Artificial cross breeding method of Jinlong hybrid spot new germplasm

Abstract

The invention relates to an artificial cross breeding method of Jinlong hybrid spot new germplasm, which belongs to the technical field of aquatic product distant cross breeding, and comprises the following steps: (1) parent breeding; (2) collecting and storing sperms of epinephelus lanceolatus; (3) carrying out reproductive regulation and ovum acquisition on the epinephelus coioides; (4) artificial preparation and incubation of hybrid fertilized eggs; according to the method, the artificial cross breeding of the female epinephelus coioides and the male epinephelus lanceolatus is realized for the first time, and the new germplasm Jinlong hybrid spots of the epinephelus coioides, which grow fast and are high in survival rate, are cultivated. The body weight of the Jinlong hybrid Epinephelus coioides of 3 months old is increased by 46.45% compared with that of pure Epinephelus coioides; and the survival rate of the new germplasm is increased by 62.50% compared with that of the epinephelus coioides. The industrial problems that artificial spawning of large groupers is difficult, the survival rate of fries is low, and fast-growing new germplasm lacks are effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic distant hybridization breeding, and specifically relates to an artificial hybridization breeding method for a new germplasm of Jinlong hybrid spot. Background Art

[0002] Grouper, a prized farmed fish, boasts an annual production of 241,500 tons, generating a value of 30 billion yuan. Hybrids are primarily used in grouper farming, and due to their advantages, such as fast growth, disease resistance, and stress tolerance, they currently account for over 80% of the market. Grouper species are plentiful, with over 160 in total. Currently, genetic improvement in grouper still primarily relies on hybrid breeding. The three approved new grouper varieties are all hybrids, but their growth rates are significantly lower than those of larger paternal species. Research on hybrid selection in grouper has found that hybrid offspring with significant growth advantages require fast-growing, large-bodied grouper species as the paternal parent. Analysis of hybrid vigor revealed that paternal subgenomic advantages regulate the growth advantage of grouper hybrid offspring. Therefore, leveraging the paternal genetic effects of large grouper species can further advance the development of fast-growing, high-quality hybrid offspring, providing suitable species for industrial and deep-sea aquaculture in my country.

[0003] The saddle-banded grouper (Epinephelus lanceolatus), a member of the Epinephelidae family, is commonly known as the dragon grouper and dragon-bearing grouper. It is primarily found in the coral reefs of the South China Sea, reaching lengths exceeding 2 meters and weights up to 400 kg, making it the largest grouper. The blue-spotted grouper (Epinephelus tukula), also known as the golden grouper, is primarily found in the Indian and Pacific Oceans. It can grow up to 2 meters and weigh up to 100 kg, making it second only to the saddle-banded grouper in size. The two species are reproductively isolated spatially, temporally, and interspecifically, and natural hybridization has not been observed. Artificial spawning techniques for these two large groupers have yet to be fully mastered. Currently, sperm from these two species is often used to crossbreed with smaller groupers, resulting in hybrid offspring that are faster than the female parent but weaker than the larger male parent. Therefore, by utilizing the genetic theory of the rapid growth effect of grouper fathers, the largest saddleback grouper is used as the father and hybridized with the second largest blue-bodied grouper, in order to cultivate hybrid offspring that surpass the large parents, which can further promote the development of grouper breeding and industry.

[0004] Both large groupers are hermaphroditic and protandrous. Through the development of temperature control and sperm cryopreservation technology, their sperm can be used year-round for artificial seed production. However, the spawning cycle of large female groupers is relatively short, and the survival rate of purebred seedlings is low. The blue-bodied grouper spawns primarily between March and April each year, producing large-sized seedlings that can be used for aquaculture in ponds and deep-sea breeding in northern China, reducing aquaculture costs and improving the quality of commercial fish. The saddleback grouper spawns primarily between July and August, and commercial fish rearing in northern China is primarily carried out in factory-style workshops, which have high heating and operating costs. Artificial breeding technology for the blue-bodied grouper has not yet been fully developed, and there is a shortage of aquaculture seedlings on the market. Therefore, leveraging the rapid growth of the large saddleback grouper paternal parent through distant hybridization can further improve the growth rate and survival rate of the blue-bodied grouper, shortening the aquaculture cycle and input costs, and effectively increasing aquaculture returns. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a breeding method for artificially hybridizing female blue-bodied big-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 big-spotted grouper, low seedling survival rate and lack of fast-growing new germplasm.

[0006] In order to solve the above technical problems, the present invention is achieved through the following technical solutions:

[0007] An artificial hybrid breeding method for a new germplasm of a golden dragon hybrid grouper comprises (1) parent selection and breeding, (2) sperm collection and preservation of saddleback grouper, (3) reproductive regulation and egg acquisition of blue-bodied giant grouper, and (4) artificial preparation and hatching of hybrid fertilized eggs.

[0008] (1) Parent selection: For blue grouper, select female fish that are over 6 years old, weigh over 20 kg, have a well-proportioned body, no deformities, and are in good physical condition as the breeding female parent; for saddle grouper, select male fish that are over 8 years old, weigh over 50 kg, have a large body, no deformities, and are in good physical condition as the breeding male parent;

[0009] (2) Collection and preservation of saddleback grouper sperm

[0010] a. Cultivation environment: Cultivation in outdoor ponds and cages, with dissolved oxygen above 6 mg / L, salinity of 28-31‰, and a cultivation temperature of 22-28°C;

[0011] b. Reproductive Regulation and Sperm Collection: In January of the following year, as water temperatures rise, feed fresh chilled fish (small fish, mackerel, etc.) mixed with nutritional supplements to promote testicular development. 24 hours before semen collection, inject mature male fish with a mixture of human chorionic gonadotropin (HCG, 300 IU / kg fish body weight) and a luteinizing hormone-releasing hormone analog (LHRH-A3, 15 μg / kg fish body weight) to improve sperm quality and production. Collect fresh semen in 50 mL cryovials and store sealed at 4°C in the dark.

[0012] (3) Reproductive regulation and egg acquisition of blue-bodied grouper

[0013] a. Rearing Environment: Female Epinephelus spp. are raised in deep-sea cages until sexual maturity and then transferred to indoor, factory-style breeding facilities. Dissolved oxygen levels are maintained above 6 mg / L, salinity levels are maintained between 28 and 31‰, and freshly-frozen fish supplemented with nutritional supplements are fed. During the breeding period, the water temperature is strictly controlled at 24-27°C. Female Epinephelus spp. with gonadal development reaching stage IV are selected and injected at the base of their pectoral fins with a mixture of human chorionic gonadotropin (HCG, 250 IU / kg fish body weight) and a luteinizing hormone-releasing hormone analog (LHRH-A3, 25 μg / kg fish body weight). Mature eggs are then collected manually.

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

[0015] a. Fertilization: artificially inseminate the collected eggs of Epinephelus serratus and sperm of Epinephelus saddleback at a ratio of 1:500, mix well, add 2 times the volume of 24-26℃ seawater, let it stand for 10 minutes, and then take the floating eggs and place them in an incubator for incubation;

[0016] b. Incubation: The incubation water temperature is 24-26℃, the salinity is 28-30‰, and the dissolved oxygen is greater than 7mg / L. At the end of the gastrula stage, the floating eggs are collected and incubated in the nursery pond. The egg density is 3.5-5.0g / m 3 .

[0017] Furthermore, after the blue-bodied grouper is transferred to an indoor factory workshop and injected with hormones, a certain number of male fish are placed at a 1:1 ratio to stimulate the maturation of the female fish's gonads through chasing behavior. The water temperature is strictly controlled below 27°C through sunshade nets and temperature control systems to prevent ovarian degeneration.

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

[0019] Furthermore, during the hatching process of the fertilized eggs, fresh seawater that has been disinfected and filtered is added to the nursery pond. In order to prevent the occurrence of viral diseases that may cause mass death of fry, the seawater is not changed during the early cultivation period. At the same time, inflation pipes are evenly arranged at the bottom and wall of the nursery pond, and liquid oxygen inflation is used to prevent water quality deterioration and bacterial growth. The early cultivation refers to 1-55 days after the fertilized eggs are hatched.

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

[0021] The principle of the present invention is: based on genome selection, through breeding technology combining multiple technologies such as group selection, environmental regulation, reproductive regulation, nutritional regulation, and distant hybridization, the interspecies, spatial and temporal reproductive isolation between female blue-bodied grouper and male saddle-banded grouper has been broken through, and a new large-scale hybrid grouper germplasm "Golden Dragon Hybrid Grouper" with fast growth and high survival rate has been artificially bred.

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

[0023] 1. Based on the comparison of grouper genomes and the hybridization principle of the fast-growing effect of large paternal parents, the present invention guides the selective hybridization breeding of male saddle grouper and female blue-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. Solving the problems of artificial spawning and a short reproductive cycle for Epinephelus spp. By stimulating the natural seawater environment, combined with group selection, nutritional regulation, and hormone control techniques, this has overcome the bottleneck of artificial spawning for Epinephelus spp. Combined with indoor, factory-style water temperature control, the spawning cycle has been extended by one month compared to pre-regulation levels, significantly improving the utilization efficiency of female Epinephelus spp.

[0025] 3. This invention, for the first time, utilizes male saddleback groupers and female blue-bodied groupers to create a new, fast-growing, high-survival large grouper germplasm, the "Golden Dragon Hybrid Grouper," effectively addressing the industry's challenge of insufficient aquaculture seedlings for large groupers. Three-month-old "Golden Dragon Hybrid Groupers" weighed 17.89±1.20g, a 46.45% increase compared to purebred blue-bodied groupers. After three months of comparative culture, 52,000 seedlings from the new germplasm survived, a 62.50% increase compared to purebred blue-bodied groupers. This provides a fast-growing, high-quality species suitable for aquaculture in northern my country's marine ponds and deep-sea cages. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Comparative analysis of the genomic relationship between Epinephelus edulis and Epinephelus saddleback;

[0027] Figure 2Farming 3-month-old grouper hybrid new germplasm Golden Dragon hybrid grouper. DETAILED DESCRIPTION

[0028] The technical solutions of the present invention are described clearly and completely below with reference to specific embodiments. It is apparent that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0029] Example 1: Broodstock screening and enhanced breeding

[0030] (1) Genome matching

[0031] The genome information of 10 different grouper species (e.g., Epinephelus saddleback, Epinephelus scabra, Epinephelus glaberys, Epinephelus scabra) and Epinephelus leucops were extracted, including saddleback grouper, blue-spotted grouper, golden tiger hybrid grouper (two haplotype genomes), brown-spotted grouper, humpback grouper, clouded grouper, red-spotted grouper, blue grouper, red nine-spined seabass and leopard gill seabass), as well as large yellow croaker, redfin pufferfish, Nile tilapia, medaka, Atlantic salmon and zebrafish. Genes of single-copy gene families were selected and the species phylogenetic tree was constructed using the maximum likelihood method using RAxML software ( Figure 1 The dominant subgenome of the saddle-banded grouper, the blue-spotted grouper, and the golden tiger hybrid grouper was found to be most closely related. This confirmed the possibility of interspecific hybridization between the two large groupers at the genomic level. Furthermore, based on the genetic theory of the rapid growth effect of large male parents, the largest saddle-banded grouper was selected as the hybrid parent.

[0032] (2) Broodstock breeding

[0033] a. Male saddleback grouper parents: 800 wild and farmed breeding stock were collected from Hainan, Shandong, and other regions, with an average weight of 43.28 kg and a coefficient of variation of phenotypic traits of 11.38%. Large, deformity-free, and robust parents were selected through artificial propagation techniques to produce large-sized seedlings. Fast-growing, deformity-free individuals were selected from these individuals to form the core breeding population, with a selection intensity of 10%. After eight years of artificial breeding, males reached sexual maturity, with an average weight of 57.36 kg and a coefficient of variation of phenotypic traits of 8.57%.

[0034] b. Female blue-bodied grouper: A basic breeding population of 1,200 wild and farmed individuals was collected in Hainan and other locations, with an average weight of 21.64 kg and a coefficient of variation of phenotypic traits of 12.57%. Parents with symmetrical body shape, no deformities, and robust physique were selected through artificial propagation techniques to produce large-sized seedlings. Fast-growing, deformity-free individuals were selected from these individuals to form the core breeding population, with a selection intensity of 13%. After six years of artificial breeding, females reached sexual maturity, with an average weight of 24.38 kg and a coefficient of variation of phenotypic traits of 9.23%.

[0035] (3) Strengthening training

[0036] a. Saddleback grouper cultivation environment: Cultivated in outdoor pond cages at a depth of 2-3 meters, with a dissolved oxygen level of 6 mg / L or higher, a salinity of 28-31‰, and a cultivation temperature of 22-28°C. Blue-spotted grouper is intensively cultivated in deep-sea cages combined with indoor factory-based conditions. Deep-sea cages are located in the open sea at a depth of 10 meters or more, at a depth of 2-3 meters. The indoor factory-based workshop is equipped with a temperature control system to maintain a dissolved oxygen level of 6 mg / L or higher, a salinity of 28-31‰, and a strictly controlled water temperature of 24-27°C during the breeding period.

[0037] b. Breeding of male saddleback grouper: In January of the following year, promote testicular development in male fish through nutritional manipulation. Feed them with chilled fish (small fish and mackerel) injected with a vitamin mixture (vitamin C and vitamin E mixed in equal proportions, containing 7-8 g / kg) at a rate of 2%-3% of their body weight. 24 hours before hybrid breeding, inject the manipulated male fish 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 the hormone effect, mature semen is obtained by squeezing the male fish's abdominal cavity. This semen is collected in 50 mL cryovials to avoid contamination with seawater and urine. Sperm with a motility of over 90% as determined by microscopy is sealed and stored in a dark environment at 4°C for use in the cultivation of new hybrid germplasm that day.

[0038] c. Cultivation of Female Blue Grouper: In late December of the same year, selected breeding parents are transferred to deep-sea cages for intensive cultivation. Tidal forces, photoperiods, water temperature, and currents are used to promote ovarian development. During this period, they are fed nutritionally fortified fresh-frozen fish at a rate of 3% to 4% of their body weight. Selected females with gonadal development reaching stage IV are 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. A number of males are placed simultaneously in a 1:1 ratio to stimulate gonadal maturation in the females through pursuit. After 48 hours of hormone effects, the females are immobilized with fish clamps, their genital pores punctured with cotton swabs, and mature eggs are collected by squeezing their abdomens. In order to prolong the spawning time of female fish, the regulated broodstock need to be transferred to an indoor factory workshop for artificial breeding in early March (when a bulge appears on the female fish's abdomen and the eggs are dug out to check whether the gonads have developed to stage III or IV). The water temperature must be strictly controlled at 24-27°C through sunshade nets and temperature control systems to prevent ovarian degeneration.

[0039] Temperature control and hormone-induced spawning have significantly prolonged the spawning cycle of blue-spotted grouper. Before the control, mature eggs could only be collected from March to April. After the control, mature eggs can be collected from March to May, extending the spawning cycle by one month. This provides a sufficient source of eggs for genetic improvement and large-scale seed production of large grouper.

[0040] Example 2: Hybrid fertilization of female blue-spotted grouper and male saddleback grouper

[0041] a. Artificial Insemination: Female blue-striped groupers with a bulging abdomen and a protruding genital pore were selected for hybrid breeding. First-time spawning females were punctured with a cotton swab and their genital pores manually squeezed. The collected eggs were divided equally and artificially inseminated with sperm from Epinephelus saddleback and Epinephelus serratus, each with a motility greater than 90%, at a volume ratio of 1:500. After mixing thoroughly, the mixture was added with two volumes of 24-26°C seawater. After 10 minutes, the floating eggs were removed, rinsed, weighed, and placed in an incubator for incubation. The hybrid eggs measured 0.90 ± 0.01 mm in diameter, and the oil globule size was 0.20 ± 0.01 mm. The fertilization rate of the hybrids was 93.62 ± 2.56%, which was not significantly different from that of purebred blue-striped groupers (96.87 ± 1.68%) (Table 1).

[0042] Table 1 Epinephelus edulis (♀) × Epinephelus saddlebackii Comparison of hatching of new hybrid germplasm Jinlong hybrid spot

[0043]

[0044] b. Incubation and cultivation: The incubation water temperature is 24-26℃, the salinity is 28-30‰, and the dissolved oxygen is greater than 7mg / L. At the end of the gastrula stage, the floating eggs are collected and incubated in the nursery pond. The egg density is 3.5-5.0g / m 3 To prevent viral diseases from causing mass mortality in fry, seawater was not replaced during the early rearing period (before the tridents completed their contraction, 1-55 days after hatching). Instead, fresh, disinfected, filtered seawater was added to the nursery ponds. Furthermore, aeration tubes were evenly distributed along the bottom and walls of the nursery ponds, using liquid oxygen to prevent water quality deterioration and the growth of pathogens. The hatchability of the hybrids was 86.59 ± 6.28%, which was not significantly different from that of purebred Epinephelus spp. (90.37 ± 3.44%) (Table 1).

[0045] c. Embryonic Development Observation: The early development of the two groups of fish was continuously observed using an optical microscope and a dissecting microscope. Fertilized eggs of the hybrid and blue-bodied grouper completed embryonic development in 25 hours 32 minutes and 25 hours 56 minutes, respectively. Newly hatched larvae of the Jinlong hybrid grouper measured 1.75 ± 0.12 mm in length, significantly larger than those of the blue-bodied grouper (1.60 ± 0.17 mm). Yolk sac consumption in the hybrid was most rapid on the first day after hatching, reaching 54.27%, and was completely absorbed by the sixth day after hatching.

[0046] d. Mitochondrial genome analysis: The mitochondrial genome of the Jinlong hybrid germplasm is 16,464 base pairs long. The base composition is: A: 28.34%, T: 26.29%, C: 29.11%, G: 16.26%, with an AT bias of 54.63% and a GC bias of 45.37%. The hybrid retains 13 protein-coding genes, two ribosomal RNA genes (one large subunit and one small subunit), and 22 transfer RNA genes. The mitochondrial composition of the new hybrid germplasm follows a maternal inheritance pattern.

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

[0048] In this case, a total of 1.5 kg of hybrid and purebred fertilized eggs were obtained. The fertilization and hatching rates of the Jinlong hybrid grouper were not significantly different from those of the purebred blue-bodied grouper. The Jinlong hybrid grouper exhibited significantly better feeding activity and disease resistance than purebred blue-bodied grouper fry. At 90 days of rearing, 52,000 hybrid fry survived, while 32,000 survived for saddleback grouper. This represents a 62.50% increase in the survival rate of the hybrid fry compared to the purebred blue-bodied grouper.

[0049] The comprehensive results show that the hybrid offspring of blue-bodied big-spotted grouper (♀) and saddle-banded grouper (♂) have high fertilization and hatching rates, normal metamorphosis and development of fry, and significantly improved survival rate of hybrid offspring compared with pure blue-bodied big-spotted grouper.

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

[0051] By constructing half-sib families, we obtained a hybrid line of Epinephelus edulis (♀) × Epinephelus saddleback (♂) and a pure line of Epinephelus edulis. A comparative breeding experiment on growth traits was carried out under the same environment, during which their growth traits were compared and analyzed.

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

[0053] Weight: The weight of the 90-day hybrid group and the control group was measured. The weight of the Jinlong hybrid grouper was 17.89±1.20g, which was significantly higher than the 12.22±1.60g of the purebred blue-bodied grouper (p<0.01). The weight trait was 46.45% higher than that of the purebred blue-bodied grouper, showing a significant hybrid growth advantage. It also has a better appearance ( Figure 2 ), the breeding prospects are broad.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. A person skilled in the art may make several improvements without departing from the principles of the present invention, and these improvements should be considered as within the scope of protection of the present invention.

Claims

1. A method for artificial hybridization breeding of a new germplasm of Jinlong hybrid spot, characterized in that: The method comprises (1) parent selection, (2) sperm collection and preservation of Epinephelus saddlebackii; (3) reproductive regulation and egg acquisition of Epinephelus scauphun; (4) artificial preparation and hatching of hybrid fertilized eggs; (1) Parent selection: Blue-bodied grouper: Select female fish that are over 6 years old, weigh over 20 kg, have a symmetrical body shape, no deformities, and are in good physical condition as breeding mothers; Saddleback grouper: Select male fish that are over 8 years old, weigh over 50 kg, have a large body shape, no deformities, and are in good physical condition as breeding fathers; (2) Saddleback grouper sperm collection and storage a. Cultivation environment: Cultivation in outdoor ponds and cages, with dissolved oxygen above 6 mg / L, salinity of 28-31‰, and a cultivation temperature of 22-28°C; b. Reproductive Regulation and Sperm Collection: In January of the following year, as water temperatures rise, feed fresh chilled fish mixed with nutritional supplements to promote testicular development. 24 hours before semen collection, inject mature male fish with a mixture of human chorionic gonadotropin and a luteinizing hormone-releasing hormone analog to improve sperm quality and production. Collect fresh semen in 50 mL cryovials and store sealed at 4°C in the dark. (3) Reproductive regulation and egg acquisition of blue-bodied grouper a. Rearing Environment: Female Epinephelus spp. are raised in deep-sea cages until sexual maturity and then transferred to indoor, factory-style breeding facilities. Dissolved oxygen levels are maintained above 6 mg / L, salinity is maintained between 28 and 31‰, and freshly-frozen fish supplemented with nutritional supplements are fed. During the breeding period, the water temperature is strictly controlled at 24-27°C. Female Epinephelus spp. with gonadal development reaching stage IV are selected and injected with a mixture of human chorionic gonadotropin and a luteinizing hormone-releasing hormone analogue at the base of their pectoral fins. Mature eggs are then collected manually. (4) artificial preparation and hatching of hybrid fertilized eggs; a. Fertilization: artificially inseminate the collected eggs of Epinephelus serratus and sperm of Epinephelus saddleback at a ratio of 1:500, mix well, add 2 times the volume of 24-26℃ seawater, let it stand for 10 minutes, and then take the floating eggs and place them in an incubator for incubation; b. Incubation: The incubation water temperature is 24-26℃, the salinity is 28-30‰, and the dissolved oxygen is greater than 7mg / L. At the end of the gastrula stage, the floating eggs are collected and incubated in the nursery pond. The egg density is 3.5-5.0g / m 3 .

2. The method according to claim 1, characterized in that After the blue-bodied grouper is transferred to an indoor factory workshop and injected with hormones, a certain number of male fish are placed at a 1:1 ratio to stimulate the maturation of the female fish's gonads through chasing behavior. The water temperature is strictly controlled below 27°C through sunshade nets and temperature control systems to prevent ovarian degeneration.

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

4. The method according to claim 1, wherein During the hatching process of the fertilized eggs, fresh seawater that has been disinfected and filtered is added to the nursery pond. In order to prevent the occurrence of viral diseases that may cause mass death of fry, the seawater is not changed during the early cultivation period. At the same time, inflation pipes are evenly arranged at the bottom and walls of the nursery pond, and liquid oxygen is used for inflation to prevent water quality deterioration and the growth of pathogens. The early cultivation refers to 1-55 days after the fertilized eggs are hatched.

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