Artificial hybridization breeding method for a new germplasm of golden dragon hybrids

Abstract

This invention relates to an artificial hybridization breeding method for a new grouper germplasm of Golden Dragon hybrids. The method belongs to the field of aquatic distant hybridization breeding technology and includes (1) parent selection; (2) sperm collection and preservation of saddle-banded grouper; (3) reproductive regulation and egg acquisition of blue-bodied large-spotted grouper; and (4) artificial preparation and hatching of hybrid fertilized eggs. This invention achieves for the first time artificial hybridization breeding of female blue-bodied large-spotted grouper and male large-saddle-banded grouper, cultivating a new grouper germplasm of Golden Dragon hybrids with fast growth and high survival rate. The weight of the 3-month-old Golden Dragon hybrids is 46.45% higher than that of purebred blue-bodied large-spotted grouper; the survival rate of the new germplasm is 62.50% higher than that of blue-bodied large-spotted grouper. This effectively solves the industrial problems of difficulty in artificial spawning of large groupers, low seedling survival, and lack of fast-growing new germplasm.

Description

Technical Field

[0001] This invention belongs to the field of aquatic distant hybridization breeding technology, specifically relating to an artificial hybridization breeding method for a new germplasm of golden dragon hybrids. Background Technology

[0002] Grouper, a prized farmed fish, has an annual production of 241,500 tons, generating a value of 30 billion yuan. Grouper farming primarily relies on hybrids, which currently account for over 80% of the market due to their rapid growth, disease resistance, and stress tolerance. There are over 160 species of grouper, and genetic improvement still mainly focuses on hybridization. The three newly approved grouper varieties are all hybrids, but their growth rate is significantly lower than that of their larger paternal parents. Research on selective hybridization of grouper has revealed that obtaining hybrid offspring with significant growth advantages requires fast-growing, large-sized grouper varieties as paternal parents. Analysis of hybrid vigor has shown that paternal subgenomic dominance regulates the formation of growth advantages in grouper hybrid offspring. Therefore, utilizing the paternal genetic effects of large grouper can further advance the breeding of fast-growing, high-quality hybrid offspring, providing suitable varieties for industrialized and deep-sea aquaculture in my country.

[0003] The saddle-tailed grouper (Epinephelus lanceolatus) belongs to the order Perciformes, family Epinephelidae, and genus Epinephelidae. It is commonly known as the giant grouper or saddle-tailed grouper. It is mainly distributed in coral reef waters, reaching a maximum length of over 2 meters and a weight of up to 400 kg, making it the largest grouper. The blue-bodied large-spotted grouper (E. tukula) belongs to the genus Epinephelidae and is commonly known as the money grouper. It is mainly distributed in the Indian and Pacific Oceans, growing up to 2 meters in length and reaching a maximum weight of 100 kg, second only to the saddle-tailed grouper in size. Both species exhibit spatial, temporal, and interspecific reproductive isolation, and no natural hybridization has been observed. Artificial spawning techniques for these two large groupers have not yet been fully developed. Currently, sperm from both species is often used to hybridize with smaller groupers to produce offspring that grow faster than the maternal parent but weaker than the larger paternal parent. Therefore, by utilizing the genetic theory of the rapid growth effect of the paternal parent in grouper, the largest grouper, the saddle-shaped grouper, can be used as the paternal parent to crossbreed with the second largest grouper, the blue-bodied large-spotted grouper, in order to cultivate hybrid offspring that surpass the large parent species. This can further promote the development of the grouper breeding and industry.

[0004] Both species of large grouper are hermaphroditic, with females maturing first. Through temperature control and sperm cryopreservation technology, their sperm can be used year-round for artificial fry production. However, the spawning cycle of female large grouper is relatively short, and the survival rate of purebred fry is low. The blue-bodied large-spotted grouper spawns mainly in March and April each year, producing large-sized fry suitable for pond and deep-sea aquaculture in northern regions, reducing aquaculture costs and improving the quality of marketable fish. The saddle-tailed grouper spawns mainly in July and August, and its marketable fish rearing in northern regions is primarily carried out in factory-style facilities, resulting in high operating costs due to temperature control. Artificial breeding technology for the blue-bodied large-spotted grouper has not yet been fully mastered, and there is a shortage of marketable fry. Therefore, utilizing the rapid growth effect of the large saddle-tailed grouper paternal parent through distant hybridization breeding technology can further improve the growth rate and survival rate of the blue-bodied large-spotted grouper, shortening the aquaculture cycle and reducing input costs, effectively increasing aquaculture profits. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a breeding method for artificially hybridizing female blue-bodied large-spotted grouper and male large saddle-banded grouper to cultivate new golden dragon hybrid grouper germplasm, aiming to solve the problems of difficulty in artificial spawning of blue-bodied large-spotted grouper, low seedling survival rate and lack of fast-growing new germplasm.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution:

[0007] A method for artificial hybridization breeding of a new germplasm of golden dragon hybrid grouper, the method comprising (1) parental selection, (2) collection and preservation of sperm from saddle-banded grouper, (3) reproductive regulation and egg acquisition of blue-bodied large-spotted grouper, and (4) artificial preparation and hatching of hybrid fertilized eggs.

[0008] The following are the parent selection criteria: Blue-bodied large-spotted grouper: female fish that are 6 years or older, weigh more than 20 kg, have a symmetrical body shape, no deformities, and a strong physique are selected as breeding mothers; Saddleband grouper: male fish that are 8 years or older, weigh more than 50 kg, have a large body shape, no deformities, and a strong physique are selected as breeding fathers.

[0009] (2) Collection and preservation of sperm from saddle-banded grouper.

[0010] a. Cultivation environment: Outdoor pond cage cultivation, dissolved oxygen above 6 mg / L, salinity 28-31‰, cultivation temperature 22-28℃;

[0011] b. Reproductive regulation and sperm collection: In January of the following year, as the water temperature rises, fresh fish (small fish, mackerel, etc.) mixed with nutritional fortifiers are fed to promote testicular development; 24 hours before sperm collection, mature male fish are injected with a mixture of human chorionic gonadotropin (HCG, 300 IU / kg fish body weight) and luteinizing hormone-releasing hormone analog (LHRH-A3, 15 μg / kg fish body weight) to improve sperm quality and sperm production; fresh semen is collected in 50 mL cryovials and stored in a sealed container at 4°C in the dark.

[0012] The reproductive regulation and egg acquisition of the blue-bodied large-spotted grouper described in (3)

[0013] a. Cultivation environment: Female blue-bodied large-spotted grouper were cultured in deep-sea net cages until sexual maturity, and then transferred to indoor factory workshops for artificial breeding. During this period, dissolved oxygen was maintained above 6 mg / L, salinity was 28-31‰, and fresh fish with nutritional fortifiers were fed. During the breeding period, the water temperature was strictly controlled at 24-27℃. Female blue-bodied large-spotted grouper with gonadal development to stage IV were selected and injected with a mixture of human chorionic gonadotropin (HCG, 250 IU / kg fish body weight) and luteinizing hormone-releasing hormone analog (LHRH-A3, 25 μg / kg fish body weight) at the base of the pectoral fin. Mature eggs were then collected artificially.

[0014] The aforementioned (4) artificial preparation and hatching of hybrid fertilized eggs;

[0015] a. Fertilization: The collected eggs of the blue-bodied large-spotted grouper and the sperm of the saddle-banded grouper were artificially inseminated at a ratio of 1:500. After stirring evenly, 2 times the volume of seawater at 24-26℃ was added. After standing for 10 minutes, the floating eggs were taken and placed in an incubator for incubation.

[0016] b. Hatching: The hatching water temperature is 24–26℃, salinity is 28–30‰, and dissolved oxygen is greater than 7 mg / L. At the end of the gastrula stage, collect the floating eggs and transfer them to the nursery pond for hatching. The egg density is 3.5–5.0 g / m³. 3 .

[0017] Furthermore, after the blue-bodied large-spotted grouper is transferred to an indoor factory workshop, after the hormone is injected, a certain number of male fish are placed at a 1:1 ratio to stimulate the female fish's gonads to mature through chasing behavior. The water temperature is strictly controlled below 27℃ through shade nets and a temperature control system to prevent ovarian degeneration.

[0018] Furthermore, the nutritional fortifier is a mixture of vitamin C and vitamin E in equal mass ratios, and the amount added to chilled fish is 7-8 g / kg.

[0019] Furthermore, during the hatching process of fertilized eggs, fresh seawater that has been disinfected and filtered is added to the nursery pond. To prevent viral diseases from causing mass mortality of fry, the seawater is not changed during the early rearing period. At the same time, air pipes are evenly arranged at the bottom and walls of the nursery pond, and liquid oxygen is used for aeration to prevent water quality deterioration and the growth of bacteria. The early rearing period refers to 1-55 days after the fertilized eggs hatch.

[0020] Furthermore, the technical solution of the present invention also includes testing the growth advantage traits of the new germplasm of the Golden Dragon hybrid.

[0021] The principle of this invention is: based on genome selection, through a combination of breeding techniques including population selection, environmental regulation, reproductive regulation, nutritional regulation, and distant hybridization, the reproductive isolation between female blue-bodied large-spotted grouper and male saddle-banded grouper, both spatially and temporally, has been overcome, and a new hybrid germplasm of large grouper, "Golden Dragon Hybrid Grouper", has been artificially cultivated with fast growth and high survival rate.

[0022] The beneficial effects of this invention compared to the prior art are as follows:

[0023] 1. Based on the comparison of grouper genomes, this invention combines the hybridization principle of the rapid growth effect of large paternal parents to guide the selection and hybridization breeding of male saddle-banded grouper and female blue-bodied large-spotted grouper from a genetic perspective, reducing the blindness of existing grouper hybridization breeding and improving the theoretical level of fish hybridization breeding.

[0024] 2. The problem of difficult artificial spawning and short breeding cycle of blue-bodied large-spotted grouper has been solved. By stimulating the natural seawater environment and combining group selection, nutritional regulation and hormone regulation technology, the bottleneck problem of difficult artificial spawning of blue-bodied large-spotted grouper has been solved; at the same time, combined with indoor factory-style water temperature control, the spawning cycle has been extended by 1 month compared with the previous regulation, which significantly improves the utilization efficiency of female blue-bodied large-spotted grouper.

[0025] 3. This invention, for the first time, utilizes male saddle-tailed grouper and female blue-bodied large-spotted grouper to cultivate a new large grouper germplasm, "Golden Dragon Hybrid Grouper," characterized by rapid growth and high survival rate, effectively solving the industry problem of insufficient culturable large grouper fry. At 3 months of age, the "Golden Dragon Hybrid Grouper" weighs 17.89±1.20g, a 46.45% increase compared to purebred blue-bodied large-spotted grouper. After 3 months of comparative rearing, the survival rate of the new germplasm fry reached 52,000, a 62.50% increase compared to purebred blue-bodied large-spotted grouper. This provides a fast-growing, high-quality, and suitable cultivar for marine ponds and deep-sea cages in northern my country. Attached Figure Description

[0026] Figure 1 Comparative analysis of the genomic relationship between the blue-bodied large-spotted grouper and the saddle-banded grouper;

[0027] Figure 2A new hybrid grouper, the Golden Dragon Hybrid Grouper, was developed from 3-month-old cultured grouper. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Selection and Enhanced Breeding of Broodstock

[0030] (1) Genome selection

[0031] Genome information was extracted from 10 different species of grouper, including *Spodoptera litura*, *Spodoptera blueina*, *Spodoptera chinensis* (two haplotype genomes), *Spodoptera brownina*, *Spodoptera humpbackii*, *Spodoptera moniliforme*, *Spodoptera redina*, *Spodoptera chinensis*, *Spodoptera nigra*, and *Spodoptera leopardina*, as well as large yellow croaker, redfin pufferfish, Nile tilapia, medaka, Atlantic salmon, and zebrafish. Genes from single-copy gene families were selected, and phylogenetic trees were constructed using the maximum likelihood method with RAxML software. Figure 1 The study found that the dominant subgenomes of the saddle-shaped grouper, the blue-bodied large-spotted grouper, and the golden tiger grouper are most closely related. The possibility of interspecific hybridization between these two large grouper species was determined at the genomic level. Furthermore, based on the genetic theory of the rapid growth effect of large paternal parents, the saddle-shaped grouper, being the largest in size, was selected as the paternal parent for hybridization.

[0032] (2) Broodstock breeding

[0033] a. Male broodstock of the saddle-banded grouper: 800 wild and farmed groupers were collected from Hainan, Shandong, and other regions as the basic breeding population. The average weight was 43.28 kg, and the phenotypic coefficient of variation was 11.38%. Large, deformed, and robust broodstock were selected and artificially bred to produce large-sized fry. From these, fast-growing, deformed individuals were selected as the core breeding population, with a selection intensity of 10%. After 8 years of artificial breeding, the males reached sexual maturity, with an average weight of 57.36 kg and a phenotypic coefficient of variation of 8.57%.

[0034] b. Female broodstock of the blue-bodied large-spotted grouper: A basic breeding population of 1200 wild and farmed individuals was collected from Hainan and other regions, with an average weight of 21.64 kg and a phenotypic coefficient of variation of 12.57%. Broodstock with symmetrical body shape, no deformities, and robust constitution were selected and artificially bred to produce large-sized fry. From these, individuals with rapid growth and no deformities were selected as the core breeding population, with a selection intensity of 13%. After 6 years of artificial breeding, the females reached sexual maturity, with an average weight of 24.38 kg and a phenotypic coefficient of variation of 9.23%.

[0035] (3) Strengthen cultivation

[0036] a. The breeding environment for saddle-shaped grouper: They are cultivated in outdoor pond cages at a depth of 2–3 m, with dissolved oxygen above 6 mg / L, salinity of 28–31‰, and a breeding temperature of 22–28℃. Blue-bodied large-spotted grouper is cultivated in a combination of deep-sea cages and indoor factory-style conditions. The deep-sea cages are set up in the open sea at a depth of over 10 m, with a water depth of 2–3 m. The indoor factory-style workshop is equipped with a temperature control system, maintaining dissolved oxygen above 6 mg / L, salinity of 28–31‰, and strictly controlling the water temperature to 24–27℃ during the breeding period.

[0037] b. Male saddleback grouper cultivation: In January of the following year, the development of testes in male fish was promoted through nutritional regulation. Fresh fish (small fish and mackerel) injected with a vitamin mixture (a mixture of vitamin C and vitamin E in equal proportions, with a content of 7-8 g / kg) were fed at a rate of 2%-3% of the fish's body weight. 24 hours before hybridization breeding, the male fish were injected with a mixture of 300 IU / kg human chorionic gonadotropin and 15 μg / kg luteinizing hormone-releasing hormone analogue to promote testicular maturation. After 24 hours of hormone effect, mature semen was obtained by squeezing the abdominal cavity of the male fish and collected in 50 mL cryovials, avoiding contamination with seawater and urine. Sperm with a motility of over 90% were stored in a sealed container at 4°C in the dark for use in the cultivation of new hybrid germplasm on the same day.

[0038] c. Cultivation of female blue-bodied large-spotted grouper: In late December of the same year, selected breeding broodstock were transferred to deep-sea net cages for intensive cultivation. Ovarian development was promoted by tidal forces, photoperiod, and changes in water temperature and current. During this period, they were fed nutritionally fortified frozen fish at a rate of 3%–4% of their body weight. Selected females with gonadal development to stage IV were injected with a mixture of HCG and LHRH-A3 oxytocin at the base of their pectoral fins, at doses of 250 IU / kg and 25 μg / kg of body weight, respectively. At the same time, a certain number of males were placed in a 1:1 ratio to stimulate gonadal maturation in the females through chasing behavior. After 48 hours of hormonal effect, the females were fixed with fish clips, and mature eggs were collected by squeezing the abdomen after piercing the genital opening with a cotton swab. To prolong the spawning period of female fish, the broodstock that have been properly regulated need to be transferred to an indoor factory-style workshop for artificial breeding in early March (when the female fish's abdomen swells and the gonads are examined after egg collection and development reaches stage III-IV). The water temperature is strictly controlled at 24-27℃ using shade nets and a temperature control system to prevent ovarian degeneration.

[0039] The spawning cycle of blue-bodied large-spotted grouper, after temperature regulation and hormone induction, was significantly extended. Before regulation, mature eggs could only be collected in March and April, but after regulation, mature eggs could be collected from March to May, extending the spawning cycle by one month. This provides a sufficient source of eggs for the genetic improvement of large grouper and the large-scale production of seedlings.

[0040] Example 2: Crossbreeding and fertilization of female blue-bodied large-spotted grouper and male saddle-banded grouper.

[0041] a. Artificial insemination: Female blue-bodied large-spotted grouper with a bulging abdomen and protruding genital opening were selected for hybridization breeding. First-time spawning females underwent artificial egg extraction by puncturing the genital opening with a cotton swab. The collected eggs were divided into two equal portions. Sperm from saddle-shaped grouper and blue-bodied large-spotted grouper with microscopically examined viability greater than 90% were used for artificial insemination at a volume ratio of 1:500. After thorough mixing, twice the volume of 24–26℃ seawater was added. After standing for 10 minutes, the floating eggs were collected, rinsed, weighed, and placed in an incubator for hatching. The hybrid egg diameter was 0.90±0.01 mm, and the oil globule diameter was 0.20±0.01 mm. The fertilization rate of the hybrid was 93.62±2.56%, which was not significantly different from that of purebred blue-bodied large-spotted grouper (96.87±1.68%) (Table 1).

[0042] Table 1 Blue-bodied large-spotted grouper (♀) × Saddleband grouper Hatching comparison of hybrid new germplasm Jinlong hybrid spots

[0043]

[0044] b. Hatching and rearing: The hatching water temperature is 24–26℃, the salinity is 28–30‰, and the dissolved oxygen is greater than 7 mg / L. At the end of the gastrula stage, collect the floating eggs and transfer them to the rearing pond for hatching, with an egg density of 3.5–5.0 g / m³. 3 To prevent viral diseases from causing mass mortality of fry, the seawater was not changed during the early rearing period (before the trident fry complete shrinkage, 1-55 days after hatching). Fresh, disinfected and filtered seawater was added to the rearing ponds. Simultaneously, aeration pipes were evenly distributed at the bottom and walls of the rearing ponds, using liquid oxygen aeration to prevent water quality deterioration and bacterial growth. The hatching rate of the hybrid fry was 86.59±6.28%, which was not significantly different from that of the purebred blue-bodied large-spotted grouper (90.37±3.44%) (Table 1).

[0045] c. Embryonic Development Observation: The early development process of the two groups of fish was continuously observed using an optical microscope and a dissecting microscope. The fertilized eggs of the hybrid new germplasm and the blue-bodied large-spotted grouper completed embryonic development at 25 h 32 min and 25 h 56 min, respectively. The total length of the newly hatched larvae of the Golden Dragon hybrid grouper was 1.75 ± 0.12 mm, significantly larger than that of the blue-bodied large-spotted grouper (1.60 ± 0.17 mm). The yolk sac of the hybrid was consumed most rapidly on day 1 after hatching, reaching 54.27%, and was completely absorbed by day 6 after hatching.

[0046] d. Mitochondrial genome analysis: The mitochondrial genome length of the Jinlong hybrid is 16464 bp. The base composition is: A: 28.34%, T: 26.29%, C: 29.11%, G: 16.26%, with AT bias (54.63%) and GC bias (45.37%). The hybrid retains 13 protein-coding genes, 2 ribosomal RNA genes (1 large subunit gene and 1 small subunit gene), and 22 transfer RNA genes. The mitochondrial composition of the new hybrid germplasm follows maternal inheritance patterns.

[0047] Example 3: Comparison of survival rates of new hybrid germplasm

[0048] In this example, a total of 1.5 kg of hybrid and purebred fertilized eggs were obtained. The fertilization rate and hatching rate of the hybrid grouper were not significantly different from those of the purebred blue-bodied large-spotted grouper. The feeding activity and disease resistance of the hybrid grouper were significantly better than those of the purebred blue-bodied large-spotted grouper fry. After 90 days of rearing, the survival rate of the hybrid fry was 52,000, and that of the saddle-banded grouper was 32,000. The survival rate of the hybrid fry was 62.50% higher than that of the purebred blue-bodied large-spotted grouper.

[0049] The results showed that the hybrid offspring of the blue-bodied large-spotted grouper (♀) × saddle-banded grouper (♂) had a high fertilization rate and hatching rate, and the larvae and juveniles underwent normal metamorphosis. The survival rate of the hybrid offspring was significantly higher than that of the pure-line blue-bodied large-spotted grouper.

[0050] Example 4: Test of growth traits of new hybrid germplasm

[0051] By constructing half-sib families, a hybrid line of blue-bodied large-spotted grouper (♀) × saddle-banded grouper (♂) and a pure line of blue-bodied large-spotted grouper were obtained. Comparative culture experiments on growth traits were carried out in the same environment, and their growth traits were compared and analyzed during the experiment.

[0052] Total length: After 90 days of comparative breeding, the total length of the hybrid group and the control group reached 10.73±0.28cm and 8.76±0.11cm respectively. The total length of the Jinlong hybrid was 1.22 times that of the purebred.

[0053] Body weight: Body weights were measured in the 90-day hybrid group and the control group. The body weight of the Jinlong hybrid grouper was 17.89±1.20g, significantly higher than that of the purebred blue-bodied large-spotted grouper (12.22±1.60g) (p<0.01). The body weight trait was 46.45% higher than that of the purebred blue-bodied large-spotted grouper, demonstrating significant hybrid growth advantage. It also exhibited a better appearance. Figure 2 The prospects for aquaculture are broad.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should be considered within the scope of protection of the present invention.

Claims

1. A method for artificial hybridization breeding of a new germplasm of golden dragon hybrids, characterized in that, The method includes (1) parental selection and breeding, (2) collection and preservation of sperm from saddle-banded grouper, (3) reproductive regulation and egg acquisition of blue-bodied large-spotted grouper, and (4) artificial preparation and hatching of hybrid fertilized eggs; The (1) parent selection: Blue-bodied large-spotted grouper: female fish aged 6 years or older, weighing over 20 kg, with a symmetrical body shape, no deformities, and robust physique were selected as breeding mothers; Saddle-banded grouper: male fish aged 8 years or older, weighing over 50 kg, with a large body shape, no deformities, and robust physique were selected as breeding fathers; The (2) saddle-banded grouper sperm collection and preservation a. Cultivation environment: Outdoor pond cage cultivation, dissolved oxygen above 6 mg / L, salinity 28-31‰, cultivation temperature 22-28℃; b. Reproductive regulation and sperm collection: In January of the following year, as the water temperature rises, fresh fish mixed with nutritional fortifiers are fed to promote testicular development; 24 hours before sperm collection, mature male fish are injected with a mixture of human chorionic gonadotropin and luteinizing hormone-releasing hormone analogues to improve sperm quality and sperm production; fresh semen is collected in 50mL cryovials and stored in a sealed container at 4°C in a light-proof environment. The reproductive regulation and egg acquisition of the blue-bodied large-spotted grouper described in (3) a. Cultivation environment: Female blue-bodied large-spotted grouper were cultured in deep-sea net cages until sexual maturity, and then transferred to indoor factory workshops for artificial breeding. During this period, dissolved oxygen was maintained above 6 mg / L, salinity was 28-31‰, and fresh fish with nutritional fortifiers were fed. During the breeding period, the water temperature was strictly controlled at 24-27℃. Female blue-bodied large-spotted grouper with gonadal development to stage IV were selected and injected with a mixture of human chorionic gonadotropin and luteinizing hormone-releasing hormone analogues at the base of the pectoral fins. Mature eggs were artificially collected. The aforementioned (4) artificial preparation and hatching of hybrid fertilized eggs; a. Fertilization: The collected eggs of the blue-bodied large-spotted grouper and the sperm of the saddle-banded grouper were artificially inseminated at a ratio of 1:

500. After stirring evenly, 2 times the volume of seawater at 24-26℃ was added. After standing for 10 minutes, the floating eggs were taken and placed in an incubator for incubation. b. Hatching: The hatching water temperature is 24–26℃, salinity is 28–30‰, and dissolved oxygen is greater than 7 mg / L. At the end of the gastrula stage, collect the floating eggs and transfer them to the nursery pond for hatching. The egg density is 3.5–5.0 g / m³. 3 .

2. The method according to claim 1, characterized in that, After the blue-bodied large-spotted grouper is transferred to an indoor factory workshop, after the hormone is injected, a certain number of male fish are placed at a 1:1 ratio to stimulate the female fish's gonads to mature through chasing behavior. The water temperature is strictly controlled below 27℃ through shade nets and a temperature control system to prevent ovarian degeneration.

3. The method according to claim 1, characterized in that, The nutritional fortifier is a mixture of vitamin C and vitamin E in equal mass ratios, and the amount added to chilled fish is 7-8 g / kg.

4. The method according to claim 1, characterized in that, During the hatching process of fertilized eggs, fresh seawater that has been disinfected and filtered is added to the nursery pond. To prevent viral diseases from causing mass mortality of fry, the seawater is not changed during the early rearing period. At the same time, air pipes are evenly arranged at the bottom and walls of the nursery pond, and liquid oxygen is used to prevent water quality deterioration and the growth of pathogens. The early rearing period refers to 1-55 days after the fertilized eggs hatch.

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