A breeding method for improving fish immunity

Abstract

The present invention provides a breeding method for improving fish immunity, comprising the following steps: S1. breeding site selection, S2. cage specifications and layout, S3. fry selection and placement, S4. fry disinfection, S5. adaptability training S6. breeding management, S7. bottom mud management. The fry disinfection of the present invention is carried out by soaking and disinfecting the fry with a composite disinfectant composed of povidone iodine, polysorbate, citric acid, and vitamin C in a specific proportion, effectively killing pathogenic microorganisms on the surface of the fish and the gills. The breeding management of the present invention comprehensively enhances the immunity of the fish by feeding an immunomodulator composed of a specific proportion of rape sterol palmitate, immunomodulatory peptide, β-glucan, and astaxanthin. The bottom mud management of the present invention reduces the content of harmful substances, inhibits the growth of pathogenic bacteria, promotes the circulation of nutrients and optimizes the water quality environment by placing a bottom mud modifier composed of a specific proportion of sodium molybdate, nitrifying bacteria, photosynthetic bacteria, tetrandrine, and sodium alginate, and promotes the healthy growth of fry.

Description

Technical Field

[0001] The invention relates to the technical field of fish breeding, and in particular to a breeding method for improving fish immunity. Background Art

[0002] Fish, especially marine fish such as golden pomfret, sea bream, and grouper, are rich in high nutritional value, mainly including high-quality protein, unsaturated fatty acids, minerals, and vitamins. These nutrients have many benefits for the human body, such as high-quality protein helps to enhance immunity and promote growth and development; unsaturated fatty acids, especially omega-3 fatty acids, are beneficial to heart and blood vessel health, help to lower cholesterol and triglyceride levels, and prevent cardiovascular diseases; at the same time, minerals and vitamins in marine fish also play an important role in maintaining bone health, enhancing immunity, and regulating metabolism. Marine fish farming is of great significance to the development of my country's fishery. It not only provides abundant aquatic products to meet market demand, but also promotes economic development in coastal areas and increases fishermen's income. There are many methods of marine aquaculture, such as pond farming and cage farming. Among them, pond farming has relatively low costs and is easy to manage, but is limited by geographical location and water quality conditions; cage farming can make more efficient use of marine resources, but the long-term lack of silt removal in cage farming areas can easily lead to silt accumulation at the bottom due to accumulation of feed and excrement, affecting water quality and the living environment of fish. At the same time, harmful substances in the sludge will continue to be released into the water, posing a threat to the health of fish. To prevent and treat fish diseases, fishermen often add antibiotics to feed. However, long-term use of antibiotics not only easily makes pathogens resistant, but may also cause an imbalance in the intestinal flora of fish and affect their immune function. In addition, antibiotic residues can also pose a potential threat to human health through the food chain. Summary of the invention

[0003] In view of this, the present invention proposes a breeding method for improving fish immunity to solve the above problems.

[0004] The technical solution of the present invention is achieved as follows: a breeding method for improving fish immunity, comprising the following steps:

[0005] S1. Selection of breeding sites: Select a sea area with a water depth of 30-40m, sandy texture and a flat bottom.

[0006] S2. Cage specifications and layout: Select 6.2m×6.2m×6.2m wind and wave resistant cages, and each group of cages shall be arranged longitudinally along the direction of tidal flow.

[0007] S3. Selection and release of fry: Select healthy and lively marine fish fry weighing 100-150g, with intact fish bodies, neat scales and no parasites on the body surface, and release 500-1200 fry into each cage.

[0008] S4. Disinfection of fish fry: Use a compound disinfectant to soak and disinfect the fish fry. Dilute the compound disinfectant 15-25 times and soak for 20-30 minutes. The compound disinfectant is composed of povidone iodine, polysorbate, citric acid, and vitamin C in a mass volume ratio of g / mL of (10-15):(2-5):(5-10):(4-6).

[0009] S5. Adaptive training: Place the fry in the training pond for adaptive training. From the 1st to the 3rd day of training, the water flow rate in the training pond is set to 20-30 cm / s, the pH value is set to 7.8-8.0, and the salinity is set to 31-32‰. From the 4th to the 7th day of training, the water flow rate in the training pond is set to 30-50 cm / s, the pH value is set to 8.0-8.4, and the salinity is set to 32-33‰. From the 8th to the 14th day of training, the water flow rate in the training pond is set to 50-70 cm / s, the pH value is set to 8.4-8.6, and the salinity is set to 33-35‰ to complete the adaptive training.

[0010] S6. Farming management: During the juvenile stage, feed the basic feed I 4 times a day, at 6:00-6:30, 10:00-10:30 in the morning and 14:00-14:30, 18:00-18:30 in the afternoon, 0.2-0.5% of the fry body weight each time, and feed the immune modulator once every 7-10 days. During the middle stage, feed the basic feed II twice a day, at 8:00-8:30 in the morning and 15:00-15:30 in the afternoon, 0.5-1.0% of the fry body weight each time, and feed the immune modulator once every 10-14 days. During the adult stage, the fish are separated and thinned, and the stocking density in each cage is 50-120 fish. The fish are fed with basic feed III once a day at 9:00-9:30 in the morning, and each time the feed is 1.0-2.0% of the weight of the fry. The fish are fed with an immune modulator once every 14-21 days. The immune modulator is composed of campesterol palmitate, immune modulator peptide, β-glucan, and astaxanthin in a mass volume g / mL ratio of (3.0-5.0):(1.5-2.5):(5.0-10.0):(0.4-0.8), and each time the feed is 0.1-0.2% of the weight of the fry.

[0011] S7. Sediment management: Add sediment improver to the sediment every 90-100 days. The sediment improver is composed of sodium molybdate, nitrifying bacteria, photosynthetic bacteria, tetrandrine, and sodium alginate in a mass ratio of (2.0-4.0):(0.01-0.03):(0.01-0.03):(4.0-6.0):(2.0-4.0).

[0012] Furthermore, the seawater velocity in the sea area of ​​S1 is 50-100 cm / s, dissolved oxygen ≥ 6 mg / L, pH value is 7.8-8.6, salinity is 31-35‰, and transparency is 8-12m.

[0013] Furthermore, the longitudinal spacing within the cage groups in S2 is 0.6-1.0 m, 3-7 cages are arranged in each group, and the lateral spacing between cage groups is 40-60 m.

[0014] Furthermore, in S3, the release is carried out on sunny days from March to May, and the marine fish fry is one of sea bream, grouper and golden pomfret.

[0015] Furthermore, the basic material I in S6 is composed of the following raw materials in parts by weight: 30-40 parts of fish meal, 20-25 parts of oil cake, 10-15 parts of wheat flour, 10-15 parts of corn starch, 5-10 parts of shrimp shell powder, 5-10 parts of bone meal, 3-5 parts of vegetable oil, 0.5-1.0 parts of choline, 0.4-0.6 parts of vitamin premix, and 0.4-0.6 parts of mineral premix.

[0016] Furthermore, the basic material II in S6 is composed of the following raw materials in parts by weight: 35-45 parts of fish meal, 20-25 parts of oil cake, 10-15 parts of wheat flour, 10-15 parts of corn starch, 3-5 parts of shrimp shell powder, 10-15 parts of bone meal, 4-6 parts of vegetable oil, 0.5-1.0 parts of choline, 0.5-0.7 parts of vitamin premix, and 0.5-0.7 parts of mineral premix.

[0017] Furthermore, the basic material III in S6 is composed of the following raw materials in parts by weight: 40-50 parts of fish meal, 15-20 parts of oil cake, 5-10 parts of wheat flour, 5-10 parts of corn starch, 15-20 parts of bone meal, 5-7 parts of vegetable oil, 0.5-1.0 parts of choline, 0.6-0.8 parts of vitamin premix, and 0.6-0.8 parts of mineral premix.

[0018] Furthermore, the vegetable oil is one of peanut oil, soybean oil, corn oil, sunflower oil, coconut oil, and sesame oil.

[0019] Furthermore, the vitamin premix is ​​composed of vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin D3, vitamin E, vitamin K3 and vitamin C in a mass ratio of (1-3):(1-3):(2-4):(0.3-0.7):(10-15):(4-8):(10-15):(1-3):(10-20).

[0020] Furthermore, the mineral premix is ​​composed of magnesium sulfate, iron sulfate, zinc sulfate, copper sulfate and sodium selenite in a mass ratio of (10-15):(5-10):(3-5):(0.06-0.1):(0.01-0.03).

[0021] Furthermore, the campesterol palmitate in S6 is prepared by the following method: campesterol and palmitic acid are mixed uniformly in a molar ratio of 1:1 to obtain mixture I; supercritical carbon dioxide is added to mixture I, stirred and dissolved to obtain mixture II, wherein the amount of supercritical carbon dioxide added is 80-120% of the volume of mixture I; lipase is added, and the reaction is carried out at 50-60°C for 60-72h, wherein the amount of lipase added is 5-10% of the volume of mixture II; the reaction is terminated, the temperature is increased to 80-90°C to inactivate the enzyme, and the supercritical carbon dioxide is removed by decompression to obtain campesterol palmitate.

[0022] Further, the immunomodulatory peptide in S6 is prepared by the following method: crushing hemp seed, adding n-hexane thereto according to a solid-liquid ratio of 1:(4-6), soaking for 3-4h, filtering out the liquid, and obtaining defatted hemp seed powder; adding water thereto according to a solid-liquid ratio of 1:(8-12), adding sodium hydroxide to adjust the pH value to 8.0-9.0, extracting for 40-60min under the conditions of ultrasonic power of 200-400W and ultrasonic frequency of 20-40kHz, filtering out the filter residue to obtain an extract, adding hydrochloric acid of 1-3% by volume to the extract, slowly stirring to precipitate protein, and placing it in a centrifuge for 4000 -6000r / min centrifugal separation for 10-20min to obtain hemp seed protein; the hemp seed protein and the composite enzyme are mixed in a mass ratio of 1:(0.2-0.4), and enzymolysis is carried out at 40-50°C for 2-4h; after the enzymolysis is completed, the temperature is increased to 80-90°C to inactivate the enzyme, and the immunomodulatory peptide is obtained by centrifugal separation at 4000-6000r / min for 10-20min; the composite enzyme is composed of neutral protease, alkaline protease, xylanase and β-glucanase in a mass ratio of (1.0-2.0):(1.0-2.0):(0.4-0.6):(0.4-0.6).

[0023] Furthermore, the amount of the bottom mud improver in S7 is 100-200g per cage, and the activity of the nitrifying bacteria and photosynthetic bacteria is 1×10 7 ≥CFU / g

[0024] Compared with the prior art, the invention has the following beneficial effects: the invention comprehensively enhances the immunity of fish by feeding an immunomodulator composed of a specific ratio of campesterol palmitate, immunomodulatory peptides, β-glucan, and astaxanthin. Among them, campesterol palmitate protects the immune cells of fish from damage by reducing oxidative stress and inflammatory response, helps maintain the balance of the immune system, and improves the disease resistance of the body. β-glucan activates the immune cells of fish, promotes the secretion of cytokines, enhances the immune response reaction and non-specific immune defense ability, thereby enhancing the resistance of fish to pathogens. Immunomodulatory peptides regulate the activity and function of immune cells by binding to immune cell receptors in fish, promote the proliferation and differentiation of immune cells, and enhance immune response. Astaxanthin reduces the damage of oxidative stress to immune cells by scavenging free radicals and protecting cells from oxidative damage, protects the normal function of immune cells, and thus improves the immune ability of fish. The immunomodulator of the invention works together through multiple mechanisms to significantly improve the immune ability of fish, comprehensively enhance the cellular immunity and humoral immunity of fish, and improve the defense ability of fish to pathogens.

[0025] In addition, the present invention uses a composite disinfectant composed of povidone iodine, polysorbate, citric acid, and vitamin C in a specific ratio to immerse and disinfect the fry, effectively killing pathogenic microorganisms on the surface of the fish body and the gills. Adaptive training is then used to reduce the stress response of the fry due to sudden environmental changes, which helps the fry maintain a stable physiological and psychological state, helps the fry strengthen their physical fitness and immunity, and improves their resistance to pathogens. The content of harmful substances is reduced by adding a sediment modifier, the growth of pathogens is inhibited, the circulation of nutrients is promoted, and the water quality environment is optimized.

[0026] The present invention improves the immunity of fish through the synergistic effect of multiple mechanisms, thereby promoting the improvement of the survival rate and weight gain rate of fry. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0030] Example 1

[0031] A breeding method for improving fish immunity comprises the following steps:

[0032] S1. Selection of breeding sites: Select a sea area with a water depth of 30m, sandy texture, flat bottom, seawater flow rate of 50-100cm / s, dissolved oxygen ≥6mg / L, pH value of 7.8-8.6, salinity of 31-35‰, and transparency of 8-12m.

[0033] S2. Cage specifications and layout: Select 6.2m×6.2m×6.2m wind and wave resistant cages. Each group of cages is arranged longitudinally along the direction of tidal flow. The longitudinal spacing within the cage group is 0.6m. Each group is equipped with 3 cages, and the horizontal spacing between cage groups is 40m.

[0034] S3. Selection and release of fry: Select healthy and lively, intact fish bodies, neatly scaled, and parasite-free 100g sea bream fry, and release 500 fry into each cage on a sunny day in March.

[0035] S4. Disinfection of fish fry: Use a compound disinfectant to soak and disinfect the fish fry. Dilute the compound disinfectant 15 times and soak for 20 minutes. The compound disinfectant is composed of povidone iodine, polysorbate, citric acid, and vitamin C in a mass volume ratio of 10:2:5:4 g / mL.

[0036] S5. Adaptive training: Place the fry in the training pond for adaptive training. From the 1st to the 3rd day of training, the water flow rate in the training pond is set to 20 cm / s, the pH value is set to 7.8, and the salinity is set to 31‰. From the 4th to the 7th day of training, the water flow rate in the training pond is set to 30 cm / s, the pH value is set to 8.0, and the salinity is set to 32‰. From the 8th to the 14th day of training, the water flow rate in the training pond is set to 50 cm / s, the pH value is set to 8.4, and the salinity is set to 33‰ to complete the adaptive training.

[0037] S6. Farming management: During the juvenile stage, feed the basic feed I 4 times a day, at 6:00 and 10:00 in the morning and 14:00 and 18:00 in the afternoon, and feed 0.2% of the fry's body weight each time. Feed immune modulators once every 7 days. During the middle-aged stage, feed the basic feed II twice a day, at 8:00 in the morning and 15:00 in the afternoon, and feed 0.5% of the fry's body weight each time. Feed immune modulators once every 10 days. During the adult stage, thinning is carried out, with a culture density of 50 fish per cage. Feed the basic feed III once a day, at 9:00 in the morning, and feed 1.0% of the fry's body weight each time. Feed immune modulators once every 14 days. The immunomodulator consists of campesterol palmitate, immunomodulatory peptide, β-glucan and astaxanthin in a mass volume g / mL ratio of 3.0:1.5:5.0:0.4, and is fed at 0.1% of the fry body weight each time. Among them, basic material I is composed of the following raw materials in parts by weight: 30 parts of fish meal, 20 parts of oil cake, 10 parts of wheat flour, 10 parts of corn starch, 5 parts of shrimp shell meal, 5 parts of bone meal, 3 parts of vegetable oil, 0.5 part of choline, 0.4 part of vitamin premix, and 0.4 part of mineral premix; basic material II is composed of the following raw materials in parts by weight: 35 parts of fish meal, 20 parts of oil cake, 10 parts of wheat flour, 10 parts of corn starch, 3 parts of shrimp shell meal, 10 parts of bone meal, 4 parts of vegetable oil, 0.5 part of choline, 0.5 part of vitamin premix, and 0.5 part of mineral premix; basic material III is composed of the following raw materials in parts by weight: 40 parts of fish meal, 15 parts of oil cake, 5 parts of wheat flour, 5 parts of corn starch, 15 parts of bone meal, 5 parts of vegetable oil, 0.5 part of choline, 0.6 part of vitamin premix, and 0.6 part of mineral premix. The vegetable oil is peanut oil, the vitamin premix is ​​composed of vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin D3, vitamin E, vitamin K3, and vitamin C in a mass ratio of 1:1:2:0.3:10:4:10:1:10, and the mineral premix is ​​composed of magnesium sulfate, iron sulfate, zinc sulfate, copper sulfate, and sodium selenite in a mass ratio of 10:5:3:0.06:0.01. Among them, rape sterol palmitate is prepared by the following method: rape sterol and palmitic acid are mixed uniformly in a molar ratio of 1:1 to obtain a mixture I; supercritical carbon dioxide is added to the mixture I, stirred and dissolved to obtain a mixture II, and the amount of supercritical carbon dioxide added is 80% of the volume of the mixture I; lipase is added, and the reaction is heated at 50°C for 72 hours, and the amount of lipase added is 5% of the volume of the mixture II; the reaction is terminated, the temperature is increased to 80°C to kill the enzyme, and the supercritical carbon dioxide is removed by decompression to obtain rape sterol palmitate.The immunomodulatory peptide is prepared by the following method: crushing hemp seeds, adding n-hexane thereto at a solid-liquid ratio of 1:4, soaking for 4 hours, filtering out the liquid, and obtaining defatted hemp seed powder; adding water thereto at a solid-liquid ratio of 1:8, adding sodium hydroxide to adjust the pH value to 8.0, extracting for 60 minutes under the conditions of an ultrasonic power of 200 and an ultrasonic frequency of 20 kHz, filtering out the filter residue to obtain an extract, adding hydrochloric acid of 1% by volume to the extract and slowly stirring to precipitate protein and The method comprises placing the hemp seed protein in a centrifuge and centrifuging at 4000 r / min for 20 minutes to obtain hemp seed protein; mixing the hemp seed protein with a composite enzyme at a mass ratio of 1:0.2, and performing enzymolysis at 40°C for 4 hours; after the enzymolysis is completed, raising the temperature to 80°C to inactivate the enzyme, and centrifuging at 4000 r / min for 20 minutes to obtain an immunomodulatory peptide; the composite enzyme consists of a neutral protease, an alkaline protease, a xylanase, and a β-glucanase in a mass ratio of 1.0:1.0:0.4:0.4.

[0038] S7. Sediment management: Every 90 days, sediment improver was added to the sediment. The amount added to each cage was 100 g. The sediment improver consisted of sodium molybdate, nitrifying bacteria, photosynthetic bacteria, tetrandrine, and sodium alginate in a mass ratio of 2.0:0.01:0.01:4.0:2.0. The activity of nitrifying bacteria and photosynthetic bacteria was 1×10 7 CFU / g.

[0039] Example 2

[0040] A breeding method for improving fish immunity comprises the following steps:

[0041] S1. Selection of breeding sites: Select a sea area with a water depth of 35m, sandy texture, flat bottom, seawater flow rate of 50-100cm / s, dissolved oxygen ≥6mg / L, pH value of 7.8-8.6, salinity of 31-35‰, and transparency of 8-12m.

[0042] S2. Cage specifications and layout: Select 6.2m×6.2m×6.2m wind and wave resistant cages. Each group of cages is arranged longitudinally along the direction of tidal flow. The longitudinal spacing within the cage group is 0.8m. Each group is equipped with 5 cages, and the horizontal spacing between cage groups is 50m.

[0043] S3. Selection and release of fry: Select healthy and lively, intact fish bodies, neatly scaled, and parasite-free 125g Gentian grouper fry, and release 800 fry into each cage on a sunny day in April.

[0044] S4. Disinfection of fish fry: Use a compound disinfectant to soak and disinfect the fish fry. Dilute the compound disinfectant 20 times and soak for 25 minutes. The compound disinfectant is composed of povidone iodine, polysorbate, citric acid, and vitamin C in a mass volume ratio of 12.5:3.5:7.5:5 g / mL.

[0045] S5. Adaptive training: Place the fry in the training pond for adaptive training. From the 1st to the 3rd day of training, the water flow rate in the training pond is set to 25 cm / s, the pH value is set to 7.9, and the salinity is set to 31.5‰. From the 4th to the 7th day of training, the water flow rate in the training pond is set to 40 cm / s, the pH value is set to 8.2, and the salinity is set to 32.5‰. From the 8th to the 14th day of training, the water flow rate in the training pond is set to 60 cm / s, the pH value is set to 8.5, and the salinity is set to 34‰ to complete the adaptive training.

[0046] S6. Farming management: During the juvenile stage, feed the basic feed I four times a day, at 6:15 am, 10:15 am, 2:15 pm, 6:15 pm, and 0.35% of the fry's body weight each time. Feed the immune modulator once every 8 days. During the middle stage, feed the basic feed II twice a day, at 8:15 am and 3:15 pm, at 0.75% of the fry's body weight each time. Feed the immune modulator once every 12 days. During the adult stage, thinning is carried out, with a culture density of 80 fish per cage. Feed the basic feed III once a day, at 9:15 am, at 1.5% of the fry's body weight each time. Feed the immune modulator once every 18 days. The immunomodulator is composed of campesterol palmitate, immunomodulatory peptide, β-glucan and astaxanthin in a mass volume g / mL ratio of 4.0:2.0:7.5:0.6, and is fed at 0.15% of the fry body weight each time. Among them, the basic feed I is composed of the following raw materials in parts by weight: 35 parts of fish meal, 22.5 parts of oil cake meal, 12.5 parts of wheat flour, 12.5 parts of corn starch, 7.5 parts of shrimp shell meal, 7.5 parts of bone meal, 4 parts of vegetable oil, 0.8 parts of choline, 0.5 parts of vitamin premix, and 0.5 parts of mineral premix. The basic feed II is composed of the following raw materials in parts by weight: 40 parts of fish meal, 22.5 parts of oil cake meal, 12.5 parts of wheat flour, and corn starch. Basic material III is composed of the following raw materials in parts by weight: 45 parts of fish meal, 17.5 parts of oil cake, 7.5 parts of wheat flour, 7.5 parts of corn starch, 17.5 parts of bone meal, 6 parts of vegetable oil, 0.7 parts of choline, 0.7 parts of vitamin premix, and 0.7 parts of mineral premix. The vegetable oil is soybean oil, the vitamin premix is ​​composed of vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin D3, vitamin E, vitamin K3, and vitamin C in a mass ratio of 2:2:3:0.5:12.5:6:12.5:2:15, and the mineral premix is ​​composed of magnesium sulfate, iron sulfate, zinc sulfate, copper sulfate, and sodium selenite in a mass ratio of 12.5:7.5:4:0.08:0.02. Among them, campesterol palmitate is prepared by the following method: campesterol and palmitic acid are uniformly mixed in a molar ratio of 1:1 to obtain mixture I; supercritical carbon dioxide is added to mixture I, stirred and dissolved to obtain mixture II, and the amount of supercritical carbon dioxide added is 100% of the volume of mixture I; lipase is added, and the reaction is carried out at 55°C for 66 hours, and the amount of lipase added is 7.5% of the volume of mixture II; the reaction is terminated, the temperature is increased to 85°C to inactivate the enzyme, and the supercritical carbon dioxide is removed by decompression to obtain campesterol palmitate.The immunomodulatory peptide is prepared by the following method: crushing hemp seeds, adding water thereto at a material-liquid ratio of 1:5 and kernel powder at a material-liquid ratio of 1:10, adding sodium hydroxide to adjust the pH value to 8.5, extracting for 50 minutes under the conditions of an ultrasonic power of 300W and an ultrasonic frequency of 30kHz, filtering out the filter residue to obtain an extract, adding 2% hydrochloric acid by volume to the extract, slowly stirring to precipitate protein, placing the extract in a centrifuge and centrifuging at 5000r / min for 15 minutes to obtain hemp seed protein; mixing the hemp seed protein with a composite enzyme at a mass ratio of 1:0.3, and performing enzymolysis at 45°C for 3 hours; after the enzymolysis is completed, raising the temperature to 85°C to inactivate the enzyme, and centrifuging at 5000r / min for 15 minutes to obtain the immunomodulatory peptide; the composite enzyme consists of a neutral protease, an alkaline protease, a xylanase, and a beta-glucanase in a mass ratio of 1.5:1.5:0.5:0.5.

[0047] S7. Sediment management: Every 95 days, sediment improver was added to the sediment. The amount added to each cage was 150 g. The sediment improver consisted of sodium molybdate, nitrifying bacteria, photosynthetic bacteria, tetrandrine, and sodium alginate in a mass ratio of 3.0:0.02:0.02:5.0:3.0. The activity of nitrifying bacteria and photosynthetic bacteria was 5×10 7 CFU / g.

[0048] Example 3

[0049] A breeding method for improving fish immunity comprises the following steps:

[0050] S1. Selection of breeding sites: Select a sea area with a water depth of 40m, sandy texture, flat bottom, seawater flow rate of 50-100cm / s, dissolved oxygen ≥6mg / L, pH value of 7.8-8.6, salinity of 31-35‰, and transparency of 8-12m.

[0051] S2. Cage specifications and layout: Select 6.2m×6.2m×6.2m wind and wave resistant cages. Each group of cages is arranged longitudinally along the direction of tidal flow. The longitudinal spacing within the cage group is 1.0m. Each group is equipped with 7 cages, and the horizontal spacing between cage groups is 60m.

[0052] S3. Selection and release of fry: Select 150g golden pomfret fry that are healthy and lively, intact, with neat scales and no parasites on the body surface. The marine fish fry can be one of sea bream, grouper, and golden pomfret. 1,200 fry are released in each cage on a sunny day in May.

[0053] S4. Disinfection of fish fry: Use a compound disinfectant to soak and disinfect the fish fry. Dilute the compound disinfectant 25 times and soak for 30 minutes. The compound disinfectant is composed of povidone iodine, polysorbate, citric acid, and vitamin C in a mass volume ratio of 15:5:10:6 g / mL.

[0054] S5. Adaptive training: Place the fry in the training pond for adaptive training. From the 1st to the 3rd day of training, the water flow rate in the training pond is set to 30 cm / s, the pH value is set to 8.0, and the salinity is set to 32‰. From the 4th to the 7th day of training, the water flow rate in the training pond is set to 50 cm / s, the pH value is set to 8.4, and the salinity is set to 33‰. From the 8th to the 14th day of training, the water flow rate in the training pond is set to 70 cm / s, the pH value is set to 8.6, and the salinity is set to 35‰ to complete the adaptive training.

[0055] S6. Farming management: During the juvenile stage, feed the basic feed I four times a day, at 6:30 and 10:30 in the morning and 14:30 and 18:30 in the afternoon, and feed 0.5% of the fry's body weight each time. Feed immune modulators once every 10 days. During the middle stage, feed the basic feed II twice a day, at 8:30 in the morning and 15:30 in the afternoon, and feed 1.0% of the fry's body weight each time. Feed immune modulators once every 14 days. During the adult stage, thinning is carried out, with a culture density of 120 fish per cage. Feed the basic feed III once a day, at 9:30 in the morning, and feed 1.0% of the fry's body weight each time. Feed immune modulators once every 21 days. The immunomodulator consists of campesterol palmitate, immunomodulatory peptide, β-glucan and astaxanthin in a mass volume g / mL ratio of 5.0:2.5:10.0:0.8, and is fed at 0.2% of the fry body weight each time. Among them, basic material I is composed of the following raw materials in parts by weight: 40 parts of fish meal, 25 parts of oil cake, 15 parts of wheat flour, 15 parts of corn starch, 10 parts of shrimp shell meal, 10 parts of bone meal, 5 parts of vegetable oil, 1.0 part of choline, 0.6 part of vitamin premix, and 0.6 part of mineral premix; basic material II is composed of the following raw materials in parts by weight: 45 parts of fish meal, 25 parts of oil cake, 15 parts of wheat flour, 15 parts of corn starch, 5 parts of shrimp shell meal, 15 parts of bone meal, 6 parts of vegetable oil, 1.0 part of choline, 0.7 part of vitamin premix, and 0.7 part of mineral premix; basic material III is composed of the following raw materials in parts by weight: 50 parts of fish meal, 20 parts of oil cake, 10 parts of wheat flour, 10 parts of corn starch, 20 parts of bone meal, 7 parts of vegetable oil, 1.0 part of choline, 0.8 part of vitamin premix, and 0.8 part of mineral premix. The vegetable oil is coconut oil, the vitamin premix consists of vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin D3, vitamin E, vitamin K3, and vitamin C in a mass ratio of 3:3:4:0.7:15:8:15:3:20, and the mineral premix consists of magnesium sulfate, iron sulfate, zinc sulfate, copper sulfate, and sodium selenite in a mass ratio of 15:10:5:0.1:0.03. Among them, campesterol palmitate is prepared by the following method: campesterol and palmitic acid are uniformly mixed in a molar ratio of 1:1 to obtain mixture I; supercritical carbon dioxide is added to mixture I, stirred and dissolved to obtain mixture II, and the amount of supercritical carbon dioxide added is 120% of the volume of mixture I; lipase is added, and the reaction is carried out at 60°C for 60 hours, and the amount of lipase added is 10% of the volume of mixture II; the reaction is terminated, the temperature is increased to 90°C to inactivate the enzyme, and the supercritical carbon dioxide is removed by decompression to obtain campesterol palmitate.The immunomodulatory peptide is prepared by the following method: crushing hemp seeds, adding n-hexane to the powder at a solid-liquid ratio of 1:6, soaking for 3 hours, filtering out the liquid, and obtaining defatted hemp seed powder; adding water to the defatted hemp seed powder at a solid-liquid ratio of 1:12, adding sodium hydroxide to adjust the pH value to 9.0, extracting for 40 minutes under the conditions of an ultrasonic power of 400W and an ultrasonic frequency of 40kHz, filtering out the filter residue to obtain an extract, adding 3% hydrochloric acid by volume to the extract, and slowly stirring to precipitate protein. The hemp seed protein is placed in a centrifuge and centrifuged at 6000r / min for 10 minutes to obtain hemp seed protein; the hemp seed protein is mixed with a composite enzyme at a mass ratio of 1:0.4, and enzymolysis is performed at 50°C for 2 hours; after the enzymolysis is completed, the temperature is increased to 90°C to inactivate the enzyme, and the mixture is centrifuged at 6000r / min for 10 minutes to obtain an immunomodulatory peptide; the composite enzyme is composed of neutral protease, alkaline protease, xylanase and β-glucanase in a mass ratio of 2.0:2.0:0.6:0.6.

[0056] S7. Sediment management: Every 100 days, sediment improver was added to the sediment. The amount added to each cage was 200 g. The sediment improver consisted of sodium molybdate, nitrifying bacteria, photosynthetic bacteria, tetrandrine, and sodium alginate in a mass ratio of 4.0:0.03:0.03:6.0:4.0. The activity of nitrifying bacteria and photosynthetic bacteria was 1×10 8 CFU / g.

[0057] Comparative Example 1

[0058] The difference between this comparative example and Example 2 is that no immunomodulator is fed in step S6.

[0059] Comparative Example 2

[0060] This comparative example is compared with Example 2, except that, in the S4 fry disinfection step, a disinfectant containing only povidone iodine is used for disinfection.

[0061] Comparative Example 3

[0062] The difference between this comparative example and Example 2 is that the adaptive training of step S5 is not performed.

[0063] Comparative Example 4

[0064] The difference between this comparative example and Example 2 is that the sediment improver consists of sodium molybdate, nitrifying bacteria, photosynthetic bacteria and sodium alginate in a mass ratio of 4.0:0.03:0.03:4.0.

[0065] 1. Determination of growth index

[0066] According to the methods of Examples 1-3 and Comparative Examples 1-3, a net cage was set up in the Xidao sea area of ​​Sanya City, Hainan Province for breeding, and the breeding period was 6 months. After the breeding was completed, the number of surviving fish was counted and the survival rate was calculated. Stop feeding for 24 hours, randomly pick 30 fish from each net cage, weigh them to calculate the average body weight, and calculate the average weight gain rate. The survival rate and average weight gain rate are shown in Table 1. The survival rate and average weight gain rate are calculated according to the following formula:

[0067] Survival rate = number of fish that survived / number of fish released × 100%

[0068] Average weight gain rate = (average weight at the end of breeding - 125) / 125 × 100%

[0069] Table 1

[0070]

[0071] It can be seen from Table 1 that Examples 1-3 are superior to Comparative Examples 1-4 in terms of survival rate and average weight gain rate. Among them, Example 2 has better performance.

[0072] By comparing Example 2 with Comparative Example 1, the survival rate increased from 62.3% to 90.3%, and the average weight gain rate increased from 265% to 380%. Example 2 feeds the immune modulator to enhance the cellular immunity and humoral immunity of the fry, thereby improving the resistance of the fry to pathogens, reducing the occurrence and spread of diseases, and thus improving the survival rate. The β-glucan therein can stimulate and activate immune cells, improve their phagocytic ability and bactericidal activity, thereby enhancing the overall immunity of the fish. Immunomodulatory peptides can regulate the immune response of the fish, enhance the activity and function of immune cells, thereby improving the defense ability of the fish against pathogens. Campesterol palmitate and astaxanthin can help remove free radicals in the body, reduce the damage of oxidative stress to the fish, thereby protecting the health of the fish and indirectly improving the survival rate. In addition, astaxanthin can also increase the activity of immune cells and promote wound healing, which helps the fish to stay healthy under stress and improve the survival rate. In addition, β-glucan can also promote the growth of beneficial intestinal bacteria and improve the structure of intestinal flora, thereby improving the utilization rate of feed and nutrient absorption rate of fish. This helps fish better absorb and utilize the nutrients in feed, promotes their growth, development and weight gain. Immunomodulatory peptides promote the growth, development and weight gain of fish by regulating the endocrine and metabolic systems of fish. Campesterol palmitate can improve the appetite and digestion ability of animals, help fish increase their feed intake, improve feed conversion rate, and thus promote weight gain. Astaxanthin promotes cell growth and division, which helps the proliferation of fish cells and the growth of tissues, thereby increasing the weight gain rate. The various components in immunomodulators work synergistically to promote the growth and development of fish. They not only improve the immunity and disease resistance of fish, but also improve the efficiency of nutrient absorption and utilization, thereby achieving an increase in survival rate and weight gain rate.

[0073] By comparing Example 2 with Comparative Example 2, immersion disinfection before fry is put in can effectively kill pathogenic microorganisms on the surface of fish body and gills, and reduce the risk of death caused by infection of pathogens in fry. Cut off the transmission route of pathogens at the same time, reduce the chance of cross infection between fry, thereby guarantee the overall health of fish school, enhance the physique of fry, reduce the occurrence of disease, make fish body can use more energy for growth rather than against pathogens, thereby promote weight gain. In Example 2, polysorbate helps to disperse povidone iodine more effectively in water, increase its solubility, thereby improve the utilization rate and disinfection effect of povidone iodine. Polysorbate can improve the wettability of povidone iodine, make it easier to penetrate into the tiny gap of fish body surface and fish scale, and enhance the killing effect of povidone iodine on pathogenic microorganisms. Citric acid provides an acidic environment, which helps to maintain the stability of povidone iodine. Vitamin C helps to protect povidone iodine from being oxidized and decomposed, thereby prolonging its effect.

[0074] By comparing Example 2 with Comparative Example 3, Example 2, through adaptive training, gradually increased water flow stimulation helps the fry to enhance muscle strength and swimming endurance, so that they can better adapt to the strong water flow conditions in the deep-sea cage, and reduce the mechanical damage and stress response caused by excessive water flow. The gradual adjustment of pH value and salinity helps the fry gradually adapt to the water quality conditions that may be encountered in the deep-sea cage, helps the fry maintain acid-base balance and osmotic pressure stability in the body, reduces physiological disorders and deaths caused by sudden changes in water quality, and thus improves the survival rate. Adaptive training allows the fry to experience a series of environmental changes before being released, so that they gradually adapt to the new environment in the deep-sea cage. This gradual adaptation process reduces the stress response of the fry caused by sudden environmental changes, helps the fry maintain a stable physiological and psychological state, helps the fry enhance physical fitness and immunity, and improves their resistance to pathogens. And adaptive training improves the swimming ability and foraging ability of the fry, and the trained fry can swim and forage more effectively, thereby increasing the food intake and nutrient intake. In addition, adaptive training enables the fry to adapt to the new environment more quickly and enter a normal growth state after being released. This reduces the growth stagnation period or growth rate reduction caused by poor environmental adaptation, thereby improving the growth efficiency and weight gain rate of the fry.

[0075] By comparing Example 2 with Comparative Example 4, the introduction of a sediment modifier can improve the sediment environment, reduce the content of harmful substances, inhibit the growth of pathogens, promote the circulation of nutrients and optimize the water quality environment, thereby promoting the survival rate and weight gain rate of fry. The tetrandrine in the sediment modifier of Example 2 can inhibit harmful microorganisms in the sediment, reduce their competitive pressure on nitrifying bacteria and photosynthetic bacteria, and thus be conducive to the reproduction and growth of beneficial microorganisms. In addition, tetrandrine can also regulate the growth hormone balance of submarine plants, stimulate cell division and growth of the root system, thereby promoting the development of the root system, thereby increasing the production of root secretions. These secretions contain a variety of organic acids and enzymes, which further decompose the organic matter in the sediment, form more pore channels, are conducive to the penetration of oxygen and the improvement of the air permeability of the sediment, and further improve the effect of the sediment modifier.

[0076] Two more comparison groups were added to further verify the effects of the immunomodulators.

[0077] Comparative Example 5

[0078] The difference between this comparative example and Example 2 is that an equal volume of campesterol is used in the immune modulator to replace campesterol palmitate.

[0079] Comparative Example 6

[0080] Compared with Example 2, the difference between this comparative example and Example 2 is that the immunomodulator does not contain immunomodulatory peptides, and is composed of campesterol palmitate, β-glucan, and astaxanthin in a mass volume g / mL ratio of 4.0:7.5:0.6.

[0081] 2. Determination of immune indicators

[0082] Two fish were taken from each of the net cages of Examples 1-3 and Comparative Examples 1, 5 and 6, placed on ice and blood was collected. After blood collection, the blood was placed in a heparin anticoagulant tube, centrifuged at 3000 r / min for 10 min, and the upper serum was taken. The alkaline phosphatase (ALP) and lysozyme (LSZ) contents were determined using a kit. The activity level of ALP can reflect the metabolic state and immune response ability of the fish body, and the activity level of LSZ can reflect the nonspecific immune defense ability of the fish body. The measured results are shown in Table 2.

[0083] Table 2

[0084]

[0085] By comparing Example 2 with Comparative Example 1 in Table 2, the alkaline phosphatase content of the immune conditioning agent of the present invention is increased from 67U / L to 109U / L, and the lysozyme content is increased from 263U / L to 389U / L. It can be seen that the immunity of fish can be effectively improved by feeding the immune conditioning agent of the present invention. Among them, campesterol palmitate protects the immune cells of the fish from damage by reducing oxidative stress and inflammatory response, helps to maintain the balance of the immune system, and improves the body's disease resistance. β-glucan activates the immune cells of the fish, promotes the secretion of cytokines, enhances the immune response and non-specific immune defense capabilities, thereby enhancing the resistance of the fish to pathogens. Immunomodulatory peptides regulate the activity and function of immune cells by binding to immune cell receptors in the fish, promote the proliferation and differentiation of immune cells, and enhance immune response. Astaxanthin reduces the damage of oxidative stress to immune cells and protects the normal function of immune cells by scavenging free radicals and protecting cells from oxidative damage, thereby improving the immune capacity of the fish. Feeding an immunomodulator composed of a specific ratio of campesterol, β-glucan, immunomodulatory peptides, and astaxanthin can work together through multiple mechanisms to significantly improve the immune capacity of the fish, comprehensively enhance the cellular immunity and humoral immunity of the fish, and improve the defense ability of the fish against pathogens. By comparing Example 2 with Comparative Example 2, the effect of campesterol palmitate is better than that of campesterol. In the present invention, campesterol palmitate is formed by esterification of campesterol and palmitic acid. This esterification reaction enhances the stability of the molecule, making it more resistant to the influence of adverse conditions such as oxidation and hydrolysis. And the absorption rate of campesterol in the intestine is low, while the esterified campesterol palmitate can significantly improve this absorption rate. This is because the esterified sterols are more likely to mix with lipids to form chylomicrons, which are then absorbed by the epithelial cells of the small intestine. By comparing Example 2 with Comparative Example 3, the lack of immunomodulatory peptides has a significant decrease in the effect of improving the immunity of fish. This is because immunomodulatory peptides can enhance the function of immune cells, promote antibody production, regulate immune balance, and play a role in other biological activities. These effects together promote the improvement of fish immunity, enabling fish to defend and eliminate pathogens more effectively when faced with invasion. Therefore, the role of immunomodulatory peptides in immunomodulators is crucial.

[0086] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A breeding method for improving fish immunity, characterized in that: The following steps are involved: S1. Selection of breeding sites: Select a sea area with a water depth of 30-40m and a flat bottom; S2. Cage specifications and layout: Select 6.2m×6.2m×6.2m wind and wave resistant cages, and each group of cages is arranged longitudinally along the direction of tidal flow; S3. Selection and placement of fry: Select healthy and lively, intact fish bodies, neatly scaled, and parasite-free marine fish fry weighing 100-150g, and place 500-1200 fry in each cage; S4. Fry disinfection: fry were immersed in a compound disinfectant and the fry were disinfected by diluting the compound disinfectant 15-25 times and soaking for 20-30 min, wherein the compound disinfectant was composed of povidone iodine, polysorbate, citric acid and vitamin C in a mass volume ratio of g / mL (10-15):(2-5):(5-10):(4-6); S5. Adaptive training: The fry were placed in a training pool for adaptive training. On the 1st to 3rd day of training, the water flow rate in the training pool was set to 20-30 cm / s, the pH value was set to 7.8-8.0, and the salinity was set to 31-32‰. On the 4th to 7th day of training, the water flow rate in the training pool was set to 30-50 cm / s, the pH value was set to 8.0-8.4, and the salinity was set to 32-33‰. On the 8th to 14th day of training, the water flow rate in the training pool was set to 50-70 cm / s, the pH value was set to 8.4-8.6, and the salinity was set to 33-35‰ to complete the adaptive training. S6. Farming management: During the juvenile stage, feed the basic feed I 4 times a day, at 6:00-6:30 in the morning, 10:00-10:30 in the morning, 14:00-14:30 in the afternoon, 18:00-18:30 in the afternoon, 0.2-0.5% of the body weight of the fry each time, and feed the immune modulator once every 7-10 days; during the middle fish stage, feed the basic feed II twice a day, at 8:00-8:30 in the morning and 15:00-15:30 in the afternoon, 0.5-1.0% of the body weight of the fry each time, and feed the immune modulator once every 10-14 days. Agent; thinning during the adult stage, the culture density of each cage is 50-120 fish, feeding basic feed III once a day, feeding at 9:00-9:30 in the morning, each feeding is based on 1.0-2.0% of the weight of the fry, and feeding once every 14-21 days. The immune modulator is composed of campesterol palmitate, immunomodulatory peptide, β-glucan, and astaxanthin in a mass volume g / mL ratio of (3.0-5.0):(1.5-2.5):(5.0-10.0):(0.4-0.8), and each feeding is based on 0.1-0.2% of the weight of the fry; S7. Sediment management: Every 90-100 days, add sediment improver to the sediment. The sediment improver is composed of sodium molybdate, nitrifying bacteria, photosynthetic bacteria, tetrandrine and sodium alginate in a mass ratio of (2.0-4.0):(0.01-0.03):(0.01-0.03):(4.0-6.0):(2.0-4.0).

2. A method for raising fish immunity according to claim 1, characterized in that: The seawater flow rate in the sea area S1 is 50-100 cm / s, dissolved oxygen is ≥ 6 mg / L, pH value is 7.8-8.6, salinity is 31-35‰, and transparency is 8-12m.

3. A method for raising fish immunity according to claim 1, characterized in that: The longitudinal spacing between the cage groups in S2 is 0.6-1.0 m, 3-7 cages are arranged in each group, and the lateral spacing between cage groups is 40-60 m.

4. A method for raising fish immunity according to claim 1, characterized in that: In the step S3, the fish fry are released on sunny days from March to May, and the marine fish fry are one of sea bream, grouper and golden pomfret.

5. A method for raising fish immunity according to claim 1, characterized in that: The basic material I in S6 is composed of the following raw materials in parts by weight: 30-40 parts of fish meal, 20-25 parts of oil cake, 10-15 parts of wheat flour, 10-15 parts of corn starch, 5-10 parts of shrimp shell powder, 5-10 parts of bone meal, 3-5 parts of vegetable oil, 0.5-1.0 parts of choline, 0.4-0.6 parts of vitamin premix, and 0.4-0.6 parts of mineral premix.

6. A method for raising fish immunity according to claim 1, characterized in that: The basic material II in S6 is composed of the following raw materials in parts by weight: 35-45 parts of fish meal, 20-25 parts of oil cake, 10-15 parts of wheat flour, 10-15 parts of corn starch, 3-5 parts of shrimp shell powder, 10-15 parts of bone meal, 4-6 parts of vegetable oil, 0.5-1.0 parts of choline, 0.5-0.7 parts of vitamin premix, and 0.5-0.7 parts of mineral premix.

7. A method for raising fish immunity according to claim 1, characterized in that: The basic material III in S6 is composed of the following raw materials in parts by weight: 40-50 parts of fish meal, 15-20 parts of oil cake, 5-10 parts of wheat flour, 5-10 parts of corn starch, 15-20 parts of bone meal, 5-7 parts of vegetable oil, 0.5-1.0 parts of choline, 0.6-0.8 parts of vitamin premix, and 0.6-0.8 parts of mineral premix.

8. A method for raising fish immunity according to claim 1, characterized in that: The campesterol palmitate in S6 is prepared by the following method: campesterol and palmitic acid are uniformly mixed in a molar ratio of 1:1 to obtain mixture I; supercritical carbon dioxide is added to mixture I, stirred and dissolved to obtain mixture II, wherein the amount of supercritical carbon dioxide added is 80-120% of the volume of mixture I; lipase is added, and the mixture is reacted at 50-60°C for 60-72h, wherein the amount of lipase added is 5-10% of the volume of mixture II; the reaction is terminated, the temperature is increased to 80-90°C to inactivate the enzyme, and the supercritical carbon dioxide is removed by decompression to obtain campesterol palmitate.

9. A method for raising fish immunity according to claim 1, characterized in that: The amount of the bottom mud improver in S7 is 100-200g per cage, and the activity of the nitrifying bacteria and photosynthetic bacteria is 1×10 7 ≥CFU / g.

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