A method for cultivating artificial hybrid seedlings of brown-spotted grouper and red nine-spined seabass

Abstract

The present invention relates to a method for cultivating artificial hybrid seedlings of brown-spotted grouper and red nine-spined seabass, and the method belongs to the technical field of distant hybridization breeding. The method comprises parent selection of brown-spotted grouper and red nine-spined seabass, parent cultivation, parent intensive cultivation, gamete collection, artificial insemination, hatching of fertilized eggs and seedling cultivation. The present invention hybridizes two species belonging to two genera of the grouper family with different geographical distributions and different reproduction times by means of reproductive regulation and distant hybridization technology, i.e., cross-generic hybridization within the grouper family, thereby breaking through the inter-generic reproductive isolation and cultivating new hybrid germplasm with excellent characteristics such as high fertilization rate, high hatching rate, high survival rate and fast growth rate.

Description

Technical Field

[0001] The invention belongs to the technical field of distant hybridization breeding, and particularly relates to a method for cultivating artificial hybrid seedlings of brown-spotted grouper and red nine-spined seabass. Background Art

[0002] Grouper is an important marine economic fish species, widely distributed in the tropical and subtropical waters of the Pacific, Atlantic, and Indian Oceans. In recent years, marine aquaculture has developed rapidly, especially grouper aquaculture. With the development of the "southern fish, northern breeding, southern fish, northern raising" model, grouper farming has spread throughout the coastal areas of northern and southern China. In addition, with the country's vigorous development of saline-alkali waters, grouper farming has also emerged. In 2023, the farming volume of grouper reached 241,000 tons, ranking among the top four marine fish aquaculture. However, there are also some problems of genetic degradation in the breeding process of grouper, such as high deformity rate, slow growth rate, and serious disease. Hybrid breeding, as an important breeding method, is widely used for the improvement of grouper germplasm. Three new grouper varieties have been bred: Tiger Dragon Hybrid Grouper, Cloud Dragon Grouper, and Golden Tiger Hybrid Grouper.

[0003] Both the brown-spotted grouper (Epinephelus fuscoguttatus) and the red nine-spined seabass (Cephalopholis sonnerati) belong to the order Perciformes and the family Epinephelidae, but they belong to different genera. The brown-spotted grouper, commonly known as tiger grouper, belongs to the genus Epinephelus and is primarily aquacultured in southern my country. While its slow growth rate and long breeding cycle allow for a large supply of high-quality eggs year-round, the red nine-spined seabass, also known as reticulated mullet or red melon-seed seabass, belongs to the genus Cephalopholis and is bright red with small brown-red or brown spots on its sides, back, and head. Its vibrant color and delicious meat make it a popular premium edible fish.

[0004] Both brown-spotted grouper and red nine-spined sea bass are hermaphrodites, with females maturing first and then, in small numbers, reversing sex to male. The low number of males in grouper populations significantly impacts sperm availability during reproduction. The diverse geographical distribution and reproductive times of different fish species present significant challenges in crossbreeding. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention relates to a method for cultivating artificial hybrid seedlings of brown-spotted grouper and red nine-spined seabass, thereby obtaining a new hybrid germplasm of grouper with high fertilization rate, high hatching rate, high survival rate and fast growth rate.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for artificially hybridizing brown grouper and red nine-spined seabass and cultivating seedlings, comprising selecting brown grouper and red nine-spined seabass parents, cultivating parents, strengthening parent cultivation, collecting gametes, artificial insemination, hatching fertilized eggs and cultivating seedlings;

[0008] The parent enhanced breeding: Brown spot grouper parent enhanced breeding: temperature control is from early November to the end of December, the temperature is reduced from 27 to 29 degrees Celsius to 21 to 22 degrees Celsius, from early January to the end of February of the following year, the water temperature is gradually increased from 21 to 22 degrees Celsius to 27 to 29 degrees Celsius; from March to October, the temperature is controlled at 27 to 29 degrees Celsius; from January to October, the feed is fed with chilled bait mixed with nutritional enhancers;

[0009] Enhanced breeding of red nine-spined sea bass: From early November to the end of December, the temperature is lowered from 25-27°C to 21-22°C. From early January to the end of February of the following year, the water temperature is gradually raised from 21-22°C to 25-27°C. From March to October, the temperature is controlled at 25-27°C. From January to October, the fish are fed with chilled bait mixed with nutritional enhancers.

[0010] The incubation conditions of the fertilized eggs are as follows: water temperature 27-28°C, salinity 28-30‰, pH 7.8-8.2, dissolved oxygen greater than 6 mg / L, continuous micro-aeration and micro-flow water; the incubation density is no more than 3.75 kg / m 3 When the eggs develop to the tail bud stage, the floating fertilized eggs are collected and placed in the nursery pond for hatching;

[0011] The seedling cultivation environment is an indoor cement pool with a water temperature of 27-28°C, a pH of 7.8-8.2, a salinity of 28-30‰, a dissolved oxygen of more than 6 mg / L, and an egg density of 4-6 g / m 3 ;

[0012] After the fry hatch, they are fed with SS rotifers as the starter bait before the yolk sac absorption is completed, followed by L rotifers, Artemia larvae, copepods, Artemia adults, and granular feed. During the feeding of rotifers, Chlorella is supplemented in the nursery water.

[0013] Preferably, in the parent selection, a female brown-spotted grouper weighing more than 5 kg and a male red nine-spined sea bass are selected as hybrid parents.

[0014] Preferably, the parent culture of brown-spotted grouper is carried out at a temperature ranging from 21 to 29°C throughout the year; a salinity of 28 to 33‰, a pH of 7.5 to 9.0, dissolved oxygen of 6 mg / L or more, a culture density of 110 to 150 fish per pond, and a male-female ratio of 3 to 6:1.

[0015] The breeding temperature of red nine-spined seabass parents ranges from 21 to 27°C throughout the year; the salinity is 28 to 33‰, the pH is 7.5 to 9.0, and the dissolved oxygen is above 6 mg / L; the breeding density is 80 to 120 fish per pond, and the male-female ratio is 3 to 6:1.

[0016] Preferably, the artificial insemination is as follows: red nine-spined seabass sperm and brown-spotted grouper eggs are fertilized at a ratio of 1 ml: 0.8-1.2 kg, and after standing for 10-15 minutes, the floating eggs are collected, and then rinsed with fresh seawater, and finally the fertilized eggs are placed in an incubation bucket for incubation.

[0017] More preferably, the artificial insemination is as follows: the semen is divided into two parts, one part of the semen is directly added to the eggs and stirred evenly, and then the other part of the semen is added with a small amount of seawater to activate it and poured into the eggs, quickly stirred evenly, and allowed to stand for 10 to 15 minutes. Then, high-salinity seawater with a salinity of 35‰ is added, and the mixture is allowed to stand for another 10 minutes. The floating eggs are collected and placed in an incubation bucket for incubation.

[0018] Preferably, the hybrid seedlings are cultured with the following feed series: SS rotifers are fed at 2 to 7 days old, with a feeding density of 4 to 8 per ml; L rotifers are fed at 7 to 18 days old, with a feeding density of 3 to 5 per ml; Artemia larvae are fed at 14 to 24 days old, with a feeding density of 2 to 3 per ml; copepods are fed at 20 to 33 days old, with a feeding density of 2 to 3 per ml; Artemia adults are fed at 30 to 50 days old, with a feeding density of 1 to 2 per ml; and pellet feed is fed 2 to 3 times a day after 50 days old.

[0019] Seedling management: For the first 20 days after frying, the light duration is 24 hours, the light intensity is 800-1200lx, and Chlorella is added once a day with micro-flow water; after 20 days, the light duration is 12 hours, the light intensity is 800-1200lx, and the amount of water change is gradually increased; according to the size differences of the fry, fry screening is started and graded cultivation is carried out.

[0020] More preferably, the water volume for seedling cultivation is 0.1-0.3 m3 for 20 days before seedling cultivation. 3 / h, then gradually increase to 3-5m 3 / h; after 40 days of age, start screening the fry by size, and the screening frequency is once every 5 to 7 days; two days before and after screening, sprinkle the fry water with health products such as electrolytic multivitamins to reduce the stress of the fry; when the fry are 50 to 60 days old, feed them with fish paste mixed with granular feed, firstly to help them quickly change bait, and secondly, fish paste is healthier for the intestines.

[0021] The principle of the present invention is: with the help of reproductive regulation and distant hybridization technology, two species belonging to two genera under the grouper family with different geographical distributions and different breeding times are hybridized, that is, cross-generic hybridization under the grouper family, breaking through the reproductive isolation between genera, thereby cultivating new hybrid germplasm with excellent characteristics such as high fertilization rate, high hatching rate and fast growth rate.

[0022] The differences and beneficial effects of the present invention compared with other marine fish breeding methods are as follows:

[0023] 1. The method of the present invention implements separate temperature control and strengthening on the two fish, so that the sexual maturity time of the two is synchronized, and eggs and sperm can be collected in the same time period, ensuring the quality of eggs and sperm, and creating excellent conditions for obtaining high-quality fertilized eggs.

[0024] 2. The sperm production of red nine-spined seabass is low, which seriously affects the large-scale production of seedlings. Through the broodstock management of the present invention, the parent breeding time can be extended and the breeding quality can be improved, which greatly extends the production cycle of the hybrid of brown-spotted grouper and red nine-spined seabass, and promotes the progress of grouper breeding.

[0025] 3. The hybrid offspring cultivated by the present invention has excellent characteristics of high fertilization rate, high hatching rate, high survival rate and fast growth rate.

[0026] 4. Brown-spotted grouper and red nine-spined seabass belong to the genera Epinephelus and Nine-spined Seabass, respectively. They are distantly related and have geographical isolation and reproductive isolation. The present invention, based on reproductive regulation and distant hybridization technology, breaks through the reproductive isolation between the two fish species of the same genera, fully utilizes hybrid advantages, and greatly promotes the cultivation of new hybrid varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The brown-spotted grouper parent of the present invention;

[0028] Figure 2 It is the parent of the red nine-spined seabass of the present invention;

[0029] Figure 3 It is a hybrid of brown-spotted grouper♀×red nine-spined seabass♂ of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention.

[0031] The artificial propagation method of the hybrid of brown grouper and red nine-spined seabass in embodiment 1 comprises the following steps:

[0032] (1) Parent selection of brown-spotted grouper and red nine-spined seabass

[0033] a. Female brown-spotted grouper: Select female brown-spotted grouper that is over 5 years old, weighs over 5 kg, is of normal size, healthy, and has good vitality;

[0034] b. Male red nine-spined seabass parent: Select male red nine-spined seabass that are over 6 years old, weigh over 5kg, have normal body shape, are healthy and have good vitality.

[0035] (2) Parent cultivation

[0036] a. Brown-spotted grouper parent breeding

[0037] a1. Cultivation Environment: Cultivation is conducted in indoor cement ponds with a temperature of 22-29°C, a salinity of 28-33‰, a pH of 7.5-9.0, and a dissolved oxygen level of at least 6 mg / L. The density is 100-150 fish per pond. The ponds should be 5m long, 5m wide, and 2m deep, with a male-to-female ratio of 4:1.

[0038] a2. Bait and feeding: Bait consists primarily of freshly-frozen fish such as mackerel and saury, at a rate of 1.5-2.0% of body weight, once every two days.

[0039] a3. Management: The water quality management of the cement pool is constant flow water, with a water inflow of 8 to 10m 3 / h, and change the water twice a day, the water replacement volume is 1 / 2 pool.

[0040] b. Red nine-spined seabass parent breeding

[0041] b1. Cultivation Environment: Cultivation should be conducted in indoor cement ponds with a temperature of 22-27°C, a salinity of 28-33‰, a pH of 7.5-9.0, and a dissolved oxygen level of at least 6 mg / L. The cultivation density should be 80-100 per pond. The pond should be 5 m long, 5 m wide, and 2 m deep, with a male-to-female ratio of 4:1.

[0042] b2. Bait and feeding: Bait consists primarily of freshly-frozen fish such as mackerel and saury, at a rate of 2-3.5% of body weight, once daily.

[0043] b3. Management: The water quality management of the cement pool is constant flow water, with a water inflow of 8 to 10m 3 / h, and change the water twice a day, the water replacement volume is 1 / 2 pool.

[0044] (3) Parental intensive cultivation

[0045] a. Enhanced breeding of brown-spotted grouper parents

[0046] a1. Water temperature for breeding: From early November to December, the parent fish are transferred to the sea for cage culture. The water temperature is lowered from 28°C to around 21°C. From early January to late February of the following year, the fish are transferred from the sea to indoor breeding ponds. The water temperature is gradually raised from 21°C to 28°C, with a 1°C increase every 8-10 days.

[0047] a2. Bait and feeding: Bait consists of fresh mackerel, saury, bonito, squid, and shelled oysters mixed with nutritional supplements (vitamin C 4g / kg, vitamin E 4g / kg). Feed the fish once every two days at a rate of 2.5-5% of their body weight. Feed the fish three times a day, with a two-day interval between feedings.

[0048] a3. Management: Water inlet is 7-10m 3 / h, and change the water twice a day, the water replacement volume is 1 / 2 pool.

[0049] b. Enhanced breeding of red nine-spined sea bass parents;

[0050] b1. Cultivation water temperature: The parent species are transferred to the sea for cage culture, and the water temperature is lowered from 25°C to around 21°C. From early January to late February of the following year, the parent species are transferred from the sea to indoor culture ponds, and the water temperature is raised from 21°C to 25°C, with a 1°C increase every 13-15 days.

[0051] b2. Bait and feeding: Bait consists of fresh mackerel, saury, bonito, squid, and shelled oysters mixed with nutritional supplements (vitamin C 4g / kg, vitamin E 4g / kg). Feed once daily at a rate of 3-5% of body weight. Feed three times a day, with two days between feedings.

[0052] b3. Management: Water inlet is 7-10m 3 / h, and change the water twice a day, the water replacement volume is 1 / 2 pool.

[0053] (4) Gamete collection

[0054] a. Collection of Brown-Dot Grouper Eggs: During the breeding season, select female brown-dot groupers every afternoon. Those with a bulging abdomen, a slightly convex and reddish genital opening, and well-developed gonads are selected and placed in the spawning pool. In the evening, the eggs are manually squeezed. Place the broodstock on the spawning bed. First, wipe the water off the fish's abdomen with a towel. Then, slide the towel from the front of the abdomen toward the genital opening and squeeze. Collect the eggs in a clean basin. Be careful not to let seawater drip into the eggs.

[0055] b. Collection of sperm from red nine-spined sea bass: During the breeding season, select male red nine-spined sea bass with mature gonads, wipe off the moisture around the genital opening with a towel, slide and squeeze from the front side of the abdomen to the genital opening, and use a straw to absorb the sperm. Then, examine the quality of the collected sperm under a microscope. When the motility is greater than 90%, wait for fertilization.

[0056] (5) Artificial insemination

[0057] First, divide the semen into two parts. Add one part of semen directly to the eggs and stir evenly. Add a small amount of seawater to activate the other part of semen and pour it into the eggs. Stir quickly and evenly. After standing for 10 minutes, add high-salinity seawater with a salinity of 35‰ and let it stand for another 10 minutes. Collect the floating eggs and place them in an incubation bucket for incubation.

[0058] (6) Hatching of fertilized eggs

[0059] The hatching bucket consists of a cylindrical barrel with a diameter of 1.5m and a conical barrel bottom. The volume of water in the hatching bucket is 0.8m 3 The incubation conditions are water temperature 27-28℃, salinity 30‰, pH 8.2, dissolved oxygen greater than 6mg / L, continuous micro-aeration and micro-flow water; the incubation density is 2kg / barrel, and the volume of water in the barrel is 0.8m 3 When the fertilized eggs develop to the tail bud stage, the floating fertilized eggs are collected and placed in the hatching pond. The hatching pond is an indoor cement pond with a length of 5m, a width of 5m and a depth of 1.5m. The egg density is 5g / m 3 , every 1~1.5m 2 An air stone was placed, and the dissolved oxygen was maintained above 6 mg / L, the pH was 8.0, the water temperature was 27-28°C, and the salinity was 30‰. In this case, the hybrid fertilized eggs hatched in 21 hours and 54 minutes (Table 1), with a fertilization rate of 94.2% and a hatching rate of 84.5%. A total of 2.5 kg of fertilized eggs were obtained.

[0060] Table 1. Time sequence of embryonic development of hybrid of brown-spotted grouper♀×red nine-spined sea bass♂

[0061]

[0062]

[0063] The artificial propagation method of Example 2 of the hybrid of brown grouper and red nine-spined seabass comprises the following steps:

[0064] (1) Parent selection of brown-spotted grouper and red nine-spined seabass

[0065] a. Female brown-spotted grouper: Select female brown-spotted grouper that is over 5 years old, weighs over 5 kg, is of normal size, healthy, and has good vitality;

[0066] b. Male red nine-spined seabass parent: Select male red nine-spined seabass that are over 7 years old, weigh over 6 kg, have normal body shape, are healthy and have good vitality.

[0067] (2) Parent cultivation

[0068] a. Brown-spotted grouper parent breeding

[0069] a1. Cultivation environment: Cultivation is carried out in factory-type cultivation ponds. The cultivation environment is an indoor cement pond with a temperature of 23-29°C, a salinity of 28-32‰, a pH of 7.8-9.0, and a dissolved oxygen of more than 6 mg / L. The cultivation density is 130-150 fish / pond. The cement pond specifications are 5m long, 5m wide, and 2m deep, and the male-female ratio is 6:1. Cage cultivation can also be used. Choose a sea area with a minimum water temperature of no less than 21°C, a salinity of 27-30‰, a pH of 8.2-8.5, a water flow of 0.5-1m / s, and a water depth of 8m. The cage specifications are 6m long, 6m wide, and 5m deep. The cultivation density is 200-250 fish / cage, and the male-female ratio is 6:1.

[0070] a2. Bait and feeding: Bait consists primarily of freshly-frozen fish such as mackerel and saury, at a rate of 1.6-2.0% of body weight, once every two days.

[0071] a3. Management: The water quality management of the cement pool is constant flow water, with a water inflow of 8 to 10m 3 / h, and water is changed twice daily, replacing half the tank. During the breeding season, brown-spotted groupers are transferred to cement tanks for artificial breeding. After the breeding season is complete, the brown-spotted groupers are transferred to sea cages to quickly recover in the natural environment and help their gonads mature again. Three days before breeding, they are transferred to cement tanks for artificial breeding.

[0072] b. Red nine-spined seabass parent breeding

[0073] b1. Breeding environment: Breeding is carried out in factory-type breeding ponds. The breeding environment is an indoor cement pool with a temperature of 23-27°C, a salinity of 28-32‰, a pH of 7.8-9.0, and a dissolved oxygen of more than 6 mg / L. The breeding density is 80-100 fish / pond. The cement pool specifications are 5m long, 5m wide, and 2m deep, and the male-female ratio is 6:1. Cage breeding can also be used. Choose a sea area with a minimum water temperature of not less than 21°C, a salinity of 27-30‰, a pH of 8.2-8.5, a water flow of 0.5-1m / s, and a water depth of 8m. The cage specifications are 6m long, 6m wide, and 5m deep. The breeding density is 200-250 fish / box, and the male-female ratio is 6:1.

[0074] b2. Bait and feeding: Bait consists primarily of freshly-frozen fish such as mackerel and saury, at a rate of 2.5-3.5% of body weight, once daily.

[0075] b3. Management: The water quality management of the cement pool is constant flow water, with a water inflow of 8 to 10m 3 / h, and change the water twice a day, changing half the water in the pond. During the breeding season, the red nine-spined sea bass is transferred to sea cages to help its gonads mature. After the breeding season, it is transferred to cement ponds.

[0076] (3) Parental intensive cultivation

[0077] a. Enhanced breeding of brown-spotted grouper parents

[0078] a1. Culture Water Temperature: Intensive culture is carried out in cement ponds at a water temperature of 28°C. The fish are then transferred to the sea for culture in early November, when the natural sea temperature is 21-22°C. In early January or early February of the following year, the fish are transferred to indoor culture ponds for culture. The culture water temperature is gradually increased from 21-22°C to 28°C, with a 1°C increase every 8-12 days.

[0079] a2. Bait and feeding: Bait consists of fresh mackerel, saury, bonito, and squid mixed with nutritional enhancers (vitamin C 5g / kg, vitamin E 5g / kg). Feed the fish once every two days at a feeding amount of 3-4.5% of their body weight.

[0080] Feed three times each time, with an interval of two days before feeding again;

[0081] a3. Management: Water inlet is 7-10m 3 / h, and change the water twice a day, the water replacement volume is 1 / 2 pool.

[0082] b. Enhanced breeding of red nine-spined seabass parents

[0083] b1. Culture Water Temperature: Intensive culture is carried out in cement pools at a water temperature of 25°C. The fish are then transferred to the sea for culture in early November, when the natural sea temperature is 21-22°C. From early January to early February of the following year, the fish are transferred to indoor culture pools for culture. The culture water temperature is gradually increased from 21-22°C to 25°C, with a 1°C increase every 20 days.

[0084] b2. Bait and feeding: Bait consists of fresh frozen fish such as mackerel, saury, bonito, and squid mixed with nutritional enhancers (vitamin C 5g / kg, vitamin E 5g / kg). Feed once a day at a feeding amount of 3-5% of body weight.

[0085] Feed three times each time, with an interval of two days before feeding again;

[0086] b3. Management: After cement pond strengthening, if the sea temperature is suitable, transfer the fish to sea cages before they reach sexual maturity.

[0087] (4) Gamete collection

[0088] a. Collection of Brown-Dot Grouper Eggs: During the breeding season, select female brown-dot groupers every afternoon. Those with a bulging abdomen, a slightly convex and reddish genital opening, and well-developed gonads are selected and placed in the spawning pool. In the evening, the eggs are manually squeezed. Place the broodstock on the spawning bed. First, wipe the water off the fish's abdomen with a towel. Then, slide the towel from the front of the abdomen toward the genital opening and squeeze. Collect the eggs in a clean basin. Be careful not to let seawater drip into the eggs.

[0089] b. Collection of sperm from red nine-spined sea bass: During the breeding season, select male red nine-spined sea bass with mature gonads, wipe off the moisture around the genital opening with a towel, slide and squeeze from the front side of the abdomen to the genital opening, and use a straw to absorb the sperm. Then, examine the quality of the collected sperm under a microscope. When the motility is greater than 95%, wait for fertilization.

[0090] (5) Artificial insemination

[0091] Brown-spotted grouper eggs were divided into two portions and inseminated with either red nine-spined sea bass sperm or brown-spotted grouper sperm, respectively, to compare fertilization and hatching rates. The sperm-to-egg ratio was 1 ml:1 kg. First, the semen was divided into two portions. One portion was added directly to the eggs and stirred thoroughly. Then, the other portion was activated by adding a small amount of seawater and poured into the eggs. The mixture was quickly stirred and allowed to stand for 15 minutes. Then, high-salinity seawater with a salinity of 35‰ was added and allowed to stand for 10 minutes. The floating eggs were collected and placed in an incubator for incubation. The insemination procedures for the two sperm types were identical.

[0092] (6) Hatching of fertilized eggs

[0093] The hatching bucket consists of a cylindrical barrel with a diameter of 1.5m and a conical barrel bottom. The incubation conditions are water temperature of 27-28℃, salinity of 30‰, pH 8.2, dissolved oxygen greater than 6mg / L, continuous micro-aeration and micro-flow of water. When the fertilized eggs develop to the tail bud stage, the floating fertilized eggs are collected and incubated in the nursery pond. The nursery pond is an indoor cement pool with specifications of 5m long, 5m wide and 1.5m deep. The egg density is 4g / m 3 , every 1~1.5m 2 Arrange an air stone, the dissolved oxygen is above 6 mg / L, pH 8.0, water temperature is 27-28℃, and salinity is 30‰.

[0094] In this example, eggs fertilized with sperm from brown-spotted grouper achieved a fertilization rate of 97.5% and a hatching rate of 89.3%. Eggs fertilized with sperm from red nine-spined sea bass achieved fertilization rates of 95.8% and hatching rates of 86.7%, respectively. The fertilization and hatching rates of the brown-spotted grouper ♀ × red nine-spined sea bass ♂ hybrid were not significantly different from those of purebred brown-spotted grouper.

[0095] Example 3: Breeding of hybrid seedlings of brown-spotted grouper ♀× red nine-spined seabass ♂;

[0096] a. Cultivation environment: Place the embryos at the tail bud stage in a cement pool with a size of 5 m long, 5 m wide and 1.5 m deep. The water temperature should be 27-28°C, the dissolved oxygen should be above 6 mg / L, the pH should be 8.0 and the salinity should be 30‰.

[0097] b. Feeding of seedlings: Hybrid seedlings do not need to be fed during the period of endogenous nutrition. In the early stage of yolk sac absorption, they are fed with SS rotifers from 2 to 7 days old, and the feeding density is 4 to 7 per ml; from 6 to 18 days old, they are fed with L rotifers, and the feeding density is 3 to 4 per ml; from 15 to 25 days old, they are fed with Artemia larvae, and the feeding density is 2 to 3 per ml; from 23 to 35 days old, they are fed with copepods, and the feeding density is 1 to 3 per ml; from 33 to 50 days old, they are fed with adult Artemia, and the feeding density is 1 to 2 per ml; from 45 to 60 days old, they are fed with fish paste mixed with pellet feed, and the feeding frequency is 2 to 3 times per day; after 55 days old, they are fed with pellet feed, 2 to 3 times per day.

[0098] c. Seedling management: Before hatching, the air stone should be used to inflate the fish with a large amount of air (the water surface ripples should be 40 cm away from the air tube). After hatching, the air stone should be used to inflate the fish with a small amount of air (the water surface ripples should be 20 cm away from the air tube). For the first 20 days of hatching, the light duration should be 24 hours, the light intensity should be 800-1200 lx, and chlorella should be added once a day. The water flow should be 0.1-0.3 m 3 / h, and in addition, photosynthetic bacteria, Bacillus subtilis and other beneficial bacteria are added to the water every 2 to 3 days; after 20 days, the illumination time is 12 hours, the light intensity is 800 to 1200 lx, and the water exchange volume is gradually increased to 3 to 5 m 3 / h; after 40 days of age, the fry size screening begins, and the screening frequency is once every 5 to 7 days; two days before and after screening, the fry water is sprayed with health products such as electrolyzed multivitamins to reduce the stress of the fry.

[0099] In this embodiment, 0.5 kg of brown-spotted grouper ♀×red nine-spined seabass ♂ hybrid eggs were cultured to 50 days of age, and a total of more than 40,000 seedlings were cultivated. The seedlings were normal in size, healthy, and vigorous.

[0100] Example 4 Comparative breeding of brown-spotted grouper ♀×red nine-spined sea bass ♂ hybrids and brown-spotted grouper purebred seedlings;

[0101] In 2025, crossbreeding of brown-spotted grouper and red nine-spined seabass was carried out, and purebred brown-spotted grouper was cultivated as a control. The specific operations such as obtaining and hatching fertilized eggs of hybrids and purebreds were the same as in Example 2. 0.7 kg of hybrid fertilized eggs and purebred fertilized eggs were obtained respectively, and there was no significant difference in fertilization rate and hatching rate between hybrids and purebreds. In a factory breeding workshop, hatching and seedling cultivation were carried out under the conditions of water temperature of 27 ° C, dissolved oxygen of 6 mg / L or more, pH 8.0, and salinity of 30‰. After screening the seedlings, hybrids and purebreds of the same specifications and quantity were selected for comparative breeding. Comparative breeding results showed that at three months of age, the hybrids averaged a weight of (24.0±3.6) g and a total length of (11.1±0.6) cm, while the brown-spotted grouper averaged a weight of (17.1±2.5) g and a total length of (9.9±0.4) cm. These hybrids were 1.4 times heavier and 1.1 times longer than the brown-spotted grouper, respectively. Furthermore, a total of 62,000 three-month-old hybrids and 55,000 brown-spotted groupers were reared, and the survival rate of the hybrid fry was 9.1% higher than that of the brown-spotted grouper. The brown-spotted grouper ♀ × red nine-spotted grouper ♂ hybrid exhibits significant advantages, including rapid growth and a high survival rate.

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for cultivating artificial hybrid seedlings of brown-spotted grouper and red nine-spined seabass, characterized in that: The method comprises parent selection of brown-spotted grouper and red nine-spined seabass, parent cultivation, parent intensive cultivation, gamete collection, artificial insemination, hatching of fertilized eggs and seed cultivation; The parent selection comprises selecting female brown-spotted grouper fish weighing more than 5 kg and male red nine-spined sea bass as hybrid parents; The parent enhanced breeding: Brown spot grouper parent enhanced breeding: temperature control is from early November to the end of December, the temperature is reduced from 27-29 degrees Celsius to 21-22 degrees Celsius; from early January to the end of February of the following year, the water temperature is gradually increased from 21-22 degrees Celsius to 27-29 degrees Celsius; from March to October, the temperature is controlled at 27-29 degrees Celsius; feeding is done with chilled bait mixed with nutritional enhancers; Enhanced breeding of red nine-spined sea bass: From early November to late December, the temperature is lowered from 25-27°C to 21-22°C. From early January to late February of the following year, the water temperature is gradually raised from 21-22°C to 25-27°C. From March to October, the temperature is maintained at 25-27°C. Feeding is done with chilled bait mixed with nutritional enhancers. The incubation conditions of the fertilized eggs are as follows: water temperature 27-28°C, salinity 28-30‰, pH 7.8-8.2, dissolved oxygen greater than 6 mg / L, continuous micro-aeration and micro-flow water; the incubation density is no more than 3.75 kg / m 3 When the eggs develop to the tail bud stage, the floating fertilized eggs are collected and placed in the nursery pond for hatching; The seedling cultivation environment is an indoor cement pool with a water temperature of 27-28°C, a pH of 7.8-8.2, a salinity of 28-30‰, a dissolved oxygen level of more than 6 mg / L, and an egg density of 4-6 g / m 3 ; After the fry hatch, they are fed with SS rotifers as the starter bait before the yolk sac absorption is completed, followed by L rotifers, Artemia larvae, copepods, Artemia adults, and granular feed. During the feeding of rotifers, Chlorella is supplemented in the nursery water.

2. The method according to claim 1, characterized in that The parent culture of brown-spotted grouper is carried out at a temperature ranging from 21 to 29°C throughout the year; the salinity is 28 to 33‰, the pH is 7.5 to 9.0, the dissolved oxygen is above 6 mg / L, the breeding density is 110 to 150 fish per pond, and the male-female ratio is 3 to 6:

1. The breeding temperature of red nine-spined seabass parents ranges from 21 to 27°C throughout the year; the salinity is 28 to 33‰, the pH is 7.5 to 9.0, and the dissolved oxygen is above 6 mg / L; the breeding density is 80 to 120 fish per pond, and the male-female ratio is 3 to 6:

1.

3. The method according to claim 1, characterized in that The artificial insemination comprises the following steps: fertilizing red nine-spined sea bass sperm with brown-spotted grouper eggs at a ratio of 1 ml: 0.8-1.2 kg; collecting the floating eggs after standing; then washing them with fresh seawater; and finally placing the fertilized eggs in an incubation bucket for incubation.

4. The method according to claim 3, characterized in that The artificial insemination comprises the following steps: dividing semen into two parts, directly adding one part of semen into eggs and stirring evenly, then adding the other part of semen into seawater for activation and pouring it into eggs, stirring evenly quickly, standing still, adding high-salinity seawater with a salinity of 35‰, standing still, collecting floating eggs, placing them in an incubation bucket for incubation.

5. The method according to claim 1, wherein The hybrid seed cultivation includes the following feed series: feeding SS rotifers at 2 to 7 days old at a feeding density of 4 to 8 per ml; feeding L rotifers at 7 to 18 days old at a feeding density of 3 to 5 per ml; feeding Artemia larvae at 14 to 24 days old at a feeding density of 2 to 3 per ml; feeding copepods at 20 to 33 days old at a feeding density of 2 to 3 per ml; feeding Artemia adults at 30 to 50 days old at a feeding density of 1 to 2 per ml; and feeding pellet feed 2 to 3 times a day after 50 days old. Seedling management: For the first 20 days, the light duration is 24 h, the light intensity is 800~1200 lx, and Chlorella is added once a day with micro-flow water; after 20 days, the light duration is 12 h, the light intensity is 800~1200 lx, and the amount of water change is gradually increased; according to the size differences of the fry, the fry are screened and graded for cultivation.

6. The method according to claim 5, characterized in that For the fry cultivation, the water volume for the first 20 days is 0.1~0.3 m 3 / h, then gradually increase to 3~5 m 3 / h; after 40 days of age, fry size screening begins, with a screening frequency of once every 5 to 7 days; when they are 50 to 60 days old, they are fed with fish paste mixed with pellet feed.

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