A method for reducing harmful vibrio in shrimp fry culture

Abstract

The application provides a shrimp fry breeding method for reducing harmful vibrio, which comprises the steps of fry pond preparation, water quality treatment, juvenile cultivation, larva cultivation, shrimp fry intensive cultivation and ponding, etc. Special adsorption substrate and phytoplankton inoculation are adopted, the adsorption substrate comprises a loaded microbial adsorbent and other components, the loaded microbial adsorbent is formed by loading microorganisms on an adsorption material; ozone treatment is combined to ensure excellent water quality. Environmental parameters are controlled and feed is fed according to different periods of the shrimp fry. The density and environment are adjusted for larva cultivation, and artificial feed is mainly used. The density is controlled, the water quality is adjusted, the water flow stimulation is increased and the feed is fed during the shrimp fry intensive cultivation, and natural ingredients such as antibacterial peptides are newly added in the feed to enhance the immunity of the shrimp fry. In particular, the water flow stimulation is performed by using an enzyme-based water treatment agent to further inhibit the harmful vibrio. The application is simple in operation, effectively reduces the influence of the vibrio on the shrimp fry, improves the survival rate of the shrimp fry, and provides a strong guarantee for the healthy cultivation of the shrimp fry.

Description

Technical Field

[0001] This invention relates to the field of shrimp larvae breeding technology, and in particular to a method for breeding shrimp larvae that reduces harmful Vibrio bacteria. Background Technology

[0002] In the process of shrimp larvae rearing, the proliferation and control of harmful Vibrio bacteria has always been a key factor affecting the success rate of shrimp farming. Traditional shrimp larvae rearing methods often face many challenges, such as rapid water quality deterioration, difficulty in effectively inhibiting harmful microorganisms (especially Vibrio), slow shrimp larvae growth, and low survival rates. These shortcomings not only increase farming costs but also seriously affect the quality of shrimp larvae and their market competitiveness. In traditional shrimp larvae rearing ponds, water quality management often relies on simple water changes and disinfection measures, making it difficult to accurately control key parameters such as dissolved oxygen, pH, and ammonia nitrogen levels. Especially under high-temperature and high-density farming conditions, water quality deteriorates rapidly, and harmful Vibrio bacteria and other microorganisms proliferate in large numbers, seriously threatening the health of shrimp larvae.

[0003] The main causes of Vibrio outbreaks are nothing more than three factors: water and bottom sediment environment, immunity, and the outbreak of pathogens. Bottom sediment is an important carrier of microorganisms and organic matter in shrimp larvae rearing ponds, but traditional methods often neglect effective treatment of sediment. The feces, feed, organic matter, and harmful microorganisms accumulated in the bottom sediment easily become a breeding ground for pathogens, further exacerbating water quality deterioration. In traditional shrimp larvae rearing, feed feeding often lacks scientific rigor and specificity, resulting in low feed utilization and easy water pollution. At the same time, the unbalanced nutritional composition of the feed fails to meet the needs of rapid shrimp larvae growth, affecting their immunity and survival rate. Faced with the threat of harmful Vibrio and other microorganisms, traditional methods often rely on chemical disinfectants, but these methods often have significant side effects and unstable effectiveness. Furthermore, the lack of targeted biological control measures fails to fundamentally solve the problem of harmful microbial proliferation. Indiscriminate disinfection disrupts the balance of pond flora and algae in the water, leading to rapid Vibrio reproduction; disinfection actually provides Vibrio with excellent opportunities for rapid growth.

[0004] Common types of Vibrio in shrimp larvae include Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus, and Vibrio cholerae. Among them, Vibrio parahaemolyticus is prone to causing gastrointestinal infections in shrimp and is the main pathogen of early mortality syndrome (EMS). Vibrio infections can easily lead to empty intestines and stomachs, red body, and sudden death in Pacific white shrimp, with mortality rates reaching over 90%. Vibrio harveyi can reduce shrimp activity, causing them to float on the bottom and mostly tumble with the water flow. They may stop feeding or eat less, and in the early stages of the disease, they may not glow, but they will fluoresce when they are dying. Vibrio anguillarum can cause symptoms such as black spots, black gills, yellow gills, and gill edema. Vibrio alginolyticus mainly causes red legs and red tail fans in shrimp, shell ulcers, and gill rot. In severe cases, it can cause enteritis, red, swollen, and atrophied liver, and bacterial white spots. Vibrio cholerae can easily cause eye rot and muscle turbidity in shrimp, often leading to secondary pathogens and shrimp death. It is evident that shrimp larvae infected with Vibrio will have poor growth and high mortality rates, resulting in reduced aquaculture yields. At the same time, it increases the cost of drug treatment and prevention, reducing the economic benefits of aquaculture. Effective prevention and control of Vibrio infection is crucial to ensuring the healthy growth of shrimp larvae and the success of aquaculture. Summary of the Invention

[0005] In view of this, the present invention proposes a method for raising shrimp larvae that reduces harmful Vibrio bacteria, thereby solving the above problems.

[0006] The technical solution of this invention is achieved as follows: A method for raising shrimp larvae that reduces harmful Vibrio bacteria:

[0007] S1. Preparation of seedling ponds: Select 3 seedling ponds with a water depth of 2-3 meters. Aerators are installed around the perimeter of the ponds, and water circulation equipment is installed at the bottom of the ponds. The bottom of the ponds is covered with an adsorption substrate with a thickness of 0.5-2.5cm, and phytoplankton are inoculated at the same time.

[0008] S2. Water treatment: Water is injected into the seedling pond and air flotation is used to remove small organic matter and impurities from the seawater. Ozone is mixed in during the water circulation and recirculation process to keep the ozone concentration in the water between 0.1-0.3 ppm. Then, the water is left to stand for 1-2 hours to kill bacteria and viruses in the water.

[0009] S3. Larval Rearing: Select healthy and vigorous nauplius shrimp larvae and release them into rearing pond No. 1 at a stocking density of 30,000-50,000 larvae / m³. 3 Adjust the pH of the pond to 7.6-8.0, the salinity to 25-30‰, the water temperature to 20-22℃, the ammonia nitrogen content to <0.25mg / L, and the dissolved oxygen to above 5mg / L. Inoculate with unicellular algae and feed Artemia nauplii as food. The daily feeding amount of unicellular algae is 50,000-100,000 cells / mL, and the daily feeding amount of Artemia nauplii is 10-50g / 10,000 shrimp. Cultivate until the shrimp are larvae.

[0010] S4. Larval shrimp rearing: Release larval shrimp into rearing pond No. 2 at a density of 0.5-0.8 million shrimp / m². 3 Adjust the pond pH to 8.0-8.2, salinity to 25-30‰, water temperature to 25-27℃, ammonia nitrogen content <0.38mg / L, and dissolved oxygen to above 5mg / L. Feed mainly with formulated artificial feed, with a daily feeding amount of 5%-7% of the juvenile shrimp's body weight. Raise the shrimp until they molt 5-8 times and reach a body length >3cm.

[0011] S5. Intensive rearing and harvesting of shrimp larvae: Transfer the molted shrimp larvae to pond No. 3, maintaining a density of 300-1000 larvae / m³. 3 Adjust the pH of the pond to 8.0-8.2, and the water quality regulation is the same as S4. Increase the water flow stimulation 5-8 times a day, with a water flow rate of 0.1-0.5m / s, for 8-20 minutes each time. Feed artificial compound feed, with a daily feeding amount of 3% to 5% of the juvenile shrimp's body weight, and cultivate until they are ready for harvest.

[0012] Furthermore, the adsorption matrix of S1 comprises the following raw materials in parts by weight: 25-50 parts of loaded microbial adsorbent, 15-25 parts of sodium carbonate, 12-20 parts of percarbonamide, 20-30 parts of quicklime, and 10-15 parts of lauric coconut oil-based quaternary phosphate.

[0013] Furthermore, the loaded microbial adsorbent is a complex formed by loading microorganisms onto the surface of the adsorbent material, specifically configured as an 8×10 6 -10 8 A bacterial suspension of CFU / mL was prepared. The adsorbent material was placed in the suspension and incubated at 35-40℃ with shaking at 150-200 rpm for 2-6 hours. After filtration and centrifugation, the microbial-loaded adsorbent material was separated from the unadsorbed microbial suspension, resulting in a loading of 3 × 10⁻⁶ microorganisms. 4 ~1×10 6 cfu / g.

[0014] Furthermore, the microorganisms are lysine-containing Bacillus, Bdellovibrio, and denitrifying Staphylococcus in a concentration ratio of 3:1-3:2-5.

[0015] Furthermore, the adsorbent material is one or a combination of several of the following: maifanite, activated carbon, zeolite, bentonite, and diatomite.

[0016] Furthermore, the phytoplankton in S1 is any one or a combination of diatoms and green algae.

[0017] Furthermore, the artificial compound feed of S4 comprises the following raw materials in parts by weight: 8-12 parts vitamin C, 3-7 parts vitamin E, 12-25 parts taurine, 1-4 parts chlorogenic acid, 10-20 parts Schizophyllum polysaccharide, 5-10 parts mannan oligosaccharide, 4-8 parts peptidoglycan, 3-7 parts canthaxanthin, 2-4 parts laurate monoglyceride, and 3-8 parts antimicrobial peptides.

[0018] Furthermore, the antimicrobial peptides are bee venom peptides, lactoferrin peptides, and giant salamander peptides in a mass ratio of (4-12):(8-15):(1-3), with a molecular weight of 2000-3000 Da.

[0019] Furthermore, the water flow stimulation in S5 uses an enzyme-based water treatment agent, which is prepared by mixing an enzyme preparation and water at a volume ratio of (1.5-5.8):100.

[0020] Furthermore, the enzyme preparation comprises: 35-45 parts tyrosinase, 20-30 parts chitinase, 8-12 parts protease, 8-10 parts lysozyme, 10-20 parts laccase, and 5-8 parts β-glucanase.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention's shrimp larvae rearing method effectively inhibits the growth of harmful Vibrio bacteria and improves the survival rate and health of shrimp larvae through the comprehensive application of innovative technologies such as microbial adsorbents, ozone treatment, enzyme-based water treatment agents, and specially formulated artificial feed. This method not only emphasizes environmental friendliness and ecological balance but also precisely controls farming conditions through a systematic cultivation program, optimizing nutrient supply and enhancing the immunity and adaptability of shrimp larvae. Furthermore, by improving the bottom sediment environment and promoting the water's self-purification capacity, this method provides a better growth environment for shrimp larvae, significantly improving farming efficiency and economic benefits. The biological control scheme using microbial communities, algae, and enzyme preparations avoids the excessive use of chemical disinfectants, protects the aquatic ecosystem, demonstrates an emphasis on sustainable development, and possesses significant practical value and broad market application potential. Detailed Implementation

[0023] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0026] Example 1

[0027] A method for raising shrimp larvae that reduces harmful Vibrio bacteria.

[0028] S1. Preparation of seedling ponds: Select three seedling ponds with a water depth of 2 meters. Aerators are installed around the perimeter of the ponds, and water circulation equipment is installed at the bottom of the ponds. An adsorption substrate with a thickness of 0.5 cm is laid on the bottom of the ponds, and diatoms are inoculated at the same time. The adsorption substrate includes the following raw materials by weight: 25 parts of loaded microbial adsorbent, 15 parts of sodium carbonate, 12 parts of percarbonamide, 20 parts of quicklime, and 10 parts of lauric coconut oil-based quaternary phosphate.

[0029] The microbial adsorbent is formed by loading microorganisms onto the surface of maifanite to form a complex, specifically an 8×10⁸ microbial load. 6 A bacterial suspension of CFU / mL was prepared by adding maifanite particles smaller than 3 mm to the suspension, incubating at 35℃ and 150 rpm for 2 hours, followed by filtration and centrifugation to separate the microbial-loaded adsorbent material from the unadsorbed microbial suspension, resulting in a loading of 3 × 10⁻⁶ CFU / mL. 4 cfu / g;

[0030] The aforementioned microorganisms are lysine-containing Bacillus, Bdellovibrio, and denitrifying Staphylococcus in a concentration ratio of 3:1:2.

[0031] S2. Water treatment: Water is injected into the seedling pond, and air flotation is used to remove impurities. Ozone is mixed in during the water circulation and recirculation process to keep the ozone concentration in the water at 0.1 ppm. Then it is left to stand for 1 hour.

[0032] S3. Larval Rearing: Select healthy and vigorous nauplius shrimp larvae and release them into rearing pond No. 1 at a stocking density of 30,000 larvae / m³. 3 The pH of the pond was adjusted to 7.6, the salinity to 25‰, the water temperature to 20℃, the ammonia nitrogen content to <0.25mg / L, and the dissolved oxygen to be controlled above 5mg / L. Unicellular algae were inoculated and Artemia nauplii were fed as food. The daily feeding amount of unicellular algae was 50,000 cells / mL, and the daily feeding amount of Artemia nauplii was 10g / 10,000 nauplii. The shrimp were then raised to the larval stage.

[0033] S4. Larval shrimp rearing: Larval shrimp are released into rearing pond No. 2 at a density of 0.5 million shrimp / m². 3 Adjust the pH of the pond to 8.0, the salinity to 25‰, the water temperature to 25℃, the ammonia nitrogen content to <0.38mg / L, and the dissolved oxygen to above 5mg / L. Feed mainly artificial compound feed, with a daily feeding amount of 5% of the juvenile shrimp's body weight. Cultivate until the shrimp have molted 5 times and reached a body length of >3cm.

[0034] S5. Intensive rearing and harvesting of shrimp larvae: Transfer the molted shrimp larvae to pond No. 3, maintaining a density of 300 larvae / m³. 3The pH of the pool was adjusted to 8.0, and the water quality was adjusted in the same way as S4. Water flow stimulation was increased 5 times a day, with a water flow rate of 0.1 m / s and 8 minutes each time. Enzyme-based water treatment agent was used for water flow stimulation. The enzyme-based water treatment agent was prepared by mixing enzyme preparation and water at a volume ratio of 1.5:100. The enzyme preparation included: 35 parts tyrosinase, 20 parts chitinase, 8 parts protease, 8 parts lysozyme, 10 parts laccase, and 5 parts β-glucanase.

[0035] At the same time, artificial feed is provided, with a daily feeding amount of 3% of the juvenile shrimp's body weight, until they are harvested.

[0036] The artificial compound feeds S4 and S5 mentioned above include the following ingredients by weight: 8 parts vitamin C, 3 parts vitamin E, 12 parts taurine, 1 part chlorogenic acid, 10 parts Schizophyllum polysaccharide, 5 parts mannan oligosaccharide, 4 parts peptidoglycan, 3 parts canthaxanthin, 2 parts laurate monoglyceride, and 3 parts antimicrobial peptides. The antimicrobial peptides are bee venom peptide, lactoferrin peptide, and giant salamander peptide in a mass ratio of 4:8:1, with a molecular weight of 2000 Da.

[0037] Example 2

[0038] A method for raising shrimp larvae that reduces harmful Vibrio bacteria.

[0039] S1. Preparation of seedling ponds: Select three seedling ponds with a water depth of 3 meters. Aerators are installed around the perimeter of the ponds, and water circulation equipment is installed at the bottom of the ponds. An adsorption substrate with a thickness of 2.5 cm is laid on the bottom of the ponds, and green algae are inoculated at the same time. The adsorption substrate includes the following raw materials by weight: 50 parts of loaded microbial adsorbent, 25 parts of sodium carbonate, 20 parts of percarbonamide, 30 parts of quicklime, and 15 parts of lauric coconut oil-based quaternary phosphate.

[0040] The microbial adsorbent is a complex formed by loading microorganisms onto the surface of activated carbon. Specifically, it is prepared with 10... 8 A bacterial suspension with a concentration of CFU / mL was prepared by adding activated carbon particles smaller than 3 mm to the suspension and incubating at 40°C with shaking at 200 rpm for 6 hours. The mixture was then filtered and centrifuged to separate the microbial-loaded adsorbent material from the unadsorbed microbial suspension, resulting in a loading of 1 × 10⁻⁶ microorganisms. 6 cfu / g;

[0041] The aforementioned microorganisms are lysine-containing Bacillus, Bdellovibrio, and denitrifying Staphylococcus in a concentration ratio of 3:3:5.

[0042] S2. Water treatment: Water is injected into the seedling pond, and air flotation is used to remove impurities. Ozone is mixed in during the water circulation and recirculation process to keep the ozone concentration in the water at 0.3 ppm. Then it is left to stand for 2 hours.

[0043] S3. Larval Rearing: Select healthy and vigorous nauplius shrimp larvae and release them into rearing pond No. 1 at a stocking density of 50,000 larvae / m³. 3 Adjust the pH of the pond to 8.0, the salinity to 30‰, the water temperature to 22℃, the ammonia nitrogen content to <0.25mg / L, and the dissolved oxygen to above 5mg / L. Inoculate with unicellular algae and feed Artemia nauplii as food. The daily feeding amount of unicellular algae is 100,000 cells / mL, and the daily feeding amount of Artemia nauplii is 50g / 10,000 larvae. Cultivate until the shrimp are juveniles.

[0044] S4. Larval shrimp rearing: Larval shrimp are released into rearing pond No. 2 at a density of 0.8 million shrimp / m². 3 Adjust the pH of the pond to 8.2, the salinity to 30‰, the water temperature to 27℃, the ammonia nitrogen content to <0.38mg / L, and the dissolved oxygen to above 5mg / L. Feed mainly artificial compound feed, with a daily feeding amount of 7% of the juvenile shrimp's body weight. Cultivate until molting 8 times and the body length is >3cm.

[0045] S5. Intensive rearing and harvesting of shrimp larvae: Transfer the molted shrimp larvae to pond No. 3, maintaining a density of 1000 larvae / m³. 3 The pH of the pool was adjusted to 8.2, and the water quality was adjusted in the same way as S4. Water flow stimulation was increased 8 times a day, with a water flow rate of 0.5 m / s and each stimulation lasting 20 minutes. Enzyme-based water treatment agent was used for water flow stimulation. The enzyme-based water treatment agent was prepared by mixing enzyme preparation and water at a volume ratio of 5.8:100. The enzyme preparation included: 45 parts tyrosinase, 30 parts chitinase, 12 parts protease, 10 parts lysozyme, 20 parts laccase, and 8 parts β-glucanase.

[0046] At the same time, artificial feed is provided, with a daily feeding amount of 5% of the juvenile shrimp's body weight, until they are harvested.

[0047] The artificial compound feeds S4 and S5 mentioned above include the following ingredients by weight: 12 parts vitamin C, 7 parts vitamin E, 25 parts taurine, 4 parts chlorogenic acid, 20 parts Schizophyllum polysaccharide, 10 parts mannan oligosaccharide, 8 parts peptidoglycan, 7 parts canthaxanthin, 4 parts monolaurate, and 8 parts antimicrobial peptides. The antimicrobial peptides are bee venom peptide, lactoferrin peptide, and giant salamander peptide in a mass ratio of 12:15:3, with a molecular weight of 3000 Da.

[0048] Example 3

[0049] A method for raising shrimp larvae that reduces harmful Vibrio bacteria.

[0050] S1. Preparation of seedling ponds: Select three seedling ponds with a water depth of 3 meters. Aerators are installed around the perimeter of the ponds, and water circulation equipment is installed at the bottom of the ponds. An adsorption substrate with a thickness of 2.5 cm is laid on the bottom of the ponds, and diatoms are inoculated at the same time. The adsorption substrate includes the following raw materials by weight: 35 parts of loaded microbial adsorbent, 20 parts of sodium carbonate, 16 parts of percarbonamide, 25 parts of quicklime, and 13 parts of lauric coconut oil-based quaternary phosphate.

[0051] The loaded microbial adsorbent is a complex formed by loading microorganisms onto the surface of bentonite, specifically an 8×10⁸ microbial adsorbent. 7 A bacterial suspension with a concentration of CFU / mL was prepared by adding bentonite particles smaller than 3 mm to the suspension and incubating at 38°C and 180 rpm for 4 hours. The mixture was then filtered and centrifuged to separate the microbial-loaded adsorbent material from the unadsorbed microbial suspension, resulting in a loading of 3 × 10⁻⁶ CFU / mL. 5 cfu / g;

[0052] The aforementioned microorganisms are lysine-containing Bacillus, Bdellovibrio, and denitrifying Staphylococcus in a concentration ratio of 3:2:3.

[0053] S2. Water treatment: Water is injected into the seedling pond, and air flotation is used to remove impurities. Ozone is mixed in during the water circulation and recirculation process to keep the ozone concentration in the water between 0.2 ppm. Then it is left to stand for 2 hours.

[0054] S3. Larval Rearing: Select healthy and vigorous nauplius shrimp larvae and release them into rearing pond No. 1 at a stocking density of 40,000 larvae / m³. 3 The pH of the pond was adjusted to 8.0, the salinity to 28‰, the water temperature to 21℃, the ammonia nitrogen content to <0.25mg / L, and the dissolved oxygen to be controlled above 5mg / L. Unicellular algae were inoculated and Artemia nauplii were fed as food. The daily feeding amount of unicellular algae was 80,000 cells / mL, and the daily feeding amount of Artemia nauplii was 40g / 10,000 larvae. The shrimp were then raised to the juvenile stage.

[0055] S4. Larval shrimp rearing: Larval shrimp are released into rearing pond No. 2 at a density of 0.7 million shrimp / m². 3 Adjust the pH of the pond to 8.2, the salinity to 28‰, the water temperature to 26℃, the ammonia nitrogen content to <0.38mg / L, and the dissolved oxygen to above 5mg / L. Feed mainly artificial compound feed, with a daily feeding amount of 6% of the juvenile shrimp's body weight. Cultivate until molting 7 times and the body length is >3cm.

[0056] S5. Intensive rearing and harvesting of shrimp larvae: Transfer the molted shrimp larvae to pond No. 3, maintaining a density of 800 larvae / m³. 3The pH of the pool was adjusted to 8.2, and the water quality was adjusted in the same way as S4. Water flow stimulation was added 7 times a day, with a water flow rate of 0.3 m / s and each stimulation lasting 12 minutes. Enzyme-based water treatment agent was used for water flow stimulation. The enzyme-based water treatment agent was prepared by mixing enzyme preparation and water at a volume ratio of 3.8:100. The enzyme preparation included: 40 parts tyrosinase, 25 parts chitinase, 10 parts protease, 9 parts lysozyme, 15 parts laccase, and 7 parts β-glucanase.

[0057] At the same time, artificial feed is provided, with a daily feeding amount of 4% of the juvenile shrimp's body weight, until they are harvested.

[0058] The artificial compound feeds S4 and S5 mentioned above include the following ingredients by weight: 10 parts vitamin C, 5 parts vitamin E, 20 parts taurine, 3 parts chlorogenic acid, 15 parts Schizophyllum polysaccharide, 8 parts mannan oligosaccharide, 6 parts peptidoglycan, 6 parts canthaxanthin, 3 parts monolaurate, and 5 parts antimicrobial peptides. The antimicrobial peptides are bee venom peptide, lactoferrin peptide, and giant salamander peptide in a mass ratio of 8:12:2, with a molecular weight of 2500 Da.

[0059] Comparative Example 1

[0060] The difference between this comparative example and Example 3 is that the microbial adsorbent loaded on the adsorption substrate at the bottom of the breeding pond in S1 of the method for reducing harmful Vibrio in shrimp fry breeding is replaced with an equal amount of microbial preparation.

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 3 is that ozone was not mixed in S2 in the method for reducing harmful Vibrio broodstock.

[0063] Comparative Example 3

[0064] The difference between this comparative example and Example 3 is that the water flow stimulation in S5 of the method for reducing harmful Vibrio bryophytes is the same as that in Example 3.

[0065] Comparative Example 4

[0066] The difference between this comparative example and Example 3 is that no antimicrobial peptides were added to the artificial compound feed in S4 and S5.

[0067] Experimental Example 1

[0068] 1. Experimental preparation:

[0069] Eight experimental zones were set up to cultivate shrimp larvae using the seedling cultivation methods of Examples 1-3 and Comparative Examples 1-4, as well as the basic traditional seedling cultivation method. Multiple culture units were set up in each experimental zone to reduce the impact of individual differences on the results. The same batch of healthy and vigorous nauplius shrimp larvae were purchased uniformly.

[0070] 2. Shrimp larvae release: According to the corresponding larval breeding methods for each group, the same number of nauplius shrimp larvae were released in each of the 8 experimental areas.

[0071] 3. Traditional seedling raising methods:

[0072] S1. Preparation of seedling pond: Select a seedling pond with a water depth of 3 meters. Aerators are installed around the pond and water circulation equipment is installed at the bottom of the pond. Inoculate diatoms.

[0073] S2. Water treatment: Add water to the seedling pond, add 10 mL / m3 of chlorine preparation, and then let it stand for 2 hours;

[0074] S3. Cultivation: Select healthy and vigorous nauplius shrimp larvae and release them into the rearing pond at a density of 40,000 larvae / m³. 3 Adjust the pond pH to 8.0, salinity to 28‰, water temperature to 21℃, ammonia nitrogen content <0.25mg / L, and dissolved oxygen to above 5mg / L. Inoculate with unicellular algae and feed Artemia nauplii as food. The daily feeding amount of unicellular algae is 80,000 cells / mL, and the daily feeding amount of Artemia nauplii is 40g / 10,000 nauplii. Cultivate until molting 7 times and the body length is >3cm. Feed with soy milk, egg yolk and shrimp meal. The daily feeding amount is 4% of the weight of the juvenile shrimp. Cultivate until harvest.

[0075] 4. Data collection and recording:

[0076] Survival rate: Throughout the entire breeding process, the growth and mortality of shrimp larvae in each experimental area were observed and recorded regularly, and the survival rate was calculated.

[0077] Infection rate: The infection rate was assessed by detecting the number of Vibrio bacteria in the intestines and hepatopancreas of shrimp larvae. PCR molecular biology methods were used to detect the presence of Vibrio.

[0078] (1) Sample collection: 100 shrimp larvae were randomly selected from each test area as test subjects to ensure that the samples were representative; the collection sites included the intestine and hepatopancreas. During the entire collection process, aseptic operation procedures must be strictly followed to prevent external contamination.

[0079] (2) Sample processing: For internal tissue samples (such as intestines, liver and pancreas), they need to be placed in a tube containing an appropriate amount of sterile saline and homogenized using a homogenizer to release the Vibrio in the cells.

[0080] (3) Vibrio detection: Using molecular biology methods, Vibrio DNA is extracted from the sample, and PCR amplification of Vibrio DNA is performed using specific primers. The PCR products are then detected by gel electrophoresis or other methods to determine whether Vibrio DNA is present.

[0081] (2) Calculate the infection rate: count the number of positive samples and calculate the infection rate of each type of Vibrio based on the total number of samples tested;

[0082] The infection rate is calculated as follows: Infection rate = (Number of shrimp infected with Vibrio / Total number of shrimp) × 100%;

[0083] 5. Results Data

[0084]

[0085]

[0086] illustrate:

[0087] 1. Experimental group number: Indicates different experimental culture ponds or batches.

[0088] 2. Not detected: This indicates that no infection with the specific Vibrio species was detected in this group of tests.

[0089] 3. Trace (<1%): This indicates that although specific Vibrio species were detected, the infection rate was very low, less than 1%.

[0090] The test results above show that the survival rate of shrimp larvae in experimental groups 1-3 (Examples 1-3) was generally higher than that in experimental groups 4-7 (Comparative Examples 1-4). Furthermore, the Vibrio infection rate in experimental groups 1-3 was relatively low, while the Vibrio infection rate in experimental groups 4-7 was relatively high. This indicates that Vibrio infection affected the shrimp larvae's immune status, leading to a decrease in survival rate. It further demonstrates that the aquaculture management measures in experimental groups 1-3 played a role in controlling Vibrio infection. The survival rate of experimental group 8 (traditional larval rearing method) was only 53%, significantly lower than the other groups.

[0091] Compared with Experimental Group 4 (Comparative Example 1), Experimental Group 3 (Example 3) demonstrated that by loading Bacillus lysine, Bdellovibrio, and Staphylococcus denitrifyingis onto the surface of the adsorbent material to form a biofilm, the large specific surface area and porous structure of the adsorbent material, through the secretion of enzymes, decomposed the cell walls of Vibrio and organic matter in the sediment, inhibiting Vibrio growth and reducing the production of harmful substances such as ammonia nitrogen and hydrogen sulfide, thereby improving water quality and reducing the risk of Vibrio infection. The combination of Bacillus lysine, Bdellovibrio, and Staphylococcus denitrifyingis can alter the microbial composition and function in the sediment, promoting the growth of beneficial microorganisms, thereby inhibiting Vibrio through ecological competition.

[0092] Compared with Experimental Group 5 (Comparative Example 2), Experimental Group 3 (Example 3) introduced ozone during the water circulation and reflux process, which stirred and flushed the sediment at the bottom of the pool, increasing the contact area between ozone and sediment. Utilizing the oxidizing effect of ozone, it can react with the double bonds of lipids in the Vibrio cell wall, penetrate into the bacterial cell, act on proteins and lipopolysaccharides, change cell permeability, and lead to cell death. It can also decompose organic matter, reduce chemical oxygen demand (COD) and biological oxygen demand (BOD), and improve the cleanliness and transparency of the water.

[0093] Compared with Experimental Group 6 (Comparative Example 3), Experimental Group 3 (Example 3) used enzyme-based water treatment agents to stimulate shrimp larvae with water flow. Under the disturbance of water flow, the biological enzyme preparation was more evenly dispersed and penetrated into all parts of the water body, including bottom mud and biofilm, increasing the opportunity for contact with Vibrio. This can destroy the formation of the biofilm protective layer of pathogens such as Vibrio, exposing Vibrio to an unfavorable environment, destroying the cell wall or cell membrane of Vibrio, leading to bacterial cell lysis, and thus directly killing Vibrio. By controlling the flow rate, the shrimp larvae's movement ability and adaptability can be trained, improving their vitality and immunity. It can also accelerate the decomposition of organic matter and the transformation of harmful substances in the water, improving the water quality environment.

[0094] Compared with Experimental Group 7 (Comparative Example 4), Experimental Group 3 (Example 3) showed that melitin peptides, a major component of bee venom, can disrupt the integrity of bacterial cell membranes, forming transmembrane pores, leading to an imbalance in the exchange of substances between the intracellular and extracellular spaces, ultimately resulting in bacterial death. Lactoferrin peptides, derived from lactoferrin, are small molecule peptides with antibacterial properties. They can deprive bacteria of the iron ions needed for growth, reducing their nutrient supply and inhibiting their growth by binding to iron ions. Giant salamander peptides, an active peptide extracted from the giant salamander (an amphibian), possess antibacterial and antiviral properties. By enhancing the immunity of shrimp larvae, they improve their resistance to pathogens. When these three peptides are combined and added to shrimp larvae feed, they can inhibit Vibrio infection through their respective antibacterial mechanisms and immunomodulatory effects. The addition of these bioactive peptides not only improves the immunity of shrimp larvae and reduces the occurrence of diseases, but also promotes the healthy growth and development of shrimp larvae.

[0095] 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 within the protection scope of the present invention.

Claims

1. A method for raising shrimp larvae with reduced harmful Vibrio bacteria, characterized in that: Includes the following steps: S1. Preparation of seedling ponds: Select 3 seedling ponds with a water depth of 2-3 meters. Aerators are installed around the perimeter of the ponds, and water circulation equipment is installed at the bottom of the ponds. The bottom of the ponds is covered with an adsorption substrate with a thickness of 0.5-2.5cm, and phytoplankton are inoculated at the same time. S2. Water treatment: Water is injected into the seedling pond, and air flotation is used to remove impurities. Ozone is mixed in during the water circulation and recirculation process to keep the ozone concentration in the water between 0.1-0.3 ppm. Then it is left to stand for 1-2 hours. S3, larva cultivation: healthy and strong nauplii shrimp fry are selected and put into the No. 1 breeding pond at a density of 30-50 thousand tails / m 3 , the pH of the water tank is adjusted to 7.6-8.0, the salinity is 25-30‰, the water temperature is 20-22℃, the ammonia nitrogen content is <0.25 mg / L, the dissolved oxygen is controlled to be more than 5 mg / L, monads and brine shrimp nauplii are inoculated and fed as bait, the daily feeding amount of monads is 50-100 thousand cells / mL, the daily feeding amount of brine shrimp nauplii is 10-50 grams / ten thousand tails, and larvae are cultivated; S4. Larval shrimp rearing: Release larval shrimp into rearing pond No. 2 at a density of 0.5-0.8 million shrimp / m². 3 Adjust the pond pH to 8.0-8.2, salinity to 25-30‰, water temperature to 25-27℃, ammonia nitrogen content <0.38mg / L, and dissolved oxygen to above 5mg / L. Feed mainly with formulated artificial feed, with a daily feeding amount of 5%-7% of the juvenile shrimp's body weight. Raise the shrimp until they molt 5-8 times and reach a body length >3cm. S5. Intensive rearing and harvesting of shrimp larvae: Transfer the molted shrimp larvae to pond No. 3, maintaining a density of 300-1000 larvae / m³. 3 Adjust the pH of the pond to 8.0-8.2, and the water quality regulation is the same as S4. Increase the water flow stimulation 5-8 times a day, with a water flow rate of 0.1-0.5m / s, for 8-20 minutes each time. Feed artificial compound feed, with a daily feeding amount of 3% to 5% of the juvenile shrimp's body weight, and cultivate until they are ready for harvest. The adsorption matrix of S1 comprises the following raw materials in parts by weight: 25-50 parts of loaded microbial adsorbent, 15-25 parts of sodium carbonate, 12-20 parts of percarbonamide, 20-30 parts of quicklime, and 10-15 parts of lauric coconut oil-based quaternary phosphate. The microbial adsorbent is formed by loading microorganisms onto the surface of the adsorbent material to form a complex, specifically an 8×10⁸ microbial load. 6 -10 8 A bacterial suspension of CFU / mL was prepared. The adsorbent material was placed in the suspension and incubated with shaking at 150-200 rpm for 2-6 hours, while maintaining a temperature of 35-40℃. After filtration and centrifugation, the microbial-loaded adsorbent material was separated from the unadsorbed microbial suspension, resulting in a loading of 3 × 10⁻⁶ microorganisms. 4 ~1×10 6 cfu / g; The microorganisms are lysine-containing Bacillus, Bdellovibrio, and denitrifying Staphylococcus in a concentration ratio of 3:1-3:2-5. The artificial compound feed of S4 comprises the following ingredients in parts by weight: 8-12 parts vitamin C, 3-7 parts vitamin E, 12-25 parts taurine, 1-4 parts chlorogenic acid, 10-20 parts Schizophyllum polysaccharide, 5-10 parts mannan oligosaccharide, 4-8 parts peptidoglycan, 3-7 parts canthaxanthin, 2-4 parts laurate monoglyceride, and 3-8 parts antimicrobial peptides; The antimicrobial peptides are bee venom peptides, lactoferrin peptides, and giant salamander peptides in a mass ratio of (4-12):(8-15):(1-3), with a molecular weight of 2000-3000 Da. The water flow stimulation in S5 uses an enzyme-based water treatment agent, which is prepared by mixing an enzyme preparation and water at a volume ratio of (1.5-5.8):

100. The enzyme preparation comprises: 35-45 parts tyrosinase, 20-30 parts chitinase, 8-12 parts protease, 8-10 parts lysozyme, 10-20 parts laccase, and 5-8 parts β-glucanase.

2. The shrimp larvae breeding method for reducing harmful Vibrio bacteria as described in claim 1, characterized in that: The adsorbent material is one or a combination of several of the following: maifanite, activated carbon, zeolite, bentonite, and diatomite.

3. The shrimp larvae breeding method for reducing harmful Vibrio bacteria as described in claim 1, characterized in that: The phytoplankton in S1 is any one or a combination of diatoms and green algae.

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