquid nitrogen includes loading 0.3 mol / L glucose + 0.1 mol / L sodium chloride protective solution, 10% dimethyl sulfoxide and grouper semen into the cryopreservation tube described in Example 1, the dilution ratio of grouper semen to cryoprotectant is 2:1, the freezing height from the liquid nitrogen surface is set to 0, 1, 3, 5, 7, 9 cm, the fumigation time is 10 min, and the thawing condition is 40°C water bath for 2 minutes. The results are as follows Figure 7 As shown, the results showed that when the freezing height from the liquid nitrogen surface was set to 3, 5, and 7 cm, the cryoprotectant had a better low-temperature preservation effect on grouper semen, and the height with the best low-temperature preservation effect was 3 cm.

[0073] Example 5

[0074] This example conducts a performance test on the low-temperature freezing storage tube provided in Example 1 and a conventional freezing storage tube without a hollow tube.

[0075] The experiment included placing 0.3 mol / L glucose + 0.1 mol / L sodium chloride protective solution, 10% dimethyl sulfoxide and 300-1800 microliters of grouper semen into the low-temperature cryopreservation tube described in Example 1 and a conventional cryopreservation tube without a hollow tube. The dilution ratio of grouper semen to cryoprotectant was 2:1, the freezing height from the liquid nitrogen surface was set to 7 cm, the fumigation time was 10 min, and the thawing condition was 40°C water bath for 2 minutes. The results are as follows Figure 8 As shown, the results show that when the cryopreservation volume is less than 700 microliters, there is no significant difference in the cryopreservation effect between the low-temperature freezing storage tube described in Example 1 and the conventional cryopreservation tube without a hollow tube; when the cryopreservation volume is greater than 900 microliters, the sperm motility of the conventional cryopreservation tube without a hollow tube will decrease significantly, while the sperm motility of the low-temperature freezing storage tube described in Example 1 has no obvious change, which shows that since there is no blockage at both ends of the hollow tube, the airflow flows smoothly, and the hollow tube also increases the surface area of ​​the hollow cryopreservation tube, thereby accelerating the cooling / heating rate and improving the uniformity of cooling / heating. When freezing a large volume of semen, for example, when the cryopreservation volume is greater than 900 μL, the hollow tube improves the smoothness of the airflow and the specific surface area, prevents the hot air from being accumulated in the hollow tubular structure, resulting in uneven cooling / heating, thereby improving the cryopreservation effect, and the sperm motility after thawing is high, which is used to solve the technical problem of poor sperm freezing effect of cryopreservation tubes in the prior art.

[0076] Example 6

[0077] The present embodiment is an application of the ultra-low temperature freezing preservation method for grouper in grouper farming, and the application specifically includes the steps of: first, taking out the low-temperature freezing preservation tube containing grouper semen and cryoprotectant from liquid nitrogen for thawing; then mixing the thawed grouper semen and grouper eggs in clean water and stirring until the grouper eggs are fertilized, wherein the thawing is to place the hollow freezing preservation tube containing grouper semen and cryoprotectant in a 40°C water bath for 2 minutes, and the grouper eggs are placed incubated at a suitable temperature after in vitro fertilization.

[0078] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A low-temperature freezing storage tube, characterized in that: Including tube body and tube cover; The bottom of the tube body is concave upward to form a hollow tube that exceeds the top of the tube body; The two ends of the hollow tube are connected; The tube cover is provided with a tube cover through hole corresponding to the hollow tube; The tube cover is threadedly connected to the tube body. The tube cover is provided with an external thread, and the top of the tube body is provided with a corresponding internal thread, so that the tube cover and the tube body are threadedly connected.

2. A low-temperature freezing storage tube according to claim 1, characterized in that: The low-temperature freezing storage tube also includes a rubber sealing ring; The rubber sealing ring is located at the top of the tube body; The outer diameter of the rubber sealing ring is the same as the outer diameter of the tube body.

3. A low-temperature freezing storage tube according to claim 1, characterized in that: The outer diameter of the tube body is 1 to 1.5 cm, the thickness of the tube body is 0.05 to 0.1 cm, and the height of the tube body is 3.5 to 4 cm; The outer diameter of the hollow tube is 0.2-0.4 cm, and the height is 4.2-4.8 cm; The diameter of the through hole of the tube cover is 0.2-0.4 cm.

4. A method for ultra-low temperature freezing and preservation of grouper, characterized in that: Includes steps: Step S1. After mixing the grouper semen with a cryoprotectant, the mixture is placed in a cryogenic storage tube as claimed in any one of claims 1 to 3 and sealed; Step S2. pre-freeze the low-temperature cryopreservation tube containing the grouper semen and the cryoprotectant above the liquid nitrogen surface; Step S3. placing the pre-frozen cryopreservation tube containing the grouper semen and the cryoprotectant into liquid nitrogen for ultra-low temperature cryopreservation; The cryoprotectant includes antifreeze and protection liquid.

5. A method for ultra-low temperature freezing and preservation of grouper according to claim 4, characterized in that: In step S1, the antifreeze agent includes dimethyl sulfoxide; The protective solution includes glucose and sodium chloride.

6. The method for ultra-low temperature freezing and preservation of grouper according to claim 5, characterized in that: In step S1, the concentration of glucose in the protective solution is 0.3 mol / L, and the concentration of sodium chloride is 0.1 mol / L; The volume ratio of dimethyl sulfoxide to the protective solution in the cryoprotectant is 1:

10.

7. The method for ultra-low temperature freezing and preservation of grouper according to claim 4, characterized in that: In step S1, the volume ratio of the grouper semen to the cryoprotectant is 2:

1.

8. The method for ultra-low temperature freezing and preservation of grouper according to claim 4, characterized in that: In step S2, the pre-freezing time is 10 minutes and the height is 3 to 7 cm.

9. The method for ultra-low temperature freezing and preservation of grouper according to claim 4, characterized in that: In S1, the total volume of the grouper semen and the cryoprotectant is 700 μL to 1800 μL.

10. Application of the method for ultra-low temperature freezing preservation of grouper according to any one of claims 4 to 9 in grouper breeding, characterized in that: The application specifically comprises the steps of: Step S4. taking out the cryopreservation tube containing the grouper semen and the cryoprotectant from the liquid nitrogen and thawing it; Step S5: mixing the thawed grouper semen and grouper eggs in clean water and stirring until the grouper eggs are fertilized.

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