A method for breeding fine-spotted grouper broodstock

Abstract

This invention provides a method for breeding broodstock of fine-spotted grouper, comprising the following steps: S1. Broodstock screening and quarantine; S2. Broodstock disinfection; S3. Transfer to a rearing pond; S4. Nutritional fortification; S5. Light control; S6. Water flow control; S7. Gonadal development monitoring; S8. Artificial spawning induction; and S9. Postpartum care. Through nutritional fortification in step S4, precise feeding by stage and sex has a transformative effect on broodstock breeding, deeply coupling nutritional supply with the reproductive physiological cycle of the broodstock: during the daily stage, a solid physical foundation is established; during the maturation stage, differentiated feeding for males and females stimulates the potential of both sexes, achieving synchronous and efficient gonadal development; and during the spawning period, a special feed focuses on protecting the liver and reducing its burden, maintaining reproductive status. This not only significantly increases spawning, fertilization rate, and hatching rate, but also ensures the sustainable use of broodstock for many years by guaranteeing their health, fundamentally solving the industry pain point of the difficulty in balancing reproductive efficiency and broodstock lifespan.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for breeding broodstock of fine-spotted grouper. Background Technology

[0002] The fine-spotted grouper, also known as the blue grouper, grows relatively quickly, shortening its breeding cycle and significantly improving capital turnover efficiency. Furthermore, this species adapts well to formulated feeds with a high feed conversion rate, effectively reducing breeding costs. In terms of disease resistance, the fine-spotted grouper exhibits superior health compared to some other grouper species, resulting in a high survival rate under standardized management and reduced production risks. Its white, tender, and flavorful flesh, rich in nutrients, meets the demands of the high-end aquatic product market, and its moderately sized marketable fish are highly favored by domestic and international restaurants and consumers. In addition, the fine-spotted grouper adapts well to various modern aquaculture models such as net cages and recirculating aquaculture systems, making its technological promotion highly feasible. Due to its outstanding economic value, the breeding of fine-spotted grouper broodstock is crucial. The physique, gonadal development quality, and reproductive performance of the broodstock (i.e., the fish used for reproduction) directly determine the survival rate, growth rate, disease resistance, and other key production traits of its offspring—the seedlings. Uncultured parent fish often have unstable reproductive cycles, large fluctuations in egg production, and inconsistent egg quality, resulting in unsatisfactory fertilization and hatching rates, which seriously restricts large-scale, standardized seedling production. Summary of the Invention

[0003] In view of this, the present invention proposes a method for breeding fine-spotted grouper broodstock to solve the above problems.

[0004] The technical solution of this invention is implemented as follows:

[0005] A method for breeding fine-spotted grouper broodstock includes the following steps:

[0006] S1. Selection and quarantine of broodstock: Select individuals aged 3-5 years, with robust physique, active swimming, no injuries or diseases, and no deformities as broodstock. Female and male fish are mixed and released at a ratio of 1-2:1. PCR testing is used to exclude nerve necrosis virus and viral hemorrhagic septicemia virus.

[0007] S2. Broodstock disinfection: Broodstock that have undergone PCR testing are disinfected by immersion in potassium permanganate solution or povidone-iodine solution;

[0008] S3. Transfer to rearing pond: The disinfected parent fish are transferred to the rearing pond for rearing. The pond water is recycled water that has been sterilized and filtered.

[0009] S4. Nutritional Fortification: During the normal feeding period, feed the fish with their usual feed twice a day, at a daily rate of 2.5-3.5% of their body weight. When ≥60% of the female fish's ovaries have developed to stage III, they enter the pre-spawning maturation period. Separate the female and male fish for this period. Feed the females with female maturation feed and the males with male maturation feed three times a day, at a daily rate of 4.0-5.0% of their body weight. During the spawning period, feed the females with female spawning feed and the males with male spawning feed twice a day, at a daily rate of 1.8-2.2% of their body weight.

[0010] S5. Light control: During the pre-ripening period, "three-color light stimulation" is used, with green light at 06:00, white light at 12:00, and yellow light at 18:00.

[0011] S6. Water flow regulation: Water flow stimulation is performed once a week during the pre-birth ripening period;

[0012] S7. Gonadal development monitoring: When the female fish's ovary develops to stage III, gonadal development is monitored weekly, and gonadal maturity is observed by abdominal palpation or microscopy.

[0013] S8. Artificial spawning: When the female fish's ovary reaches stage IV maturity and the egg diameter is 0.45-0.55mm, and the male fish's semen is milky white, it enters the spawning period from the maturation period. Oxytocin is injected into both the female and male fish.

[0014] S9. Postpartum care: After the last spawning, transfer the female and male fish to a medicated bath tank under aeration and oxygenation conditions for 60-90 minutes. Feed them postpartum feed for 15-20 days after the last spawning, three times a day, with a daily feed amount of 2.5-3.5% of the parent fish's body weight.

[0015] Furthermore, in S1, female fish weigh 1.0-1.5 kg and male fish weigh ≥1.5 kg; in S2, the potassium permanganate solution concentration is 0.01-0.02%, and the soaking time is 10-15 min; the povidone-iodine solution concentration is 10-15 ppm, and the soaking time is 20-30 min; in S3, the pond water depth is 1.2-1.8 m, and the depth of a single pond is ≥50 m. 3 The temperature should be controlled at 23-25℃, salinity at 28-32‰, dissolved oxygen ≥5mg / L, ammonia nitrogen ≤0.5mg / L, pH at 7.8-8.2, and stocking density in the rearing pond at 4-8kg / m². 3 .

[0016] Furthermore, the daily feed in S4 includes the following ingredients by weight: 40-50 parts fish meal, 20-25 parts soybean meal, 13-17 parts wheat flour, 4-6 parts fish oil, 3-5 parts brewer's yeast, and 0.3-0.5 parts premix I. The female fish growth promoter includes the following ingredients by weight: 50-55 parts fish meal, 10-15 parts squid viscera powder, 8-12 parts krill powder, 6-10 parts Schizochyptella powder, 4-6 parts soybean lecithin, 3-5 parts kudzu root fermentation powder, 0.5-1.5 parts red clover extract, and 0.4-0.6 parts premix II. The male fish growth promoter includes the following ingredients by weight: 48-52 parts fish meal, 8-12 parts squid viscera powder, 5 parts fish oil, 3-5 parts brewer's yeast, and 0.3-0.5 parts premix I. The female fish spawning feed comprises the following ingredients by weight: 35-40 parts fish meal, 13-17 parts enzymatically hydrolyzed fish syrup, 9-11 parts puffed soybeans, 3-5 parts alfalfa powder, 0.5-0.7 parts premix III, 0.2-0.4 parts bile acids, and 0.1-0.2 parts curcumin. The male fish spawning feed comprises the following ingredients by weight: 38-42 parts fish meal, 12-16 parts enzymatically hydrolyzed fish syrup, 10-14 parts puffed soybeans, 3-5 parts brewer's yeast, 0.15-0.25 parts bile acids, and 0.5-0.7 parts premix III.

[0017] Furthermore, each serving of premix I includes the following ingredients: 7000-8000 IU Vitamin A, 2500-3500 IU Vitamin D3, 60-100 mg Vitamin E, 100-150 mg Vitamin C, 10-15 mg Folic Acid, 150-200 mg Niacin, 9-11 mg Vitamin K3, 40-45 mg Vitamin B2, 15-20 mg Vitamin B6, 60-80 mg Ferrous Sulfate, 80-120 mg Zinc Sulfate, 35-45 mg Manganese Sulfate, and 30-50 g zeolite powder as a carrier. Each serving of premix II includes the following ingredients: 200-250 mg Vitamin E, 100-150 mg Vitamin C, and 8000-10000 IU Vitamin D3. Each portion of premix III contains the following ingredients: Vitamin A, 3000-4000 IU Vitamin D3, 400-600 mg Inositol, 100-150 mg Zinc Methionine, 120-160 mg β-glucan, 0.3-0.4 mg Sodium Selenite, 800-1000 mg Choline Chloride, and 40-50 g Zeolite Powder as a carrier.

[0018] Furthermore, kudzu root fermented powder is prepared by the following method: dried kudzu root slices are pulverized to 40-60 mesh to obtain kudzu root powder; the kudzu root powder and wheat bran are mixed at a weight ratio of 7-9:2 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 105-110℃ for 35-45 minutes, then cooled to 24-30℃ for later use; brown sugar is dissolved in sterile water to prepare a 4-6% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 120-125℃ for 15-20 minutes to obtain sterilized brown sugar solution; Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum are inoculated into the sterilized brown sugar solution, and Bacillus subtilis is steam-sterilized at 35-39℃. Activated Bacillus subtilis was obtained by shaking culture at 150-200 rpm for 23-26 hours at 28-32℃ for 33-39 hours to obtain activated Saccharomyces cerevisiae. Activated Lactobacillus plantarum was obtained by static culture at 35-40℃ for 45-50 hours. The activated Bacillus subtilis, activated Saccharomyces cerevisiae, and activated Lactobacillus plantarum were mixed at a volume ratio of (1.8-2.2):(0.9-1.1):(1.9-2.1) to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterilized brown sugar solution at an inoculum volume of 4-6% and cultured at 30-32℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent is prepared by mixing CFU / mL. The working fermentation agent is then evenly sprayed onto the mixture at 8-12% of the weight of the mixture while stirring. After spraying, the mixture is fermented at 30-35℃ and 80-85% humidity for 70-75 hours, with stirring every 12 hours. After fermentation, the mixture is transferred to a dryer and dried at 60-65℃ until the moisture content is ≤10%. The mixture is then passed through a 60-100 mesh sieve to obtain kudzu root fermentation powder.

[0019] Furthermore, the red clover extract is prepared by the following method: dried red clover is pulverized to 40-60 mesh to obtain clover powder; the clover powder is mixed with 70-80 wt% ethanol solution at a material-to-liquid ratio of 1:8-10 in an extraction tank, heated to 75-80℃, and refluxed twice for 1.5-2.0 h each time. After extraction, the mixture is filtered through a 150-200 mesh sieve, and the two extracts are combined. The combined extract is centrifuged at 6000-8000 rpm for 15-25 min, and the supernatant is collected; the supernatant is transferred to a concentration device and concentrated to a relative density of 50-60℃ and a vacuum of -0.08 to -0.10 MPa. A concentrated extract with a density of 1.10-1.15 was obtained and set aside. β-Cyclodextrin and purified water were mixed at a ratio of 1:14-16 and stirred at 58-62℃ to prepare a β-cyclodextrin solution. The concentrated extract was added to the β-cyclodextrin solution while stirring at a weight ratio of 1:1.4-1.6 (1:1.4-1.6). The mixture was stirred continuously at 80-150 rpm for 1.8-2.2 hours, and then refrigerated at 3-5℃ for 10-15 hours to obtain an inclusion complex. The inclusion complex was transferred to a vacuum drying device and dried at 60-65℃ and -0.06 to -0.08 MPa until the moisture content was ≤5%. The solution was then passed through an 80-100 mesh sieve to obtain the red clover extract.

[0020] Furthermore, the fenugreek seed fermentation powder is prepared by the following method: dried fenugreek seeds are pulverized to 40-60 mesh to obtain fenugreek seed powder; the fenugreek seed powder is mixed with wheat bran at a weight ratio of 8-10:1 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 105-110℃ for 35-45 minutes, then cooled to 24-30℃ for later use; brown sugar is dissolved in sterile water to prepare a 4-6% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 120-125℃ for 15-20 minutes to obtain sterilized brown sugar. The following steps were performed: *Aspergillus niger* and *Lactobacillus plantarum* were inoculated separately into a sterile brown sugar solution. *Aspergillus niger* was cultured at 28-32℃ with shaking at 150-200 rpm for 35-40 hours to obtain activated *Aspergillus niger*, and *Lactobacillus plantarum* was cultured statically at 35-40℃ for 45-50 hours to obtain activated *Lactobacillus plantarum*. The activated *Aspergillus niger* and activated *Lactobacillus plantarum* were mixed at a volume ratio of 1:(1.8-2.2) to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterile brown sugar solution at an inoculation rate of 4-6% and cultured at 31-34℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9Prepare a working fermentation agent at CFU / mL; spray the working fermentation agent evenly onto the mixture at 10-15% of the weight of the mixture while stirring. After spraying, ferment at 30-32℃ and 85-90% humidity for 70-75 hours, turning it over once every 12 hours. After fermentation, transfer it to a dryer and dry it at 60-65℃ until the moisture content is ≤10%. Then pass it through a 60-100 mesh sieve to obtain fenugreek seed fermentation powder.

[0021] Furthermore, in S5, the green light intensity is 900-1100 lux, irradiation time is 20-30 minutes; the white light intensity is 1400-1600 lux, irradiation time is 30-40 minutes; and the yellow light intensity is 1300-1500 lux, irradiation time is 15-25 minutes.

[0022] Furthermore, the water flow velocity in S6 is 0.3-0.4 m / s, maintained for 1.5-2.0 h.

[0023] Furthermore, in S8, female fish were injected with HCG 800-1000 IU / kg + LHRH-A3 4-6μg / kg, divided into two injections. The first injection was 2 / 3 of the dose, and the remaining dose was injected 24 hours later. The male fish were injected with half the dose simultaneously with the second injection of the female fish.

[0024] Furthermore, the S9 herbal bath contains the following ingredients in parts by weight: 3.0-5.0 parts hydrolyzed sodium hyaluronate, 1.0-2.0 parts γ-aminobutyric acid, 0.5-1.0 parts L-theanine, 1.0-2.0 parts lysozyme, 1.0-1.5 parts D-calcium pantothenate, 0.1-0.2 parts astragaloside A, and 90-100 parts purified water.

[0025] Furthermore, the S9 post-harvest feed includes the following ingredients by weight: 35-45 parts fish meal, 10-15 parts fermented soybean meal, 5-8 parts krill meal, 15-20 parts high-gluten flour, 4-6 parts fish oil, 2-3 parts soybean lecithin, 0.1-0.2 parts mannan oligosaccharide, 0.3-0.5 parts glutamine, 0.1-0.2 parts eyebright extract, 1.0-2.0 parts hydrolyzed collagen peptides, and 0.4-0.6 parts premix I.

[0026] Furthermore, eyebright extract is prepared by the following method: Dried eyebright whole herb is pulverized using a pulverizer and passed through a 20-40 mesh sieve to obtain eyebright powder, which is then set aside. The eyebright powder and 40-50 wt% ethanol solution are added to an extraction tank at a material-to-liquid ratio of 1:8-10 (g / mL). Ultrasonic extraction is performed at a power of 300-500 W, a frequency of 20-40 kHz, and a temperature of 50-60℃ for 25-35 min. After extraction, the mixture is filtered through a 150-200 mesh sieve, and the filtrate is collected. The residue is extracted once more, and the two filtrates are combined. The combined filtrate is centrifuged at 5500-6500 rpm for 12-18 min, and the supernatant is collected. The supernatant was transferred to a vacuum concentration tank and concentrated to a relative density of 1.10-1.15 at a temperature of 50-60℃ and a vacuum degree of -0.08 to -0.09 MPa to obtain a concentrated extract. The concentrated extract was placed in a vacuum drying oven and dried at 65-70℃ and -0.07 to -0.08 MPa until the moisture content was ≤5%. Then it was passed through an 80-100 mesh sieve to obtain eyebright extract.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. This invention uses the signal of "female ovary development to stage III" to synchronously switch to the maturation period, supplemented by "three-color light stimulation" and periodic water flow regulation, to construct an environmental and physiological synchronization system. This effectively guides the parent fish population into a unified reproductive state, promoting a high degree of matching in the gonadal development rhythms of male and female parent fish. This, in turn, promotes highly concentrated spawning behavior, effectively avoiding the management difficulties and resource waste caused by sporadic spawning, thereby significantly increasing the spawning volume and fertilized egg acquisition efficiency per unit time, laying a solid foundation for large-scale seedling production.

[0029] 2. This invention utilizes separate male and female feeding based on reproductive differences and precise nutrition. The fermented kudzu root powder and red clover extract in the female fish's ripening feed work synergistically to promote efficient yolk deposition and provide antioxidant protection, ensuring plump, uniform eggs with strong embryonic viability. The fermented fenugreek seed powder in the male fish's ripening feed acts as a natural androgen precursor, promoting testicular development and increasing sperm volume and motility. This simultaneous improvement in the quality of both male and female gametes leads to higher fertilization and hatching rates, and enhances the initial health of the fry from the source.

[0030] 3. This invention balances the principles of "reproduction and health," transforming broodstock from "consumables" into reusable "production resources." The bile acids and curcumin added to the spawning feed, the anti-stress and mucosal repair components (hydrolyzed sodium hyaluronate) in the postpartum medicated bath formula, and the intestinal health (mannan oligosaccharides) and body condition recovery (hydrolyzed collagen peptides) design in the postpartum feed together constitute a comprehensive health protection system. This significantly alleviates reproductive stress, prevents liver and gallbladder diseases and postpartum infections, greatly reduces postpartum mortality in broodstock, promotes rapid recovery of physical strength, supports the start of the next reproductive cycle, and significantly extends the effective lifespan of high-quality broodstock. Detailed Implementation

[0031] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0034] Example 1

[0035] A method for breeding fine-spotted grouper broodstock includes the following steps:

[0036] S1. Selection and quarantine of broodstock: Select 3-year-old, robust, active, injury-free, disease-free, and deformed individuals as broodstock. Mix female and male fish at a 1:1 ratio, with females weighing 1.0 kg and males weighing 1.5 kg. Use PCR testing to exclude nerve necrosis virus and viral hemorrhagic septicemia virus.

[0037] S2. Broodstock disinfection: Broodstock that have undergone PCR testing are disinfected by immersing in a 0.01% potassium permanganate solution for 15 minutes;

[0038] S3. Transfer to rearing tank: The disinfected broodstock are transferred to the rearing tank, which uses sterilized and filtered circulating water. The tank depth is 1.8m, and each tank is 50m deep. 3 The temperature was controlled at 23℃, salinity at 28‰, dissolved oxygen at 5mg / L, ammonia nitrogen at 0.5mg / L, pH at 7.8, and the stocking density in the rearing pond was 4kg / m². 3 ;

[0039] S4. Nutritional Fortification: During the daily feeding period, feed the following ingredients by weight: 40 parts fish meal, 20 parts soybean meal, 13 parts wheat flour, 4 parts fish oil, 3 parts brewer's yeast, and 0.3 parts premix I. Feed twice a day, with a daily feeding amount of 2.5% of the broodstock's body weight. When 60% of the female fish's ovaries reach stage III, they enter the pre-spawning maturation period. Separate the female and male fish for rearing. During the pre-spawning maturation period, feed the female fish with female maturation feed and the male fish with male maturation feed. The female maturation feed includes the following ingredients by weight: 50 parts fish meal, 10 parts squid viscera powder, 8 parts krill powder, 6 parts Schizochytrium powder, 4 parts soybean lecithin, 3 parts kudzu root fermentation powder, 0.5 parts red clover extract, and 0.4 parts premix II. The male maturation feed includes the following ingredients by weight: 48 parts fish meal, 20 parts soybean meal, 13 parts wheat flour, 4 parts fish oil, 3 parts brewer's yeast, and 0.3 parts premix II. The broodstock are fed three times a day, with a daily feed amount of 4.0% of their body weight. During the spawning period, female broodstock are fed a female spawning feed, and male broodstock are fed a male spawning feed. The female spawning feed consists of the following ingredients by weight: 35 parts fish meal, 13 parts enzymatically hydrolyzed fish slurry, 9 parts extruded soybeans, 3 parts alfalfa powder, 0.5 parts premix III, 0.2 parts bile acids, and 0.1 parts curcumin. The male spawning feed consists of the following ingredients by weight: 38 parts fish meal, 12 parts enzymatically hydrolyzed fish slurry, 10 parts extruded soybeans, 3 parts brewer's yeast, 0.15 parts bile acids, and 0.57 parts premix III. The broodstock are fed twice a day, with a daily feed amount of 1.8% of their body weight.

[0040] S5. Light control: During the pre-ripening period, "three-color light stimulation" is used, with green light irradiation at 06:00, white light irradiation at 12:00, and yellow light irradiation at 18:00; among them, the green light intensity is 900 lux and the irradiation time is 30 minutes, the white light intensity is 1400 lux and the irradiation time is 40 minutes, and the yellow light intensity is 1300 lux and the irradiation time is 25 minutes.

[0041] S6. Water flow regulation: Water flow stimulation is performed once a week during the pre-ripening period, with a water flow rate of 0.3 m / s, maintained for 2.0 h.

[0042] S7. Gonadal development monitoring: When the female fish's ovary develops to stage III, gonadal development is monitored weekly, and gonadal maturity is observed by abdominal palpation or microscopy.

[0043] S8. Artificial spawning: When the female fish's ovary reaches stage IV maturity and the egg diameter is 0.45 mm, and the male fish's semen is milky white, it enters the spawning period from the maturation period. Inject oxytocin into both the female and male fish. The female fish is injected with HCG 800 IU / kg + LHRH-A3 4μg / kg, divided into two injections. The first injection is 2 / 3 of the dose, and the remaining dose is injected 24 hours later. The male fish is injected with half the dose simultaneously with the second injection of the female fish.

[0044] S9. Postpartum Care: After the last spawning, transfer the spawning female and male fish to a medicated bath tank under aerated conditions for 60 minutes. The medicated bath consists of the following ingredients by weight: 3.0 parts hydrolyzed sodium hyaluronate, 1.0 part γ-aminobutyric acid, 0.5 parts L-theanine, 1.0 part lysozyme, 1.0 part D-calcium pantothenate, 0.1 part astragaloside A, and 90 parts purified water. Feed the fish with postpartum feed for 15 days after spawning. The postpartum feed consists of the following ingredients by weight: 35 parts fishmeal, 10-15 parts fermented soybean meal, 5 parts krill meal, 15 parts high-gluten flour, 4 parts fish oil, 2 parts soybean lecithin, 0.1 part mannan oligosaccharide, 0.3 parts glutamine, 0.1 parts eyebright extract, 1.0 part hydrolyzed collagen peptides, and 0.4 parts premix I. Feed three times a day, with a daily feed amount of 2.5% of the broodstock's body weight.

[0045] Each premix I contains the following ingredients: 7000 IU Vitamin A, 2500 IU Vitamin D3, 60 mg Vitamin E, 100 mg Vitamin C, 10 mg Folic Acid, 150 mg Niacin, 9 mg Vitamin K3, 40 mg Vitamin B2, 15 mg Vitamin B6, 60 mg Ferrous Sulfate, 80 mg Zinc Sulfate, 35 mg Manganese Sulfate, and 30 g zeolite powder as a carrier. Each premix II contains the following ingredients: 200 mg Vitamin E, 100 mg Vitamin C, 8000 IU Vitamin D3, 10 mg Vitamin D4, 15 mg Vitamin D5, 15 mg Vitamin D6, 60 mg Ferrous Sulfate, 80 mg Zinc Sulfate, 10 mg Zinc Sulfate, 150 mg Folic Acid, 150 mg Niacin ... Each premix III contains the following ingredients as a carrier: Vitamin A, 3000 IU Vitamin D3, 400 mg Inositol, 100 mg Zinc Methionine, 120 mg β-glucan, 0.3 mg Sodium Selenite, 800 mg Choline Chloride, and 40 g Zeolite Powder.

[0046] The kudzu root fermented powder is prepared by the following method: dried kudzu root slices are pulverized to 40 mesh to obtain kudzu root powder; the kudzu root powder and wheat bran are mixed at a weight ratio of 7:2 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 105℃ for 45 minutes, then cooled to 24℃ for later use; brown sugar is dissolved in sterile water to prepare a 4% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 120℃ for 20 minutes to obtain sterilized brown sugar solution; Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum are inoculated into the sterilized brown sugar solution respectively. Bacillus subtilis was cultured at 35℃ and 150 rpm for 26 h with shaking to obtain activated Bacillus subtilis; Saccharomyces cerevisiae was cultured at 28℃ and 140 rpm for 39 h with shaking to obtain activated Saccharomyces cerevisiae; and Lactobacillus plantarum was cultured statically at 35℃ for 50 h to obtain activated Lactobacillus plantarum. The activated Bacillus subtilis, activated Saccharomyces cerevisiae, and activated Lactobacillus plantarum were mixed at a volume ratio of 1.8:0.9:1.9 to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterilized brown sugar solution at a 4% inoculum and cultured at 30℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent was prepared by mixing CFU / mL. The working fermentation agent was evenly sprayed onto the mixture at 8% of the weight of the mixture while stirring. After spraying, the mixture was fermented at 30℃ and 80% humidity for 75 hours, and stirred once every 12 hours. After fermentation, the mixture was transferred to a dryer and dried at 60℃ until the moisture content was 10%. Then it was passed through a 60-mesh sieve to obtain kudzu root fermentation powder.

[0047] The red clover extract was prepared by the following method: dried red clover was pulverized to 40 mesh to obtain clover powder; the clover powder and 70wt% ethanol solution were mixed in an extraction tank at a material-to-liquid ratio of 1:8 (g / mL), heated to 75℃, and refluxed twice for 1.5 hours each time. After extraction, the mixture was filtered through a 150-mesh sieve, and the two extracts were combined. The combined extract was centrifuged at 6000 rpm for 25 minutes, and the supernatant was collected; the supernatant was transferred to a concentration device and concentrated to the required concentration at 50℃ and a vacuum of -0.08 MPa. A concentrated extract with a density of 1.10 was obtained and set aside. β-cyclodextrin and purified water were mixed at a ratio of 1:14 and stirred at 58°C to prepare a β-cyclodextrin solution. The concentrated extract was added to the β-cyclodextrin solution while stirring at a weight ratio of 1:1.4 (concentrated extract to β-cyclodextrin). The mixture was stirred continuously at 80 rpm for 2.2 h, and then refrigerated at 3°C ​​for 15 h to obtain an inclusion complex. The inclusion complex was transferred to a vacuum drying device and dried at 60°C and -0.06 MPa until the moisture content was 5%. The solution was then passed through an 80-mesh sieve to obtain the red clover extract.

[0048] The fenugreek seed fermentation powder is prepared by the following method: dried fenugreek seeds are pulverized to 40 mesh to obtain fenugreek seed powder; the fenugreek seed powder is mixed with wheat bran at a weight ratio of 8:1 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 105℃ for 45 minutes, then cooled to 24℃ for later use; brown sugar is dissolved in sterile water to prepare a 4% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 120℃ for 20 minutes to obtain sterilized brown sugar solution. Aspergillus niger and Lactobacillus plantarum were inoculated separately into sterile brown sugar solution. Activated Aspergillus niger was obtained by incubating at 28℃ and 200 rpm with shaking for 40 h, and activated Lactobacillus plantarum was obtained by static incubation at 350℃ for 50 h. The activated Aspergillus niger and activated Lactobacillus plantarum were mixed at a volume ratio of 1:1.8 to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterile brown sugar solution at a 4% inoculation rate and incubated at 31℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent was prepared by mixing CFU / mL. The working fermentation agent was evenly sprayed onto the mixture at 10% of the weight of the mixture while stirring. After spraying, the mixture was fermented at 30℃ and 85% humidity for 70 hours, and stirred once every 12 hours. After fermentation, the mixture was transferred to a dryer and dried at 60℃ until the moisture content was 10%. Then it was passed through a 60-mesh sieve to obtain fenugreek seed fermentation powder.

[0049] The eyebright extract was prepared by the following method: Dried eyebright whole herb was pulverized using a pulverizer and passed through a 20-mesh sieve to obtain eyebright powder, which was set aside. The eyebright powder and 40wt% ethanol solution were added to an extraction tank at a material-to-liquid ratio of 1:8 (g / mL). Ultrasonic extraction was performed at 300W power, 20kHz frequency, and 50℃ for 35 minutes. After extraction, the mixture was filtered through a 150-mesh sieve, and the filtrate was collected. The residue was extracted once more, and the two filtrates were combined. The combined filtrate was centrifuged at 5500rpm for 18 minutes, and the supernatant was collected. The supernatant was transferred to a vacuum concentration tank and concentrated to a relative density of 1.10 at 50℃ and a vacuum of -0.08MPa to obtain a concentrated extract. The concentrated extract was placed in a vacuum drying oven and dried to 5% moisture content at 65℃ and -0.07MPa, then passed through an 80-mesh sieve to obtain the eyebright extract.

[0050] Example 2

[0051] A method for breeding fine-spotted grouper broodstock includes the following steps:

[0052] S1. Selection and quarantine of broodstock: Select 4-year-old, robust, active, injury-free, disease-free, and deformed individuals as broodstock. Mix female and male fish at a ratio of 1.5:1, with females weighing 1.2 kg and males weighing 1.8 kg. Use PCR testing to exclude nerve necrosis virus and viral hemorrhagic septicemia virus.

[0053] S2. Broodstock disinfection: Broodstock that have undergone PCR testing are disinfected by immersing them in a 12 ppm povidone-iodine solution for 25 minutes.

[0054] S3. Transfer to rearing tank: The disinfected broodstock are transferred to the rearing tank, which uses sterilized and filtered circulating water. The tank depth is 1.5m, and each tank is 75m deep. 3 The temperature was controlled at 24℃, salinity at 30‰, dissolved oxygen at 5.5 mg / L, ammonia nitrogen at 0.4 mg / L, pH at 8.0, and the stocking density in the rearing pond was 6 kg / m³. 3 ;

[0055] S4. Nutritional Fortification: During the daily feeding period, feed the following ingredients by weight: 45 parts fishmeal, 22 parts soybean meal, 15 parts wheat flour, 5 parts fish oil, 4 parts brewer's yeast, and 0.4 parts premix I. Feed twice a day, with a daily feed amount of 3.0% of the broodstock's body weight. When 60% of the female fish's ovaries reach stage III, they enter the pre-spawning maturation period. Separate the female and male fish for rearing. During the pre-spawning maturation period, feed the female fish with female maturation feed and the male fish with male maturation feed. The female maturation feed includes the following ingredients by weight: 53 parts fishmeal, 12 parts squid viscera powder, 10 parts krill powder, 8 parts Schizochytridium powder, 5 parts soybean lecithin, 4 parts kudzu root fermentation powder, 1.0 part red clover extract, and 0.5 parts premix II. The male maturation feed includes the following ingredients by weight: 50 parts fishmeal... 10 parts squid viscera powder, 6.5 parts krill powder, 5 parts brewer's yeast, 1.5 parts fenugreek seed fermentation powder, and 0.6 parts premix II. Feed three times a day, with a daily feed amount of 4.5% of the broodstock's body weight. During the spawning period, female fish are fed female spawning feed and male fish are fed male spawning feed. The female spawning feed consists of the following ingredients by weight: 38 parts fish meal, 15 parts enzymatically hydrolyzed fish slurry, 10 parts extruded soybeans, 4 parts alfalfa powder, 0.6 parts premix III, 0.5 parts bile acids, and 0.15 parts curcumin. The male spawning feed consists of the following ingredients by weight: 40 parts fish meal, 14 parts enzymatically hydrolyzed fish slurry, 12 parts extruded soybeans, 4 parts brewer's yeast, 0.2 parts bile acids, and 0.6 parts premix III. Feed twice a day, with a daily feed amount of 2.0% of the broodstock's body weight.

[0056] S5. Light Control: During the pre-ripening period, a "three-color light stimulation" method is used: green light at 06:00, white light at 12:00, and yellow light at 18:00. Specifically, the green light intensity is 1000 lux for 25 minutes, the white light intensity is 1500 lux for 35 minutes, and the yellow light intensity is 1400 lux for 20 minutes.

[0057] S6. Water flow regulation: Water flow stimulation is performed once a week during the pre-ripening period, with a water flow rate of 0.4 m / s, maintained for 1.8 hours.

[0058] S7. Gonadal development monitoring: When the female fish's ovary develops to stage III, gonadal development is monitored weekly, and gonadal maturity is observed by abdominal palpation or microscopy.

[0059] S8. Artificial spawning: When the female fish's ovaries reach stage IV maturity and the egg diameter is 0.50 mm, and the male fish's semen is milky white, the fish enters the spawning period from the maturation period. Inject oxytocin into both the female and male fish. The female fish is injected with HCG 900 IU / kg + LHRH-A3 5μg / kg, divided into two injections. The first injection is 2 / 3 of the dose, and the remaining dose is injected 24 hours later. The male fish is injected with half the dose simultaneously with the second injection of the female fish.

[0060] S9. Postpartum Care: After the final spawning, transfer the spawning female and male fish to a medicated bath for 75 minutes under aerated conditions. The medicated bath consists of the following ingredients by weight: 4.0 parts hydrolyzed sodium hyaluronate, 1.5 parts γ-aminobutyric acid, 0.8 parts L-theanine, 1.5 parts lysozyme, 1.3 parts D-calcium pantothenate, 0.15 parts astragaloside A, and 95 parts purified water. Feed the fish with postpartum feed for 18 days after spawning. The postpartum feed consists of the following ingredients by weight: 40 parts fishmeal, 13 parts fermented soybean meal, 7 parts krill meal, 18 parts high-gluten flour, 5 parts fish oil, 2.5 parts soybean lecithin, 0.15 parts mannan oligosaccharide, 0.4 parts glutamine, 0.15 parts eyebright extract, 1.5 parts hydrolyzed collagen peptides, and 0.5 parts premix I. Feed three times a day, with a daily feed amount of 3.0% of the broodstock's body weight.

[0061] Each premix I contains the following ingredients: 7500 IU Vitamin A, 3000 IU Vitamin D3, 80 mg Vitamin E, 130 mg Vitamin C, 12 mg Folic Acid, 180 mg Niacin, 10 mg Vitamin K3, 42 mg Vitamin B2, 18 mg Vitamin B6, 70 mg Ferrous Sulfate, 100 mg Zinc Sulfate, 40 mg Manganese Sulfate, and 40 g zeolite powder as a carrier. Each premix II contains the following ingredients: 220 mg Vitamin E, 130 mg Vitamin C, 9000 IU... Each serving of premix III contains the following ingredients: 230mg vitamin E, 400mg vitamin C, 9000IU vitamin A, 3500IU vitamin D3, 500mg inositol, 120mg zinc methionine, 140mg β-glucan, 0.4mg sodium selenite, 900mg choline chloride, and 45g zeolite powder as a carrier.

[0062] The kudzu root fermented powder is prepared by the following method: dried kudzu root slices are pulverized to 50 mesh to obtain kudzu root powder; the kudzu root powder and wheat bran are mixed at a weight ratio of 8:2 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 108℃ for 40 minutes, then cooled to 27℃ for later use; brown sugar is dissolved in sterile water to prepare a 5% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 122℃ for 18 minutes to obtain sterilized brown sugar solution; Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum are inoculated into the sterilized brown sugar solution respectively. Bacillus subtilis was cultured at 37℃ and 180 rpm for 24 h to obtain activated Bacillus subtilis; Saccharomyces cerevisiae was cultured at 30℃ and 150 rpm for 36 h to obtain activated Saccharomyces cerevisiae; and Lactobacillus plantarum was cultured at 38℃ for 48 h to obtain activated Lactobacillus plantarum. The activated Bacillus subtilis, activated Saccharomyces cerevisiae, and activated Lactobacillus plantarum were mixed at a volume ratio of 2.0:1.0:2.0 to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterilized brown sugar solution at a 5% inoculum and cultured at 31℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent was prepared by mixing CFU / mL. The working fermentation agent was evenly sprayed onto the mixture at 10% of the weight of the mixture while stirring. After spraying, the mixture was fermented at 32℃ and 83% humidity for 72 hours, and stirred once every 12 hours. After fermentation, the mixture was transferred to a dryer and dried at 63℃ until the moisture content was 8%. Then it was passed through an 80-mesh sieve to obtain kudzu root fermentation powder.

[0063] The red clover extract was prepared by the following method: dried red clover was pulverized to 50 mesh to obtain clover powder; the clover powder and 75wt% ethanol solution were mixed in an extraction tank at a material-to-liquid ratio of 1:9 (g / mL), heated to 78℃, and refluxed twice for 1.8 hours each time. After extraction, the mixture was filtered through an 180-mesh sieve, and the two extracts were combined. The combined extract was centrifuged at 7000 rpm for 20 minutes, and the supernatant was collected; the supernatant was transferred to a concentration device and concentrated to a relative density of 55℃ and a vacuum of -0.09 MPa. A concentrated extract with a density of 1.13 was obtained and set aside. β-cyclodextrin and purified water were mixed at a ratio of 1:15 and stirred at 60°C to prepare a β-cyclodextrin solution. The concentrated extract was added to the β-cyclodextrin solution while stirring at a weight ratio of 1:1.5 (concentrated extract to β-cyclodextrin). The mixture was stirred continuously at 120 rpm for 2.0 h, and then refrigerated at 4°C for 12 h to obtain an inclusion complex. The inclusion complex was transferred to a vacuum drying device and dried at 62°C and -0.07 MPa until the moisture content was 4.5%. The solution was then passed through a 90-mesh sieve to obtain the red clover extract.

[0064] The fenugreek seed fermentation powder is prepared by the following method: dried fenugreek seeds are pulverized to 50 mesh to obtain fenugreek seed powder; the fenugreek seed powder is mixed with wheat bran at a weight ratio of 9:1 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 108℃ for 40 minutes, then cooled to 27℃ for later use; brown sugar is dissolved in sterile water to prepare a 5% brown sugar solution, dispensed into Erlenmeyer flasks, and steam-sterilized at 122℃ for 18 minutes to obtain sterilized brown sugar. Sugar solution: *Aspergillus niger* and *Lactobacillus plantarum* were separately inoculated into sterile brown sugar solution. *Aspergillus niger* was cultured at 30℃ with shaking at 180 rpm for 38 h to obtain activated *Aspergillus niger*, and *Lactobacillus plantarum* was cultured statically at 38℃ for 48 h to obtain activated *Lactobacillus plantarum*. The activated *Aspergillus niger* and activated *Lactobacillus plantarum* were mixed at a volume ratio of 1:2 to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterile brown sugar solution at a 5% inoculation rate and cultured at 32℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent was prepared by mixing CFU / mL. The working fermentation agent was evenly sprayed onto the mixture at 12% of the weight of the mixture while stirring. After spraying, the mixture was fermented at 31℃ and 88% humidity for 72 hours, and stirred once every 12 hours. After fermentation, the mixture was transferred to a dryer and dried at 62℃ until the moisture content was 8%. Then it was passed through an 80-mesh sieve to obtain fenugreek seed fermentation powder.

[0065] The eyebright extract was prepared by the following method: Dried eyebright whole herb was pulverized using a pulverizer and passed through a 30-mesh sieve to obtain eyebright powder, which was set aside. The eyebright powder and 45wt% ethanol solution were added to an extraction tank at a material-to-liquid ratio of 1:9 (g / mL). Ultrasonic extraction was performed at 400W power, 30kHz frequency, and 55℃ for 30 minutes. After extraction, the mixture was filtered through an 180-mesh sieve, and the filtrate was collected. The residue was extracted once more, and the two filtrates were combined. The combined filtrate was centrifuged at 6000rpm for 15 minutes, and the supernatant was collected. The supernatant was transferred to a vacuum concentration tank and concentrated to a relative density of 1.13 at 55℃ and a vacuum of -0.08MPa to obtain a concentrated extract. The concentrated extract was placed in a vacuum drying oven and dried to a moisture content of 4.5% at 68℃ and -0.08MPa, and then passed through a 90-mesh sieve to obtain the eyebright extract.

[0066] Example 3

[0067] A method for breeding fine-spotted grouper broodstock includes the following steps:

[0068] S1. Selection and quarantine of broodstock: Select 5-year-old, robust, active, injury-free, disease-free, and deformed individuals as broodstock. Mix female and male fish at a ratio of 2:1, with females weighing 1.5 kg and males weighing 2.0 kg. Use PCR testing to exclude nerve necrosis virus and viral hemorrhagic septicemia virus.

[0069] S2. Broodstock disinfection: Broodstock that have undergone PCR testing are disinfected by immersing them in a 15 ppm povidone-iodine solution for 20 minutes.

[0070] S3. Transfer to rearing tank: The disinfected broodstock are transferred to the rearing tank, which uses sterilized and filtered circulating water. The tank depth is 1.2m, and each tank is 100m³. 3 The temperature was controlled at 25℃, salinity at 32‰, dissolved oxygen at 6mg / L, ammonia nitrogen at 0.3mg / L, pH at 8.2, and the stocking density in the rearing pond was 8kg / m². 3 ;

[0071] S4. Nutritional Fortification: During the routine feeding period, feed the fish with a daily diet consisting of the following ingredients by weight: 50 parts fish meal, 25 parts soybean meal, 17 parts wheat flour, 6 parts fish oil, 5 parts brewer's yeast, and 0.5 parts premix I. Feed twice a day, with a daily feeding amount of 3.5% of the broodstock's body weight. When 60% of the female fish's ovaries reach stage III, they enter the pre-spawning maturation period. Separate the female and male fish for rearing. During the pre-spawning maturation period, feed the female fish with a female maturation feed and the male fish with a male maturation feed. The female maturation feed consists of the following ingredients by weight: 55 parts fish meal, 15 parts squid viscera powder, 12 parts krill powder, 10 parts Schizochytrium powder, 6 parts soybean lecithin, 5 parts kudzu root fermentation powder, 1.5 parts red clover extract, and 0.6 parts premix II. The male maturation feed consists of the following ingredients by weight: 52 parts... Fish meal, 12 parts squid viscera meal, 8 parts krill meal, 6 parts brewer's yeast, 2.0 parts fenugreek seed fermentation powder, and 0.7 parts premix II were fed three times a day, with a daily feed amount of 5.0% of the broodstock's body weight. During the spawning period, female fish were fed female spawning feed and male fish were fed male spawning feed. The female spawning feed consisted of the following ingredients by weight: 40 parts fish meal, 17 parts enzymatically hydrolyzed fish slurry, 11 parts extruded soybeans, 5 parts alfalfa meal, 0.7 parts premix III, 0.4 parts bile acids, and 0.2 parts curcumin. The male spawning feed consisted of the following ingredients by weight: 42 parts fish meal, 16 parts enzymatically hydrolyzed fish slurry, 14 parts extruded soybeans, 5 parts brewer's yeast, 0.25 parts bile acids, and 0.7 parts premix III. The male fish were fed twice a day, with a daily feed amount of 2.2% of the broodstock's body weight.

[0072] S5. Light Control: During the pre-ripening period, a "three-color light stimulation" method is used, with green light irradiation at 06:00, white light irradiation at 12:00, and yellow light irradiation at 18:00. Specifically, the green light intensity is 1100 lux for 20 minutes, the white light intensity is 1600 lux for 30 minutes, and the yellow light intensity is 1500 lux for 15 minutes.

[0073] S6. Water flow regulation: During the pre-ripening period, water flow stimulation is performed once a week at a flow rate of 0.4 m / s for 1.5 hours.

[0074] S7. Gonadal development monitoring: When the female fish's ovary develops to stage III, gonadal development is monitored weekly, and gonadal maturity is observed by abdominal palpation or microscopy.

[0075] S8. Artificial spawning: When the female fish's ovaries reach stage IV maturity and the egg diameter is 0.55 mm, and the male fish's semen is milky white, the fish enters the spawning period from the maturation period. Inject oxytocin into both the female and male fish. The female fish is injected with HCG 1000 IU / kg + LHRH-A3 6μg / kg, divided into two injections. The first injection is 2 / 3 of the dose, and the remaining dose is injected 24 hours later. The male fish is injected with half the dose simultaneously with the second injection of the female fish.

[0076] S9. Postpartum Care: After the final spawning, transfer the spawning female and male fish to a medicated bath tank under aerated conditions for 90 minutes. The medicated bath consists of the following ingredients by weight: 5.0 parts hydrolyzed sodium hyaluronate, 2.0 parts γ-aminobutyric acid, 1.0 part L-theanine, 2.0 parts lysozyme, 1.5 parts D-calcium pantothenate, 0.2 parts astragaloside A, and 100 parts purified water. Feed the fish with postpartum feed for 20 days after spawning. The postpartum feed consists of the following ingredients by weight: 45 parts fishmeal, 15 parts fermented soybean meal, 8 parts krill meal, 20 parts high-gluten flour, 6 parts fish oil, 3 parts soybean lecithin, 0.2 parts mannan oligosaccharide, 0.5 parts glutamine, 0.2 parts eyebright extract, 2.0 parts hydrolyzed collagen peptides, and 0.6 parts premix I. Feed three times a day, with a daily feed amount of 3.5% of the broodstock's body weight.

[0077] Each premix I contains the following ingredients: 8000 IU Vitamin A, 3500 IU Vitamin D3, 100 mg Vitamin E, 150 mg Vitamin C, 15 mg Folic Acid, 200 mg Niacin, 11 mg Vitamin K3, 45 mg Vitamin B2, 20 mg Vitamin B6, 80 mg Ferrous Sulfate, 120 mg Zinc Sulfate, 45 mg Manganese Sulfate, and 50 g zeolite powder as a carrier. Each premix II contains the following ingredients: 250 mg Vitamin E, 150 mg Vitamin C, and 10000 IU Vitamin D3. Each serving of premix III contains the following ingredients: Vitamin A, 4000 IU Vitamin D3, 600 mg Inositol, 150 mg Zinc Methionine, 160 mg β-glucan, 0.4 mg Sodium Selenite, 1000 mg Choline Chloride, and 50 g Zeolite Powder as a carrier.

[0078] The kudzu root fermented powder is prepared by the following method: dried kudzu root slices are pulverized to 60 mesh to obtain kudzu root powder; the kudzu root powder and wheat bran are mixed at a weight ratio of 9:2 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 110℃ for 35 minutes, then cooled to 30℃ for later use; brown sugar is dissolved in sterile water to prepare a 6% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 125℃ for 15 minutes to obtain sterilized brown sugar solution; Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum are inoculated into the sterilized brown sugar solution respectively. Bacillus subtilis was cultured at 39℃ and 200 rpm for 23 h with shaking to obtain activated Bacillus subtilis; Saccharomyces cerevisiae was cultured at 32℃ and 160 rpm for 33 h with shaking to obtain activated Saccharomyces cerevisiae; and Lactobacillus plantarum was cultured at 40℃ for 45 h with static incubation to obtain activated Lactobacillus plantarum. The activated Bacillus subtilis, activated Saccharomyces cerevisiae, and activated Lactobacillus plantarum were mixed at a volume ratio of 2.2:1.1:2.1 to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterilized brown sugar solution at a 6% inoculum and cultured at 32℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent was prepared by mixing CFU / mL. The working fermentation agent was evenly sprayed onto the mixture at 8-12% of the weight of the mixture while stirring. After spraying, the mixture was fermented at 35℃ and 85% humidity for 70 hours, and stirred once every 12 hours. After fermentation, the mixture was transferred to a dryer and dried at 65℃ until the moisture content was 6%. Then it was passed through a 100-mesh sieve to obtain kudzu root fermentation powder.

[0079] The red clover extract was prepared by the following method: dried red clover was pulverized to 60 mesh to obtain clover powder; the clover powder and 80wt% ethanol solution were mixed in an extraction tank at a material-to-liquid ratio of 1:10 (g / mL), heated to 80℃, and refluxed twice for 2.0 h each time. After extraction, the mixture was filtered through a 200-mesh sieve, and the two extracts were combined. The combined extracts were centrifuged at 8000 rpm for 15 min, and the supernatant was collected; the supernatant was transferred to a concentration device and concentrated to the required concentration at 60℃ and a vacuum of -0.10 MPa. A concentrated extract with a density of 1.15 was obtained and set aside. β-cyclodextrin and purified water were mixed at a ratio of 1:16 and stirred at 62°C to prepare a β-cyclodextrin solution. The concentrated extract was added to the β-cyclodextrin solution while stirring, according to a weight ratio of 1:1.6 (concentrated extract to β-cyclodextrin). The mixture was stirred continuously at 150 rpm for 1.8 h, and then refrigerated at 5°C for 10 h to obtain an inclusion complex. The inclusion complex was transferred to a vacuum drying device and dried at 65°C and -0.08 MPa until the moisture content was 4%. The solution was then passed through a 100-mesh sieve to obtain the red clover extract.

[0080] The fenugreek seed fermentation powder is prepared by the following method: dried fenugreek seeds are pulverized to 60 mesh to obtain fenugreek seed powder; the fenugreek seed powder is mixed with wheat bran at a weight ratio of 10:1 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 110℃ for 35 minutes, then cooled to 30℃ for later use; brown sugar is dissolved in sterile water to prepare a 6% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 125℃ for 15 minutes to obtain sterilized brown sugar. The following steps were performed: *Aspergillus niger* and *Lactobacillus plantarum* were inoculated separately into sterile brown sugar solution. *Aspergillus niger* was cultured at 32℃ with shaking at 200 rpm for 35 h to obtain activated *Aspergillus niger*, and *Lactobacillus plantarum* was cultured statically at 40℃ for 45 h to obtain activated *Lactobacillus plantarum*. The activated *Aspergillus niger* and activated *Lactobacillus plantarum* were mixed at a volume ratio of 1:2.2 to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterile brown sugar solution at a 6% inoculation rate and cultured at 34℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent was prepared by mixing CFU / mL. The working fermentation agent was evenly sprayed onto the mixture at 15% of the weight of the mixture while stirring. After spraying, the mixture was fermented at 32℃ and 90% humidity for 70 hours, and stirred once every 12 hours. After fermentation, the mixture was transferred to a dryer and dried at 65℃ until the moisture content was 6%. Then it was passed through a 100-mesh sieve to obtain fenugreek seed fermentation powder.

[0081] The eyebright extract was prepared by the following method: Dried eyebright whole herb was pulverized using a pulverizer and passed through a 40-mesh sieve to obtain eyebright powder, which was set aside. The eyebright powder and 50wt% ethanol solution were added to an extraction tank at a material-to-liquid ratio of 1:10 (g / mL). Ultrasonic extraction was performed at 500W power, 40kHz frequency, and 50℃ for 25 minutes. After extraction, the mixture was filtered through a 200-mesh sieve, and the filtrate was collected. The residue was extracted once more, and the two filtrates were combined. The combined filtrate was centrifuged at 6500rpm for 12 minutes, and the supernatant was collected. The supernatant was transferred to a vacuum concentration tank and concentrated to a relative density of 1.15 at 60℃ and a vacuum of -0.09MPa to obtain a concentrated extract. The concentrated extract was placed in a vacuum drying oven and dried to 4% moisture content at 70℃ and -0.08MPa, then passed through a 100-mesh sieve to obtain the eyebright extract.

[0082] Comparative Example 1

[0083] Compared with Example 2, the difference in this comparative example is that in the S4 nutritional fortification stage, all participants were fed a regular diet.

[0084] Comparative Example 2

[0085] Compared with Example 2, the difference in this comparative example is that the female fish growth-promoting feed raw materials in the S4 nutritional fortification step do not contain kudzu root fermentation powder or red clover extract.

[0086] Comparative Example 3

[0087] Compared with Example 2, the difference in this comparative example is that in the S5 light control step, a single white light irradiation is used instead of "three-color light cycle stimulation". A white light with a light intensity of 1500 lux is used for 35 minutes, and the irradiation is carried out 3 times a day.

[0088] Broodstock were cultured according to the methods described in Examples 1-3 and Comparative Examples 1-3, respectively. During the spawning period, 20 female fish were randomly selected from the culture tanks of Examples 1-3 and Comparative Examples 1-3, respectively. Oxytocin was injected, and eggs were collected and weighed immediately. The egg weight of each fish was recorded, and the average egg weight was calculated. Simultaneously, male semen was collected and immediately mixed with eggs at a 1:2 ratio, with the addition of 0.65% physiological saline. The mixture was gently stirred with a feather for 5 minutes. After standing for 10 minutes, excess semen was removed by rinsing with clean water. 10,000 eggs were randomly placed in seawater at 25°C and a salinity of 3‰. The buoyancy of the eggs was observed; those that floated were fertilized eggs. The fertilization rate was calculated as: (Number of floating eggs / 10,000) × 100%. Subsequently, 1,000 fertilized eggs were randomly selected and incubated under the same conditions. After incubation, the hatching rate was calculated as: (Number of hatched fry / 1,000) × 100%. The results are shown in Table 1.

[0089] Table 1

[0090] Average egg weight / kg Fertilization rate / % Hatching rate / % Example 1 350 86.7 95.3 Example 2 358 87.5 96.4 Example 3 351 86.4 95.6 Comparative Example 1 257 65.5 69.9 Comparative Example 2 309 75.2 81.9 Comparative Example 3 290 69.1 75.2

[0091] As can be seen from Table 1, Examples 1-3 are significantly better than Comparative Examples 1-3 in terms of average egg weight, fertilization rate, and hatching rate. This shows that the broodstock breeding method of the present invention produces broodstock with high egg production and excellent egg quality, thereby achieving high fertilization rate and high hatching rate.

[0092] By comparing Comparative Example 1 and Example 2, this invention achieves precise regulation of gonadal development, spawning quality, and broodstock health through phased feeding with different formulated feeds (daily feed, growth promoter feed, and spawning feed), resulting in significant nutritional fortification. Daily feed provides protein, energy, amino acids, vitamins, and minerals to maintain the basal metabolism and daily activity needs of the broodstock, ensuring stable health and laying the foundation for subsequent gonadal development. Growth promoter feed, rich in taurine, EPA, DHA, and trace elements from squid viscera powder, krill powder, and Schizochytrium powder, directly participates in sex hormone synthesis and oocyte maturation. Soybean lecithin contains phosphatidylcholine, which regulates lipid metabolism, promotes gonadal cell membrane stability, and acts as a signaling molecule in gonadal development regulation. Furthermore, different feed formulations are tailored to the differences in reproductive nutritional needs between male and female fish, avoiding nutrient waste and interference, and maximizing their respective potential. The female fish maturation feed incorporates specific ingredients—kudzu root fermented powder and red clover extract. Kudzu root fermented powder contains daidzein, which has phytoestrogen activity and can promote gonadal development and ovulation in female fish. Red clover extract is rich in clover extract, which has antioxidant and anti-inflammatory effects, reducing oxidative damage to the gonads and improving egg quality. The male fish maturation feed incorporates fenugreek seed fermented powder, which contains diosgenin, a high-quality raw material for androgen synthesis, directly promoting testicular interstitial cell development and spermatogenesis. During spawning, the female fish are fed a separate spawning feed. The alfalfa meal in the female spawning feed contains daidzein and genistein, whose structures are similar to endogenous estrogens in fish. This can mimic estrogen effects, regulating the endocrine system of the parent fish, promoting ovarian development, inducing ovulation hormone secretion, and improving spawning synchronicity and egg quantity. The female spawning feed promotes egg development through alfalfa meal, protects egg quality through curcumin, and supports high metabolism through enzymatic hydrolysis of fish lysate, thereby increasing both the quantity and quality of eggs produced. The male spawning feed enhances sperm production and motility through brewer's yeast and maintains metabolism through enzymatic hydrolysis of fish lysate, ensuring sperm quality. Together, they balance amino acids, supplement vitamins and minerals, and promote lipid absorption to synergistically meet the basic reproductive needs of both male and female broodstock, ultimately improving the reproductive efficiency of the breeding population.

[0093] Comparing Comparative Example 2 with Example 2, it can be seen that the lack of kudzu root fermented powder and red clover extract in the female fish ripening feed significantly weakens its effects. This is because the daidzein, puerarin, and daidzein contained in kudzu root fermented powder undergo fermentation, which significantly enhances the bioavailability of their active ingredients. These components target and regulate ovarian development, improve the metabolic state of broodstock, stimulate the proliferation of ovarian granulosa cells, and promote estrogen secretion from theca cells, thus driving ovarian development from stage III to stage IV. Furthermore, puerarin possesses strong antioxidant capabilities, reducing the damage to the ovaries caused by reactive oxygen species generated during the high metabolism of the pre-spawning ripening period (protecting mitochondrial function of follicular cells), reducing oocyte DNA fragmentation, and thereby improving fertilization rates. Simultaneously, its anti-inflammatory effect can reduce prostaglandin E2 levels in ovarian tissue, alleviating ovarian inflammation caused by pre-spawning stress. Red clover extract contains genistein, daidzein, and chickpea sprout A, which induce theca cells to secrete progesterone. Through the synergistic effect of progesterone and estrogen, it promotes the development of follicles from the secondary stage to the mature stage. Genistein also increases the fluidity of the oocyte membrane by regulating the cholesterol content of the oocyte membrane, which facilitates sperm penetration and thus improves the fertilization rate.

[0094] By comparing Comparative Example 3 with Example 2, the broodstock breeding method of the present invention, through the use of "tricolor light stimulation," uses green light to stimulate the visual system and hypothalamus of the broodstock, relieving the inhibitory effect of melatonin at night, initially activating the reproductive endocrine axis, and synchronizing the physiological rhythms of the broodstock population to the same starting point, thus promoting synchronous development. White light stimulates the hypothalamus to release gonadotropin-releasing hormone, which in turn prompts the pituitary gland to secrete gonadotropins. Yellow light stimulation then helps the broodstock smoothly transition from a high-intensity "anabolism" state and consolidates the signal of "preparing for reproduction today" within the biological clock, preventing drastic fluctuations in physiological state. By employing "tricolor light stimulation," the synchronous and high-quality development of the gonads in the broodstock population is promoted.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for breeding broodstock of fine-spotted grouper, characterized in that, Includes the following steps: S1. Selection and quarantine of broodstock: Select individuals aged 3-5 years, with robust physique, active swimming, no injuries or diseases, and no deformities as broodstock. Female and male fish are mixed and released at a ratio of 1-2:

1. PCR testing is used to exclude nerve necrosis virus and viral hemorrhagic septicemia virus. S2. Broodstock disinfection: Broodstock that have undergone PCR testing are disinfected by immersion in potassium permanganate solution or povidone-iodine solution; S3. Transfer to rearing pond: The disinfected parent fish are transferred to the rearing pond for rearing. The pond water is recycled water that has been sterilized and filtered. S4. Nutritional Fortification: During the normal feeding period, feed the fish with their usual feed twice a day, at a daily rate of 2.5-3.5% of their body weight. When ≥60% of the female fish's ovaries have developed to stage III, they enter the pre-spawning maturation period. Separate the female and male fish for this period. Feed the females with female maturation feed and the males with male maturation feed three times a day, at a daily rate of 4.0-5.0% of their body weight. During the spawning period, feed the females with female spawning feed and the males with male spawning feed twice a day, at a daily rate of 1.8-2.2% of their body weight. S5. Light control: During the pre-ripening period, "three-color light stimulation" is used, with green light at 06:00, white light at 12:00, and yellow light at 18:

00. S6. Water flow regulation: Water flow stimulation is performed once a week during the pre-ripening period; S7. Gonadal development monitoring: When the female fish's ovary develops to stage III, gonadal development is checked weekly, and gonadal maturity is observed by abdominal palpation or microscopy. S8. Artificial spawning: When the female fish's ovary reaches stage IV maturity and the egg diameter is 0.45-0.55mm, and the male fish's semen is milky white, it enters the spawning period from the maturation period. Oxytocin is injected into both the female and male fish. S9. Postpartum care: After the last spawning, transfer the female and male fish to a medicated bath tank under aeration and oxygenation conditions for 60-90 minutes. Feed them postpartum feed for 15-20 days after the last spawning, three times a day, with a daily feed amount of 2.5-3.5% of the parent fish's body weight.

2. The method for breeding fine-spotted grouper broodstock as described in claim 1, characterized in that, In S1, the female fish weigh 1.0-1.5 kg and the male fish weigh ≥1.5 kg. In S2, the potassium permanganate solution concentration is 0.01-0.02%, and the soaking time is 10-15 min; the povidone-iodine solution concentration is 10-15 ppm, and the soaking time is 20-30 min. In S3, the pond water depth is 1.2-1.8 m, and the depth of a single pond is ≥50 m. 3 The temperature should be controlled at 23-25℃, salinity at 28-32‰, dissolved oxygen ≥5mg / L, ammonia nitrogen ≤0.5mg / L, pH at 7.8-8.2, and stocking density in the rearing pond at 4-8kg / m². 3 .

3. The method for breeding fine-spotted grouper broodstock as described in claim 1, characterized in that, The daily feed in S4 includes the following ingredients by weight: 40-50 parts fish meal, 20-25 parts soybean meal, 13-17 parts wheat flour, 4-6 parts fish oil, 3-5 parts brewer's yeast, and 0.3-0.5 parts premix I. The female fish growth promoter includes the following ingredients by weight: 50-55 parts fish meal, 10-15 parts squid viscera meal, 8-12 parts krill meal, 6-10 parts Schizochyptella meal, 4-6 parts soybean lecithin, 3-5 parts kudzu root fermentation powder, and 0.5-1.5 parts [unclear - possibly a ingredient name or ingredient]. The feed for male fish to mature includes the following ingredients by weight: 48-52 parts fish meal, 8-12 parts squid viscera powder, 5-8 parts krill powder, 4-6 parts brewer's yeast, 1.0-2.0 parts fenugreek seed fermentation powder, and 0.5-0.7 parts premix II. The feed for female fish to spawn includes the following ingredients by weight: 35-40 parts fish meal, 13-17 parts enzymatically hydrolyzed fish slurry, 9-11 parts puffed soybean, and 3-5 parts alfalfa powder. The male fish spawning feed comprises the following ingredients by weight: 38-42 parts fishmeal, 12-16 parts enzymatically hydrolyzed fish slurry, 10-14 parts extruded soybean, 3-5 parts brewer's yeast, 0.15-0.25 parts bile acids, and 0.5-0.7 parts premix III; each part of premix I comprises the following ingredients: 7000-8000 IU vitamin A, 2500-35... Each premix II contains the following ingredients: 00 IU Vitamin D3, 60-100 mg Vitamin E, 100-150 mg Vitamin C, 10-15 mg Folic Acid, 150-200 mg Niacin, 9-11 mg Vitamin K3, 40-45 mg Vitamin B2, 15-20 mg Vitamin B6, 60-80 mg Ferrous Sulfate, 80-120 mg Zinc Sulfate, 35-45 mg Manganese Sulfate, and 30-50 g zeolite powder as a carrier. Each portion of premix III contains the following ingredients: 200-250mg vitamin E, 300-500mg vitamin C, 8000-10000IU vitamin A, 3000-4000IU vitamin D3, 400-600mg inositol, 100-150mg zinc methionine, 120-160mg β-glucan, 0.3-0.4mg sodium selenite, 800-1000mg choline chloride, and 40-50g zeolite powder as a carrier.

4. The method for breeding fine-spotted grouper broodstock as described in claim 3, characterized in that, The kudzu root fermented powder is prepared by the following method: dried kudzu root slices are pulverized to 40-60 mesh to obtain kudzu root powder; the kudzu root powder and wheat bran are mixed at a weight ratio of 7-9:2 to obtain a mixture; the mixture is placed in an autoclave and steam-sterilized at 105-110℃ for 35-45 minutes, then cooled to 24-30℃ for later use; brown sugar is dissolved in sterile water to prepare a 4-6% brown sugar solution, which is dispensed into Erlenmeyer flasks and steam-sterilized at 120-125℃ for 15-20 minutes to obtain sterilized brown sugar solution; Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum are inoculated into the sterilized brown sugar solution, and Bacillus subtilis is sterilized at 35-39℃... Activated Bacillus subtilis was obtained by shaking and culturing at 150-200 rpm for 23-26 hours; activated Saccharomyces cerevisiae was obtained by shaking and culturing at 28-32℃ and 140-160 rpm for 33-39 hours; and activated Lactobacillus plantarum was obtained by static culturing at 35-40℃ for 45-50 hours. The activated Bacillus subtilis, activated Saccharomyces cerevisiae, and activated Lactobacillus plantarum were mixed at a volume ratio of (1.8-2.2):(0.9-1.1):(1.9-2.1) to obtain a mixed bacterial solution. This mixed bacterial solution was inoculated into fresh sterilized brown sugar solution at an inoculum volume of 4-6% and cultured at 30-32℃ until the total viable count reached ≥1.0 × 10⁻⁶. 9 The working fermentation agent is prepared by mixing CFU / mL. The working fermentation agent is then evenly sprayed onto the mixture at 8-12% of the weight of the mixture while stirring. After spraying, the mixture is fermented at 30-35℃ and 80-85% humidity for 70-75 hours, with stirring every 12 hours. After fermentation, the mixture is transferred to a dryer and dried at 60-65℃ until the moisture content is ≤10%. The mixture is then passed through a 60-100 mesh sieve to obtain kudzu root fermentation powder.

5. The method for breeding fine-spotted grouper broodstock as described in claim 3, characterized in that, The red clover extract is prepared by the following method: dried red clover is pulverized to 40-60 mesh to obtain clover powder; the clover powder is mixed with 70-80wt% ethanol solution at a material-to-liquid ratio of 1:8-10 in an extraction tank, heated to 75-80℃, and refluxed twice for 1.5-2.0 h each time. After extraction, the mixture is filtered through a 150-200 mesh sieve, and the two extracts are combined. The combined extract is centrifuged at 6000-8000 rpm for 15-25 min, and the supernatant is collected; the supernatant is transferred to a concentration device and concentrated to a relative density at 50-60℃ and a vacuum of -0.08 to -0.10 MPa. The concentration was 1.10-1.15, and a concentrated extract was obtained for later use. β-Cyclodextrin and purified water were mixed at a ratio of 1:14-16 and stirred at 58-62℃ to prepare a β-cyclodextrin solution. The concentrated extract was added to the β-cyclodextrin solution while stirring at a weight ratio of 1:1.4-1.

6. The mixture was stirred continuously at 80-150 rpm for 1.8-2.2 h, and then refrigerated at 3-5℃ for 10-15 h to obtain an inclusion complex. The inclusion complex was transferred to a vacuum drying device and dried at 60-65℃ and -0.06 to -0.08 MPa until the moisture content was ≤5%. The solution was then passed through an 80-100 mesh sieve to obtain the red clover extract.

6. The method for breeding fine-spotted grouper broodstock as described in claim 1, characterized in that, In S5, the green light intensity is 900-1100 lux and the irradiation time is 20-30 minutes; the white light intensity is 1400-1600 lux and the irradiation time is 30-40 minutes; and the yellow light intensity is 1300-1500 lux and the irradiation time is 15-25 minutes.

7. The method for breeding fine-spotted grouper broodstock as described in claim 1, characterized in that... The water flow velocity in S6 is 0.3-0.4 m / s, and is maintained for 1.5-2.0 h.

8. The method for breeding fine-spotted grouper broodstock as described in claim 1, characterized in that, In the S8 method, female fish are injected with HCG 800-1000 IU / kg + LHRH-A3 4-6μg / kg in two doses. The first dose is 2 / 3 of the total dose, and the remaining dose is injected 24 hours later. The dose for male fish is halved and injected simultaneously with the second dose of the female fish.

9. The method for breeding fine-spotted grouper broodstock as described in claim 1, characterized in that, The S9 herbal bath comprises the following ingredients in parts by weight: 3.0-5.0 parts hydrolyzed sodium hyaluronate, 1.0-2.0 parts γ-aminobutyric acid, 0.5-1.0 parts L-theanine, 1.0-2.0 parts lysozyme, 1.0-1.5 parts D-calcium pantothenate, 0.1-0.2 parts astragaloside A, and 90-100 parts purified water.

10. The method for breeding fine-spotted grouper broodstock as described in claim 1, characterized in that, The post-harvest feed in S9 includes the following raw materials in parts by weight: 35-45 parts fish meal, 10-15 parts fermented soybean meal, 5-8 parts krill meal, 15-20 parts high-gluten flour, 4-6 parts fish oil, 2-3 parts soybean lecithin, 0.1-0.2 parts mannan oligosaccharide, 0.3-0.5 parts glutamine, 0.1-0.2 parts eyebright extract, 1.0-2.0 parts hydrolyzed collagen peptides, and 0.4-0.6 parts premix I.