Methods for breeding and raising seedlings of hybrids between two large grouper species

Abstract

The present invention relates to a method for breeding and seedling cultivation of two large grouper species hybridization, belonging to the technical field of distant hybridization breeding, and the method comprises: (1) breeding of large grouper parent fish groups; (2) reproductive regulation and sperm collection of blue-bodied large-spotted grouper; (3) parent fish cultivation and egg acquisition of saddle-banded grouper; (4) artificial hybridization fertilization and hatching; (5) hybrid seedling cultivation; the present invention realizes distant hybridization breeding of two large grouper species for the first time, breaks through the reproductive isolation between species, space and time, and cultivates new grouper germplasm with fast growth rate and high survival rate. The weight of the 3-month-old hybrid new germplasm is 31.15% higher than that of the purebred saddle-banded grouper; 98.02% higher than that of the same cultured species, golden tiger hybrid grouper, and 168.19% higher than that of pearl gentian. When cultivated to 5cm, the survival rate of hybrid seedlings is 68,000, which is 30.77% higher than that of purebred saddle grouper. It can effectively solve the industry problems of insufficient utilization of advantageous traits of large grouper and lack of culturable seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of distant hybridization breeding, and in particular relates to a method for breeding and seedling cultivation of hybridization between two large grouper species. Background Art

[0002] Grouper is an important high-end marine aquaculture fish in my country, with an annual aquaculture volume of more than 240,000 tons and an output value of 20 billion yuan. With the continuous expansion of the grouper aquaculture industry, the problem of insufficient fast-growing and stress-resistant aquaculture seedlings is particularly prominent. Taking advantage of the growth advantages of large groupers (saddle-banded grouper and blue-bodied big-spotted grouper), three new hybrid varieties have been bred through hybrid breeding, but the growth rate of the new varieties is significantly lower than that of the large parents. So far, no hybrid grouper combination with super-parent growth advantages has been found, and the number of new variety seedlings is restricted by the germplasm and maturity of the parent large grouper. Therefore, breaking through the factory-based artificial breeding technology of large groupers and cultivating new germplasm with super-parent growth advantages can further expand the scale of grouper aquaculture and provide sufficient large-sized seedlings for my country's land-based factory-based and deep-sea aquaculture.

[0003] The saddle-banded grouper (Epinephelus lanceolatus) and the blue-bodied grouper (Epinephelidae) belong to the order Perciformes, the family Epinephelidae, and the genus Epinephelidae. They are the two largest groupers. The saddle-banded grouper, commonly known as the dragon grouper and dragon-bearing grouper, is mainly distributed in Southeast Asia, Australia, and the South my country Sea. It is known for its large size and fast growth. The weight of a one-year-old fish can reach 1.5 to 3.0 kg, the maximum body length is over 2 meters, and the weight can reach 400 kg. It is the largest grouper. The blue-bodied grouper, commonly known as the golden grouper, is distributed in the Indian Ocean to the Pacific Ocean, and is distributed in the southern waters of my country and the Taiwan waters. It has delicious meat, can grow to 2 meters, and weigh up to 100 kg. It is second only to the saddle-banded grouper in size.

[0004] Both large groupers have the characteristics of sex reversal, being hermaphrodites, with females first and males later. At present, the artificial breeding technology of large groupers in northern factories is not yet mature, and there are certain difficulties in spawning of broodstock, and the survival rate of seedlings is low; at the same time, the proportion of mature males in the blue-bodied grouper population is low, and mature semen can only be collected within 4 months of the year, and the amount of semen produced is extremely small. Therefore, the large-scale breeding of large groupers is greatly restricted, and there is a shortage of cultivatable seedlings on the market. In the natural environment, there is a certain degree of reproductive isolation between the blue-bodied grouper and the saddle-banded grouper in terms of geographical distribution, breeding time and cultivation temperature, and no natural hybridization has been found. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for hybrid breeding and seedling cultivation of blue-bodied big-spotted grouper and saddle-banded grouper, aiming to solve the problems of low maturity rate and small amount of semen of male blue-bodied big-spotted grouper; difficulty in breeding purebred saddle-banded grouper under factory conditions and low survival rate of seedlings; and lack of fast-growing new germplasm of large grouper and insufficient culturable seedlings.

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

[0007] The first object of the present invention is to provide a breeding method for interspecific hybridization of two large groupers, the method comprising: (1) breeding of large grouper broodstock populations; (2) reproductive regulation and sperm collection of blue-bodied big-spotted groupers; (3) breeding of broodstock and egg collection of saddle-banded groupers; (4) artificial hybridization fertilization and hatching; and (5) breeding of hybrid seedlings.

[0008] (1) Selection of large grouper broodstock populations: selecting female saddle groupers aged 7 or above, weighing more than 40 kg and having a healthy physique as the female broodstock; selecting male blue groupers aged 8 or above, weighing more than 25 kg and having a healthy physique as the male broodstock;

[0009] (2) Reproductive regulation and sperm collection of blue-bodied grouper

[0010] a. Cultivation environment: Factory workshop cultivation, water flow exchange volume 8m 3 / h, dissolved oxygen above 6mg / L, salinity 28-31‰, and cultivation temperature 20-28℃;

[0011] b. Reproductive regulation: Use environmental temperature control to regulate the maturation of the gonads of blue-bodied big spotted grouper. In late October, the culture water temperature is reduced by 1°C every 8 to 10 days. The water temperature drops to 21°C by the end of December and is maintained at this temperature for 15 to 20 days. After that, the temperature is gradually increased until the water temperature reaches 26°C in late March of the following year. After the environmental temperature is controlled by first cooling and then heating, the gonads of male blue-bodied big spotted grouper gradually mature, and fresh semen can be collected in early April.

[0012] c. Sperm collection: 2 days before sperm collection, inject a mixture of 200 IU / kg human chorionic gonadotropin (HCG) and 10 μg / kg luteinizing hormone-releasing hormone analog (LHRH-A3) into mature male fish to promote sperm production;

[0013] (3) Breeding and egg collection of broodstock of Epinephelus saddleback

[0014] a. Cultivation environment: Factory workshop cultivation, water flow exchange volume is 8m 3 / h, dissolved oxygen above 6mg / L, salinity 28-31‰, and cultivation temperature 22-30℃;

[0015] b. Enhanced cultivation before reproduction: The parents will start intensive cultivation in January of the following year, and the water temperature will gradually increase from 22℃ to 28℃, with an increase of 1℃ every 8-12 days; female saddle grouper fish with gonad development to stage IV will be selected for ripening and induced spawning. The oxytocin used is a mixture of HCG and LHRH-A3, with the dosage of 350IU / kg and 20μg / kg fish body weight respectively, and mature eggs will be collected manually;

[0016] (4) Artificial hybridization fertilization and hatching

[0017] a. Artificial insemination: dry insemination of blue-bodied grouper sperm with a microscopic activity of more than 90% and saddle-banded grouper eggs at a volume ratio of 1:500, stirring evenly, adding 2 times the volume of 23-25°C seawater, letting it stand, taking the floating eggs, washing them, and placing them in an incubator for incubation;

[0018] b. Incubation: Incubation conditions are water temperature 24-26°C, water flow 0.2-0.3m 3 / h, salinity 28-30‰, dissolved oxygen greater than 6mg / L, when the eggs develop to the tail bud stage, collect the floating eggs and move them to the nursery pond for incubation, with an egg density of 7.0-8.0 g / m 3 , continuous micro-inflation and micro-flow water incubation.

[0019] Furthermore, in the above (3) breeding of broodstock of grouper and obtaining of eggs, fresh fish supplemented with 3% VC and 2% VE are fed during the temperature rise period of step b, and 350 IU / kg of HCG and 20 μg / kg of LHRH-A3 are injected 2 days before egg collection, and the water exchange rate needs to be increased to 10 m 3 / h, stimulate the maturation of female fish gonads.

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

[0021] The second object of the present invention is to provide a method for cultivating interspecific hybrid seedlings of two large groupers, which is as follows: collecting the floating eggs from the hybrid fertilized eggs of the blue-bodied big-spotted grouper and the saddle-banded grouper and moving them to a nursery pond for incubation, wherein the water temperature of the nursery pond is 24-27° C.; maintaining the dissolved oxygen content at 6-10 mg / L; and maintaining the water flow exchange rate at 0.2-0.3 m / s before the yolk sac disappears (0-6 days). 3 / h, and the water flow gradually increases to 5m / h after the 9th day (when fin spines grow on the back and abdomen) as the seedlings grow. 3 / h; the feeding bait series is as follows:

[0022] On the 0th to 4th day, before opening, add concentrated Chlorella, and the final concentration of Chlorella in the water is 5 to 8×10 5 cells / mL;

[0023] On the 5th to 15th day, small (SS) rotifers were fed to a final concentration of 5 to 8 ind / mL in the water. Oyster larvae were fed twice a day to a final concentration of 10 to 15 ind / mL in the water.

[0024] From 16th to 23rd day, L-type rotifers were fed twice a day, and the final concentration of L-type rotifers in the water was 4-6ind / mL;

[0025] From the 24th to the 30th day, Artemia nauplii were fed twice a day, and the final concentration of Artemia nauplii in the water was 2-3 ind / mL;

[0026] After the 30th day, artificial compound feed was fed.

[0027] Preferably, during the critical period from yolk sac absorption to external feeding, the fry have just opened their mouths to eat and have weak swimming ability. During this stage, the fry grow slowly and have a high mortality rate. They should be fed with algae liquid and small rotifers at a high density, and the water exchange rate should be maintained at 0.2-0.3m 3 / h, the dissolved oxygen content is maintained at 6-8 mg / L, and the environmental disturbance caused by water flow and oxygenation is controlled to a minimum.

[0028] Preferably, 7 to 34 days after hatching, i.e., the late larval period, the second dorsal fin and pelvic fin naturally open into a triangular cone shape. During this period, the fish mainly feed on small and large Artemia, and the first accelerated growth occurs. Due to problems such as disease and nutritional deficiencies, large-scale mortality is prone to occur. Nutrition should be strengthened, feed should be changed in time according to fish development and mouth size, and the aquaculture water quality should be kept fresh.

[0029] Preferably, 47 to 86 days after hatching, i.e., the juvenile period, the mouth cleft is significantly enlarged and the growth is accelerated. Due to the large growth differences between individuals, the phenomenon of residual food in fry is serious. This period is the third dangerous period. The feeding amount should be increased appropriately during the day, and each feeding should be stopped until more than 2 / 3 of the fry do not eat, and the feeding frequency should be increased to 3 times / d. The light source in the workshop should be turned off after 30 minutes of feeding. At the same time, tiles, PVC pipes and other shelters should be added to the pool, and the fry should be separated regularly with a screen for graded cultivation.

[0030] The principle of the present invention is: through the breeding technology combining multiple technologies such as group selection, reproductive regulation, and distant hybridization, the reproductive isolation between two large grouper species is broken, and a new hybrid grouper germplasm with super-parent growth advantage and high survival rate is cultivated for the first time.

[0031] The beneficial effects of the present invention compared with the prior art are as follows:

[0032] 1. The present invention solves the problem of difficulty in artificial breeding of saddle grouper in the north and low survival rate of seedlings. Through group breeding combined with nutrition regulation, hormone regulation and water flow stimulation, the industrial problem of difficulty in artificial spawning of saddle grouper is solved.

[0033] 2. The present invention solves the industrial problem of low male maturity rate, short breeding cycle and low semen volume of blue-bodied grouper under factory farming conditions in the north. The reproductive regulation technology combining environmental temperature control with hormone induced production is used to improve the male maturity rate of blue-bodied grouper, extend the semen collection cycle by 2 months compared with that before regulation, and increase the single semen production by 1 to 2 times compared with that of unregulated male fish, which greatly improves the utilization efficiency of the excellent traits of blue-bodied grouper.

[0034] 3. The present invention solves the bottleneck problems of lack of new germplasm of large grouper and insufficient cultivarable seedlings. The present invention realizes the distant hybridization breeding of two large groupers for the first time, breaks through the reproductive isolation between species, space and time, and cultivates new germplasm of grouper with fast growth rate and high survival rate. The body weight of the 3-month-old hybrid new germplasm is 80.00±1.41g, which is 31.15% higher than that of purebred saddle grouper; 98.02% higher than that of the similar bred species Golden Tiger hybrid grouper, and 168.19% higher than that of pearl gentian, with significant super-parent growth advantages. When cultivated to 5cm, the survival rate of hybrid seedlings is 68,000, which is 30.77% higher than that of purebred saddle grouper. It can effectively solve the industry problems of insufficient utilization of the advantageous traits of large groupers and lack of cultivarable seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 New hybrid germplasm of 6-month-old Epinephelus saddleback (♀) × Epinephelus ocellaris (♂) was cultured. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1: Broodstock screening and enhanced breeding

[0038] (1) Broodstock breeding

[0039] a. Saddle-banded grouper: In Hainan, Shandong, Fujian and other places, 800 breeding fish were selected and collected from wild and farmed populations, with an average weight of 35.64 kg, to construct the basic breeding population (G0) of saddle-banded grouper. In the same year, seedlings were cultivated through artificial propagation. Early and mid-term selection was carried out at 8 months and 14 months of age, and individuals with fast growth, bright body color, no deformity and no symptoms were selected. The selection intensity was 5%, and a G1 breeding population of 500 was constructed. After 6 years of cultivation, they reached sexual maturity, with an average weight of 37.24 kg and an average coefficient of variation of phenotypic traits of 7.27%. The G1 population was used to artificially breed seedlings, and early and mid-term selection was carried out at 5 months and 15 months of age, with a selection intensity of 4.5% and 4.8% respectively. The seedlings reached sexual maturity after 6 years of cultivation, and a G2 generation breeding population of 400 was constructed, with an average weight of 39.68 kg and an average coefficient of variation of phenotypic traits of 6.95%.

[0040] The G2 generation breeding population was further selected, and the screening criteria were fast-growing, normal-shaped, disease-free, over 7 years old, and weighing over 40 kg in the same population. The selection intensity was 8%; and the phenotypic traits of the breeding population were measured (n=30), among which the total length / body length was 1.16±0.02, the head length / body length was 0.35±0.03, the body height / body length was 0.36±0.02, the caudal peduncle length / body length was 0.14±0.02, and the caudal peduncle height / body length was 0.13±0.01.

[0041] b. Blue-bodied big-spotted grouper: In Hainan, Fujian, Taiwan and other places, 500 breeding fish were selected and collected from wild and farmed groups, with an average weight of 22.68kg, to construct the basic breeding population (G0) of blue-bodied big-spotted grouper. In the same year, sexually mature individuals were used for artificial reproduction and seedling cultivation. The seedlings grew to 12 months old, and fast-growing, deformed and disease-free seedlings were screened and cultivated. The selection intensity was 5%, forming a G1 early group of 800, with an average weight of 1.18kg; the 6-year-old group reached 168, with an average weight of 23.57kg, and the average coefficient of variation of phenotypic traits was 10.71%, to construct the G1 breeding population. Artificial breeding was carried out using mature G1 groups, and early and mid-term selection was carried out on seedlings at 12 and 15 months of age, with selection intensities of 2.5% and 5% respectively, to cultivate 1,100 G2 candidate groups with an average weight of 1.86kg. After 6 years of breeding, the G2 mature population reached 269, with an average body weight of 24.64 kg and an average coefficient of variation of phenotypic traits of 9.46%.

[0042] The G2 generation breeding population was further selected, and the screening criteria were fast-growing, normal-shaped, disease-free, over 8 years old, and weighing over 25 kg in the same population, with a selection intensity of 10%; and the phenotypic traits of the breeding population were measured (n=30), among which the total length / body length was 1.17±0.03, the head length / body length was 0.38±0.05, the body height / body length was 0.34±0.02, the caudal peduncle length / body length was 0.12±0.02, and the caudal peduncle height / body length was 0.12±0.01.

[0043] (2) Strengthening training

[0044] a. Cultivation environment: All the above selected broodstock are intensively cultivated in the factory workshop in the north. The cultivation pond is 6m×8m×2.5m, with a water exchange volume of 8m 3 / h, dissolved oxygen above 6mg / L, salinity 28-31‰. The culture temperature of blue-spotted grouper is 20-28℃, the culture density is 30 per pond, and fresh mackerel is fed once in the morning, with the feeding amount being 2%-3% of the fish body weight; the culture temperature of saddle-banded grouper is 22-30℃, the culture density is 25 per pond, and fresh small fish are mainly fed, twice a day, with the feeding amount being 3%-5% of the fish body weight.

[0045] b. Enhanced cultivation before breeding of saddle-banded grouper: The saddle-banded grouper parents began to be intensively cultivated in January of the following year. The water temperature gradually increased from 22℃ to 28℃, increasing by 1℃ every 8 to 12 days; the water exchange volume increased to 10m 3 / h, stimulating the gonadal development of female fish by changing the water flow; feeding fresh small fish and mackerel, and adding 3% VC and 2% VE; selecting female saddle grouper with gonadal development to stage IV for ripening and inducing spawning. The oxytocin used is a mixture of HCG and LHRH-A3, with the dosage of 350 IU / kg and 20 μg / kg fish body weight respectively. 48 hours after injection, the mature eggs are collected by squeezing the abdomen.

[0046] c. Strengthening cultivation of blue-bodied big-spotted grouper before reproduction: In late October each year, reproductive regulation experiments were carried out on selected blue-bodied big-spotted grouper by controlling the environmental temperature. The water temperature was reduced by 1°C every 8 to 10 days. The water temperature dropped to 20 to 21°C by the end of December. This temperature was maintained for 15 to 20 days, and then the temperature was gradually increased until the water temperature gradually rose to 25 to 26°C in March of the following year. During this period, fresh mackerel was fed once a day in the morning, and 2% VC and 1% VE were added. 2 days before semen collection, 200 IU / kg of human chorionic gonadotropin (HCG) and 10 μg / kg of luteinizing hormone-releasing hormone analog (LHRH-A3) were injected into the regulated male fish to promote the maturation of male fish. 24 hours after the injection, the milky white semen was squeezed out of the abdominal cavity, collected in 10 mL cryopreservation tubes and stored at low temperature and light-shielded. The sperm motility was tested to be more than 90%.

[0047] After temperature control and hormone induction, the sperm production cycle of blue-bodied grouper was significantly extended. Before the control, mature semen could only be collected from May to September. After the control, fresh semen could be collected from April to October of the same year, and the sperm collection cycle was extended by 2 months. Before the control, the amount of semen collected from a single fish was 0.5-1mL, and after the control, the amount of semen collected increased to 1-2mL, and the sperm production increased by 1-2 times. It provides a sufficient sperm source for the genetic improvement of large grouper and large-scale seed production.

[0048] Example 2: Hybrid fertilization of blue-bodied grouper and saddle-banded grouper

[0049] a. Artificial insemination: Select female saddle grouper with swollen abdomen to maturity for artificial egg squeezing. Divide the collected eggs into two parts and put them in dry plastic basins. At the same time, collect appropriate amount of fresh sperm from saddle grouper and blue grouper, and dry fertilize them with saddle grouper eggs at a volume ratio of 1:500. After stirring evenly, add 2 times the volume of seawater (water temperature 23-25℃), let it stand for 10 minutes, take the floating eggs, rinse and weigh them, and place them in an incubator for incubation.

[0050] b. Incubation: Incubation conditions are water temperature 24-26°C, water flow 0.2-0.3m 3 / h, salinity 28-30‰, dissolved oxygen greater than 6mg / L, during the incubation period, the embryonic development of the two groups of fish was continuously observed using an optical microscope, and the fertilization rate and hatching rate were calculated. When the eggs developed to the tail bud stage, they were collected and moved to the nursery pond for incubation, and the egg density was 7.0-8.0 g / m 3 , continuous micro-inflation and micro-flow water incubation.

[0051] c. Observation of embryonic development: The fertilized eggs of the hybrid group and the saddle grouper completed embryonic development in 21h 24min and 21h 32min respectively. The consumption of yolk sac and oil ball in embryonic development of the hybrid new germplasm was 29.45%, and that of oil ball was 20.75%. The yolk sac was consumed fastest at 1d after hatching, reaching 58.70%; the oil ball was consumed fastest at 3d after hatching, reaching 32.08%; the yolk sac and oil ball of the fry were basically absorbed on the 5th day after hatching.

[0052] d. Observation of morphological development: The second dorsal fin spine and pelvic fin spine of the hybrid germplasm reached their longest length at 29 days after hatching, which were 8.15±0.02mm and 5.80±0.10mm respectively. They completely degenerated at 47 days and metamorphosis was completed. The eye diameter of the 1st day larvae was 0.16±0.01mm, which increased 22 times at 86 days; the length of the mouth cleft was 0.09±0.02mm at 4 days, which increased 99 times at 86 days. According to the morphological changes of yolk sac, second dorsal fin spine, pelvic fin spine, scales, body color, etc., the post-embryonic metamorphosis of the hybrids was divided into early larvae (0-6 days after hatching), late larvae (7-34 days after hatching), juvenile stage (35-46 days after hatching) and juvenile stage (47-86 days after hatching). The early larvae grew slowly, and the growth rate of the late larvae to juvenile stage gradually accelerated.

[0053] Example 3: Cultivation of new hybrid germplasm and comparison of survival rates

[0054] a. Seedling cultivation: The factory-based workshop cultivation pool is 6m×6m×1.5m, with a water depth of 1m and a cultivation water temperature of 24-27℃. As the seedlings grow, the water flow gradually increases from 0.3m 3 / h increased to 5m 3 / h. Before opening on the 4th day, add concentrated Chlorella (8×10 5 cells / mL); from 5th to 15th day, small rotifers (5-8ind / mL) were fed, and oyster larvae (10-15ind / mL) were fed twice a day; from 16th to 23rd day, L-type rotifers (4-6ind / mL) were fed twice a day; from 24th to 30th day, Artemia nauplii (2-3ind / mL) were fed twice a day; after 30th day, artificial compound feed was fed.

[0055] b. Comparison of survival rate: In this example, a total of 2.6 kg of hybrid and purebred fertilized eggs were obtained, and the fertilization rate and hatching rate of the hybrid were 89.09% ± 0.08% and 62.59% ± 10.70%, respectively, which were not significantly different from those of the purebred saddle grouper (Table 1). The feeding vitality and stress resistance of the hybrid new germplasm were significantly better than those of the purebred saddle grouper seedlings. When cultivated to 5 cm, the survival rate of the hybrid seedlings was 68,000, and that of the saddle grouper was 52,000. The survival rate of the hybrid seedlings was 30.77% higher than that of the purebred saddle grouper (Table 1).

[0056] The comprehensive results showed that the hybrid offspring of Epinephelus saddleback (♀) × Epinephelus cyanobacteria (♂) had higher fertilization and hatching rates, normal metamorphosis of larvae and juveniles, and significantly higher survival rates than pure Epinephelus saddleback.

[0057] Table 1 Comparison of hatching between the hybrid F1 of Epinephelus saddleback (♀) × Epinephelus leucocephalus (♂) and the control group

[0058]

[0059] Example 4: Testing of growth traits of new hybrid germplasm

[0060] Through the construction of half-sib families, the hybrid lines of Epinephelus saddleback (♀) × Epinephelus brevis (♂) and the pure lines of Epinephelus saddleback were obtained. A comparative breeding experiment on growth traits was carried out under the same environment, during which a comparative analysis of their growth traits was conducted.

[0061] Total length: From 0 to 52 days, there was no significant difference between the hybrid group and the control group; from 58 to 86 days, the hybrid group began to be significantly longer than the control group, and at 86 days, the total lengths of the hybrid group and the control group reached 77.82±2.79mm and 68.09±1.19mm, respectively. The total length of the hybrid was 1.14 times that of the purebred.

[0062] Body weight: The body weight of the 86-day hybrid group and the control group was measured (n=50). The body weight of the hybrid was 80.00±1.41g, which was significantly higher than the 61.00±1.45g of the purebred saddle grouper (p<0.01), and the body weight trait was 31.15% higher than that of the purebred saddle grouper. Compared with the hybrid groupers cultured for 90 days in the same period: Golden Tiger Hybrid Grouper (40.40±1.28g) and Pearl Gentian (29.83±0.51g) (Chen et al., Aquaculture, 2023), the hybrid germplasm cultivated by the present invention has a body weight increase of 98.02% and 168.19% respectively compared with the above similar cultured varieties, showing significant hybrid growth advantages.

[0063] In this example, after 6 months of comparative breeding, the weight of the hybrid new germplasm can reach 500g and the total length is 29.5cm. The weight of similar breeding species, Golden Tiger Hybrid Grouper and Pearl Gentian, reaches 500g, which requires 12 months and 14 months of breeding respectively (Chen et al., Aquaculture, 2023). The hybrid new germplasm of grouper cultivated by the present invention can significantly shorten the breeding cycle and provide high-quality large-sized seedlings for factory-based and deep-sea breeding in my country. In addition, the hybrid is slender and well-proportioned, with a better appearance ( Figure 1 ), the breeding prospects are broad.

[0064] 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 principle of the present invention, and these improvements shall be deemed as the protection scope of the present invention.

Claims

1. A breeding method for interspecific hybridization of two large grouper species, characterized in that: The method comprises: (1) breeding of large grouper broodstock; (2) reproductive regulation and sperm collection of blue-bodied grouper; (3) breeding of broodstock and egg collection of saddle-banded grouper; (4) artificial hybrid fertilization and hatching; and (5) hybrid seed breeding. (1) Selection of large grouper broodstock populations: selecting female saddle groupers aged 7 or above, weighing more than 40 kg and having a healthy physique as the female broodstock; selecting male blue groupers aged 8 or above, weighing more than 25 kg and having a healthy physique as the male broodstock; The (2) reproduction regulation and sperm collection of blue-bodied grouper: a. Cultivation environment: Factory workshop cultivation, water flow exchange volume 8m 3 / h, dissolved oxygen above 6mg / L, salinity 28-31‰, and cultivation temperature 20-28℃; b. Reproductive regulation: In late October, the water temperature is lowered by 1°C every 8 to 10 days. By the end of December, the water temperature drops to 21°C and is maintained at this temperature for 15 to 20 days. Then the temperature is gradually raised until the water temperature reaches 26°C in late March of the following year. The gonads of male blue-bodied grouper gradually mature, and fresh semen can be collected in early April. c. Sperm collection: 2 days before sperm collection, inject a mixture of 200 IU / kg of chorionic gonadotropin and 10 μg / kg of luteinizing hormone-releasing hormone analogue into mature male fish to promote sperm production; (3) breeding of broodstock of Epinephelus saddleback and obtaining of eggs: a. Cultivation environment: Factory workshop cultivation, water flow exchange volume is 8m 3 / h, dissolved oxygen above 6mg / L, salinity 28-31‰, and cultivation temperature 22-30℃; b. Enhanced cultivation before reproduction: The parents will start intensive cultivation in January of the following year, and the water temperature will gradually increase from 22℃ to 28℃, with an increase of 1℃ every 8-12 days; female saddle grouper fish with gonad development to stage IV will be selected for ripening and induced spawning. The oxytocin used is a mixture of HCG and LHRH-A3, with the dosage of 350IU / kg and 20μg / kg fish body weight respectively, and mature eggs will be collected manually; The (4) artificial hybridization fertilization and hatching: a. Artificial insemination: dry insemination of blue-bodied grouper sperm with a microscopic activity of more than 90% and saddle-banded grouper eggs at a volume ratio of 1:500, stirring evenly, adding 2 times the volume of 23-25°C seawater, letting it stand, taking the floating eggs, washing them, and placing them in an incubator for incubation; b. Incubation: Incubation conditions are water temperature 24-26°C, water flow 0.2-0.3m 3 / h, salinity 28-30‰, dissolved oxygen greater than 6mg / L, when the eggs develop to the tail bud stage, collect the floating eggs and move them to the nursery pond for incubation, with an egg density of 7.0-8.0 g / m 3 , continuous micro-inflation and micro-flow water incubation.

2. The breeding method for interspecific hybridization of two large groupers according to claim 1, characterized in that: In the breeding of broodstock and egg collection of saddle grouper, fresh fish with a mass ratio of 3% VC and 2% VE are fed during the temperature rise period of step b, and 350 IU / kg of chorionic gonadotropin and 20 μg / kg of luteinizing hormone releasing hormone analogues are injected 2 days before egg collection. During this period, the water exchange rate needs to be increased to 10m 3 / h, stimulate the maturation of female fish gonads.

3. The breeding method for interspecific hybridization of two large groupers according to claim 1, characterized in that: The method also includes testing the growth advantage traits of the new hybrid germplasm.

4. A method for cultivating interspecific hybrid seedlings of two large grouper species, characterized in that: The method is as follows: collecting the floating eggs developed to the tail bud stage according to claim 1 and moving them to a nursery pond for incubation; the water temperature of the nursery pond is 24-27°C; the dissolved oxygen content is maintained at 6-10 mg / L; before the yolk sac disappears, the water flow exchange speed is maintained at 0.2-0.3 m / s. 3 / h, and the water flow gradually increases to 5m / h after the 9th day as the seedlings grow. 3 / h; the feeding bait series is as follows: On the 0th to 4th day, before opening, add concentrated Chlorella, and the final concentration of Chlorella in the water is 5 to 8×10 5 cells / mL; On the 5th to 15th day, small rotifers were fed to a final concentration of 5 to 8 ind / mL in the water. Oyster larvae were fed twice a day to a final concentration of 10 to 15 ind / mL in the water. From 16th to 23rd day, L-type rotifers were fed twice a day, and the final concentration of L-type rotifers in the water was 4-6ind / mL; From the 24th to the 30th day, Artemia nauplii were fed twice a day, and the final concentration of Artemia nauplii in the water was 2-3 ind / mL; After the 30th day, artificial compound feed was fed.

5. The method for cultivating interspecific hybrid seedlings of two large groupers according to claim 4, characterized in that: During the critical period from yolk sac absorption to external feeding, the water exchange rate is maintained at 0.2-0.3m 3 / h, the dissolved oxygen content is maintained at 6-8 mg / L, and the environmental disturbance caused by water flow and oxygenation is controlled to a minimum.

6. The method for cultivating interspecific hybrid seedlings of two large groupers according to claim 4, characterized in that: During the 47-86 days after hatching, which is the juvenile period, the feeding amount should be increased appropriately during the day. Feed until more than 2 / 3 of the fry stop eating each time. Increase the feeding frequency to 3 times / day and turn off the light source in the workshop 30 minutes after feeding. At the same time, add shelters in the pond and use screens to separate the fry regularly for graded cultivation.

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