A method for artificial hybridization breeding of brown-spotted grouper Epinephelus sp. male and Epinephelus lanceolatus female

Abstract

This invention relates to a method for artificial hybridization breeding of male brown-spotted grouper and female humpback bass. The method belongs to the field of distant hybridization breeding technology. It includes the selection of brown-spotted grouper and humpback bass parents, parent maturation cultivation, gamete collection, artificial insemination and hatching, and seedling cultivation. This invention uses reproductive regulation and distant hybridization technology to hybridize two species belonging to two genera within the grouper family with different geographical distributions and different breeding times, that is, intergeneric hybridization within the grouper family, which breaks through the reproductive isolation between genera, thereby cultivating superior varieties with fast growth and high survival rates.

Description

Technical Field

[0001] This invention belongs to the field of distant hybridization breeding technology, specifically relating to an artificial hybridization breeding method for male brown-spotted grouper and female humpback bass. Background Technology

[0002] Grouper is an important economic fish species in my country. According to data from the China Fisheries Statistical Yearbook in recent years, as of 2024, grouper production reached 267,488 tons, a year-on-year increase of 10.77% compared to 241,480 tons in 2023. It is mainly distributed in Guangdong, Hainan, and Fujian provinces. Among them, Hainan Province's grouper production was 102,276 tons, accounting for 38.2%. In recent years, the production of grouper in Hainan Province has generally shown a continuous upward trend. However, in recent years, the degradation of grouper germplasm resources and the shortage of high-quality seedlings have become increasingly prominent problems. The lack of genetically improved varieties and the emergence of serious diseases pose significant challenges to the development of the grouper aquaculture industry, limiting its growth. Solving these problems solely through traditional artificial breeding techniques is no longer sufficient. Therefore, reducing the difficulty of grouper breeding and cultivation, and obtaining high-quality, high-yield, and stress-resistant new aquaculture varieties has become an urgent goal for the grouper aquaculture industry.

[0003] Hybrid breeding combines the superior traits of the parents to obtain new hybrid varieties with excellent characteristics. In grouper, excellent hybrid varieties such as the Pearl Giant Grouper (female Brown Spotted Grouper x Male Giant Grouper), the Golden Tiger Grouper (female Brown Spotted Grouper x Male Blue-bodied Giant Grouper), and the Flower Dragon Grouper (Humpbacked Grouper (Cromileptes altivelis♀) and Saddleback Grouper (Epinephelus lanceolatus♂)) have been obtained and have a good market share.

[0004] The humpback sea bass (Chromileptes altivelis) belongs to the order Perciformes, family Serranidae, subfamily Chromileptesinae, and genus Chromileptes. Commonly known as the mouse sea bass, it is a high-grade marine economic fish. It has a complete range of amino acids, low levels of unsaturated fatty acids, and delicious meat. It has high edible and ornamental value and is loved by consumers. However, it grows slowly and has a low population density.

[0005] The brown-spotted grouper (Epinephelus fuscoguttatus) belongs to the order Perciformes, family Epinephelidae, and genus Epinephelus. Commonly known as the tiger grouper, it is distributed in the Indo-Pacific region and is a warm-water reef-dwelling fish. The brown-spotted grouper is an important economically important fish species for both fishing and aquaculture. It is highly nutritious and delicious. Artificial breeding is relatively easy, and it exhibits strong disease resistance. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention relates to a method for artificial hybridization breeding of male brown-spotted grouper and female humpback bass, resulting in a new hybrid grouper germplasm with high fertilization rate, high hatching rate, high survival rate, fast growth rate, and high stress resistance.

[0007] This invention is achieved through the following technical solution:

[0008] A method for artificial hybridization breeding of male brown-spotted grouper and female humpback bass includes selection of brown-spotted grouper and humpback bass parents, parent maturation cultivation, gamete collection, artificial insemination and hatching, and seedling cultivation.

[0009] The parent selection criteria are as follows: select male brown-spotted grouper that are 7 years or older, weigh over 12 kg, have a normal body shape, are healthy, and have good vitality; select female humpback bass that are 4 years or older, weigh over 3 kg, are active, have a regular shape, are brightly colored, and are healthy.

[0010] The aforementioned parental maturation-promoting breeding:

[0011] Brown-spotted grouper broodstock breeding: In March, the temperature is controlled at 25-26℃, maintained until May. Then, the temperature is increased to 28-29℃, maintained until October. Afterward, the temperature is slowly decreased to 24-26℃, maintained until December. Then, the temperature is increased to 27-28℃, maintained until March of the following year. During this period, excellent water quality is maintained, and nutrition is fortified to support gonadal development. The salinity is 28-33‰, pH is 7.5-9.0, and dissolved oxygen is 6... For strains with a concentration of mg / L or higher, the stocking density is 110-150 tails / pond, with a female-to-male ratio of 3-6:1. When preparing semen, slowly lower the temperature to 21-23℃ over 2-3 days, and maintain this temperature for 4-6 days. Then, slowly raise the temperature to 28-29℃ over 3-5 days, and maintain this temperature for 4-6 days. After that, slowly lower the temperature to 24-25℃ over 2-3 days, and maintain this temperature for 5 days. Finally, slowly raise the temperature to 28℃ over 3-5 days, and maintain this temperature at 28℃. The photoperiod is white:black = 16:8, the light intensity is 10000-15000 lx, and the nighttime light intensity is 200-500 lx. During this period, the fish are continuously fortified with nutrients. After about a month, try sliding and squeezing from the front of the abdomen towards the genital opening to see if semen is produced. When semen is needed, inject the mature humpback bass with human chorionic gonadotropin (HCG) at 40 μg / kg and releasing hormone analog (LRH-A) at 30 μg / kg of fish body weight, diluted with 0.6% physiological saline, every other day, until they give birth.

[0012] Furthermore, the breeding of brown-spotted grouper broodstock was enhanced: the broodstock were fed a diet including fresh shrimp, crab, squid, and miscellaneous fish once a day, with the amount being about 4% of the broodstock's body weight. At the same time, fortifiers such as vitamin E, vitamin V, cod liver oil, and lecithin were added to the diet, with each addition being 0.5% of the fish's body weight, in order to promote the gonadal development of the broodstock.

[0013] Humpback grouper broodstock breeding: To better promote gonad development in brown-spotted grouper, the temperature was controlled at 23-24℃ in January, maintained until March, then increased to 25-26℃, maintained until June, then slowly decreased to 22-23℃, maintained until October, then increased to 25-26℃, maintained until January of the following year. Excellent water quality was maintained, and nutritional supplementation was provided to support gonad development. The seawater salinity was 28‰~32‰, water temperature 20~28℃, pH 7.5~9.0, and dissolved oxygen 6... For strains with a concentration of mg / L or higher, the stocking density is 120-180 individuals per pond, with a female-to-male ratio of 1-5:1. When egg production is desired, the temperature is slowly lowered to 20-22℃ over 2-3 days and maintained for 9-11 days. Then, the temperature is slowly raised to around 28℃ over 5-6 days and maintained for 9-11 days. The temperature is then slowly lowered to 22-23℃ over 2-3 days and maintained for 10 days. Finally, the temperature is slowly raised to around 26℃ over 3-5 days and maintained at 26℃. The photoperiod is white:black = 14:10, with a light intensity of 10000-15000 lx and a nighttime light intensity of 200-500 lx. Nutritional supplementation is continuously provided. Egg production typically occurs around 45 days. The parents' condition is monitored; if their food intake decreases while they are in good health, the female's abdomen is observed to indicate bulging, and the egg cell maturity is assessed before egg production.

[0014] Further strengthening the breeding of humpback bass broodstock: The broodstock are fed with feed including fresh shrimp, crab, squid, and miscellaneous fish once a day, with the amount of feed being about 4% of the broodstock's body weight. At the same time, fortifiers such as vitamin E, vitamin V, cod liver oil, and lecithin are added to the feed, with the amount of fortifier added each time being 0.5% of the fish's body weight, in order to promote the gonadal development of the broodstock.

[0015] Gamete collection: Humpback perch eggs and brown-spotted grouper sperm are collected manually, taking care to avoid seawater dripping in.

[0016] As a preferred technical solution, the semen is diluted with a sperm-enhancing diluent at a volume ratio of 1:9. 50 ml of the sperm-enhancing diluent contains 0.4 g NaCl, 0.1 g KHCO3, 0.08 g Tris, 0.5 g glucose, 5 ml bovine fetal serum, and the remainder is water. When sperm motility is above 90%, the semen is stored at low temperature, awaiting fertilization.

[0017] Artificial insemination and hatching: Insemination is performed according to the ratio of original semen: egg cells = 1ml: 1-1.2kg. After stirring evenly, the mixture is transferred to a hatching container. The hatching water temperature is 26-28℃, the salinity is 29-32, the dissolved oxygen is ≥6 mg / L, and the pH is 8.0-8.2. After hatching for 4 hours, oxygen is stopped and the mixture is allowed to stand. The upper layer of eggs is then collected and transferred to another hatching container. Once the fry hatch, they are transferred to a nursery pond.

[0018] As a preferred technical solution, artificial insemination employs dry fertilization.

[0019] Seedling cultivation: SS rotifers are inoculated into the seedling cultivation pond, and amino acid fertilizer is used in combination with Bacillus subtilis for water cultivation, maintaining a transparency of 1m-1.1m;

[0020] As a preferred technical solution: For the first 1-3 days, use SS rotifers (100-150µm) at a density of 800-850 rotifers / L; for the 4th-5th days, introduce SS rotifers + S rotifers (150-200µm) at a ratio of 2:1, ensuring an effective food density of 700-750 rotifers / L; for the 6th-7th days, introduce S rotifers, achieving an effective food density of 600-650 rotifers / L; for the 8th-9th days, introduce S rotifers + L rotifers (170-300µm) at a ratio of 2:1, achieving an effective density of 500-550 rotifers / L; for the 10th-12th days, introduce indoor L rotifers, achieving an effective food density of 500-550 rotifers / L; for the 13th-15th days, introduce indoor L rotifers + newly hatched Artemia larvae (350-450µm) at a ratio of 4:6, ensuring an effective density of 400-450 rotifers / L; for the 16th-17th days... Introduce indoor L-rotifers and newly hatched Artemia at a ratio of 2:8, ensuring an effective density of 400-450 individuals / L. From day 18-22, introduce newly hatched Artemia, ensuring an effective density of 300-350 individuals / L, with a morning to afternoon ratio of 7:3. From day 23-25, feed Artemia larvae and Daphnia mongolica (500-600µm) at a ratio of 3:1, maintaining an effective food density of 300-350 individuals / L. Feeding density: 7:3 (morning:afternoon); 26-28 days after the start of feeding, feed brine shrimp larvae + Daphnia mongolica larvae in a 1:1 ratio, maintaining an effective feed density of 300-350 individuals / L, with a morning:afternoon ratio of 7:3; 29-30 days after the start of feeding, feed Daphnia mongolica larvae, maintaining an effective feed density of 200-250 individuals / L, with a morning:afternoon ratio of 7:3; after 30 days, transfer to a rearing pond for nurserying.

[0021] Do not change the water for days 1-7. On day 8, you can add a small amount of water, adding 10cm of new water each day. On day 11, you can change the water, adding and draining 15cm of water each day.

[0022] Seedling management: The seedlings are raised in flowing water in the breeding pond. Adult Mongolian naked-bellied daphnia are fed for 30-40 days. Adult brine shrimp are used for 40-50 days. Fish paste is mixed with pellet feed for acclimation for 50-60 days. After 60 days, compound pellet feed is fed. When the seedlings are 6-8cm long, they are transferred to net cages for breeding.

[0023] The principle of this invention is to use reproductive regulation and distant hybridization techniques to hybridize two species belonging to two families within the order Perciformes that have different geographical distributions and different breeding times, i.e., interfamilial hybridization within the order Perciformes, thereby breaking through the reproductive isolation between families and cultivating new hybrid germplasm with excellent characteristics such as high fertilization rate, high hatching rate and fast growth rate.

[0024] The differences and beneficial effects of this invention compared to other marine fish breeding methods are as follows:

[0025] 1. The semen of brown-spotted grouper is low and of unstable quality. This invention uses a sperm-enhancing diluent to enhance the sperm of humpback bass, thereby improving the fertilization rate.

[0026] 2. The hybrids cultivated in this invention have excellent traits such as strong resistance to adverse conditions, fast growth rate, delicious meat, and high survival rate;

[0027] 3. This invention enables the parental gonads to mature synchronously by regulating temperature, photoperiod, and light intensity.

[0028] 4. Brown-spotted grouper and humpback perch belong to the Grouperidae and Serranidae families respectively. They are distantly related and are geographically and reproductively isolated. This invention, based on reproductive regulation and distant hybridization technology, breaks through the reproductive isolation between the two fish species, makes full use of hybrid vigor, and greatly promotes the cultivation of new hybrid varieties. Attached Figure Description

[0029] Figure 1 This diagram illustrates the developmental process of a hybrid egg of a humpback perch (female) and a brown-spotted grouper (male). Stages are: 1. 2-cell stage; 2. 16-cell stage; 3. Mulberry stage; 4. High blastocyst stage; 5. Gastrula stage; 6. Embryo formation stage; 7. Embryo closure stage; 8. Auditory sac formation stage; 9. Lens formation stage; 10. Hatching stage; 11. Newly hatched larvae; 12. First day larvae.

[0030] Figure 2 A comparison image of the growth of tiger-spotted and hybrid spotted tiger-spotted ...

[0031] Figure 3 A comparison chart of the weights of tiger-spotted and hybrid tiger-spotted ...

[0032] Figure 4 This is a picture of a hybrid of brown-spotted grouper and humpback perch. Detailed Implementation

[0033] To make the objectives, 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 only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 to represent selected embodiments of the invention.

[0034] Example 1

[0035] A method for artificial hybridization breeding of brown-spotted grouper and humpback perch, comprising: selection of brown-spotted grouper and humpback perch parents, parent cultivation, parent enhancement cultivation, gamete collection, artificial insemination, hatching of fertilized eggs and seedling cultivation;

[0036] (1) Parental selection:

[0037] Male broodstock of brown-spotted grouper: Select male brown-spotted groupers that are 7 years or older, weigh over 12 kg, have a normal body shape, are healthy, and have good vitality.

[0038] Female parent humpback bass: Select female humpback bass that are 4 years or older, weigh over 3kg, are active, have a regular shape, bright colors, and are healthy;

[0039] (2) Parental maturation promotion

[0040] a. Breeding of Brown Spotted Grouper Parents: To better promote gonadal development in brown spotted grouper, the temperature is controlled at 25-26℃ in March, maintained until May, then increased to 28-29℃, maintained until October, then slowly decreased to 24-26℃, maintained until December, then increased to 27-28℃, maintained until March of the following year. Excellent water quality is maintained throughout this period, with nutritional fortification to support gonadal development. The salinity is 28-33‰, pH is 7.5-9.0, and dissolved oxygen is 6... For strains with a concentration of mg / L or higher, the stocking density is 110-150 tails / pond, with a female-to-male ratio of 3-6:1. When preparing semen, slowly lower the temperature to 21-23℃ over 2-3 days, and maintain this temperature for about 5 days. Then, slowly raise the temperature to 28-29℃ over 3-5 days, and maintain this temperature for about 5 days. Next, slowly lower the temperature to 24-25℃ over 2-3 days, and maintain this temperature for 5 days. Finally, slowly raise the temperature to about 28℃ over 3-5 days, and maintain the final temperature at around 28℃. The photoperiod is white:black = 14:10, the light intensity is 10000-15000 lx, and the nighttime light intensity is 200-500 lx. During this period, the fish are continuously fortified with nutrients. After about a month, try sliding and squeezing from the front of the abdomen towards the genital opening to see if semen is produced. When semen is needed, inject the mature humpback bass with human chorionic gonadotropin (HCG) at 40 μg / kg and releasing hormone analog (LRH-A) at 30 μg / kg of fish body weight, diluted with 0.6% physiological saline, every other day, until they give birth.

[0041] As a preferred implementation method, the broodstock of brown-spotted grouper is enhanced by feeding the broodstock with feed including fresh shrimp, crab, squid, and miscellaneous fish once a day, at a rate of about 4% of the broodstock's body weight. At the same time, fortifiers such as vitamin E, vitamin V, cod liver oil, and lecithin are added to the feed, at a rate of 0.5% of the fish's body weight each time, to promote the gonadal development of the broodstock.

[0042] b. Broodstock rearing of humpback grouper: To better promote gonad development in brown-spotted grouper, the temperature was controlled at 23-24℃ in January, maintained until March, then increased to 25-26℃, maintained until June, then slowly decreased to 22-23℃, maintained until October, then increased to 25-26℃, maintained until January of the following year. Excellent water quality was maintained, and nutritional support was provided to support gonad development. The seawater salinity was 28‰~32‰, pH 7.5~9.0, and dissolved oxygen 6... For strains with a concentration of mg / L or higher, the stocking density is 120-180 individuals per pond, with a female-to-male ratio of 1-5:1. When egg production is desired, the temperature is slowly lowered to 20-22℃ over 2-3 days and maintained for about 10 days. Then, the temperature is slowly raised to about 28℃ over 5-6 days and maintained for about 10 days. The temperature is then slowly lowered to 22-23℃ over 2-3 days and maintained for 10 days. Finally, the temperature is slowly raised to about 26℃ over 3-5 days and maintained at approximately 26℃. The photoperiod is white:black = 14:10, with a light intensity of 10000-15000 lx and a nighttime light intensity of 200-500 lx. Nutritional supplementation is continuously provided. Egg production typically occurs around 45 days. The parents are observed for reduced food intake if they are in good health; in this case, the mother's abdomen is observed for swelling, and the egg cell maturity is assessed before egg production.

[0043] As a preferred implementation method, the broodstock of humpback perch are enhanced by feeding them with a diet consisting of fresh shrimp, crab, squid, and miscellaneous fish once a day at a rate of about 4% of their body weight. At the same time, fortifiers such as vitamin E, vitamin V, cod liver oil, and lecithin are added to the feed at a rate of 0.5% of the fish's body weight each time to promote the gonadal development of the broodstock.

[0044] (3) Gamete collection:

[0045] a. Collection of humpback bass eggs: During the breeding season, female humpback bass are selected every afternoon, choosing those with a bulging abdomen, slightly protruding and reddish genital opening, and well-developed gonads. These are then placed in the spawning tank, and the eggs are manually squeezed out at night. The parent fish are placed on the spawning bed, and the abdomen is first dried with a towel. Then, the eggs are squeezed out from the front of the abdomen towards the genital opening, and collected in a clean basin. Care is taken to avoid seawater dripping into the eggs.

[0046] b. Collection of sperm from brown-spotted grouper: During the breeding season, oxytocin is injected at a dose of 40 μg / kg hCG + 30 μg / kg LRH-A (releasing hormone analogue) per kg of fish body weight, diluted with 0.6% saline, and injected into the pectoral and dorsal fins. When production is needed, mature male brown-spotted grouper are selected. The area around the genital opening is wiped clean with a towel. Sperm is collected by sliding and squeezing from the anterior side of the abdomen towards the genital opening using a pipette. The quality of the collected sperm is then examined under a microscope. When the sperm motility is above 80%, it is divided into two portions. One portion is directly stored at low temperature, and the other portion is placed in the prepared diluent. 1 ml of sperm is diluted with 9 ml of diluent to enhance the sperm, and then stored at low temperature until fertilization. The diluent is prepared as follows: 0.4 g NaCl, 0.1 g KHCO3, 0.08 g Tris, 0.5 g glucose, and ultrapure water are brought to a final volume of 45 ml, then 5 ml bovine fetal serum is added.

[0047] (4) Artificial insemination:

[0048] Clean, fully aerated seawater was injected into the hatching tank, with the water temperature adjusted to 28℃ and salinity to 32‰. The collected sperm and eggs were divided into two portions, each with a sperm:egg ratio of 1ml:1-1.2kg. One portion underwent dry fertilization (sperm and eggs were thoroughly mixed before adding an appropriate amount of hatching water to activate the sperm), while the other portion underwent wet fertilization (sperm was added to the eggs, followed immediately by seawater activation). See Table 1 for details. After stirring for approximately 1 minute at the start of fertilization, the eggs were transferred to hatching tanks. The hatching tanks maintained a water temperature of 26-28℃, salinity of 29-32, dissolved oxygen ≥6 mg / L, and pH of 8.0-8.2. A maximum of 2kg of hybrid eggs could be placed in a 400L hatching tank. After 4 hours of incubation, aeration was stopped, and the tanks were allowed to stand for 10 minutes. The high-quality eggs from the top layer were then transferred to another clean hatching tank. Once the fry hatched, they were transferred to the nursery pond. Three batches of hybrid eggs were produced in May 2025.

[0049] Table 1. Fertilization rates of hybrid eggs under different fertilization methods

[0050] ;

[0051] As shown in Table 1, the use of diluent + dry insemination is more suitable for hybridization of male brown-spotted grouper and female red-spined bass.

[0052] Example 2

[0053] (1) Parental selection:

[0054] Female parent humpback bass: Select female humpback bass that are 3 years, 4 years, or older, weigh over 5 kg, have a normal body shape, are healthy, and have good vitality.

[0055] Male broodstock of brown-spotted grouper: Select male brown-spotted groupers that are 7, 8, or older, weigh over 12kg, are active, have a regular shape, bright colors, and are healthy.

[0056] (2) Parental maturation promotion breeding:

[0057] Breeding of Brown Spotted Grouper Parents: To better promote gonadal development in Brown Spotted Grouper, the temperature was controlled at 25-26℃ in March, maintained until May, then increased to 28-29℃, maintained until October, then slowly decreased to 24-26℃, maintained until December, then increased to 27-28℃, maintained until March of the following year. Excellent water quality was maintained throughout this period, with nutrient fortification to support gonadal development. The salinity was 28-33‰, pH 7.5-9.0, and dissolved oxygen 6... For strains with a concentration of mg / L or higher, the stocking density is 110-150 tails / pond, with a female-to-male ratio of 3-6:1. When preparing semen, slowly lower the temperature to 21-23℃ over 2-3 days, and maintain this temperature for about 5 days. Then, slowly raise the temperature to 28-29℃ over 3-5 days, and maintain this temperature for about 5 days. Next, slowly lower the temperature to 24-25℃ over 2-3 days, and maintain this temperature for 5 days. Finally, slowly raise the temperature to about 28℃ over 3-5 days, and maintain the final temperature at around 28℃. The photoperiod is white:black = 16:8, the light intensity is 10000-15000 lx, and the nighttime light intensity is 200-500 lx. During this period, the fish are continuously fortified with nutrients. After about a month, try sliding and squeezing from the front of the abdomen towards the genital opening to see if semen is produced. When semen is needed, inject the mature humpback bass with human chorionic gonadotropin (HCG) at 40 μg / kg and releasing hormone analog (LRH-A) at 30 μg / kg of fish body weight, diluted with 0.6% physiological saline, every other day, until they give birth.

[0058] As a preferred implementation method, the broodstock of brown-spotted grouper is enhanced by feeding the broodstock with feed including fresh shrimp, crab, squid, and miscellaneous fish once a day, with the amount of feed being about 4% of the broodstock's body weight. At the same time, fortifiers such as vitamin E, vitamin V, cod liver oil, and lecithin are added to the feed, with each addition being 0.5% of the fish's body weight, in order to promote the gonadal development of the broodstock.

[0059] Humpback grouper broodstock breeding: To better promote gonad development in brown-spotted grouper, the temperature was controlled at 23-24℃ in January, maintained until March, then increased to 25-26℃, maintained until June, then slowly decreased to 22-23℃, maintained until October, then increased to 25-26℃, maintained until January of the following year. Excellent water quality was maintained throughout this period, with nutrient enhancement to support gonad development. The seawater salinity was 28‰~32‰, pH 7.5~9.0, and dissolved oxygen 6... For strains with a concentration of mg / L or higher, the stocking density is 120-180 individuals per pond, with a female-to-male ratio of 1-5:1. When egg production is desired, the temperature is slowly lowered to 20-22℃ over 2-3 days and maintained for about 10 days. Then, the temperature is slowly raised to about 28℃ over 5-6 days and maintained for about 10 days. The temperature is then slowly lowered to 22-23℃ over 2-3 days and maintained for 10 days. Finally, the temperature is slowly raised to about 26℃ over 3-5 days and maintained at approximately 26℃. The photoperiod is white:black = 14:10, with a light intensity of 10000-15000 lx and a nighttime light intensity of 200-500 lx. Nutritional supplementation is continuously provided. Egg production typically occurs around 45 days. The parents are observed for reduced food intake if they are in good health; in this case, the mother's abdomen is observed for swelling, and the egg cell maturity is assessed before egg production.

[0060] As a preferred implementation method, the broodstock of humpback perch are enhanced by feeding them with a diet consisting of fresh shrimp, crab, squid, and miscellaneous fish once a day at a rate of about 4% of their body weight. At the same time, fortifiers such as vitamin E, vitamin V, cod liver oil, and lecithin are added to the feed at a rate of 0.5% of the fish's body weight each time to promote the gonadal development of the broodstock.

[0061] (3) Gamete collection:

[0062] a. Collection of humpback bass eggs: During the breeding season, female humpback bass are selected every afternoon, choosing those with a bulging abdomen, slightly protruding and reddish genital opening, and well-developed gonads. These are then placed in the spawning tank, and the eggs are manually squeezed out at night. The parent fish are placed on the spawning bed, and the abdomen is first dried with a towel. Then, the eggs are squeezed out from the front of the abdomen towards the genital opening, and collected in a clean basin. Care is taken to avoid seawater dripping into the eggs.

[0063] b. Collection of sperm from brown-spotted grouper: During the breeding season, inject oxytocin at a dose of 40 μg / kg hCG + 30 μg / kg body weight of releasing hormone analogue (LRH-A), diluted with 0.6% saline. The injection volume should be 0.9 ml per fish, using the pectoral and dorsal fins. When production is needed, select male brown-spotted groupers with mature gonads. Wipe the area around the genital opening with a towel, slide and squeeze from the anterior abdomen towards the genital opening, and collect the sperm with a pipette. Examine the collected sperm under a microscope. If sperm motility is above 80%, dilute 1 ml of sperm with 9 ml of diluent, store at low temperature, and await fertilization.

[0064] (4) Artificial insemination and incubation:

[0065] Prepare the incubation water by thoroughly aerating clean seawater and adjusting the temperature to 28℃ and salinity to 32‰. Use a ratio of 1ml sperm to 1-1.2kg egg cells for dry fertilization (first thoroughly mix the sperm and egg cells, then add an appropriate amount of incubation water to activate the sperm). After about 1 minute, transfer the mixture to an incubation tank. The incubation tank water temperature should be 26-28℃, salinity 29-32, dissolved oxygen ≥6 mg / L, and pH 8.0-8.2. A maximum of 2kg of hybrid eggs can be placed in a 400L incubation tank. After 4 hours of incubation, stop aeration and let the mixture stand for 10 minutes. Remove the best eggs from the top layer and transfer them to another clean incubation tank. The entire incubation process is described in [link to incubation process]. Figure 1 After the fish fry break through the membrane, they are transferred to the nursery pond;

[0066] Table 2 Comparison of fertilization rate and deformity rate of hybrid eggs produced at different ages

[0067] ;

[0068] For humpback bass, choose females aged 4 years or older, and for brown-spotted grouper, choose males aged 7 years or older. The resulting eggs and sperm are better used to produce hybrid eggs.

[0069] Example 3

[0070] 1. Seedlings are cultivated for 1-30 days to obtain 1.2 kg of hybrid eggs, which are then placed in a 26m... 2 Ten seedlings, each weighing 1.2 kg, were placed in a 1.5m circular nursery pond for hatching. The water used for hatching was disinfected and filtered seawater. The nursery ponds were inoculated with *Stellaria media* rotifers, and an amino acid fertilizer (0.5 ml / m²) was applied. 3 Combined with Bacillus subtilis (0.2g / m) 3 Water should be prepared to maintain a water transparency of 1m-1.1m in the nursery pond. The feed during this period is as follows:

[0071] (1) For the first 1-3 days after opening, the density of SS rotifers (100-150um) in the feed room should be maintained at 800-850 per L;

[0072] (2) After 4-5 days of opening, introduce indoor SS rotifers + S rotifers (150-200), with a ratio of 2:1, to ensure an effective food density of 700-750 rotifers / L;

[0073] (3) Introduce S rotifers 6-7 days after the start of feeding, with an effective feed density of 600-650 rotifers / L;

[0074] (4) After 8-9 days of opening, introduce S rotifers + L rotifers (170-300um) in a ratio of 2:1 to ensure an effective density of 500-550 rotifers / L;

[0075] (5) Introduce indoor L rotifers 10-12 days after opening, with an effective feed density of 500-550 rotifers / L;

[0076] (6) After 13-15 days of opening, introduce indoor L rotifers + newly hatched Artemia (350-450um) in a ratio of 4:6 to ensure an effective density of 400-450 insects / L;

[0077] (7) After 16-17 days of opening, introduce indoor L rotifers + newly hatched Artemia in a ratio of 2:8 to ensure an effective density of 400-450 insects / L;

[0078] (8) After 18-22 days of opening, introduce newly hatched Artemia larvae to ensure an effective density of 300-350 larvae / L. The ratio of morning to afternoon introduction is 7:3.

[0079] (9) Feed brine shrimp larvae + Daphnia mongolica (500-600um) for 23-25 ​​days after the start of feeding, with a feeding ratio of 3:1. The effective feed density should be maintained at 300-350 insects / L. The ratio of morning feeding to afternoon feeding should be 7:3.

[0080] (10) Feed brine shrimp larvae + Daphnia mongolica in a ratio of 1:1 for 26-28 days after the start of feeding, and maintain an effective feed density of 300-350 larvae / L. The ratio of morning feeding to afternoon feeding is 7:3.

[0081] (11) Feed Mongolian naked-bellied daphnia on the first 29-30 days, and keep the effective feed density at 200-250 daphnia / L. The ratio of morning feeding to afternoon feeding is 7:3.

[0082] (12) After about 30 days, the seedlings can be pulled out of the nursery pond by netting and taken to the cement pond for seedling raising.

[0083] The water in the seedling pond should not be changed for the first 1-7 days. On the 8th day, a small amount of water can be added, with a daily water depth of 10cm. On the 11th day, the water can be changed, with a daily water depth of 15cm for both inlet and outlet to maintain stability. Clean seawater should be used for both replenishment and replacement.

[0084] 2. Seedling Management: Cultivate the seedlings in flowing water in cement ponds. Continue feeding with Mongolian naked-bellied daphnia for 30-40 days, four times a day. Close the water outlet before feeding, and reopen it after the fry's abdomens swell with the sight of them having eaten their fill. Half an hour after feeding, begin a large-scale water change, siphoning off feces and uneaten feed. After draining to a suitable water level, refill with water and add 2ml / m³ of [unspecified ingredient]. 3 The lactic acid bacteria help maintain the gut health of fish fry.

[0085] Feed the fry with adult brine shrimp (or adult artichokes) four times a day for 40-50 days. Close the water outlet before feeding, and reopen it when the fry's abdomens swell up from eating. Half an hour after the fry start feeding, perform a large water change, sucking up feces and uneaten food. After draining to a suitable water level, add water again and add 2ml / m³ of [unspecified ingredient]. 3 The lactic acid bacteria help maintain the gut health of fish fry.

[0086] For 50-60 days, use a mixture of fish paste and pelleted feed for acclimation, reducing the amount of live food. Acclimate the fry until their bellies swell, then stop feeding for half an hour. After this, perform a large water change, sucking up feces and uneaten food. Once the water level is appropriate, add water again, adding 2ml / m³ of feed twice a day, morning and evening. 3 The lactic acid bacteria help maintain the gut health of fish fry.

[0087] After 60 days, feed 2-3 times a day. Before feeding, mix probiotics and vitamins (1% of body weight) into the feed to improve the health of the fish fry.

[0088] When the fish fry reach 6-8cm in length, they can be transferred to net cages for rearing if conditions permit. It is important to prevent them from escaping to avoid genetic contamination. Using this method, 50,000 hybrid fry were obtained.

[0089] Example 4

[0090] 1.0 kg of hybrid eggs were obtained and cultured at 26 m 2 Ten seedlings, each weighing 1.0 kg, were placed in a 1.5m circular nursery pond for hatching. Clean, disinfected, and filtered seawater was added to the pond. SS rotifers were inoculated into the pond, and amino acid fertilizer (0.5ml / m²) was applied. 3) Combined with Bacillus subtilis (0.2g / m²) 3 The water should be prepared to maintain a transparency of 1-1.1m. The following are the bait options:

[0091] 1. For the first 1-3 days after feeding, maintain the density of SS rotifers (100-150um) in the indoor feed at 800-850 per L;

[0092] 2. After 4-5 days of initial feeding, introduce indoor SS rotifers + S rotifers (150-200um) at a ratio of 2:1 to ensure an effective food density of 700-750 rotifers / L;

[0093] 3. Introduce S rotifers 6-7 days after the start of feeding, with an effective feed density of 600-650 rotifers / L;

[0094] 4. After 8-9 days of opening, introduce S rotifers + L rotifers (170-300um) at a ratio of 2:1; ensure an effective density of 500-550 rotifers / L. On the 8th day, add a small amount of water, 10cm per day. On the 11th day, change the water. Maintain a stable water inflow and outflow of 15cm per day, and use clean seawater for replenishment and replacement.

[0095] 5. After 10-12 days of initial feeding, introduce indoor L-shaped rotifers at an effective feed density of 500-550 rotifers / L;

[0096] 6. After 13-15 days of opening, introduce indoor L rotifers + newly hatched Artemia (350-450um) in a ratio of 4:6 to ensure an effective density of 400-450 insects / L;

[0097] 7. After 16-17 days of opening, introduce indoor L rotifers + newly hatched Artemia at a ratio of 2:8 to ensure an effective density of 400-450 insects / L;

[0098] 8. Introduce newly hatched Artemia larvae 18-22 days after opening to ensure an effective density of 300-350 larvae / L;

[0099] 9. 23-25 ​​days after starting feeding, transfer the fry to the nursery pond. Lower the water level to 40cm, then administer a stress-reducing medication. Continue to move the fry to the nursery pond without leaving the water, and raise them in a slightly flowing water environment. Feed them brine shrimp larvae + Daphnia mongolica (500-600um) at a ratio of 3:1, four times a day. Close the water outlet before feeding, and reopen it when the fry's abdomens swell and they have eaten their fill. Half an hour after feeding, begin a large-scale water change, siphoning off feces and uneaten food. After draining to a suitable water level, refill the pond and add 5ml / m³ of water. 3 Lactic acid bacteria help maintain the gut health of fish fry;

[0100] 10. Feed brine shrimp larvae + Daphnia mongolica in a 1:1 ratio for 26-28 days after feeding. Close the water outlet before feeding 4 times a day. When the fry's abdomen swells and they are full, reopen the outlet. Half an hour after the fry start feeding, begin changing a large amount of water, sucking up feces and uneaten food, etc. After draining to a suitable water level, add water again and add 5ml / m3 of lactic acid bacteria to maintain the fry's intestines.

[0101] 11. Feed Mongolian naked-bellied daphnia on days 29-30 after feeding, maintaining an effective food density of 200-250 fish / L. Feed four times a day. Close the water outlet before feeding, and reopen it after the fry have eaten until their bellies bulge. Half an hour after the fry start feeding, perform a large water change, siphoning off feces and uneaten food. After draining to a suitable water level, add fresh water and add 5ml / m³ of supplemental fertilizer. 3 Lactic acid bacteria help maintain the gut health of fish fry;

[0102] 12. Propagate the fish in a cement pond with flowing water. Continue feeding with Mongolian naked-bellied daphnia for 30-40 days, four times a day. Close the outlet before feeding, and reopen it after the fry have eaten until their bellies bulge. Half an hour after feeding, begin a large-scale water change, siphoning off feces and uneaten food. After draining to a suitable water level, add fresh water and add 2ml / m³ of [unspecified ingredient]. 3 Lactic acid bacteria help maintain the gut health of fish fry;

[0103] 13. Feed the fry with adult brine shrimp (or adult artemia) 4 times a day for 40-50 days. Close the water outlet before feeding, and reopen it when the fry's abdomens swell up from eating. Half an hour after the fry start feeding, begin a large water change, sucking up feces and uneaten food. After draining to a suitable water level, add water again and add 2ml / m³ of [unspecified ingredient]. 3 Lactic acid bacteria help maintain the gut health of fish fry;

[0104] 14. For 50-60 days, use a mixture of fish paste and pelleted feed for acclimatization, reducing the amount of live food. Acclimatize the fry until their bellies swell, then stop feeding for half an hour. Afterward, perform a large water change, sucking up feces and uneaten food. Once the water level is appropriate, add fresh water, adding 2ml / m³ of feed twice a day, morning and evening. 3 Lactic acid bacteria help maintain the gut health of fish fry;

[0105] After 15 or 60 days, feed 2-3 times a day. Before feeding, mix probiotics and vitamins (1% of body weight) into the feed to improve the health of the fish fry.

[0106] 16. When the fish fry reach 6-8cm in length, they can be transferred to net cages for rearing if conditions permit. It is important to prevent them from escaping to avoid genetic contamination.

[0107] Example 5: Comparative culture of male brown-spotted grouper × female humpback bass hybrid and purebred brown-spotted grouper fry;

[0108] In 2025, hybridization breeding of brown-spotted grouper and humpback perch was carried out, while purebred brown-spotted grouper was bred as a control. The hybrids were 7-year-old brown-spotted grouper and 4-year-old humpback perch. Fertilized eggs were obtained using a diluent + dry insemination method. The purebred fertilized eggs were obtained using the same method and from the same batch as the hybrids, and the specific hatching operations were the same as in Example 3. 1.2 kg of hybrid fertilized eggs and 1.2 kg of purebred fertilized eggs were obtained. There was no significant difference in fertilization rate and hatching rate between the hybrids and the purebreds. The results are shown in Table 3.

[0109] Table 3 Comparison of fertilization and hatching effects between hybrid and purebred tiger stripes

[0110] .

[0111] Hatching and fry cultivation were conducted in a factory-style aquaculture facility under conditions of 28.5℃ water temperature, dissolved oxygen above 6 mg / L, pH 8.0, and salinity 32‰. After screening, hybrids and purebreds of the same size and quantity were selected for comparative rearing. At 6 months of age, the comparative rearing results showed that the average weight of the hybrids was 155.6g and the average total length was 21.0cm, while the average weight of the brown-spotted grouper was 114.8g and the average total length was 18.5cm. (See details...) Figure 2 , Figure 3 The appearance of hybrids can be seen Figure 4 The hybrid of male brown-spotted grouper and female humpback bass exhibits excellent characteristics such as rapid growth and high survival rate.

Claims

1. A method for artificial hybridization breeding of male brown-spotted grouper and female humpback bass, characterized in that, The method includes selection of brown-spotted grouper and humpback perch as broodstock, broodstock maturation cultivation, gamete collection, artificial insemination and hatching, and seedling cultivation. The parent selection criteria are as follows: select male brown-spotted grouper that are 7 years or older, weigh over 12 kg, have a normal body shape, are healthy, and have good vitality; select female humpback bass that are 4 years or older, weigh over 3 kg, are active, have a regular shape, are brightly colored, and are healthy. The aforementioned parental maturation-promoting breeding: (a) Breeding of Brown Spotted Grouper Parents: In March, the temperature is controlled at 25-26℃, and maintained until May. Then, the temperature is raised to 28-29℃ and maintained until October. After that, the temperature is slowly lowered to 24-26℃ and maintained until December. Then, the temperature is raised to 27-28℃ and maintained until March of the following year. When sperm preparation is needed, the temperature is slowly lowered to 21-23℃ over 2-3 days and maintained for 4-6 days. Then, the temperature is slowly raised to 28-29℃ over 3-5 days and maintained for 4-6 days. After that, ... The temperature is slowly lowered to 24-25℃ over 2-3 days and maintained for 5 days. Finally, it is slowly raised to 28℃ over 3-5 days and the final temperature is controlled at 28℃. The photoperiod is white:black = 16:8, the light intensity is 10000-15000 lx, and the nighttime light intensity is 200-500 lx. When semen is needed, the gonads of mature brown-spotted grouper are injected with 40 μg / kg of human chorionic gonadotropin and 30 μg / kg of releasing hormone analogue 7 days in advance, once every other day, until spawning. (b) Breeding of broodstock of humpback bass: In January, the temperature is controlled at 23-24℃, and then raised to 25-26℃ in March. After that, the temperature is slowly lowered to 22-23℃ in June, and then raised to 25-26℃ in October, and then raised to 25-26℃ in January of the following year. When oocyte production is required, the temperature is slowly lowered to 20-22℃ in 2-3 days and held for 9-11 days. Then the temperature is slowly raised to 28℃ in 5-6 days and held for 9-11 days. After that, the temperature is slowly lowered to 22-23℃ in 2-3 days and held for 10 days. Finally, the temperature is slowly raised to 26℃ in 3-5 days and the final temperature is controlled at 26℃. The photoperiod is white:black = 14:10, the light intensity is 10000-15000lx, and the nighttime light intensity is 200-500lx. The artificial insemination and hatching process is as follows: The collected semen is diluted with a sperm-enhancing diluent at a volume ratio of 1:

9. 50 ml of the sperm-enhancing diluent contains 0.4 g NaCl, 0.1 g KHCO3, 0.08 g Tris, 0.5 g glucose, and 5 ml bovine fetal serum, with the remainder being water. Insemination is performed at a ratio of original semen to egg cells = 1 ml: 1-1.2 kg. After stirring evenly, the mixture is transferred to an incubation container. The incubation water temperature is 26-28℃, the salinity is 29-32, the dissolved oxygen is ≥6 mg / L, and the pH is 8.0-8.

2. After 4 hours of incubation, oxygen is removed and the mixture is allowed to stand still. The upper layer of eggs is then removed and transferred to another incubation container. Once the fry hatch, they are transferred to a nursery pond. Seedling cultivation: SS rotifers are inoculated into the seedling cultivation pond, and amino acid fertilizer is used in combination with Bacillus subtilis for water cultivation, maintaining a transparency of 1m-1.1m; Do not change the water from day 1 to day 7. Add a small amount of water on day 8, with a daily water depth of 10cm. Change the water on day 11, with a daily water depth of 15cm for both inlet and outlet. In the breeding pond, the fish are raised in flowing water. For 30-40 days, they are fed adult Daphnia mongholica. For 40-50 days, they are fed adult Artemia spp. For 50-60 days, they are trained to eat fish paste mixed with pellet feed. After 60 days, they are fed compound pellet feed. When the fry reach 6-8cm, they are transferred to net cages for further culture. The feed series for seedling cultivation is as follows: For the first 1-3 days, maintain a density of SS rotifers at 800-850 individuals / L; for the 4th-5th days, introduce SS rotifers + S rotifers at a ratio of 2:1, ensuring an effective feed density of 700-750 individuals / L; for the 6th-7th days, introduce S rotifers, achieving an effective feed density of 600-650 individuals / L; for the 8th-9th days, introduce S rotifers + L rotifers at a ratio of 2:1, achieving an effective density of 500-550 individuals / L; for the 10th-12th days, introduce indoor L rotifers, achieving an effective feed density of 500-550 individuals / L; for the 13th-15th days, introduce indoor L rotifers + newly hatched Artemia larvae at a ratio of 4:6, ensuring an effective density of 400-450 individuals / L; for the 16th-17th days, introduce indoor... The feed consisted of rotifers and newly hatched Artemia in a ratio of 2:8, maintaining an effective density of 400-450 individuals / L. Newly hatched Artemia were introduced 18-22 days after the start of the breeding season, maintaining an effective density of 300-350 individuals / L. From 23-25 ​​days after the start of the breeding season, Artemia larvae and Daphnia mongolica were fed in a ratio of 3:1, maintaining an effective feed density of 300-350 individuals / L. From 26-28 days after the start of the breeding season, Artemia larvae and Daphnia mongolica were fed in a ratio of 1:1, maintaining an effective feed density of 300-350 individuals / L. From 29-30 days after the start of the breeding season, Daphnia mongolica were fed, maintaining an effective feed density of 200-250 individuals / L. After 30 days, the animals were transferred to a rearing pond for nurserying. The ratio of morning to afternoon feeding amounts was 7:

3.

2. The breeding method according to claim 1, characterized in that, Enhanced breeding of brown-spotted grouper and humpback perch broodstock: The broodstock were fed a diet including fresh shrimp, crab, squid, and miscellaneous fish once a day at a rate of 4% of their body weight. At the same time, fortifiers including vitamin E, vitamin V, cod liver oil, and lecithin were added to the diet at a rate of 0.5% of the fish's body weight each time.

3. The breeding method according to claim 2, characterized in that, Artificial insemination uses dry fertilization.

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