A method for hatching of litopenaeus vannamei fertilized eggs

Abstract

The application provides a method for hatching Litopenaeus vannamei fertilized eggs, which comprises S1 parent shrimp selection and cultivation, S2 mating and spawning, S3 in vitro hatching, S4 egg peeling, and S5 egg washing. The method can inhibit the generation of pathogenic bacteria in the hatching process by adding the hatching water of the application in the in vitro hatching process, can reduce the agglomeration and adhesion of egg membranes by using intensive water flow stimulation and controlling the water flow rate, can make the egg membranes brittle and easy to break, can improve the hatching rate of fertilized eggs, and can effectively separate multiple egg membranes without damaging the egg membrane cells, thereby avoiding agglomeration, facilitating hatching, further reducing the risk of infection of fertilized eggs with pathogenic bacteria in the hatching process, and ensuring the full hatching of fertilized eggs. The method for hatching Litopenaeus vannamei fertilized eggs has high hatching efficiency, high survival rate, low malformation rate, short membrane emergence time, and low viral infection rate, and the average hatching rate is 92.5-97.2%, the malformation rate is 0, the survival rate is 94.2-96.6%, and the membrane emergence time is 35-41 days. The prevention and treatment effects of IHHNV, WSSV and TSV are obvious.

Description

Technical Field

[0001] The invention relates to the field of artificial propagation of shrimps in aquaculture, and in particular to a method for hatching fertilized eggs of Litopenaeus vannamei. Background Art

[0002] Litopenaeus vannamei, also known as White Prawn, Whiteleg Shrimp, and Vannamei Shrimp, is native to Ecuador and the Pacific coast of Central and South America. It thrives in tropical, subtropical, warm temperate, and temperate climate zones, from the west coast of the Pacific to the central Gulf of Mexico. Its life cycle is one year. Litopenaeus vannamei is a tropical shrimp that thrives in wide temperature and salinity zones. It is characterized by rapid growth, strong adaptability, low feed requirements, and the ability to be cultured at high densities. However, in recent years, due to eutrophication in coastal waters, a high number of diseases have emerged during the nursery process, which are difficult to control. During artificial propagation, the fertilized egg membrane softens and loses its elasticity, and the embryos are prone to rupture and die. Low hatchability, embryonic malformations, and larval slime disease are common symptoms, seriously impacting the quality, yield, and profitability of larval shrimp production in shrimp hatcheries.

[0003] During in vitro hatching, fertilized eggs of Penaeus vannamei may be infected with a variety of diseases. Common pathogens include bacteria, fungi, viruses, and parasites. These pathogens can cause death, deformities, and growth retardation in the fertilized eggs, thereby affecting the quality and quantity of shrimp seedlings. To reduce the occurrence of these diseases, a series of preventive measures are necessary, such as strengthening water quality management, disinfecting incubators, and using sterile culture media. Currently, the most common viruses involved are infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot disease virus (WSSV), and taura syndrome virus (TSV). Therefore, research on in vitro hatching techniques for Penaeus vannamei fertilized eggs is urgently needed to reduce the spread of viral infections and improve the yield and quality of shrimp seedlings. Summary of the Invention

[0004] In view of this, the present invention provides a method for hatching fertilized eggs of Litopenaeus vannamei to solve the above problems.

[0005] The technical solution of the present invention is achieved as follows: A method for hatching fertilized eggs of Litopenaeus vannamei comprises the following steps:

[0006] S1. Selection and cultivation of broodstock: Select broodstock that are over 8 months old, weigh 20-30g, are healthy, free of disease and injury, and have strong mobility. Stock the broodstock into the pond at a density of 30-40 per cubic meter. 2 , change the water once a day and feed the animals every day;

[0007] S2. Mating and spawning: Transfer sexually mature broodstock to the breeding pond with a male-female ratio of 1-3:1 and a stocking density of 5-20 per cubic meter. 2 , change the water once a day, take out the mature female shrimp, remove its ovaries, disinfect it with iodine tincture with a mass concentration of 0.2-1%, put the ovaries in a sterile container, add sterile artificial seawater with a salt content of 1-3%, and gently rub it with your hands to make the fertilized eggs combine with the sperm to obtain fertilized eggs;

[0008] S3. In vitro incubation: Place the fertilized eggs in a sterile incubator, add incubation water, maintain the water temperature at 26-28°C, pH 7.5-8.0, dissolved oxygen 6-8 mg / L, salinity 25-35‰, and light intensity 2000-3000 LUX, stimulate the eggs with water flow every 20-40 minutes, and repeatedly stimulate for 30-60 minutes before returning them to the incubation tank. Clean out bad eggs regularly and continue incubation until hatching is complete.

[0009] S4, egg peeling: take out the above fertilized eggs, put them into the debonding agent and stir them at a constant speed to debond them. The debonding temperature is 20-30°C and the stirring rate is 120-300 rpm. Stir continuously until the fertilized eggs are debonded.

[0010] S5. Egg washing: Place the debonded fertilized eggs into a phosphate buffered saline solution with a mass concentration of 3-10%, let it stand for 10-30 minutes, rinse, and wait for the shrimp eggs to detach from the membrane.

[0011] Furthermore, the daily water exchange volume in S1 is 10-15% of the pond volume.

[0012] Furthermore, the daily water exchange volume in S2 is 15-35% of the volume of the breeding pond.

[0013] Furthermore, the hatching water in S3 comprises the following raw materials in parts by weight: 40-60 parts of sterile water, 0.3-0.8 parts of triiodothyronine, 0.5-1.0 parts of poly(lactide-glycolide), 1-5 parts of povidone iodine, 0.8-1.2 parts of allicin, 0.2-0.8 parts of butylphthalide, and 0.1-0.5 parts of microbial preparation.

[0014] Furthermore, the microbial preparation is any one or a combination of Saccharomyces cerevisiae, Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus, Alcaligenes faecalis, Aspergillus niger and Azospirillum brasiliensis.

[0015] Furthermore, the hatching density of the fertilized eggs in S3 is 800-1000 eggs / L.

[0016] Furthermore, the stimulation water in S3 is seawater at 17-20°C and with a salinity of 8-10‰.

[0017] Further, the water stimulation flow rate in S3 is 0.4-0.6 mL / s.

[0018] Further, the debonding agent in S4 is a urea-sodium chloride mixed aqueous solution with a mass concentration of 3-5%, and the mass ratio of urea to sodium chloride is 3-8:15.

[0019] Further, the ratio of fertilized eggs to debonding agent in S4 is 1-3:5.

[0020] Compared with the prior art, the method has the following beneficial effects:

[0021] The method has high hatching efficiency, high survival rate, low deformity rate, short membrane emergence time, and low viral infection rate, with an average hatching rate of 92.5-97.2%, a deformity rate of 0, a survival rate of 94.2-96.6%, and a membrane emergence time of 35-41 days. The prevention and control effect of IHHNV, WSSV, and TSV is obvious, and the infection rates in Examples 1-5 are all negative. The method uses sterile water as the main hatching water in the hatching water, and the raw materials are mutually complementary, which has good immunoprotective effect, can inhibit the bacteria generated during hatching, and enhance the antiviral performance of the fertilized eggs during hatching. The method uses intensive water flow stimulation to control the water flow rate, and selects specific seawater to stimulate the fertilized eggs, combines water flow speed and water pressure control repeated stimulation, causes the swelling and shrinking of the egg membrane, reduces the agglomeration and adhesion of the egg membrane, makes the egg membrane brittle and easy to break the membrane, and significantly improves the hatching rate of the fertilized eggs. The selected debonding agent can effectively separate multiple egg membranes and does not easily damage the egg membrane cells, avoids agglomeration, helps hatching, reduces the deformity rate, further reduces the risk of infection of the fertilized eggs with bacteria during hatching, ensures the full hatching of the fertilized eggs, and thus improves the hatching rate of the fertilized eggs. The method plays a role in laying the foundation for comprehensive prevention and control measures for multiple pathogens in disease prevention and control work. DETAILED DESCRIPTION

[0022] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0023] The experimental methods used in the examples of the present application are conventional methods unless otherwise specified.

[0024] The materials, reagents, etc. used in the examples of the present application can be obtained from commercial channels unless otherwise specified.

[0025] Example 1

[0026] A method for hatching fertilized eggs of Litopenaeus vannamei: comprising the following steps:

[0027] S1. Selection and cultivation of broodstock: Select broodstock that are over 8 months old, weigh 20-30g, are healthy, free of disease and injury, and have strong mobility. Stock the broodstock into the pond at a density of 30 per cubic meter. 2 , change the water once a day, the amount of water changed is 10% of the pond volume, and feed is given every day;

[0028] S2. Mating and spawning: Transfer sexually mature broodstock to the breeding pond with a male-female ratio of 1:1 and a stocking density of 5 per cubic meter. 2 , change the water once a day, the water change volume is 15% of the volume of the breeding pond, take out the mature female shrimp, remove their ovaries, disinfect them with iodine tincture with a mass concentration of 0.2%, put the ovaries into a sterile container, add sterile artificial seawater with a salt content of 1%, and gently rub them with your hands to make the fertilized eggs combine with the sperm to obtain fertilized eggs;

[0029] S3, in vitro hatching: the fertilized eggs were placed in a sterile incubator, and hatching water was added at a hatching density of 800 eggs / L. The water temperature was maintained at 26 ° C, the pH value was 7.5, the dissolved oxygen was 6 mg / L, the salinity was 25‰, and the light intensity was 2000 LUX. Water flow stimulation was performed every 20 minutes. The stimulation water was 17 ° C, the salinity was 8‰, and the water stimulation flow rate was 0.4 mL / s. After repeated stimulation for 30 minutes, the eggs were returned to the incubation tank, the bad eggs were regularly cleaned, and the incubation was continued until the hatching was completed. The above-mentioned hatching water comprises the following raw materials in parts by weight: 40 parts of sterile water, 0.3 parts of triiodothyronine, 0.5 parts of poly(lactide-co-glycolide), 1 part of povidone iodine, 0.8 parts of allicin, 0.2 parts of butylphthalide, and 0.1 parts of saccharomyces cerevisiae.

[0030] S4, egg peeling: take out the above fertilized eggs, put them into the detackifying agent and stir them at a uniform speed to detackify, the ratio of fertilized eggs to detackifying agent particles / mL is 1:5, the detackifying agent is a 3% urea-sodium chloride mixed aqueous solution with a mass ratio of urea to sodium chloride of 3:15, the detackifying temperature is 20 ° C, the stirring rate is 120 rpm, and continuous stirring is continued until the fertilized eggs are detackified;

[0031] S5. Egg washing: Place the debonded fertilized eggs into a 3% phosphate buffered saline solution, let it stand for 10 minutes, rinse, and wait for the eggs to detach from the membrane.

[0032] Example 2

[0033] A method for hatching fertilized eggs of Litopenaeus vannamei comprises the following steps:

[0034] S1. Selection and cultivation of broodstock: Select broodstock that are over 8 months old, weigh 20-30g, are healthy, free of disease and injury, and have strong mobility. Stock the broodstock into the pond at a density of 40 per cubic meter. 2, change the water once a day, the amount of water changed is 15% of the pond volume, and feed is given every day;

[0035] S2. Mating and spawning: Transfer sexually mature broodstock to the breeding pond with a male-female ratio of 3:1 and a stocking density of 20 per cubic meter. 2 , change the water once a day, the water change volume is 35% of the volume of the breeding pond, take out the mature female shrimp, remove their ovaries, disinfect them with iodine tincture with a mass concentration of 0.2-1%, put the ovaries in a sterile container, add sterile artificial seawater with a salt content of 3%, and gently rub them with your hands to make the fertilized eggs combine with the sperm to obtain fertilized eggs;

[0036] S3, in vitro hatching: the fertilized eggs were placed in a sterile incubator, and hatching water was added at a hatching density of 1000 eggs / L. The water temperature was maintained at 28 ° C, the pH value was 8.0, the dissolved oxygen was 8 mg / L, the salinity was 35‰, and the light intensity was 3000 LUX. Water flow stimulation was performed every 40 minutes. The stimulation water was 20 ° C, the salinity was 10‰, and the water stimulation flow rate was 0.6 mL / s. After repeated stimulation for 60 minutes, the eggs were returned to the hatching tank, bad eggs were regularly cleaned, and the incubation was continued until the hatching was completed. The above-mentioned hatching water comprises the following raw materials in parts by weight: 60 parts of sterile water, 0.8 parts of triiodothyronine, 1.0 parts of poly(lactide-co-glycolide), 5 parts of povidone iodine, 1.2 parts of allicin, 0.8 parts of butylphthalide, and 0.5 parts of saccharomyces cerevisiae.

[0037] S4, egg peeling: take out the above fertilized eggs, put them into the detackifying agent and stir them at a uniform speed to detackify, the ratio of fertilized eggs to detackifying agent particles / mL is 3:5, the detackifying agent is a 5% urea-sodium chloride mixed aqueous solution with a mass ratio of urea to sodium chloride of 8:15, the detackifying temperature is 30°C, the stirring rate is 300 rpm, and continuous stirring is carried out until the fertilized eggs are detackified;

[0038] S5. Egg washing: Place the debonded fertilized eggs into a 10% phosphate buffered saline solution, let it stand for 30 minutes, rinse, and wait for the eggs to detach from the membrane.

[0039] Example 3

[0040] A method for hatching fertilized eggs of Litopenaeus vannamei comprises the following steps:

[0041] S1. Selection and cultivation of broodstock: Select broodstock that are over 8 months old, weigh 20-30g, are healthy, free of disease and injury, and have strong mobility. Stock the broodstock into the pond at a density of 35 per cubic meter. 2 , change the water once a day, the amount of water changed is 12% of the pond volume, and feed is given every day;

[0042] S2. Mating and spawning: Transfer sexually mature broodstock to the breeding pond with a male-female ratio of 2:1 and a stocking density of 12 per cubic meter.2 , change the water once a day, the water change volume is 25% of the volume of the breeding pond, take out the mature female shrimp, remove their ovaries, disinfect them with iodine tincture with a mass concentration of 0.6%, put the ovaries in a sterile container, add sterile artificial seawater with a salt content of 2%, and gently rub them with your hands to make the fertilized eggs combine with the sperm to obtain fertilized eggs;

[0043] S3, in vitro hatching: the fertilized eggs were placed in a sterile incubator, and hatching water was added to the hatching density of 900 eggs / L. The water temperature was maintained at 27 ° C, the pH value was 7.8, the dissolved oxygen was 7 mg / L, the salinity was 30‰, and the light intensity was 2500 LUX. Water flow stimulation was performed every 30 minutes. The stimulation water was 18 ° C, the salinity was 9‰, and the water stimulation flow rate was 0.5 mL / s. After repeated stimulation for 40 minutes, the eggs were returned to the hatching tank, the bad eggs were regularly cleaned, and the incubation was continued until the hatching was completed. The above-mentioned hatching water comprises the following raw materials in parts by weight: 50 parts of sterile water, 0.5 parts of triiodothyronine, 0.8 parts of poly(lactide-co-glycolide), 3 parts of povidone iodine, 1.0 parts of allicin, 0.5 parts of butylphthalide, and 0.3 parts of Bacillus subtilis.

[0044] S4, egg peeling: take out the above fertilized eggs, put them into the detackifying agent and stir them at a uniform speed to detackify, the ratio of fertilized eggs to detackifying agent particles / mL is 2:5, the detackifying agent is a 4% urea-sodium chloride mixed aqueous solution with a mass ratio of urea to sodium chloride of 5:15, the detackifying temperature is 25 ° C, the stirring rate is 200 rpm, and continuous stirring is continued until the fertilized eggs are detackified;

[0045] S5. Egg washing: Place the debonded fertilized eggs into a 6% phosphate buffered saline solution, let it stand for 20 minutes, rinse, and wait for the eggs to detach from the membrane.

[0046] Example 4

[0047] A method for hatching fertilized eggs of Litopenaeus vannamei comprises the following steps:

[0048] S1. Selection and cultivation of broodstock: Select broodstock that are over 8 months old, weigh 20-30g, are healthy, free of disease and injury, and have strong mobility. Stock the broodstock into the pond at a density of 35 per cubic meter. 2 , change the water once a day, the amount of water changed is 12% of the pond volume, and feed is given every day;

[0049] S2. Mating and spawning: Transfer sexually mature broodstock to the breeding pond with a male-female ratio of 2:1 and a stocking density of 12 per cubic meter. 2, change the water once a day, the water change volume is 25% of the volume of the breeding pond, take out the mature female shrimp, remove their ovaries, disinfect them with iodine tincture with a mass concentration of 0.6%, put the ovaries in a sterile container, add sterile artificial seawater with a salt content of 2%, and gently rub them with your hands to make the fertilized eggs combine with the sperm to obtain fertilized eggs;

[0050] S3. In vitro hatching: The fertilized eggs were placed in a sterile incubator, and hatching water was added at a hatching density of 900 eggs / L. The water temperature was maintained at 27°C, the pH value was 7.8, the dissolved oxygen was 7 mg / L, the salinity was 30‰, and the light intensity was 2500LUX. Water flow stimulation was performed every 30 minutes. The stimulation water was 18°C, the salinity was 9‰, and the water stimulation flow rate was 0.5 mL / s. After repeated stimulation for 40 minutes, the eggs were returned to the hatching tank, bad eggs were regularly cleaned, and the incubation was continued until the hatching was completed. The above-mentioned hatching water comprises the following raw materials in parts by weight: 40 parts of sterile water, 0.3 parts of triiodothyronine, 0.5 parts of poly(lactide-co-glycolide), 1 part of povidone iodine, 0.8 parts of allicin, 0.2 parts of butylphthalide, and 0.1 parts of saccharomyces cerevisiae.

[0051] S4, egg peeling: take out the above fertilized eggs, put them into the detackifying agent and stir them at a uniform speed to detackify, the ratio of fertilized eggs to detackifying agent particles / mL is 2:5, the detackifying agent is a 4% urea-sodium chloride mixed aqueous solution with a mass ratio of urea to sodium chloride of 5:15, the detackifying temperature is 25 ° C, the stirring rate is 200 rpm, and continuous stirring is continued until the fertilized eggs are detackified;

[0052] S5. Egg washing: Place the debonded fertilized eggs into a 6% phosphate buffered saline solution, let it stand for 20 minutes, rinse, and wait for the eggs to detach from the membrane.

[0053] Example 5

[0054] A method for hatching fertilized eggs of Litopenaeus vannamei comprises the following steps:

[0055] S1. Selection and cultivation of broodstock: Select broodstock that are over 8 months old, weigh 20-30g, are healthy, free of disease and injury, and have strong mobility. Stock the broodstock into the pond at a density of 35 per cubic meter. 2 , change the water once a day, the amount of water changed is 12% of the pond volume, and feed is given every day;

[0056] S2. Mating and spawning: Transfer sexually mature broodstock to the breeding pond with a male-female ratio of 2:1 and a stocking density of 12 per cubic meter. 2 , change the water once a day, the water change volume is 25% of the volume of the breeding pond, take out the mature female shrimp, remove their ovaries, disinfect them with iodine tincture with a mass concentration of 0.6%, put the ovaries in a sterile container, add sterile artificial seawater with a salt content of 2%, and gently rub them with your hands to make the fertilized eggs combine with the sperm to obtain fertilized eggs;

[0057] S3, incubation in vitro: the fertilized eggs are placed in a sterile incubator, and incubation water is added, the incubation density is 900 eggs / L, the water temperature is kept at 27℃, the pH value is 7.8, the dissolved oxygen is 7 mg / L, the salinity is 30‰, the light intensity is 2500 LUX, the water flow stimulation is performed every 30 min, the stimulation water is 18℃, the salinity is 9‰, the water stimulation flow rate is 0.5 mL / s, after repeated stimulation for 40 min, the eggs are put back into the incubator, the bad eggs are cleaned regularly, and the incubation is continued until the incubation is completed; the above-mentioned incubation water comprises the following raw materials by weight: 60 parts of sterile water, 0.8 parts of triiodothyronine, 1.0 part of polyglycolide, 5 parts of povidone-iodine, 1.2 parts of allicin, 0.8 parts of butyl phthalide, and 0.5 parts of saccharomyces cerevisiae;

[0058] S4, egg peeling: the above-mentioned fertilized eggs are taken out and put into a debonding agent for uniform stirring debonding, the ratio of the fertilized eggs to the debonding agent is 2:5, the debonding agent is a urea-sodium chloride mixed aqueous solution with a mass concentration of 4%, the mass ratio of urea to sodium chloride is 5:15, the debonding temperature is 25℃, and the stirring rate is 200 rpm, and the fertilized eggs are continuously stirred until debonding;

[0059] S5, egg washing: the above-mentioned debonded fertilized eggs are put into a phosphate buffered saline solution with a mass concentration of 6%, and are placed for 20 min for rinsing, and the shrimp eggs are peeled.

[0060] Comparative Example 1

[0061] The difference between this comparative example and Example 3 is that the incubation water in S3 is only sterile water.

[0062] Comparative Example 2

[0063] The difference between this comparative example and Example 3 is that the water flow stimulation is not performed in S3.

[0064] Comparative Example 3

[0065] The difference between this comparative example and Example 3 is that the debonding agent in S4 is replaced with an equal amount of mud.

[0066] Test Example:

[0067] The test area is selected in a breeding farm in Tiantou Village, Wengtian Town, Wenchang City, Hainan Province, 400 Penaeus vannamei parent shrimps are randomly selected, 8 test areas are set, each area has 50 shrimps, the fertilized eggs of Penaeus vannamei are hatched by the fertilized egg hatching methods of Examples 1-5 and Comparative Examples 1-3, the conditions in the process of artificial hatching of the examples and the comparative examples are observed and recorded, and the clutch weight, the average hatching rate, the peeling time and the infection rate are counted respectively.

[0068] Hatching rate (%) = average number of hatched eggs / average number of fertilized eggs × 100%;

[0069] Deformity rate (%) = average number of deformed embryos / average number of fertilized eggs × 100%;

[0070] Survival rate (%) = number of surviving seedlings / total number of emerging seedlings × 100%;

[0071] Here are the results:

[0072] Number of eggs held (egg) Average hatching rate% Deformity rate (%) Survival rate% Film-forming time (d) Test Area 1 <![CDATA[14.7×10 4 ]]> 96.2 0 94.2 36 Test Area 2 <![CDATA[14.5×10 4 ]]> 95.0 0 95.8 36 Test Area 3 <![CDATA[14.8×10 4 ]]> 97.2 0 96.6 35 Test Area 4 <![CDATA[14.2×10 4 ]]> 94.7 0 95.1 41 Test Area 5 14.4 x 10 4 ]] 92.5 0 94.2 39 Test Area 6 <![CDATA[14.6×10 4 ]]> 81.9 0 85.6 42 Test Area 7 <![CDATA[14.7×10 4 ]]> 82.6 0.02 87.2 41 Test Area 8 <![CDATA[14.3×10 4 ]]> 83.4 0.04 835.6 43

[0073] Viral infections included infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot disease virus (WSSV), and taura syndrome virus (TSV). The infection rates in the above-mentioned test areas and the blank area (natural hatching) were determined by PCR detection. The following is the infection rate:

[0074] Infection rate = (number of positive tests / number of tests) × 100%

[0075]

[0076]

[0077] Note: “+” indicates a positive result, “-” indicates a negative result

[0078] From the above results, it can be seen that the incubation method of the present invention has high hatching efficiency, high survival rate, low deformity rate, short hatching time, and low virus infection rate. Among them, the prevention and control effects of IHHNV, WSSV, and TSV are obvious. Compared with natural incubation, the infection rates in test areas 1-5 are all negative, and test areas 6-8 have different degrees of infection rates. Test area 6 corresponds to proportion 1. This shows that the present invention uses sterile water as the main incubation water in the incubation water, supplemented by the synergistic cooperation between the various raw materials, has good immune protection effect, can inhibit the pathogens generated during the incubation process, and enhance the antiviral performance of the fertilized eggs during the incubation process;

[0079] Test area 7 corresponds to corresponding proportion 2. The present invention adopts intensive water flow stimulation, controls the water flow rate, and selects specific seawater for water flow stimulation on the fertilized eggs. Combined with the water flow rate and water pressure to control the repeated stimulation, the egg membrane expands and contracts, reducing the clumping and adhesion of the egg membrane, making the egg membrane brittle and easy to rupture, and significantly improving the hatching rate of the fertilized eggs;

[0080] Test area 8 corresponds to proportion 3, indicating that the debonding agent selected by the present invention can effectively separate multiple egg membranes and is not easy to damage the egg membrane cells, avoid agglomeration, help hatching, reduce the deformity rate, and further reduce the risk of fertilized eggs being infected with pathogens during the hatching process, ensuring that the fertilized eggs are fully hatched, thereby improving the hatching rate of the fertilized eggs.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for hatching fertilized eggs of Litopenaeus vannamei, characterized in that: The following steps are involved: S1. Selection and cultivation of broodstock: Select broodstock that are over 8 months old, weigh 20-30g, are healthy, free of disease and injury, and have strong mobility. Stock the broodstock into the pond at a density of 30-40 per cubic meter. 2 , change the water once a day and feed the animals every day; S2. Mating and spawning: Transfer sexually mature broodstock to the breeding pond with a male-female ratio of 1-3:1 and a stocking density of 5-20 per cubic meter. 2 , change the water once a day, take out the mature female shrimp, remove its ovaries, disinfect it with iodine tincture with a mass concentration of 0.2-1%, put the ovaries in a sterile container, add sterile artificial seawater with a salt content of 1-3%, and gently rub it with your hands to make the fertilized eggs combine with the sperm to obtain fertilized eggs; S3. In vitro incubation: Place the fertilized eggs in a sterile incubator, add incubation water, maintain the water temperature at 26-28°C, pH 7.5-8.0, dissolved oxygen 6-8 mg / L, salinity 25-35‰, and light intensity 2000-3000 LUX, stimulate the eggs with water flow every 20-40 minutes, and repeatedly stimulate for 30-60 minutes before returning them to the incubation tank. Clean out bad eggs regularly and continue incubation until hatching is complete. S4, egg peeling: take out the above fertilized eggs, put them into the debonding agent and stir them at a constant speed to debond them. The debonding temperature is 20-30°C and the stirring rate is 120-300 rpm. Stir continuously until the fertilized eggs are debonded. S5, egg washing: placing the above-mentioned debonded fertilized eggs into a phosphate buffered saline solution containing a mass concentration of 3-10%, standing for 10-30 minutes, rinsing, and waiting for the shrimp eggs to detach from the membrane; The hatching water in S3 comprises the following raw materials in parts by weight: 40-60 parts of sterile water, 0.3-0.8 parts of triiodothyronine, 0.5-1.0 parts of poly(lactide-glycolide), 1-5 parts of povidone iodine, 0.8-1.2 parts of allicin, 0.2-0.8 parts of butylphthalide, and 0.1-0.5 parts of a microbial preparation; The microbial preparation is any one or a combination of several of the following: saccharomyces cerevisiae, bacillus subtilis, lactobacillus, alcaligenes faecalis, aspergillus niger and azospirillum brasiliensis.

2. The method for hatching fertilized eggs of Litopenaeus vannamei according to claim 1, wherein: The amount of water exchanged per day in S1 was 10-15% of the pond volume.

3. The method for hatching fertilized eggs of Litopenaeus vannamei according to claim 1, wherein: In S2, the daily water exchange rate is 15-35% of the breeding tank volume.

4. The method for hatching fertilized eggs of Litopenaeus vannamei according to claim 1, wherein: The hatching density of the fertilized eggs in S3 is 800-1000 eggs / L.

5. The method for hatching fertilized eggs of Litopenaeus vannamei according to claim 1, wherein: The stimulation water in S3 is seawater at 17-20°C and with a salinity of 8-10‰.

6. The method for hatching fertilized eggs of Litopenaeus vannamei according to claim 1, wherein: The water stimulation flow rate in S3 is 0.4-0.6 mL / s.

7. The method for hatching fertilized eggs of Litopenaeus vannamei according to claim 1, wherein: The detackifying agent in S4 is a urea-sodium chloride mixed aqueous solution with a mass concentration of 3-5%, and the mass ratio of urea to sodium chloride is 3-8:

15.

8. The method for hatching fertilized eggs of Litopenaeus vannamei according to claim 1, wherein: The ratio of fertilized eggs to debonding agent in the S4 is 1-3:5.

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