Method for breeding vannamei shrimp seedlings by using bio-floc

Abstract

This invention belongs to the field of shrimp farming technology, specifically relating to a method for raising Litopenaeus vannamei larvae using bioflocs. The method includes the following steps: (1) preparing raceway-shaped bioflocs; (2) preparing functional bioflocs; and (3) raising mysids and larvae; and raising adult larvae. This invention provides an effective method for raising Litopenaeus vannamei larvae without the use of antibiotics, solving the problem of antibiotic abuse during the larval stage, and is of great significance for promoting the green and healthy development of the shrimp larval industry.

Description

Technical Field

[0001] This invention belongs to the field of shrimp farming technology, specifically relating to a method for raising Litopenaeus vannamei larvae using bioflocs. Background Technology

[0002] Currently, bioflocs are mainly used in the Litopenaeus vannamei shrimp farming industry. This model, with beneficial bacteria at its core, can effectively decompose organic matter such as uneaten feed and feces, thereby significantly reducing the accumulation of harmful substances such as ammonia nitrogen and nitrite in the water, improving water stability and buffering capacity, alleviating shrimp stress responses, and reducing the risk of diseases caused by water quality deterioration. The beneficial bacteria in bioflocs can activate immune cells in shrimp, enhance their non-specific immunity, and thus inhibit the reproduction of pathogens, further reducing the incidence of disease. Therefore, it has broad application prospects in shrimp farming.

[0003] However, the application of bioflocs in Litopenaeus vannamei seedling production is extremely rare, and reports of their use in treating vibrio diseases in larvae are even scarcer. In the shrimp seedling stage, besides viral and enterosporadic diseases, vibrio infection is the most common and extremely harmful problem. Among them, Vibrio harveyi and Vibrio parahaemolyticus are the most prevalent pathogenic Vibrio species. Infections with Vibrio harveyi and Vibrio parahaemolyticus cause lesions in the liver and intestines of shrimp larvae, severely impairing digestion and absorption, significantly slowing growth, affecting metamorphosis, leading to inconsistent larvae size, weak constitution, and in severe cases, even acute or chronic death. Pathogenic Vibrio infections cause enormous losses to the Litopenaeus vannamei seedling industry.

[0004] Chinese Patent 201910857941.X discloses a novel biofloc and its application and method for raising Litopenaeus vannamei. The biofloc significantly improves the water quality of Litopenaeus vannamei raising ponds and increases the survival rate, growth indicators and immunity of the shrimp.

[0005] Chinese Patent 201710048682.7 discloses a method for cultivating bioflocs and its application in Litopenaeus vannamei shrimp farming. The method involves applying a mixture of three preparations—Bacillus powder, yeast powder, and lactic acid bacteria liquid—to aquaculture to promote the formation of bioflocs in the water. This is beneficial for Litopenaeus vannamei, a cash crop, by enhancing its immunity and disease resistance, inhibiting diseases caused by Vibrio, increasing feed intake, and improving the survival rate of Litopenaeus vannamei shrimp.

[0006] However, the two invention patents mentioned above only utilize the functions of bioflocs in improving water quality and inhibiting Vibrio to prevent disease during the breeding and nursery stages, without mentioning their effective treatment in cases where Vibrio disease has already occurred. Furthermore, it is impractical to cultivate bioflocs with disease prevention functions in a timely manner during the seedling stage. First, because shrimp larvae are very small and sensitive, their immune systems are not yet fully developed and are extremely sensitive to changes in the external environment. Any slight change in water quality or pathogen invasion can lead to high mortality rates. Due to their immature immune systems, shrimp larvae are more susceptible to infection by bacteria, viruses, and other pathogens. Once disease occurs, it spreads rapidly, is difficult to treat, and often results in mass mortality. Second, the seedling cycle of Litopenaeus vannamei is short, reaching marketable size in only 12-15 days. It is difficult to cultivate stable bioflocs with functions of improving water quality and inhibiting harmful Vibrio within such a short time, let alone treating Vibrio disease. This indicates that the existing application of bioflocs in Litopenaeus vannamei farming is not suitable for seedling cultivation and lacks the ability to treat Vibrio disease in larvae.

[0007] Therefore, there is an urgent need for a method to cultivate Litopenaeus vannamei larvae using bioflocs. Summary of the Invention

[0008] The purpose of this invention is to provide a method for culturing Litopenaeus vannamei larvae using bioflocs. This method can successfully treat Litopenaeus vannamei larvae infected with Vibrio harveyi and Vibrio parahaemolyticus, with significant effects, and simultaneously achieves the goal of antibiotic-free larval rearing. It effectively treats and avoids the problems of slow growth, uneven size, and even mass mortality caused by Vibrio parahaemolyticus and Vibrio harveyi diseases, thus achieving the goal of antibiotic-free Litopenaeus vannamei larval rearing.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for culturing Litopenaeus vannamei larvae using bioflocs, comprising the following steps:

[0010] (1) Preparation of racetrack-shaped bioflocs, the operation steps are as follows:

[0011] a. Three days before stocking the shrimp fry, add brown sugar and compound bacteria A to the water in the raceway pond;

[0012] b. On the 4th day, shrimp fry were released into the raceway pool.

[0013] c. During the first month of breeding, add brown sugar at 4 to 6 times the weight of the feed, and add 1 to 2 ppm of compound bacteria A and 0.5 to 1 ppm of nitrifying bacteria daily.

[0014] d. During the second month of aquaculture, after the water in the raceway pool is allowed to settle in an Inhofe conical tube, the content of floc precipitate is less than 10 mL.

[0015] e. After two months of cultivation, a sudden and sharp decrease in nitrite content was detected, eventually stabilizing near zero, indicating the formation of stable bioflocs. After Inhofe conical sedimentation and centrifugation, precipitate A was obtained. Plate count analysis showed a Bacillus content greater than 1.0 × 10⁻⁶. 6 cfu / g precipitate A;

[0016] f. After the water in the raceway aquaculture pond is filtered, it is pumped into an empty seedling pond, aerated and ready for use, to obtain biological flocs in the seedling pond.

[0017] (2) Preparation of functional bioflocs: The operation steps are as follows: Add a mixture containing compound bacteria B to the bioflocs prepared in step (1) in the seedling pond, and continue aeration. After the water in the raceway pond is allowed to settle through an Inhofe conical tube, the content of the floc precipitate should reach 10~15mL. Take the floc precipitate for testing, and after centrifugation and dehydration, precipitate B is obtained. The content of Bacillus spores is greater than 1.0×10⁻⁶ using the plate count method. 7 CFU / g precipitate B yielded functional bioflocs;

[0018] (3) The farming methods for mysid shrimp and juvenile shrimp include the following steps:

[0019] a. During the mysid and juvenile shrimp stages, a drainage net frame is placed in the nursery pond, and the water level in the original nursery pond is lowered to 15-20cm using the siphon method.

[0020] b. Pump the functional bioflocs into the original seedling pond to increase the water level in the seedling pond by 15-20cm and maintain this for 24 hours without feeding.

[0021] c. After 24 hours, pump the functional bioflocs into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time, until the water level in the seedling pond reaches 80 to 85 cm, and continue not to feed.

[0022] d. After 48 hours, take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, the feeding can be resumed as usual; if the average content of Vibrio parahaemolyticus and Vibrio harveyi is 1.0 × 10⁻⁶, the feeding can be resumed as usual. 1 cfu / g ~1.0×10 3 cfu / g, every 6-8 hours use a sieve to concentrate functional bioflocs to a water level height of 10-12cm (floc amount = floc content × bottom area × water level height), dilute with water and stir well before pouring into the seedling pond.

[0023] e. After 72 hours, take seedling samples for further testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. When the detected content is 1.0 × 10⁻⁶, the result is considered satisfactory. 0 cfu / g ~1.0×10 1A concentration of CFU / g is considered a successful treatment for Vibrio infection; a detection level of 1.0 × 10⁻⁶ CFU / g is also considered a successful treatment. 2 cfu / g ~1.0×10 3 cfu / g, repeat step d until treatment is successful;

[0024] The rearing method for seedlings includes the following steps:

[0025] a. Large seedling stage, when the shrimp seedlings reach a length of 0.8-1cm, place a seedling collection frame in the seedling collection pit on the side of the seedling pond, add water to the seedling collection frame to a height of 30-35cm, connect the seedling collection net frame to the drainage outlet of the seedling pond, and use a siphon to drain the water in the seedling pond into the seedling collection frame until the water level in the seedling pond drops to 15-20cm. During this process, use a net to scoop the large seedlings into the seedling collection bucket for temporary rearing every 5 minutes.

[0026] b. After the seedling collection is completed, disinfect the seedlings in the collection buckets;

[0027] c. Pump the functional bioflocs into the empty seedling pond, add water to a height of 40-45cm, move the large seedlings from the seed collection bucket into the seedling pond, and keep them without feeding for 24 hours.

[0028] d. After 24 hours, pump the functional bioflocs into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time, until the water level in the seedling pond reaches 80 to 85 cm, and continue not to feed.

[0029] e. After 48 hours, take samples of large seedlings to test the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, feeding can be resumed without changing the water; if the detected content is 1.0 × 10⁻⁶, the feeding can be resumed. 1 cfu / g ~1.0×10 3 cfu / g, every 6-8 hours use a sieve to concentrate 10-12cm of functional bioflocs to a water level height (floc amount = floc content × bottom area × water level height), mix with water and pour into the seedling pond.

[0030] f. After 72 hours, samples were taken for further testing of the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the test results, when the detected content was 1.0 × 10⁻⁶, the result was considered satisfactory. 0 cfu / g ~1.0×10 1 A concentration of CFU / g is considered a successful treatment for Vibrio infection; a detected concentration of 1.0 × 10⁻⁶ CFU / g is considered a successful treatment. 2 cfu / g ~1.0×10 3 cfu / g, repeat step e until treatment is successful.

[0031] Furthermore, the compound bacteria A comprises Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1:(1.2-1.4):(0.5-0.7).

[0032] Furthermore, the components of the mixture containing compound bacteria B and their usage in the biofloc of the seedling pond are as follows: 1~2 ppm compound bacteria B, 1~2 ppm vitamin C, 10~20 ppm baking soda, 5~10 ppm brown sugar, 2~3 ppm Vibrio harveyi phage freeze-dried powder, 2~3 ppm Vibrio parahaemolyticus phage freeze-dried powder, 1~2 ppm shrimp seedling chips, and 1~2 ppm Artemia spp.

[0033] Furthermore, the compound bacteria B comprises Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus in a weight ratio of (1.5-1.7):1:(0.2-0.5).

[0034] Furthermore, after two months of culture in step (1), quicklime, dolomite powder or sodium bicarbonate is added to the culture water according to the pH and total alkalinity test results. For every 10 ppm decrease in total alkalinity, 20-30 ppm is added. When the nitrite content is detected to suddenly drop sharply and eventually remain close to 0, it indicates that the bioflocs have formed a stable morphology.

[0035] Furthermore, after measuring with an Inhofe conical tube in step (2), if the biofloc content is less than 10 mL, continue to add 2-5 ppm of brown sugar and 10-20 ppm of baking soda.

[0036] Furthermore, in step (1), shrimp fry are selected that have been raised to a size of 1.5-2cm in length and are stocked at a density of 300-500 fry per cubic meter of water.

[0037] Furthermore, in step (1), 3 days before the shrimp fry are released, brown sugar at a concentration of 5-10 ppm and compound bacteria A at a concentration of 2-3 ppm are added to the water in the raceway pond.

[0038] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0039] (1) The long-term use of antibiotics to treat Vibrio parahaemolyticus and Vibrio harveyi in traditional shrimp breeding has led to bacterial resistance, increased antibiotic dosage, and antibiotic abuse. The method of the present invention can perfectly solve this serious problem in treating vibrio diseases. The method of the present invention uses a mixture of compound bacteria A and compound bacteria B, which has a good therapeutic effect on Vibrio parahaemolyticus and Vibrio harveyi.

[0040] (2) In this invention, it is used as a component of functional bioflocs, works synergistically with other components, requires a low dosage and can proliferate in the nutrient environment provided by the bioflocs, and can ultimately effectively treat vibrio diseases while greatly reducing costs.

[0041] (3) When conventionally treating shrimp larvae with Vibrio parahaemolyticus and Vibrio harveyi, the bacteriophages are directly applied to the nursery pond, which is affected by the water quality environment of the nursery pond. In this invention, after the bacteriophages are used as components of functional bioflocs, most of the original nursery pond water is drained during treatment, and the synergistic inhibitory effect of the bioflocs on Vibrio provides stable water quality conditions and environment, greatly enhancing the treatment effect;

[0042] (4) Conventional bioflocs can only improve water quality and prevent Vibrio diseases. Although they greatly reduce the chance of disease occurrence, they are not very effective in treating Vibrio diseases. Therefore, they are mostly used for aquaculture rather than seedling cultivation. However, the functional bioflocs of this invention form functional biofloc particles that are ingested by seedlings and enter their bodies, thus enhancing nutrition and providing treatment.

[0043] (5) This invention provides an effective method for treating vibrio disease in shrimp larvae without the use of antibiotics, filling this technological gap and solving the problem of antibiotic abuse in the seedling process. The method of this invention has been used in practice and is a mature biofloc culture technology, which is of great significance for promoting the green and healthy development of the shrimp seedling industry. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] All raw materials used in this invention are commercially available products:

[0046] Bacillus pumilus, product number: SMHCC D11043, purchased from Shanghai Preservation Microbiology Co., Ltd.

[0047] Bacillus licheniformis, product number: SMHCC D10742, purchased from Shanghai Baocang Microbial Co., Ltd.

[0048] Bacillus subtilis, product number: SMHCC D10486, purchased from Shanghai Baocang Microbiology Co., Ltd.

[0049] Bacillus mucilaginosus, product number: SMHCC D12019, purchased from Shanghai Baocang Microbiology Co., Ltd.

[0050] Pichia pastoris, product number: SHMCC D20457, purchased from Shanghai Preservation Microbial Co., Ltd.

[0051] Lactobacillus acidophilus, product number: SHMCC D24720, purchased from Shanghai Preservation Microbiology Co., Ltd.

[0052] Inhofe conical tube, brand: VITLAB Inhofe tube, Germany. After placing the sample in the Inhofe conical tube for 15 minutes, the vertical height of easily sinking solids is directly read.

[0053] Example 1

[0054] This embodiment provides a method for culturing Litopenaeus vannamei larvae using bioflocs, including the following steps:

[0055] (1) Preparation of racetrack-shaped bioflocs: The cultivation steps are as follows:

[0056] a. Three days before stocking the shrimp larvae, fill the raceway pond with 60cm of water. Use a jet injector to provide high dissolved oxygen and propel the water in a circular flow within the raceway, controlling the flow rate at 0.3 m / s. Add 8 ppm of brown sugar and 3 ppm of compound bacteria A directly to the raceway pond water, mix well, and then sprinkle. Add once daily for three consecutive days. The compound bacteria A consists of Bacillus pumilus, Bacillus subtilis, and Bacillus licheniformis in a weight ratio of 1:1.3:0.6.

[0057] b. Stocking shrimp larvae: Select shrimp larvae that have been grown to a size of 1.5-2cm in length, and stock them at a density of 400 larvae per cubic meter of water.

[0058] c. Within one month of breeding, add brown sugar at 5 times the weight of the feed, and add 2 ppm of compound bacteria A and 0.5 ppm of nitrifying bacteria daily. Test the ammonia nitrogen and nitrite content in the water daily.

[0059] d. During the second month of aquaculture, the pH, total alkalinity, ammonia nitrogen, and nitrite content of the water should be tested daily. The total weight of brown sugar added should be determined by multiplying the ammonia nitrogen test value by 12 times the total water volume. The floc content should be tested daily using an Inhofe cone tube, and the content of biological flocs should be controlled to be within 10 mL.

[0060] e. After two months of culture, quicklime was added to the culture water based on pH and total alkalinity test results. For every 10 ppm decrease in total alkalinity, 25 ppm was added. When a sudden and sharp drop in nitrite content was detected, eventually stabilizing near 0, it indicated the formation of bioflocs. Water from the raceway was taken, subjected to Inhofe conical sedimentation, centrifuged, and dehydrated to obtain the floc precipitate. The Bacillus content was then tested using the plate count method, adhering to standard GB20287-2006, with a content greater than 1.0 × 10⁻⁶. 6 CFU / g flocculent precipitate;

[0061] f. After the water in the raceway aquaculture pond is filtered through a 120-mesh filter, it is pumped into an empty seedling pond with a water level of 1 meter. Nanotubes are used for aeration and the seedling pond bioflocs are obtained.

[0062] (2) Preparation of functional bioflocs, the specific operation steps are as follows: Then add a mixture containing compound bacteria B to the biofloc water prepared in step (1), the ratio is 1.5 ppm vitamin C, 14 ppm baking soda, 7 ppm brown sugar, 1.6 ppm compound bacteria B, 2.3 ppm Vibrio harveyi phage freeze-dried powder (Qingdao Nuoan Biotech Co., Ltd.), 2.5 ppm Vibrio parahaemolyticus phage freeze-dried powder (Qingdao Nuoan Biotech Co., Ltd.), 1.3 ppm shrimp seedling chips (Boshan Biotechnology Co., Ltd.), 1.6 ppm Artemia pulp (Artemia hatched for 24 hours, collected, drained and pulped); the compound bacteria B includes Bacillus subtilis, Bacillus licheniformis and Bacillus mucilaginosus in a weight ratio of 1.6:1:0.4. Except for Artemia spores, all other ingredients need to be rubbed through a 100-mesh silk screen until the mesh size is less than 100 mesh before adding water and pouring it into the pool; increase the aeration rate of the nanotubes and continue aeration for 13 hours; the required biofloc content in the water is 12 mL. If the biofloc content is less than 10 mL after Inhofe conical tube testing, add 3 ppm of brown sugar and 15 ppm of baking soda; use the plate count method to test the Bacillus content, following standard GB20287-2006, with a content requirement of 1.0 × 10⁻⁶. 7 Functional bioflocs were obtained when the cfu / g concentration was above a certain level.

[0063] (3) The farming methods for mysids and juvenile shrimp include the following steps:

[0064] a) During the mysid and larval stages, the larvae are relatively weak. A drainage net frame with a 60-mesh mesh is placed in the nursery pond. Several shrimp with a diameter of 63mm are then placed inside. ＃ The siphon tubes are placed inside the net frame, and the water level in the original seedling pond is lowered to 18cm by siphoning. The number of siphon tubes is increased or decreased to control the drainage speed and prevent the seedlings from being sucked into the drainage net.

[0065] b. Pump the functional bioflocs into the original seedling pond to increase the water level in the seedling pond by 17cm and maintain this for 24 hours without feeding.

[0066] c. After 24 hours, pump the functional bioflocs into the seedling pond every 6 hours, increasing the water level by 10cm each time, until the water level in the seedling pond reaches 80cm, and continue not to feed.

[0067] d. After 48 hours, visually inspect the seedlings for their condition and vigor. Take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the vigor and Vibrio test results, plan the next steps. If Vibrio is not detected, gradually resume regular feeding; if the detected content is 1.0 × 10⁻⁶, [further steps can be taken]. 1cfu / g ~1.0×10 3 cfu / g, then every 6 hours use a 300-mesh silk screen to collect functional bioflocs at a water level of 10cm, mix with water and pour into the seedling pond;

[0068] e. After 72 hours, take seedling samples to further test the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the test results, when the detected content is 1.0 × 10⁻⁶, the result is considered acceptable. 0 cfu / g ~1.0×10 1 A cfu / g level is considered a successful treatment; a detected level of 1.0 × 10⁻⁶ is considered a successful treatment. 2 cfu / g ~1.0×10 3 A cfu / g level is considered a treatment failure.

[0069] (4) The breeding method for seedlings includes the following steps:

[0070] a. Large larvae stage: When the shrimp larvae reach a body length of 0.8cm or more, place a collection frame in the collection pit at the edge of the larvae pond, add 30cm of water to the collection frame, and connect the collection frame to the drain outlet of the larvae pond. Initially, use 3 63mm diameter wire mesh nets. ＃ The siphon pipe drains into the seedling collection frame. When the water level in the seedling pond drops to 18cm, the drain pipe is pulled out. During this process, every 5 minutes, the large seedlings are scooped into the 500-liter seedling collection bucket with a 40-mesh net for temporary rearing.

[0071] b. After the seedling collection is completed, add povidone-iodine at a concentration of 1.5 ppm to the 500-liter seedling collection bucket and disinfect for 2 minutes;

[0072] c. Pump 40cm functional bioflocs into the empty seedling pond, then move the large seedlings from the seed collection bucket into it, and keep it without feeding for 24 hours.

[0073] d. After 24 hours, pump the functional bioflocs into the seedling pond every 6 hours, increasing the water level by 10cm each time, until the water level in the seedling pond reaches 80cm, and continue not to feed.

[0074] e. After 48 hours, visually inspect the condition and vitality of the seedlings. Take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the vitality and Vibrio test results, plan the next steps. If Vibrio is not detected, gradually resume feeding without changing the water; if the detected content is 1.0 × 10⁻⁶, [further steps can be taken]. 1 cfu / g ~1.0×10 3 cfu / g, then every 6 hours use a 300-mesh silk screen to collect functional bioflocs at a water level of 10cm, mix with water and pour into the seedling pond;

[0075] f. After 72 hours, samples were taken for further testing of the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the test results, when the detected content was 1.0 × 10⁻⁶, the result was considered satisfactory. 0cfu / g ~1.0×10 1 A cfu / g level is considered a successful treatment; a detected level of 1.0 × 10⁻⁶ is considered a successful treatment. 2 cfu / g ~1.0×10 3 A level of cfu / g is considered a treatment failure.

[0076] Example 2

[0077] The difference between this embodiment and Embodiment 1 is that: the compound bacteria A includes Bacillus pumilus, Bacillus subtilis, and Bacillus licheniformis in a weight ratio of 1:1.2:0.5. The compound bacteria B includes Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus in a weight ratio of 1.7:1:0.2.

[0078] Comparative Example 1

[0079] The difference between this comparative example and Example 1 is that the compound bacteria A includes Pichia pastoris, Bacillus subtilis, and Lactobacillus acidophilus in a weight ratio of 1:1.3:0.6. The compound bacteria B includes Bacillus subtilis, Pichia pastoris, and Lactobacillus acidophilus in a weight ratio of 1.6:1:0.4.

[0080] Comparative Example 2

[0081] The difference between this comparative example and Example 1 is that the compound bacteria A includes Pichia pastoris, Bacillus subtilis and Lactobacillus acidophilus in a weight ratio of 1:1.3:0.6.

[0082] Comparative Example 3

[0083] The difference between this comparative example and Example 1 is that the ratio of compound bacteria A and compound bacteria B is different.

[0084] The compound bacteria A comprises Bacillus pumilus, Bacillus subtilis, and Bacillus licheniformis in a weight ratio of 1.3:0.6:1. The compound bacteria B comprises Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus in a weight ratio of 0.4:1.6:1.

[0085] Comparative Example 4

[0086] This comparative example provides a method for culturing Litopenaeus vannamei larvae using bioflocs, including the following steps:

[0087] (1) Preparation of bioflocs in the seedling pond, the cultivation operation steps are as follows:

[0088] a. Three days before stocking the shrimp larvae, fill the raceway pond with 60cm of water. Use a jet injector to provide high dissolved oxygen and propel the water in a circular flow within the raceway, controlling the flow rate at 0.3 m / s. Add 8 ppm of brown sugar and 3 ppm of powdered compound bacteria A directly to the raceway pond water, mix well, and then sprinkle. Add once daily for three consecutive days. The compound bacteria A consists of Bacillus pumilus, Bacillus subtilis, and Bacillus licheniformis in a weight ratio of 1:1.3:0.6.

[0089] b. Stocking shrimp larvae: Select shrimp larvae that have been grown to a size of 1.5-2cm in length, and stock them at a density of 400 larvae per cubic meter of water.

[0090] c. Within one month of breeding, add brown sugar at 5 times the weight of the feed, and add 2 ppm of compound bacteria and 0.5 ppm of nitrifying bacteria daily. Test the ammonia nitrogen and nitrite content in the water daily.

[0091] d. During the second month of aquaculture, the pH, total alkalinity, ammonia nitrogen, and nitrite content of the water should be tested daily. The total weight of brown sugar added should be determined by multiplying the ammonia nitrogen test value by 12 times the total water volume. The floc content should be tested daily using an Inhofe cone tube, and the content of biological flocs should be controlled to be within 10 mL.

[0092] e. After two months of culture, add quicklime, dolomite powder, or sodium bicarbonate to the culture water based on pH and total alkalinity test results. Add 25 ppm of quicklime for every 10 ppm decrease in total alkalinity. When a sudden and sharp drop in nitrite content is detected, eventually stabilizing near 0, it indicates the formation of bioflocs. Take samples of the biofloc precipitate from the raceway pond and use the plate count method to test the Bacillus content, adhering to standard GB20287-2006, with a content requirement of 1.0 × 10⁻⁶. 6 CFU / g or higher;

[0093] f. After the water in the raceway aquaculture pond is filtered through a 120-mesh filter, it is pumped into an empty seedling pond with a water level of 1 meter. Nanotubes are used for aeration and the seedling pond bioflocs are obtained.

[0094] (2) The farming methods for mysids and juvenile shrimp include the following steps:

[0095] a) During the mysid and larval stages, the larvae are relatively weak. A drainage net frame with a 60-mesh mesh is placed in the nursery pond. Several shrimp with a diameter of 63mm are then placed inside. ＃ The siphon tubes are placed inside the net frame, and the water level in the original seedling pond is lowered to 18cm by siphoning. The number of siphon tubes is increased or decreased to control the drainage speed and prevent the seedlings from being sucked into the drainage net.

[0096] b. Pump the prepared functional bioflocs along with the pond water into the original seedling pond to increase the water level by 17cm and maintain this for 24 hours without feeding.

[0097] c. After 24 hours, pump the functional bioflocs along with the pond water into the nursery every 6 hours, increasing the water level by 10cm each time, until the water level in the nursery reaches 80cm, and continue not to feed.

[0098] d. After 48 hours, visually inspect the seedlings for their condition and vigor. Take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the vigor and Vibrio test results, plan the next steps. If Vibrio is not detected, gradually resume regular feeding; if the detected content is 1.0 × 10⁻⁶, [further steps can be taken]. 1 cfu / g ~1.0×10 3 cfu / g, then every 6 hours use a 300-mesh silk screen to collect functional bioflocs at a water level of 10cm, mix with water and pour into the seedling pond;

[0099] e. After 72 hours, take seedling samples to further test the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the test results, when the detected content is 1.0 × 10⁻⁶, the result is considered acceptable. 0 cfu / g ~1.0×10 1 A concentration of CFU / g is considered a successful treatment; a detection value of 1.0 × 10⁻⁶ CFU / g is also considered a successful treatment. 2 cfu / g ~1.0×10 3 A cfu / g level is considered a treatment failure.

[0100] (3) The breeding method for seedlings includes the following steps:

[0101] a. Large larvae stage: When the shrimp larvae reach a body length of 0.8-1cm or more, place a collection frame in the collection pit at the edge of the larvae pond, add 30cm of water to the collection frame, and connect the collection frame to the drain outlet of the larvae pond. Initially, use 3 63mm diameter wire mesh nets. ＃ The siphon pipe drains into the seedling collection frame. When the water level in the seedling pond drops to 18cm, the drain pipe is pulled out. During this process, every 5 minutes, the large seedlings are scooped into the 500-liter seedling collection bucket with a 40-mesh net for temporary rearing.

[0102] b. After the seedling collection is completed, add povidone-iodine at a concentration of 1.5 ppm to the 500-liter seedling collection bucket and disinfect for 2 minutes;

[0103] c. Pump 40cm functional bioflocs into the empty seedling pond, then move the large seedlings from the seed collection bucket into it, and keep it without feeding for 24 hours.

[0104] d. After 24 hours, pump the functional bioflocs into the seedling pond every 6 hours, increasing the water level by 10cm each time, until the water level in the seedling pond reaches 80cm, and continue not to feed.

[0105] e. After 48 hours, visually inspect the condition and vitality of the seedlings. Take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the vitality and Vibrio test results, plan the next steps. If Vibrio is not detected, gradually resume feeding without changing the water; if the detected content is 1.0 × 10⁻⁶, [further steps can be taken]. 1 cfu / g ~1.0×10 3 cfu / g, then every 6 hours use a 300-mesh silk screen to concentrate the functional bioflocs to a height of 10cm in water, mix with water and pour into the seedling pond;

[0106] f. After 72 hours, samples were taken for further testing of the average content of Vibrio parahaemolyticus and Vibrio harveyi. Based on the test results, when the detected content was 1.0 × 10⁻⁶, the result was considered satisfactory. 0 cfu / g ~1.0×10 1 A cfu / g level is considered a successful treatment; a detected level of 1.0 × 10⁻⁶ is considered a successful treatment. 2 cfu / g ~1.0×10 3 A level of cfu / g is considered a treatment failure.

[0107] Comparative Example 5

[0108] This comparative study used bacteriophages (Vibrio parahaemolyticus bacteriophage and Vibrio harveyi bacteriophage in a 1:1 weight ratio, purchased from Qingdao Nuoan Biotech Biotechnology Co., Ltd.) for culture. The dosage of bacteriophages in the nursery pond was 2.5 ppm, twice a day, without draining the pond water. The bacteriophages were sprinkled into the original pond once in the morning and once in the afternoon, for a total of 3 days.

[0109] Performance testing

[0110] 1. At the Jijia Base of Guangdong Haiwei Agricultural Group, seedlings diagnosed with Vibrio infection in the nursery ponds were cultured using the methods of Examples 1-2 and Comparative Examples 1-3, with 3 parallel groups (3 nursery ponds) per group. The average Vibrio content was recorded on days 1-3 (D1, D2, and D3). After the experiment, based on the test results, the average Vibrio content was determined. Treatment is considered successful if the average Vibrio content is [missing information]. If the result is not satisfactory, it is considered a treatment failure, and the results are shown in Table 1.

[0111]

[0112] Experimental results show that, in Examples 1-2, using specific ratios of compound bacteria A and compound bacteria B, the method of the present invention achieves a high success rate in treating Vibrio-infected seedlings within 72 hours. In particular, the success rate in Example 1 is consistently above 90%. However, in Comparative Examples 1-3, changing the types or ratios of compound bacteria A and compound bacteria B resulted in varying degrees of decreased treatment success rates. In Comparative Example 1, changing the species of compound bacteria A and compound bacteria B led to treatment failure in all cases. In Comparative Example 2, changing the species of compound bacteria A resulted in partial treatment failure, indicating that the compound bacteria A and compound bacteria B of the present invention have a synergistic therapeutic effect. In Comparative Example 3, changing the ratio of compound bacteria A and compound bacteria B decreased the therapeutic effect, demonstrating that the method of the present invention can only achieve a high success rate when using specific types and ratios of bacteria working synergistically.

[0113] 2. Two years of treatment application for shrimp larvae were carried out at the Jijia Base of Guangdong Haiwei Agricultural Group. From March to May 2023, Vibrio treatment experiments were conducted using Example 1, Comparative Example 4 and Comparative Example 5.

[0114] 72 hours later, samples were taken for further testing of the average levels of Vibrio parahaemolyticus and Vibrio harveyi. After treatment, based on the test results, when the detected levels were... If the content is not detected, it is considered a successful treatment. This is considered a treatment failure.

[0115] The treatment results are shown in Tables 2 and 3.

[0116]

[0117]

[0118]

[0119] In the table, the Vibrio content is the average content of Vibrio parahaemolyticus and Vibrio harveyi in the total infected seedling ponds. D3 and D4 refer to the average content of Vibrio parahaemolyticus and Vibrio harveyi in the infected seedling ponds on the third and fourth days of treatment, respectively. The number of ponds discharged represents the number of seedling ponds that failed treatment. The cure rate is the number of seedling ponds that were successfully treated. The survival rate is the average survival rate of the successfully treated seedling ponds.

[0120] ; .

[0121] The results showed that from March to May 2023, a total of 30 shrimp larvae ponds were treated using the method in Comparative Example 4. Before treatment, the Vibrio content was 5.6 × 10⁻⁶. 4 After treatment, the cfu / g concentration of Vibrio d3 was 5.5 × 10⁻⁶. 2 cfu / g, D4 Vibrio content was 2.0×10 3The cfu / g concentration was 68.1%, the cure rate was 68.1%, and the seedling survival rate was 62.9%. From March to May 2023, a total of 29 shrimp larvae ponds were treated using the method in Comparative Example 5. Before treatment, the Vibrio content was 6.8 × 10⁻⁶. 4 After treatment, the cfu / g concentration of Vibrio d3 was 6.8 × 10⁻⁶. 2 cfu / g, D4 Vibrio content was 1.5×10 1 The cfu / g concentration was 81.9%, the cure rate was 73.5%, and the seedling survival rate was 73.5%. From March to October 2024, a total of 116 shrimp larvae ponds were treated using the method in Example 1. Before treatment, the Vibrio content was 3.8 × 10⁻⁶. 5 After treatment, the cfu / g level of Vibrio D3 decreased to 9.0 × 10⁻⁶. 2 cfu / g, D4 Vibrio content decreased to 1.5×10 1 The cfu / g level was 94.7%, the cure rate was 94.7%, and the seedling survival rate was 88.7%.

[0122] The results showed that all three treatment methods reduced Vibrio levels to varying degrees. Comparative Example 4 showed the worst cure and survival rates, while Comparative Example 5, using bacteriophages, showed better results, although there were variations in treatment effectiveness across different batches in different months, indicating unstable treatment outcomes. Example 1 demonstrated the best treatment effect, showing an exponential decrease in Vibrio levels, high cure and survival rates, and no significant differences between batches in different months. This suggests that the use of functional bioflocs is highly effective in treating Vibrio disease in Litopenaeus vannamei larvae.

[0123] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for culturing Litopenaeus vannamei larvae using bioflocs, characterized in that, Includes the following steps: (1) Preparation of racetrack-shaped bioflocs, the operation steps are as follows: a. Three days before stocking the shrimp fry, add brown sugar and compound bacteria A to the water in the raceway pond; b. On the 4th day, shrimp fry were released into the raceway pool. c. During the first month of breeding, add brown sugar to the water in the raceway pool and add 1-2 ppm of compound bacteria A and 0.5-1 ppm of nitrifying bacteria daily; the compound bacteria A includes Bacillus pumilus, Bacillus subtilis and Bacillus licheniformis in a weight ratio of 1:(1.2-1.4):(0.5-0.7). d. During the second month of aquaculture, after the water in the raceway pool is allowed to settle in an Inhofe conical tube, the content of floc precipitate is less than 10 mL. e. After two months of culture, when a sudden and sharp drop in nitrite content was detected, water from the raceway pool was taken, subjected to Inhofe conical sedimentation, centrifuged, and dehydrated to obtain precipitate A. The precipitate A was then analyzed using the plate count method, and the Bacillus content was found to be greater than 1.0 × 10⁻⁶. 6 cfu / g precipitate A; f. After the water in the raceway aquaculture pond is filtered, it is pumped into an empty seedling pond, aerated and ready for use, to obtain biological flocs in the seedling pond. (2) Preparation of functional bioflocs: The operation steps are as follows: Add a mixture containing compound bacteria B to the bioflocs prepared in step (1) in the seedling pond, and continue aeration. After the water in the raceway pond is allowed to settle through an Inhofe conical tube, the content of the floc precipitate should reach 10~15mL. Take the floc precipitate for testing, and after centrifugation and dehydration, precipitate B is obtained. The content of Bacillus spores is greater than 1.0×10⁻⁶ using the plate count method. 7 CFU / g precipitate B yielded functional bioflocs; The components of the mixture containing compound bacteria B and their usage in the biofloc of the seedling pond are as follows: 1-2 ppm compound bacteria B, 1-2 ppm vitamin C, 10-20 ppm baking soda, 5-10 ppm brown sugar, 2-3 ppm Vibrio harveyi phage freeze-dried powder, 2-3 ppm Vibrio parahaemolyticus phage freeze-dried powder, 1-2 ppm shrimp seedlings, and 1-2 ppm Artemia spp.; the compound bacteria B includes Bacillus subtilis, Bacillus licheniformis, and Bacillus mucilaginosus in a weight ratio of (1.5-1.7):1:(0.2-0.5); (3) The farming methods for mysid shrimp and juvenile shrimp include the following steps: a. During the mysid and juvenile shrimp stages, place a drainage net frame in the nursery pond to lower the water level of the original nursery pond to 15-20cm. b. Pump the functional bioflocs prepared in step (2) into the original seedling pond to increase the water level of the seedling pond by 15-20cm and maintain it for 24 hours without feeding. c. After 24 hours, pump the functional bioflocs prepared in step (2) into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time, until the water level in the seedling pond reaches 80 to 85 cm, and continue not to feed. d. After 48 hours, take seedling samples for testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, the feeding can be resumed as usual; if the average content of Vibrio parahaemolyticus and Vibrio harveyi is 1.0 × 10⁻⁶, the feeding can be resumed as usual. 1 cfu / g ~1.0×10 3 cfu / g, add the functional bioflocs prepared in step (2) to the seedling pond; e. After 72 hours, take seedling samples for further testing and calculate the average content of Vibrio parahaemolyticus and Vibrio harveyi. When the detected content is 1.0 × 10⁻⁶, the result is considered satisfactory. 0 cfu / g ~1.0×10 1 A concentration of CFU / g is considered a successful treatment for Vibrio infection; a detection level of 1.0 × 10⁻⁶ CFU / g is also considered a successful treatment. 2 cfu / g ~1.0×10 3 cfu / g, repeat step d until treatment is successful; The rearing method for seedlings includes the following steps: a. During the seedling stage, place a seedling collection frame in the seedling collection pit next to the seedling pond, add water to the seedling collection frame, connect the seedling collection net frame to the drainage outlet of the seedling pond, and drain the water in the seedling pond into the seedling collection frame until the water level in the seedling pond drops to 15~20cm. During this process, use a net to scoop the large seedlings into the seedling collection bucket for temporary rearing every 5 minutes. b. After the seedling collection is completed, disinfect the seedlings in the collection buckets; c. Pump the functional bioflocs prepared in step (2) into the empty seedling pond, add water to a height of 40-45cm, move the large seedlings from the seed collection bucket into the seedling pond, and keep them without feeding for 24 hours. d. After 24 hours, pump the functional bioflocs prepared in step (2) into the seedling pond every 5 to 7 hours, increasing the water level by 8 to 10 cm each time, until the water level in the seedling pond reaches 80 to 85 cm, and continue not to feed. e. After 48 hours, take samples of large seedlings to test the average content of Vibrio parahaemolyticus and Vibrio harveyi. If Vibrio parahaemolyticus and Vibrio harveyi are not detected, feeding can be resumed without changing the water; if the detected content is 1.0 × 10⁻⁶, the feeding can be resumed. 1 cfu / g ~1.0×10 3 cfu / g, add the functional bioflocs prepared in step (2) to the seedling pond every 6-8 hours; f. After 72 hours, samples were taken for further testing of the average content of Vibrio parahaemolyticus and Vibrio harveyi. At the end of the test, based on the results, a detection value of 1.0 × 10⁻⁶ was considered acceptable. 0 cfu / g ~1.0×10 1 A concentration of CFU / g is considered a successful treatment for Vibrio infection; a detected concentration of 1.0 × 10⁻⁶ CFU / g is considered a successful treatment. 2 cfu / g ~1.0×10 3 cfu / g, repeat step e until treatment is successful.

2. The method for culturing Litopenaeus vannamei larvae using bioflocs according to claim 1, characterized in that, After two months of culture in step (1), quicklime, dolomite powder or sodium bicarbonate are added to the culture water according to the pH and total alkalinity test results. For every 10 ppm decrease in total alkalinity, 20-30 ppm is added. When the nitrite content is detected to suddenly drop sharply and eventually remain close to 0, it indicates that the bioflocs have formed a stable form.

3. The method for culturing Litopenaeus vannamei larvae using bioflocs according to claim 1, characterized in that, After measuring with an Inhofe conical tube in step (2), if the biofloc content is less than 10 mL, continue to add 2-5 ppm of brown sugar and 10-20 ppm of baking soda.

4. The method for culturing Litopenaeus vannamei larvae using bioflocs according to claim 1, characterized in that, In step (1), shrimp fry are stocked by selecting shrimp fry that have been raised to a size of 1.5-2cm in length, and stocking density is 300-500 shrimp per cubic meter of water.

5. The method for culturing Litopenaeus vannamei larvae using bioflocs according to claim 1, characterized in that, In step (1), three days before stocking the shrimp larvae, add 5-10 ppm of brown sugar and 2-3 ppm of compound bacteria A to the water in the raceway pond.

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