A large-scale breeding method of tiger stripe and mouse stripe hybrid

Abstract

The application relates to a large-scale breeding method of tiger and mouse hybrid fry. The method comprises the following steps: transferring the initial hatched fry of the tiger and mouse hybrid to a breeding pond, and feeding oyster fertilized eggs; feeding SS type rotifers which are intensively cultured by Chlorella liquid at the age of 4-15 days; feeding Artemia nauplii which are intensively cultured by DHA intensifier and Neurospora crassa at the age of 16-30 days; Neurospora crassa ); feeding Mongolian limnadia and Artemia at the age of 31-35 days; gradually replacing the live bait with micro-fermentation micro-pellet feed and finally completely converting to the micro-fermentation micro-pellet feed at the age of 36-60 days, wherein the micro-fermentation micro-pellet feed is prepared by solid-state micro-fermentation of micro-pellet feed by Geobacillus stearothermophilus; and water quality management of the breeding pond: maintaining salinity of 30-32‰, temperature of 25-28 DEG C, dissolved oxygen of greater than or equal to 6 mg / L, and ammonia nitrogen of less than 0.5 mg / L. The method can significantly improve the survival rate, growth performance and low-salt tolerance of the hybrid fry, the survival rate can reach 92.0%-94.5%, and the lower limit of salt tolerance can reach 10.0-10.7‰, and the method is suitable for large-scale popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of fish seedling technology, specifically relating to a method for large-scale seedling production of a hybrid of tiger grouper and mouse grouper. Background Technology

[0002] Mouse-shaped grouper, scientific name: humpback perch ( Chromileptes altivelis (This refers to a species of fish belonging to the order Perciformes, family Serranidae, subfamily Scutinae, and genus Perciformes.) The mouse grouper (Siniperca maculatus) has delicious flesh, high nutritional value, and extremely high economic value, making it one of the most prized marine aquaculture fish. The tiger grouper (Siniperca maculatus) is also known as the brown-spotted grouper (Siniperca maculatus). Epinephelus fuscoguttatus (Grouper), a fish belonging to the order Perciformes, family Serranidae, and genus Leptodontidae, is widely distributed in the Indo-West Pacific and Red Sea regions and is one of the important aquaculture species in the southern coastal areas of my country.

[0003] Grouper hybridization breeding utilizes the genetic complementarity between different grouper species or genera to cultivate offspring that combine the superior traits of both parents. The applicant previously successfully developed a breeding method for crossbreeding tiger grouper and mouse grouper, producing hybrid offspring that effectively combine the superior traits of both parents, exhibiting advantages in aquaculture such as rapid growth, high survival rate, low deformity rate, and high nutritional value. High-temperature and low-salt tolerance has also been improved. However, there is still room for improvement in various indicators, and the main technical challenges currently are as follows: First, in the breeding of hybrid grouper fry, the first three months after the fry begin feeding are a period of high mortality. With traditional breeding methods, the survival rate is difficult to exceed 80%. The survival rate of fry from reciprocal crosses is particularly low.

[0004] Second, although the salt tolerance of the orthogonal offspring has improved, it still cannot meet the requirements of low-salinity seawater aquaculture, which limits its promotion and application in low-salinity estuarine areas or areas with large salinity fluctuations.

[0005] Therefore, there is an urgent need for a method for large-scale breeding of tiger grouper and mouse grouper hybrids that can systematically solve the above problems and further improve the survival rate, growth performance and salt tolerance of hybrid fry. Summary of the Invention

[0006] The purpose of this invention is to provide a method for large-scale seedling production of tiger-spotted and mouse-spotted hybrids to solve the above-mentioned problems.

[0007] The technical solution of this invention mainly includes the following: A method for large-scale seedling production of a hybrid of tiger stripe and mouse stripe includes the following steps: S1. Transfer the newly hatched fry of the tiger grouper and mouse grouper hybrid to the nursery pond and feed them with oyster fertilized eggs; S2. Feed SS-type rotifers cultured in Chlorella solution from 4 to 15 days old; S3. Feeding infants aged 16-30 days with DHA-fortified bacteria and Neurospora crassa ( Neurospora crassa Artemia nauplii that are jointly reinforced; S4. Feed Daphnia mongolica and Artemia var. mongolica at 31-35 days old; S5. Gradually replace live bait with micro-fermented microparticle feed from 36 to 60 days of age and eventually completely convert to micro-fermented microparticle feed. The micro-fermented microparticle feed is prepared by solid-state micro-fermentation of microparticle feed by Bacillus stearothermophilus.

[0008] S6. Seedling pond water quality management: maintain salinity 30~32‰, temperature 25~28℃, dissolved oxygen ≥6mg / L, ammonia nitrogen <0.5mg / L.

[0009] Preferably, in step S1, the density of newly hatched fry is 10,000 to 30,000 fry / m³, and the feeding density of oyster fertilized eggs is 5 to 8 eggs / mL, fed twice a day; in step S2, the feeding density of SS-type rotifers is 5 to 8 eggs / mL, fed twice a day; in step S3, the feeding density of Artemia nauplii is 1.0 to 1.5 eggs / mL, fed three times a day; in step S4, the feeding density of Daphnia mongolica and Artemia salina is 400 to 500 eggs / L, the feeding ratio of Daphnia mongolica to Artemia salina is 1:1, and fed three times a day.

[0010] Preferably, the method for enhancing the nauplius larvae in step S3 is as follows: transferring the nauplius larvae into an enhancement tank, adjusting the density to 100-200 nauplius / mL, and adding DHA enhancer and Neurospora crassa (… Neurospora crassa The bacterial solution was subjected to enhancement treatment for 12-24 hours.

[0011] Preferably, the amount of DHA fortifier added is 0.2~0.4 g / L, and the final concentration of the rough Neurospora spp. solution is 1×10⁻⁶. 6 ~1×10 7 cfu / mL.

[0012] Preferably, the DHA fortifier is added at a concentration of 0.2 g / L, and the final concentration of the rough Neurospora spp. solution is 1 × 10⁻⁶. 7 The concentration of CFU / mL and the fortification time are 12 hours; or the amount of DHA fortifier added is 0.4 g / L, and the final concentration of the rough Neurospora spp. solution is 1 × 10⁻⁶. 6 cfu / mL, enhancement time was 24 hours.

[0013] Preferably, the preparation method of the micro-fermented microparticle feed in step S5 is as follows: mixing the components of the microparticle feed and inoculating with activated thermophilic Bacillus steatis (Bacillus steatis). Geobacillus stearothermophilus Bacterial solution, final concentration 4×10 6 ~5×10 6Mix the CFU / g material thoroughly and evenly; place the inoculated material in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 12~24 hours, turning it over every 2~3 hours during the period; after micro-fermentation, dry it until the moisture content is <10%, sieve and grade it to obtain micro-fermented microparticle feeds of different particle sizes.

[0014] Preferably, the microparticle feed formula, by mass percentage, is as follows: 55%~60% fish meal, 10%~15% wheat flour, 5%~8% soybean meal, 8%~10% fish oil, 5%~8% brewer's yeast powder, 1%~1.5% calcium dihydrogen phosphate, 0.1%~0.2% choline chloride, 0.8%~1.5% seaweed powder, 0.1%~0.2% Tween 80, 0.03%~0.05% phytase, with the remainder being water.

[0015] Preferably, the method for replacing live bait with micro-fermented microparticle feed in step S5 is as follows: 10% (w / w) of micro-fermented microparticle feed is mixed into the live bait to replace an equal amount of live bait, and the proportion is increased by 10% every 2 days until it is completely converted to micro-fermented microparticle feed.

[0016] Preferably, the tiger-spotted and mouse-spotted hybrid is a direct or inverse cross: the direct cross uses a male tiger-spotted fish as the father and a female mouse-spotted fish as the mother; the inverse cross uses a female tiger-spotted fish as the mother and a male mouse-spotted fish as the father.

[0017] Preferably, in step S2, the SS-type rotifers are cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours before being fed.

[0018] The beneficial effects of this invention are: (1) This invention introduces Neurospora crassa (a type of bacteria) into the nauplius stage of Artemia var. crassa (a type of bacteria) during the strengthening stage of Artemia var. crassa. Neurospora crassa In conjunction with DHA fortifiers, *Neurospora crassa* significantly enhances the nutritional value of *Artemia nauplii* nauplii, thereby improving the growth performance and survival rate of fish fry.

[0019] (2) This invention uses *Bacillus stearothermophilus* (…). Geobacillus stearothermophilus Solid-state micro-fermentation of microparticle feed effectively degrades anti-nutritional factors in the feed, improves the bioavailability of protein and minerals, enhances feed palatability and digestibility, and further promotes the growth and development of fish fry, as well as their tolerance to low salinity and survival rate.

[0020] (3) The synergistic application of two technologies—Neurospora crassa fortified Artemia and Bacillus thuringiensis micro-fermentation feed—significantly increased the survival rate of hybrid fish fry to 92.0%–94.5%. This was significantly higher than conventional fry raising methods, methods using only micro-fermented feed without Artemia fortification, methods using only Artemia fortification without micro-fermented feed, and methods using Bacillus subtilis instead of Bacillus thuringiensis for micro-fermentation. This indicates that the two technologies have a significant synergistic effect.

[0021] (4) The hybrid fish fry cultivated by the method of the present invention have the advantages of fast growth, high survival rate and strong tolerance to low salt, which can meet the requirements of low salt seawater aquaculture.

[0022] (5) The method of the present invention has standardized process, clear parameters, and strong operability. It is suitable for large-scale seedling production of tiger stripe and mouse stripe hybrids and has broad prospects for promotion and application. Detailed Implementation

[0023] To facilitate a clearer understanding of the technical content of this invention by those skilled in the art, the invention will be further described below in conjunction with specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] The following feeding density of live bait refers to the target concentration of bait in the seedling water after feeding.

[0026] The tiger-spotted and mouse-spotted hybrids described in the examples or comparative examples were derived from the following methods: (1) Parent breeding Parent fish were separated into different ponds for rearing, with a rearing density of 2 humpback bass per m. 3 Brown-spotted grouper stocking density: 0.5 fish / m² 3 The seawater salinity is 30‰~32‰, dissolved oxygen is ≥6mg / L, and pH is 8.0~8.2.

[0027] Phase 1: Parent fish are introduced into the pond. The water temperature for raising humpback bass is 23-24℃, and the water temperature for raising brown-spotted grouper is 25-26℃. The photoperiod is 10L:14D, and the light intensity is 500-800lx.

[0028] Feed the fish primarily with fresh mixed fish (sharpfish) in feed A, at a daily rate of 2%–3% of body weight, twice a day, once in the morning and once in the afternoon. By weight percentage, feed A contains 3% fish oil, 0.02% vitamin E, 0.03% vitamin C, and the remainder is fresh mixed fish.

[0029] The humpback bass were continuously cultured for 7 days, and the brown-spotted grouper was continuously cultured for 14 days.

[0030] Phase Two: The water temperature for humpback bass rearing is raised to 25-26℃, and the water temperature for brown-spotted grouper rearing is raised to 28-29℃. The photoperiod is extended to 12L:12D, and the light intensity is 800-1000lx.

[0031] Feed the fish with feed B, which mainly consists of fresh miscellaneous fish (anchovies), at a daily rate of 3% to 4% of body weight, twice a day, once in the morning and once in the afternoon. By weight percentage, feed B contains 2% phospholipids, 0.5% cholesterol, 1.5% eicosapentaenoic acid, and 0.03% vitamin E, with the remainder being fresh miscellaneous fish.

[0032] Both humpback bass and brown-spotted grouper were continuously cultured for 7 days.

[0033] Phase 3: The water temperature for humpback bass rearing is reduced to 22-23℃, and the water temperature for brown-spotted grouper rearing is reduced to 24-26℃. The photoperiod is extended to 14L:10D.

[0034] Feed the fish with feed C, which is mainly composed of fresh anchovies, at a daily rate of 3% to 4% of body weight, twice a day, once in the morning and once in the afternoon. By weight percentage, feed C contains 3% lecithin, 0.04% vitamin E, 0.005% astaxanthin, 2% docosahexaenoic acid, and the remainder is fresh anchovies.

[0035] The humpback bass and brown-spotted grouper were cultured for 7 days each.

[0036] Phase Four: Initiate temperature pulse stimulation, maintain 24°C for 48 hours, then lower to 20°C and maintain for 24 hours, then raise to 28°C and maintain for 48 hours. This cycle is repeated twice. Humpback sea bass and brown-spotted grouper synchronously perform the temperature pulse cycle.

[0037] During this period, feed was provided with feed D, which, by weight percentage, contained 3% docosahexaenoic acid, 3% lecithin, and 0.05% vitamin E, with the remainder being fresh miscellaneous fish (anchovy). The daily feed amount was reduced to 1.5%–2% of body weight.

[0038] Hormone-induced labor: The mother receives an intramuscular injection of hCG in her back at a dose of 500-800 IU / kg. Observe the mother's abdominal changes and ovulation behavior for 36-48 hours post-injection. Collect oocytes when the mother's abdomen is significantly distended, the genital pore is dilated, and clear oocytes flow out upon gentle abdominal pressure.

[0039] (2) Optimization of the fertilization fluid system Prepare an enhancement solution containing BSA (bovine serum albumin): 0.1% (w / v), taurine 10 mmol / L, melibiose 0.5% (w / v), caffeine 5 mmol / L, sodium cromoglycate 2 mmol / L, with the remainder being the base solution.

[0040] Base solution: artificial seawater, salinity 31‰, pH: 8.2~8.4, maintained using a 20 mmol / L Tris-HCl buffer system.

[0041] (3) Artificial insemination The eggs (approximately 500,000) were placed in a basin and spread out in a single layer.

[0042] Collect 2-3 mL of the father's original semen by gently pressing the abdomen. Use semen and eggs at a ratio of 1 mL of original semen to 1-1.2 kg of eggs.

[0043] Add the original semen to the enhancement solution (using a semen-enhancing solution volume ratio of 1:5), mix gently, and immediately pour in the eggs. Gently stir for 30 seconds to ensure full contact between the sperm and eggs, and let stand for 5 minutes. Wash the fertilized eggs three times with fresh artificial seawater.

[0044] (4) Hatching Fertilized eggs are transferred to an incubation tank (500L volume) at a density of ≤300 eggs / L. Incubation conditions: seawater salinity 30~32‰, temperature 26~28℃, dissolved oxygen ≥6mg / L, pH 8.0~8.2, with micro-aeration to ensure uniform suspension of the eggs.

[0045] Example 1: A method for large-scale seedling production of a hybrid of tiger stripe and mouse stripe 0-3 days old: Newly hatched fry (offspring of a 4-year-old female mouse-spotted fish and a 6-year-old male tiger-spotted fish) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0046] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0047] 16-30 days old: Feed with DHA fortifier (Algamac 2000) and Neurospora crassa ( Neurospora crassa Artemia nauplii larvae after 12 hours of SHBCC D82167 enhancement were fed at a density of approximately 1.0-1.5 nauplii / mL, and were fed 3 times daily.

[0048] Artemia nauplii enrichment method: Transfer Artemia nauplii to an enrichment tank and adjust the density to 100-200 nauplii / mL. Add DHA fortifier (Algamac 2000) at 0.2 g / L, at a concentration of 1×10⁻⁶. 7 Enhancement treatment was performed by adding coarse Neurospora solani solution to a final concentration of cfu / mL. Micro-aeration was initiated to maintain DO > 5 mg / L.

[0049] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0050] 36-60 days old: Mix 10% (w / w) of micro-fermented microparticle feed into the live bait (i.e., Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to micro-fermented microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, and the remainder is water.

[0051] Preparation method of micro-fermented microparticle feed: Mix the various components of the microparticle feed and inoculate with activated Bacillus stearothermophilus at an inoculation rate of 5% (v / w). Geobacillus stearothermophilus SMHCC D80490 bacterial culture (final concentration approximately 5×10⁻⁶) 6 (CFU / g of material), mix thoroughly. Spread the inoculated material evenly on a sterile stainless steel tray to a thickness of 3-5 cm. Place in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 12 hours, turning it over every 2-3 hours. After micro-fermentation, dry with hot air at 55℃ until the moisture content is <10%, then sieve and grade to obtain micro-fermented microparticle feeds of different particle sizes.

[0052] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30‰-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / ml, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0053] Example 2: A method for large-scale seedling production of a hybrid of tiger-spotted and mouse-spotted cultivars 0-3 days old: Newly hatched fry (offspring of a 4-year-old female tiger grouper and a 6-year-old male mouse grouper) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0054] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0055] 16-30 days old: Feed with DHA fortifier (Algamac 2000) and Neurospora crassa ( Neurospora crassa Artemia nauplii larvae after 12 hours of SHBCC D82167 enhancement were fed at a density of approximately 1.0-1.5 nauplii / mL, and were fed 3 times daily.

[0056] Artemia nauplii enrichment method: Transfer Artemia nauplii to an enrichment tank and adjust the density to 100-200 nauplii / mL. Add DHA fortifier (Algamac 2000) at 0.2 g / L, at a concentration of 1×10⁻⁶. 7 Enhancement treatment was performed by adding coarse Neurospora solani solution to a final concentration of cfu / mL. Micro-aeration was initiated to maintain DO > 5 mg / L.

[0057] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0058] 36-60 days old: Mix 10% (w / w) of micro-fermented microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to micro-fermented microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, and the remainder is water.

[0059] Preparation method of micro-fermented microparticle feed: Mix the various components of the microparticle feed and inoculate with activated Bacillus stearothermophilus at an inoculation rate of 5% (v / w). Geobacillus stearothermophilus SMHCC D80490 bacterial culture (final concentration approximately 5×10⁻⁶) 6 (CFU / g of material), mix thoroughly. Spread the inoculated material evenly on a sterile stainless steel tray to a thickness of 3-5 cm. Place in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 12 hours, turning it over every 2-3 hours during this period. After micro-fermentation, dry with hot air at 55℃ until the moisture content is <10%, then sieve and grade to obtain micro-fermented microparticle feeds of different particle sizes.

[0060] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / mL, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0061] Example 3: A method for large-scale seedling production of a hybrid of tiger-spotted and mouse-spotted cultivars 0-3 days old: Newly hatched fry (offspring of a 4-year-old female tiger grouper and a 6-year-old male mouse grouper) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0062] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0063] 16-30 days old: Feed with DHA fortifier (Algamac 2000) and Neurospora crassa ( Neurospora crassa Artemia nauplii larvae, after being enhanced with SHBCC D82167 for 24 hours, were fed at a density of approximately 1.0-1.5 nauplii / mL, three times a day.

[0064] Artemia nauplii enrichment method: Transfer Artemia nauplii to an enrichment tank and adjust the density to 100-200 nauplii / mL. Add DHA fortifier (Algamac 2000) at 0.4 g / L, at a concentration of 1×10⁻⁶. 6 Enhancement treatment was performed by adding coarse Neurospora solani solution to a final concentration of cfu / mL. Micro-aeration was initiated to maintain DO > 5 mg / L.

[0065] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0066] 36-60 days old: Mix 10% (w / w) of micro-fermented microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to micro-fermented microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, and the remainder is water.

[0067] Preparation method of micro-fermented microparticle feed: Mix the components of the microparticle feed and inoculate with activated Bacillus stearothermophilus at an inoculation rate of 8% (v / w). Geobacillus stearothermophilus SMHCC D80490 bacterial culture (final concentration approximately 4×10⁻⁶) 6(CFU / g of material), mix thoroughly. Spread the inoculated material evenly on a sterile stainless steel tray to a thickness of 3-5 cm. Place in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 24 hours, turning it over every 2-3 hours. After micro-fermentation, dry with hot air at 55℃ until the moisture content is <10%, then sieve and grade to obtain micro-fermented microparticle feeds of different particle sizes.

[0068] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30‰-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / ml, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0069] Example 4: A method for large-scale seedling production of a hybrid of tiger-spotted and mouse-spotted cultivars 0-3 days old: Newly hatched fry (offspring of a 4-year-old female tiger grouper and a 6-year-old male mouse grouper) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0070] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0071] 16-30 days old: Feed with DHA fortifier (Algamac 2000) and Neurospora crassa ( Neurospora crassa Artemia nauplii larvae after 12 hours of SHBCC D82167 enhancement were fed at a density of approximately 1.0-1.5 nauplii / mL, and were fed 3 times daily.

[0072] Artemia nauplii enrichment method: Transfer Artemia nauplii to an enrichment tank and adjust the density to 100-200 nauplii / mL. Add DHA fortifier (Algamac 2000) at 0.2 g / L, at a concentration of 1×10⁻⁶. 7 Enhancement treatment was performed by adding coarse Neurospora solani solution to a final concentration of cfu / mL. Micro-aeration was initiated to maintain DO > 5 mg / L.

[0073] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0074] 36-60 days old: Mix 10% (w / w) of micro-fermented microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to micro-fermented microparticle feed. Microparticle feed formula (by weight percentage): fish meal 55%, wheat flour 10%, soybean meal 8%, fish oil 10%, brewer's yeast powder 8%, calcium dihydrogen phosphate 1.5%, choline chloride 0.1%, seaweed powder 1.5%, Tween 80 0.2%, phytase 0.05%, balance water.

[0075] Preparation method of micro-fermented microparticle feed: Mix the various components of the microparticle feed and inoculate with activated Bacillus stearothermophilus at an inoculation rate of 5% (v / w). Geobacillus stearothermophilus SMHCC D80490 bacterial culture (final concentration approximately 5×10⁻⁶) 6 (CFU / g of material), mix thoroughly. Spread the inoculated material evenly on a sterile stainless steel tray to a thickness of 3-5 cm. Place in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 12 hours, turning it over every 2-3 hours during this period. After micro-fermentation, dry with hot air at 55℃ until the moisture content is <10%, then sieve and grade to obtain micro-fermented microparticle feeds of different particle sizes.

[0076] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / ml, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0077] Comparative Example 1: A method for large-scale seedling production of a tiger-spotted and mouse-spotted hybrid 0-3 days old: Newly hatched fry (offspring of a 4-year-old female mouse-spotted fish and a 6-year-old male tiger-spotted fish) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0078] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0079] 16-30 days old: Feed Artemia nauplii fortified with DHA fortifier (Algamac 2000) for 12 hours, at a density of about 1.0-1.5 nauplii / mL, 3 times a day.

[0080] Artemia nauplii enhancement method: Transfer Artemia nauplii to an enhancement tank and adjust the density to 100-200 nauplii / mL. Add DHA enhancer (Algamac 2000) at 0.2 g / L for enhancement treatment. Turn on micro-aeration to maintain DO > 5 mg / L.

[0081] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0082] 36-60 days old: Mix 10% (w / w) microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, balance water.

[0083] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / ml, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0084] Comparative Example 2: A method for large-scale seedling production of a hybrid of tiger stripe and mouse stripe 0-3 days old: Newly hatched fry (offspring of a 4-year-old female tiger grouper and a 6-year-old male mouse grouper) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0085] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0086] 16-30 days old: Feed Artemia nauplii fortified with DHA fortifier (Algamac 2000) for 12 hours, at a density of about 1.0-1.5 nauplii / mL, 3 times a day.

[0087] Artemia nauplii enhancement method: Transfer Artemia nauplii to an enhancement tank and adjust the density to 100-200 nauplii / mL. Add DHA enhancer (Algamac 2000) at 0.2 g / L for enhancement treatment. Turn on micro-aeration to maintain DO > 5 mg / L.

[0088] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0089] 36-60 days old: Mix 10% (w / w) microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, balance water.

[0090] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / ml, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0091] Comparative Example 3: A method for large-scale seedling production of a tiger-spotted and mouse-spotted hybrid. 0-3 days old: Newly hatched fry (offspring of a 4-year-old female tiger grouper and a 6-year-old male mouse grouper) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0092] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0093] 16-30 days old: Feed Artemia nauplii fortified with DHA fortifier (Algamac 2000) for 12 hours, at a density of about 1.0-1.5 nauplii / mL, 3 times a day.

[0094] Artemia nauplii enhancement method: Transfer Artemia nauplii to an enhancement tank and adjust the density to 100-200 nauplii / mL. Add DHA enhancer (Algamac 2000) at 0.2 g / L for enhancement treatment. Turn on micro-aeration to maintain DO > 5 mg / L.

[0095] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0096] 36-60 days old: Mix 10% (w / w) of micro-fermented microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to micro-fermented microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, and the remainder is water.

[0097] Preparation method of micro-fermented microparticle feed: Mix the various components of the microparticle feed and inoculate with activated Bacillus stearothermophilus at an inoculation rate of 5% (v / w). Geobacillus stearothermophilus SMHCC D80490 bacterial culture (final concentration approximately 5×10⁻⁶) 6 (CFU / g of material), mix thoroughly. Spread the inoculated material evenly on a sterile stainless steel tray to a thickness of 3-5 cm. Place in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 12 hours, turning it over every 2-3 hours during this period. After micro-fermentation, dry with hot air at 55℃ until the moisture content is <10%, then sieve and grade to obtain micro-fermented microparticle feeds of different particle sizes.

[0098] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / ml, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0099] Comparative Example 4: A method for large-scale seedling production of a tiger-spotted and mouse-spotted hybrid. 0-3 days old: Newly hatched fry (offspring of a 4-year-old female tiger grouper and a 6-year-old male mouse grouper) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0100] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0101] 16-30 days old: Feed with DHA fortifier (Algamac 2000) and Neurospora crassa ( Neurospora crassa Artemia nauplii larvae after 12 hours of SHBCC D82167 enhancement were fed at a density of approximately 1.0-1.5 nauplii / mL, and were fed 3 times daily.

[0102] Artemia nauplii enrichment method: Transfer Artemia nauplii to an enrichment tank and adjust the density to 100-200 nauplii / mL. Add DHA fortifier (Algamac 2000) at 0.2 g / L, at a concentration of 1×10⁻⁶. 7 Enhancement treatment was performed by adding coarse Neurospora solani solution to a final concentration of cfu / mL. Micro-aeration was initiated to maintain DO > 5 mg / L.

[0103] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0104] 36-60 days old: Mix 10% (w / w) microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, balance water.

[0105] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / ml, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0106] Comparative Example 5: A method for large-scale seedling production of a tiger-spotted and mouse-spotted hybrid. 0-3 days old: Newly hatched fry (offspring of a 4-year-old female tiger grouper and a 6-year-old male mouse grouper) are transferred to a nursery pond (5m×3m×1.2m) for rearing at a density of 20,000 fry / m². 3 Once the newly hatched fry begin to eat, they can be fed fertilized oyster eggs at a density of about 5-8 eggs / mL, twice a day.

[0107] 4-15 days old: Feed SS type rotifers cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours at a density of about 5-8 rotifers / mL, twice a day.

[0108] 16-30 days old: Feed with DHA fortifier (Algamac 2000) and Neurospora crassa ( Neurospora crassa Artemia nauplii larvae after 12 hours of SHBCC D82167 enhancement were fed at a density of approximately 1.0-1.5 nauplii / mL, and were fed 3 times daily.

[0109] Artemia nauplii enrichment method: Transfer Artemia nauplii to an enrichment tank and adjust the density to 100-200 nauplii / mL. Add DHA fortifier (Algamac 2000) at 0.2 g / L, at a concentration of 1×10⁻⁶. 7 Enhancement treatment was performed by adding coarse Neurospora solani solution to a final concentration of cfu / mL. Micro-aeration was initiated to maintain DO > 5 mg / L.

[0110] 31-35 days old: Feed Daphnia mongolica and Artemia salina at a density of 400-500 per L, with a feeding ratio of 1:1, and feed 3 times a day.

[0111] 36-60 days old: Mix 10% (w / w) of micro-fermented microparticle feed into the live bait (Daphnia mongolica + Artemia salina) to replace an equal amount of live bait. Increase the proportion by 10% every 2 days until completely switched to micro-fermented microparticle feed. Microparticle feed formula (by weight percentage): fish meal 60%, wheat flour 15%, soybean meal 5%, fish oil 8%, brewer's yeast powder 5%, calcium dihydrogen phosphate 1%, choline chloride 0.2%, seaweed powder 0.8%, Tween 80 0.1%, phytase 0.03%, and the remainder is water.

[0112] Preparation method of micro-fermented microparticle feed: Mix the components of the microparticle feed and inoculate with Bacillus subtilis at an inoculation rate of 5% (v / w). Bacillus subtilis SMHCC D83124 bacterial culture (final concentration approximately 5×10⁻⁶) 6 (CFU / g of material), mix thoroughly. Spread the inoculated material evenly on a sterile stainless steel tray to a thickness of 3-5 cm. Place in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 12 hours, turning it over every 2-3 hours. After micro-fermentation, dry with hot air at 55℃ until the moisture content is <10%, then sieve and grade to obtain micro-fermented microparticle feeds of different particle sizes.

[0113] Water quality management in the nursery pond: For seedlings aged 0-35 days, change 30% of the water daily; from 36 days onwards, change 50% of the water daily. Maintain salinity at 30-32‰, temperature at 25-28℃, dissolved oxygen ≥6mg / L, and ammonia nitrogen <0.5mg / L. Test the Vibrio count in the nursery water every 3 days; if it exceeds 1×10⁻⁶, [further action should be taken]. 4 If CFU / mL, increase the water exchange rate and apply 2 mg / L povidone-iodine.

[0114] Evaluation of seedling cultivation effect in experimental cases Data detection methods for each indicator: 1. The total length (mm) was measured directly with vernier calipers, and the weight (g) was weighed using an electronic balance. Sample size: 30 tails were randomly selected from each group.

[0115] 2. Survival rate (%), direct count statistics, sample size: all individuals in each group.

[0116] Survival rate (%) = N t / N0× 100%, initial number of fish tails N0, number of surviving individuals N t Calculate the mean ± standard deviation for each group.

[0117] 3. The lower limit of salt tolerance was determined using the gradient salt reduction-half-tolerance method. Sample size: 15 tails were randomly selected from each group, with 3 replicates per group.

[0118] Operating steps: (1) Start with a salinity of 32‰ and allow the fish to adapt to this salinity for 24 hours; (2) Decrease the salinity at a rate of 2‰ / 12 hours (by adding aerated freshwater); (3) Observe every 12 hours and record any abnormal behavior of each fish (frantic swimming, accelerated breathing, darkening of body color); (4) Use the salinity at which 50% of the individuals show obvious stress response (continuous surfacing, rolling over, loss of emergency reflex) as the lower limit of salt tolerance; (5) Once the critical salinity is reached, immediately return to normal seawater.

[0119] The 60-day-old fish fry obtained after cultivation in each example were cultured under the same conditions (salinity 31‰, temperature 26~28℃, density 20 fish / m³). 3 The seedlings were raised to 6 months of age, during which time they were fed the microparticle feed of Comparative Example 1. The seedling raising effects of each example and comparative example are shown in Tables 1 and 2 below.

[0120] Table 1

[0121] Table 2

[0122] The results above show that the survival rates of Examples 1-4 all reached over 92%, and were significantly higher than those of the comparative example. This indicates that the technical solution of the present invention has a significant effect on improving the survival rate of hybrid fish fry.

[0123] Comparing Example 2 and Comparative Example 3, the difference lies in that Example 2 used *Neurospora crassa* in combination with a DHA enhancer to strengthen Artemia nauplii, while Comparative Example 3 only used a DHA enhancer. The survival rate of Example 2 was significantly higher than that of Comparative Example 3. This indicates that *Neurospora crassa* plays an irreplaceable role in the strengthening process of Artemia nauplii and makes a significant contribution to the survival rate in the later seedling stage.

[0124] Comparing Example 2 and Comparative Example 4, the difference lies in that Example 2 used Bacillus stearothermophilus micro-fermented microparticle feed during the 36-60 day age stage, while Comparative Example 4 used unfermented ordinary microparticle feed. The survival rate of Example 2 was 12.7 percentage points higher than that of Comparative Example 4, and the lower salt tolerance limit of Example 2 (8‰) was lower than that of Comparative Example 4 (11.3‰). This indicates that the various digestive enzymes and immunostimulants in the micro-fermented microparticle feed can significantly improve the immunity and stress resistance of the larvae.

[0125] Comparative Example 2 (neither using Neurospora crassa fortification nor micro-fermented feed) had the lowest survival rate, at only 70.0%; Comparative Example 3 (using only micro-fermented feed, without Neurospora crassa fortification) had a survival rate of 80.2%, an increase of 10.2 percentage points compared to Comparative Example 2; Comparative Example 4 (using only Neurospora crassa fortification, without micro-fermented feed) had a survival rate of 78.3%, an increase of 8.3 percentage points compared to Comparative Example 2; Example 2 (using both technologies in combination) achieved a survival rate of 92.0%, an increase of 22 percentage points compared to Comparative Example 2. These data indicate that the combined effect of the two technologies is far greater than the sum of their individual effects, demonstrating a significant synergistic effect.

[0126] Comparing Example 2 and Comparative Example 5, both used *Neurospora crassa* to enhance the production of Artemia nauplius larvae and micro-fermented microparticle feed. However, Example 2 used *Bacillus thermophilus*, while Comparative Example 5 used *Bacillus subtilis*. Example 2 showed better survival rate and lower salt tolerance than Comparative Example 5. This indicates that *Bacillus thermophilus* has a species-specific advantage in solid-state microfermentation.

[0127] The lower salt tolerance limits of all embodiments were 8.0–10.7‰, which was superior to 14.7‰ in Comparative Example 1 and 12.0‰ in Comparative Example 2. This indicates that micro-fermented microparticle feed and Neurospora crassa fortification contribute to improved salt tolerance. The improved low-salt tolerance may be related to beneficial metabolites (such as short-chain fatty acids and antimicrobial peptides) produced during micro-fermentation and immunostimulants provided by Neurospora crassa, which enhance gill ion regulation and osmolarity regulation.

[0128] In summary, this invention achieves a comprehensive improvement in the survival rate, growth performance, and salt tolerance of tiger grouper and mouse grouper hybrid seedlings through two core technological innovations: the combined use of Neurospora crassa and DHA fortifier to enhance Artemia nauplius larvae and solid micro-fermentation microparticle feed from Bacillus stearothermophilus. These improvements are further supported by scientific feed transition strategies and water quality management measures. All indicators are significantly superior to traditional seedling methods.

[0129] The above description is only a partial embodiment of the technical solution of the present invention. These embodiments are merely illustrative examples intended to help those skilled in the art understand the core concept of the present invention, and are not exhaustive enumerations of all embodiments of the present invention. The scope of protection of the present invention should be determined by the claims. Any adaptive modifications, equivalent substitutions of technical features, further optimizations and improvements made based on the basic principles and concepts disclosed in the present invention are all within the scope of protection of the present invention.

Claims

1. A method for large-scale seedling cultivation of a hybrid of tiger stripe and mouse stripe, characterized in that, Includes the following steps: S1. Transfer the newly hatched fry of the tiger grouper and mouse grouper hybrid to the nursery pond. Feed the newly hatched fry with oyster fertilized eggs after they start to eat. S2. Feed SS-type rotifers cultured in Chlorella solution from 4 to 15 days old; S3. Feeding infants aged 16-30 days with DHA-fortified bacteria and Neurospora crassa ( Neurospora crassa Artemia nauplii that are jointly reinforced; S4. Feed Daphnia mongolica and Artemia var. mongolica at 31-35 days old; S5. Gradually replace live bait with micro-fermented microparticle feed from 36 to 60 days of age and eventually completely convert to micro-fermented microparticle feed. The micro-fermented microparticle feed is prepared by solid-state micro-fermentation of microparticle feed by Bacillus stearothermophilus.

2. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 1, characterized in that, In step S1, the density of newly hatched fry is 10,000 to 30,000 fry / m³, and the feeding density of oyster fertilized eggs is 5 to 8 eggs / mL, fed twice a day; in step S2, the feeding density of SS-type rotifers is 5 to 8 eggs / mL, fed twice a day; in step S3, the feeding density of Artemia nauplii is 1.0 to 1.5 eggs / mL, fed three times a day; in step S4, the feeding density of Daphnia mongolica and Artemia salina is 400 to 500 eggs / L, with a feeding ratio of Daphnia mongolica to Artemia salina of 1:1, fed three times a day.

3. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 1, characterized in that, The method for enhancing the nauplius larvae in step S3 is as follows: The nauplius larvae are transferred to an enhancement tank, the density is adjusted to 100-200 larvae / mL, and DHA enhancer and Neurospora crassa (…) are added. Neurospora crassa The bacterial solution was subjected to enhancement treatment for 12-24 hours.

4. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 3, characterized in that, The DHA fortifier is added at a concentration of 0.2~0.4 g / L, and the final concentration of the rough Neurospora spp. solution is 1×10⁻⁶ g / L. 6 ~1×10 7 cfu / mL.

5. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 4, characterized in that, The DHA fortifier was added at a rate of 0.2 g / L, and the final concentration of the rough Neurospora spp. solution was 1 × 10⁻⁶. 7 The concentration of CFU / mL and the fortification time are 12 hours; or the amount of DHA fortifier added is 0.4 g / L, and the final concentration of the rough Neurospora spp. solution is 1 × 10⁻⁶. 6 cfu / mL, enhancement time was 24 hours.

6. The method for large-scale seedling production of tiger-spotted and mouse-spotted hybrids according to claim 1, characterized in that, The preparation method of micro-fermented microparticle feed in step S5 is as follows: mix the components of the microparticle feed, and inoculate with activated thermophilic Bacillus stearothermophilus (Bacillus stearothermophilus). Geobacillus stearothermophilus Bacterial solution, final concentration 4×10 6 ~5×10 6 Mix the CFU / g material thoroughly and evenly; place the inoculated material in a constant temperature incubator at 50±2℃ for solid-state micro-fermentation for 12~24 hours, turning it over every 2~3 hours during the period; after micro-fermentation, dry it until the moisture content is <10%, sieve and grade it to obtain micro-fermented microparticle feeds of different particle sizes.

7. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 1, characterized in that, The microparticle feed formula, by weight percentage, is as follows: fish meal 55%~60%, wheat flour 10%~15%, soybean meal 5%~8%, fish oil 8%~10%, brewer's yeast powder 5%~8%, calcium dihydrogen phosphate 1%~1.5%, choline chloride 0.1%~0.2%, seaweed powder 0.8%~1.5%, Tween 80 0.1%~0.2%, phytase 0.03%~0.05%, with the remainder being water.

8. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 1, characterized in that, The method for replacing live bait with micro-fermented microparticle feed in step S5 is as follows: 10% (w / w) of micro-fermented microparticle feed is mixed into the live bait to replace an equal amount of live bait, and the proportion is increased by 10% every 2 days until it is completely converted to micro-fermented microparticle feed.

9. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 1, characterized in that, The hybrid of tiger grouper and mouse grouper is either a direct cross or a reciprocal cross: the direct cross uses male tiger grouper as the father and female mouse grouper as the mother; the reciprocal cross uses female tiger grouper as the mother and male mouse grouper as the father.

10. The method for large-scale seedling cultivation of tiger-spotted and mouse-spotted hybrids according to claim 1, characterized in that, In step S2, the SS-type rotifers are cultured in Chlorella solution with a mass concentration of 15‰ for 6 hours before being fed.

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