Crossbreeding method for groupers

Abstract

The invention relates to a grouper crossbreeding method. The method comprises the four steps of parent breeding, temperature-sensitive domestication, artificial spawning and insemination, incubation and fry breeding, specifically, cold water-based clear water stone spots and hot water-based brown spot stone spots are selected as parents, the clear water stone spots are used as female parents, the brown spot stone spots are used as male parents, and the middle water temperature is adapted through gradient temperature-sensitive domestication; stress-resistant feed containing hilsa herring egg source peptide and haematococcus pluvialis astaxanthin ester and a temperature-sensitive blender are matched, a reproductive care agent is supplemented to strengthen sperm and egg performance, and normative spawning induction, insemination and graded cultivation processes are adopted. According to the method, the problems of poor water temperature adaptation and low hatching rate of grouper hybrid sperms and eggs in different temperature areas are solved, the wide-temperature property of filial generations is improved, the method adapts to the water temperature of 18-28 DEG C, the hatching rate, the fry survival rate, the adult fish survival rate and the average weight of single fish are remarkably improved, and technical support is provided for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of fish hybridization breeding technology, and specifically relates to a method for hybridization breeding of grouper. Background Technology

[0002] Grouper is an important marine aquaculture fish in my country, prized for its delicious meat, rapid growth, and high market value, holding a significant position in the aquaculture industry. However, traditional grouper farming and breeding still faces numerous technical bottlenecks that need to be overcome. The core issue is the narrow temperature adaptability of single grouper species, which severely limits the expansion of farming areas: tropical and subtropical grouper species (such as brown-spotted grouper, with a natural growth temperature range of 25℃~32℃) have weak cold resistance and are prone to frostbite and death in low-temperature environments, making it impossible for them to overwinter in northern waters; cold-water grouper species (such as clear-water grouper, with a natural growth temperature range of 15℃~22℃) lack high-temperature resistance, experiencing growth stagnation and decreased disease resistance during high summer temperatures, making it difficult to extend their farming range to high-temperature areas.

[0003] To address the aforementioned issues, existing technologies have attempted to conduct hybridization research among grouper species. However, these studies primarily focus on hybridization between grouper species from the same temperature zone, failing to fully utilize the complementary advantages of temperature adaptability genes between tropical and cold-water grouper species. This results in the hybrid offspring's limited temperature adaptability and fails to fundamentally solve the problem of narrow temperature adaptability in grouper. Furthermore, when hybridizing grouper species with significant temperature differences, the mismatch in the adaptability of the maternal and paternal sperm and eggs to incubation water temperatures leads to a significant decrease in the hatching rate of the hybrid eggs, further hindering the industrial application of hybridization breeding technology for grouper from different temperature zones. Current hatching rates for hybrid grouper from the same temperature zone are typically 60%–70%, and the temperature adaptability range of the hybrid offspring only covers the overlap between the parent temperature zones, failing to achieve a wide temperature adaptability range of 18℃–28℃.

[0004] Therefore, achieving efficient hybridization of grouper from different temperature zones, improving the wide temperature range of hybrid offspring, and increasing the hatching rate of hybrid fish eggs are key technical issues that urgently need to be addressed in the current grouper aquaculture and breeding field. Summary of the Invention

[0005] This invention provides a method for hybrid breeding of grouper, which solves the related technical problems of hybridization between grouper with large temperature differences.

[0006] The technical solution adopted in this invention:

[0007] This invention provides a method for hybrid breeding of grouper, the specific steps of which are as follows:

[0008] Parent breeding: Two types of adult grouper, one cold-water and one hot-water, were selected as parents. Clear-water grouper (cold-water) and brown-spotted grouper (hot-water) were preferred. Clear-water grouper was used as the female parent and brown-spotted grouper as the male parent. Individuals with excellent growth performance, no diseases, and sexual maturity were selected. The brown-spotted grouper females weighed ≥2.5kg and the males weighed ≥3kg, while the clear-water grouper females weighed ≥2kg and the males weighed ≥2.5kg. The temporary holding density was 1-2 fish per cubic meter of water. The two types of parents were temporarily held in separate ponds. The criteria for judging sexual maturity were that the female fish had a swollen and soft abdomen and a slightly open genital pore, and that a milky white semen flowed out when the abdomen of the male fish was gently pressed.

[0009] Temperature-sensitive acclimatization: The two parent fish were subjected to gradient temperature-sensitive acclimatization. The water temperature was gradually adjusted to the midpoint of the natural growth water temperature of the two parent fish (23℃~24℃) at a rate of 0.5℃~1℃ per day, and the acclimatization period was 7~8 days. During the acclimatization period, the dissolved oxygen in the water was maintained at ≥6mg / L and the pH value was 7.8~8.5. Resistant feed was used to feed the fish 1~2 times a day, and the amount of feed was 2%~3% of the body weight of the parent fish. A temperature-sensitive conditioner was added to the culture water every day. The temperature-sensitive conditioner should be prepared and used immediately and added within 2 hours after preparation.

[0010] The stress-resistant feed comprises, by weight: 480-500 parts fish meal, 180-200 parts soybean meal, 100-120 parts wheat flour, 60-80 parts fish oil, 1.5-2.5 parts betaine, 0.8-1 parts sodium hyaluronate, 0.6-0.9 parts shad egg peptide, and 0.4-0.7 parts Haematococcus pluvialis astaxanthin ester. The fish meal is imported white fish meal with a crude protein content ≥65%; the shad egg peptide has a molecular weight ≤3000 Da and a purity ≥95%; and the Haematococcus pluvialis astaxanthin ester has a purity ≥5% and is a natural extract.

[0011] The temperature-sensitive blending agent is formulated as follows: per liter of water, add: 45-55 mg Vitamin C, 18-24 mg Betaine, 13-18 mg Glutamine, 8-11 mg Licorice Extract, and 8-12 mg Garlic Extract. The garlic extract is prepared by selecting fresh, mold-free garlic, peeling and crushing it into garlic paste, adding sterile distilled water at a solid-liquid ratio of 1:8, anaerobic fermenting at 35-40℃ for 24-36 hours, filtering through an 80-mesh filter, and freeze-drying the filtrate into powder. Before use, dilute with isothermal water at a ratio of 1:5. The licorice extract used is a product with a glycyrrhizic acid content ≥20%. All components are weighed according to the formula and fully dissolved, with a stirring rate controlled at 300 r / min and a dissolution time of 15 minutes.

[0012] Artificial ovulation and insemination: During the reproductive period care stage, add reproductive care agent to the water daily, and keep the water still for 2 hours after adding the agent. The reproductive care agent formula is: 27~32mg Astragalus polysaccharide, 23~27mg Vitamin E, 18~22mg Egg yolk phosphoprotein, 8~12mg Hyaluronic acid and 0.5~1mg Chitosan per liter of water. During spawning induction, luteinizing hormone-releasing hormone (LRH-A3) is injected into the dorsal fin base muscles of the parent fish in two separate injections, 12 hours apart. The dosage for female (clear water grouper) is 5-8 μg / kg body weight, and the dosage for male (brown spot grouper) is half that of the female. For artificial fertilization, the abdomen is gently pressed along the genital opening to expel the eggs and sperm. Dry fertilization is performed using a 5-8:1 egg-to-sperm volume ratio. After gently stirring for 1-2 minutes, the mixture is allowed to stand for 5-8 minutes. Adhesive eggs are gently dispersed with a sterile soft brush in water at a constant temperature of 23-24℃. Direct sunlight is avoided throughout the process. Astragalus polysaccharide purity is ≥70%, and egg yolk phosphoprotein is derived from egg yolk with a purity ≥90%. Insemination is performed on a sterile operating table at an ambient temperature controlled between 23-25℃ to prevent temperature fluctuations from affecting the fertilization rate.

[0013] Hatching and Seedling Cultivation: Fertilized fish eggs are placed in a temperature-controlled environment for hatching. The water temperature is maintained at a moderate level. Micro-nano aeration discs (aeration intensity 0.3~0.5 m³ / (h·m³)) are used in conjunction with a low-speed magnetic stirrer (speed 30~50 r / min) to provide intermittent oxygenation by stirring for 10 minutes and letting it stand for 20 minutes. Water quality is controlled throughout the hatching and cultivation process, with ammonia nitrogen ≤0.1 mg / L and nitrite ≤0.02 mg / L. Water is changed once a day, with 1 / 3 to 1 / 2 of the water being changed. During water changes, the aeration intensity is reduced and stirring is paused. After hatching, the fish are graded and raised as fry, juveniles, and young fish. The fry are fed rotifers and brine shrimp larvae at a water temperature of 23℃~24℃. The juveniles and young fish are fed crushed stress-resistant feed and copepods, and wide-temperature acclimatization is carried out simultaneously. In the later stage, the water temperature fluctuates to 18℃~28℃, specifically 18℃ in the early morning and 28℃ at noon. The increase and decrease of temperature are linear.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] 1. Solving the Temperature Adaptation Challenge: By combining gradient temperature-sensitive acclimatization with a temperature-sensitive modifier, and relying on the synergistic effect of the modifier and stress-resistant feed, the temperature compatibility of cold-water and hot-water grouper parent sperm and eggs is significantly improved. The functional components in the stress-resistant feed can strengthen the parent's constitution and optimize sperm and egg quality from within, while the temperature-sensitive modifier can alleviate acclimatization stress and regulate the temperature-sensitive adaptation environment of the water body from the outside. The combined effect of these two approaches breaks through the limitations of traditional same-temperature-zone hybridization, fully leveraging the complementary advantages of the temperature-adaptive genes of the two parents, enabling the hybrid offspring to maintain a stable wide-temperature range of 18℃~28℃, significantly expanding the aquaculture area.

[0016] 2. Improved Breeding Efficiency: The stress-resistant feed contains shad egg-derived peptides and Haematococcus pluvialis astaxanthin esters, which work synergistically. It also forms a highly efficient synergy with a temperature-sensitive regulator, and together with reproductive care agents, constructs an "internal supplementation + external regulation" breeding support system. The stress-resistant feed thickens the surface of the eggs and sperm at a nutritional level, improving their elasticity and resilience. The temperature-sensitive regulator, through water environment regulation, promotes the absorption and conversion of nutrients from the feed by the parent fish, reducing stress damage during egg and sperm development. Together, they reduce the egg wall breakage rate during hatching. Combined with standardized spawning and fertilization processes, this significantly improves the hatching rate of fish eggs and the survival rate of fry, completely solving the core bottleneck of low hatching rates in hybrid grouper from different temperature zones. Experimental verification shows that the hatching rate of fish eggs using this invention can reach over 93%, and the survival rate of fry exceeds 92%, an improvement of over 30% compared to existing technologies.

[0017] 3. Enhance Seedling Stress Resistance: A combined approach of "oral administration of stress-resistant feed + external application of temperature-sensitive modifier + graded acclimatization" is employed. The synergistic effect of the stress-resistant feed and the temperature-sensitive modifier provides crucial support for enhancing seedling stress resistance. The stress-resistant feed provides the seedlings with the antioxidant and antibacterial functional nutrients needed for growth, while the temperature-sensitive modifier optimizes the microenvironment of the aquaculture water and alleviates temperature-sensitive stress in the seedlings. Together, they enhance the seedlings' tolerance to temperature fluctuations and disease resistance, significantly reducing the incidence of diseases during the aquaculture process. Simultaneously, they ensure stable growth of seedlings at different temperatures, greatly improving the economic benefits of aquaculture.

[0018] 4. Coordination of Fish Egg Adhesion and Sperm / Egg Surface Structure: Utilizing the synergistic effect of shad egg-derived peptides and sodium hyaluronate in the stress-resistant feed, and phospholipids and hyaluronic acid in the reproductive care agent, significantly increases the viscosity of fish eggs, leading to increased viscous secretions on the egg surface. This allows fertilization to occur without de-adhesion, facilitating sperm attachment to the egg and greatly improving the fertilization success rate. Since increased viscosity of fertilized eggs can easily lead to clumping and oxygen deficiency, this problem is addressed during the incubation stage using aeration and agitation for oxygen supply. Simultaneously, the aforementioned components synergistically strengthen the cell membrane integrity and toughness of the egg and sperm epithelium, giving them high resistance to mechanical impact. This effectively resists mechanical damage during aeration and agitation, achieving a process adaptation of "viscosity regulation - uniform oxygen supply - damage resistance," further improving fertilization and hatching rates and overcoming the technical contradictions between viscosity, oxygen supply, and damage resistance in traditional hybridization incubation. Detailed Implementation

[0019] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0020] Example 1

[0021] (a) Parental selection and breeding

[0022] Adult cold-water clear-water grouper and warm-water brown-spotted grouper were selected as broodstock, with the clear-water grouper as the female and the brown-spotted grouper as the male. Individuals with excellent growth performance, no disease, and reaching sexual maturity were selected. The criteria for sexual maturity were: the female's abdomen was swollen and soft, and the genital pore was slightly open; in the male, milky white semen flowed out when the abdomen was gently pressed. The standard weight for the broodstock was: female brown-spotted grouper ≥ 2.5 kg and male ≥ 3 kg; female clear-water grouper ≥ 2 kg and male ≥ 2.5 kg. The female-to-male ratio was 1:1, and the temporary holding density was 1 fish per cubic meter of water. The two types of broodstock were temporarily held in separate ponds.

[0023] (ii) Temperature-sensitive domestication

[0024] The two parent plants were subjected to gradient temperature-sensitive acclimatization, with the water temperature gradually adjusted to 23℃, the midpoint of the natural growth water temperature of the two parent plants, at a rate of 0.5℃ per day, for a acclimatization period of 7 days.

[0025] During the acclimatization period, the dissolved oxygen in the water was maintained at 6 mg / L and the pH value at 7.8. A stress-resistant feed was used, fed once daily at 3% of the broodstock's body weight. The stress-resistant feed consisted of the following components by weight: 480 parts fishmeal, 180 parts soybean meal, 100 parts wheat flour, 60 parts fish oil, 1.5 parts betaine, 0.8 parts sodium hyaluronate, 0.6 parts shad egg peptide, and 0.4 parts Haematococcus pluvialis astaxanthin ester.

[0026] Add a temperature-sensitive conditioner to the aquaculture water daily. The conditioner should be prepared and used immediately, and added within 2 hours after preparation. The formula is as follows: per liter of water, add: 45mg vitamin C, 18mg betaine, 13mg glutamine, 8mg licorice extract and 8mg garlic extract.

[0027] The preparation process of garlic extract is as follows: Select fresh garlic that is free from mold and rot, peel it and crush it into garlic paste, add sterile distilled water at a solid-liquid ratio of 1:8, and place it in a constant temperature environment of 35℃ for anaerobic fermentation for 24 hours. After fermentation, filter it through an 80-mesh filter to remove residue, take the filtrate and freeze dry it under vacuum to make powder, and dilute it with isothermal water at a ratio of 1:5 before use.

[0028] The advantages of this temperature-sensitive conditioning agent and its preparation process are that the requirement of preparing and using it immediately and adding it within 2 hours can avoid the degradation of active ingredients. The combination of vitamin C, betaine, and glutamine can specifically alleviate osmotic stress during temperature-sensitive acclimatization. Combined with the natural antibacterial and anti-inflammatory effects of licorice extract and fermented garlic extract, it not only strengthens the temperature-sensitive adaptability of the parent feed but also reduces the risk of disease during acclimatization. The vacuum freeze-drying process preserves the active ingredients of garlic extract. Compared with the component failure caused by traditional high-temperature drying, it further improves the compatibility between the conditioning agent and stress-resistant feed, overcoming the technical shortcomings of existing conditioning agents that only focus on temperature-sensitive regulation and neglect immune protection.

[0029] (III) Artificial induction of labor and insemination

[0030] During the breeding season care phase, a reproductive care agent is added to the water daily. After addition, the water is left to stand for 2 hours to ensure the active ingredients fully penetrate the surface of the sperm and eggs. The reproductive care agent formula is as follows: per liter of water: 27 mg Astragalus polysaccharide, 23 mg Vitamin E, 18 mg Egg vitellin, 8 mg Hyaluronic acid, and 0.5 mg Chitosan. Breeding season care continues for 3 days, with the reproductive care agent added daily at 9:00 AM. After addition, the circulation system is shut off, and normal operation resumes after 2 hours. The advantages of this breeding season care process are that the 3-day continuous care and 2-hour resting operation allow the reproductive care agent to fully penetrate into the parent reproductive organs. Astragalus polysaccharides and vitamin E work synergistically for antioxidant effects, while egg yolk phosphoprotein and hyaluronic acid specifically optimize the surface structure of sperm and eggs. Combined with the antibacterial and preservative effects of chitosan, this process solves the problem of traditional hybridization focusing only on inducing spawning and lacking pretreatment before sperm and egg development. Together with the stress-resistant feed and temperature-sensitive modifier mentioned above, it forms a closed loop of "nutritional fortification - temperature-sensitive adaptation - reproductive optimization", which significantly improves the quality of sperm and eggs and fertilization activity, ensuring a high hatching rate.

[0031] Artificial spawning was induced using luteinizing hormone-releasing hormone (LRH-A3). The injection was administered intramuscularly at the base of the dorsal fin of the parent fish, in two separate injections 12 hours apart. The dosage was 5 μg / kg body weight for females and half that of males (2.5 μg / kg body weight). During spawning, the parent fish was slowly moved along the path towards the genital opening while pressure was applied to the abdomen to expel the sperm and eggs.

[0032] The dry insemination method was used to complete the fertilization of sperm and eggs. The eggs and sperm were mixed at a volume ratio of 5:1, gently stirred for 1 minute and then left to stand for 5 minutes. If the eggs were stuck together after insemination, they were gently dispersed in 23°C water with a sterile soft brush to avoid the formation of an ovum and oxygen deficiency. Direct sunlight was avoided throughout the insemination and dispersion process.

[0033] (iv) Hatching and Seedling Cultivation

[0034] After fertilization, the fish eggs are placed in a temperature-controlled environment for incubation. The incubation water temperature is controlled at 22℃. Aeration is carried out using micro-nano aeration discs (aeration intensity 0.3m³ / (h·m³)) and a low-speed magnetic stirrer (speed 30r / min). Oxygen is supplied in an intermittent mode of "stirring for 10 minutes and letting stand for 20 minutes".

[0035] Water quality indicators during the entire incubation and seedling cultivation process are controlled as follows: ammonia nitrogen content ≤0.1mg / L, nitrite content ≤0.02mg / L; water is changed once a day, with the water volume being 1 / 3 of the water body. During water change, the aeration intensity is reduced to 0.1m³ / (h·m³) and stirring is suspended.

[0036] After hatching, the fish are raised in three stages: larvae, juveniles, and young fish. During the larval stage, the water temperature is kept stable at 23℃, and the fish are fed rotifers and brine shrimp larvae. During the juvenile and young fish stages, the fish are fed crushed stress-resistant feed with an appropriate amount of copepods. At the same time, the fish are subjected to intensive acclimatization training. In the later stage of acclimatization, the water temperature fluctuation range is gradually expanded to 18℃~28℃. Specifically, the water temperature is lowered to 18℃ at midnight and raised to 28℃ at noon. The increase and decrease of temperature are carried out linearly.

[0037] The advantages of this graded breeding and wide-temperature acclimatization process are that the phased feeding is adapted to the feeding and nutritional needs of the fry at different developmental stages, and the crushed stress-resistant feed continues the nutritional system of the parent fish in the early stage, ensuring the continuity of hybrid traits; the linear and gradual wide-temperature acclimatization mode starts from the juvenile stage and controls the daily temperature difference, allowing the fry to gradually adapt to a wide temperature range of 18℃~28℃, breaking through the limitations of traditional hybrid fry that are only acclimatized in the adult stage and have poor temperature adaptability.

[0038] Example 2

[0039] (a) Parental selection and breeding

[0040] Adult cold-water clear-water grouper and hot-water brown-spotted grouper were selected as broodstock. Individuals with excellent growth performance, no disease, and reaching sexual maturity were selected. The criteria for sexual maturity were: females had a swollen and soft abdomen with a slightly open genital pore; and males released milky white semen when the abdomen was gently pressed. The broodstock weight standards were: female brown-spotted grouper ≥ 2.5 kg, males ≥ 3 kg; female clear-water grouper ≥ 2 kg, males ≥ 2.5 kg; the female-to-male ratio was 2:1, and the temporary holding density was 2 fish per cubic meter of water. The two types of broodstock were temporarily held in separate ponds.

[0041] (ii) Temperature-sensitive domestication

[0042] The two parent plants were subjected to gradient temperature-sensitive acclimatization, with the water temperature gradually adjusted to 24℃, the midpoint of the natural growth water temperature for both parent plants, at a rate of 1℃ per day, for a period of 8 days.

[0043] During the acclimatization period, the dissolved oxygen in the water was maintained at 8 mg / L and the pH value at 8.5. A stress-resistant feed was used, fed twice daily at 2% of the broodstock's body weight. The stress-resistant feed consisted of the following components by weight: 500 parts fishmeal, 200 parts soybean meal, 120 parts wheat flour, 80 parts fish oil, 2.5 parts betaine, 1 part sodium hyaluronate, 0.9 parts shad egg peptide, and 0.7 parts Haematococcus pluvialis astaxanthin ester.

[0044] Add a temperature-sensitive conditioner to the aquaculture water daily. The conditioner should be prepared and used immediately, and added within 2 hours after preparation. The formula is as follows: per liter of water, add: 55mg vitamin C, 24mg betaine, 18mg glutamine, 11mg licorice extract and 12mg garlic extract.

[0045] The preparation process of garlic extract is as follows: Select fresh garlic that is free from mold and rot, peel it and crush it into garlic paste, add sterile distilled water at a solid-liquid ratio of 1:10, and place it in a constant temperature environment of 40℃ for anaerobic fermentation for 36 hours. After fermentation, filter it through an 80-mesh filter to remove residue, take the filtrate and freeze dry it under vacuum to make powder, and dilute it with isothermal water at a ratio of 1:5 before use.

[0046] (III) Artificial induction of labor and insemination

[0047] During the breeding season care phase, add a reproductive care agent to the water daily. After adding the agent, let the water stand for 2 hours to ensure that the active ingredients fully act on the surface of the sperm and eggs. The reproductive care agent formula is as follows: per liter of water, add: 32mg Astragalus polysaccharide, 27mg Vitamin E, 22mg Vitellin, 12mg Hyaluronic Acid, and 1mg Chitosan.

[0048] Artificial spawning was induced using luteinizing hormone-releasing hormone (LRH-A3). The injection was administered intramuscularly at the base of the dorsal fin of the parent fish, in two doses 12 hours apart. The dosage was 8 μg / kg body weight for females and half that of males (4 μg / kg body weight). During spawning, the parent fish was slowly moved along the genital opening while pressure was applied to the abdomen to expel the sperm and eggs.

[0049] The dry insemination method was used to complete the fertilization of sperm and eggs. The eggs and sperm were mixed at a volume ratio of 8:1, gently stirred for 2 minutes, and then left to stand for 8 minutes. If the eggs were stuck together after insemination, they were gently dispersed in 24°C water with a sterile soft brush to avoid the formation of an ovum and oxygen deficiency. Direct sunlight was avoided throughout the insemination and dispersion process.

[0050] (iv) Hatching and Seedling Cultivation

[0051] After fertilization, the fish eggs are placed in a temperature-controlled environment for incubation. The incubation water temperature is controlled at 24℃. Aeration is carried out using micro-nano aeration discs (aeration intensity 0.5m³ / (h·m³)) and a low-speed magnetic stirrer (speed 50r / min). Oxygen is supplied in an intermittent mode of "stirring for 10 minutes and letting stand for 20 minutes".

[0052] Water quality indicators during the entire incubation and seedling cultivation process are controlled as follows: ammonia nitrogen content ≤0.1mg / L, nitrite content ≤0.02mg / L; water is changed once a day, with the water volume being 1 / 2 of the water body. During water change, the aeration intensity is reduced to 0.1m³ / (h·m³) and stirring is suspended.

[0053] After hatching, the fish are raised in three stages: larvae, juveniles, and young fish. During the larval stage, the water temperature is kept stable at 24℃, and the fish are fed rotifers and brine shrimp larvae. During the juvenile and young fish stages, the fish are fed crushed stress-resistant feed with an appropriate amount of copepods. At the same time, the fish are subjected to intensive acclimatization training. In the later stage of acclimatization, the water temperature fluctuation range is gradually expanded to 18℃~28℃. Specifically, the water temperature is lowered to 18℃ at midnight and raised to 28℃ at noon. The increase and decrease of temperature are carried out linearly.

[0054] Example 3

[0055] (a) Parental selection and breeding

[0056] Adult cold-water clear-water grouper and hot-water brown-spotted grouper were selected as broodstock. Individuals with excellent growth performance, no disease, and reaching sexual maturity were selected. The criteria for sexual maturity were: female fish had a swollen and soft abdomen with a slightly open genital pore; and male fish showed milky white semen discharge when the abdomen was gently pressed. The standard weight for broodstock was: female brown-spotted grouper ≥ 2.5 kg and male ≥ 3 kg; female clear-water grouper ≥ 2 kg and male ≥ 2.5 kg. The female-to-male ratio was 1.5:1, and the temporary holding density was 2 fish per cubic meter of water. The two types of broodstock were temporarily held in separate ponds.

[0057] (ii) Temperature-sensitive domestication

[0058] The two parent plants were subjected to gradient temperature-sensitive acclimatization. The water temperature was gradually adjusted to 23.5℃, the midpoint of the natural growth water temperature of the two parent plants, at a rate of 0.5℃ per day, for a acclimatization period of 7 days.

[0059] During the acclimatization period, the dissolved oxygen in the water was maintained at 8 mg / L and the pH value at 8. A stress-resistant feed was used, fed twice daily at 2% of the broodstock's body weight. The stress-resistant feed consisted of the following components by weight: 490 parts fishmeal, 190 parts soybean meal, 110 parts wheat flour, 70 parts fish oil, 2 parts betaine, 0.9 parts sodium hyaluronate, 0.75 parts shad egg peptide, and 0.55 parts Haematococcus pluvialis astaxanthin ester.

[0060] Add a temperature-sensitive conditioner to the aquaculture water daily. The conditioner must be prepared and used immediately. Add it to the aquaculture water within 2 hours after preparation. The formula is as follows: 50mg Vitamin C, 21mg Betaine, 16mg Glutamine, 10mg Licorice Extract and 10mg Garlic Extract per liter of water.

[0061] The preparation process of garlic extract is as follows: Select fresh garlic that is free from mold and rot, peel it and crush it into garlic paste, add sterile distilled water at a solid-liquid ratio of 1:9, and place it in a constant temperature environment of 37℃ for anaerobic fermentation for 30 hours. After fermentation, filter it through an 80-mesh filter to remove residue, take the filtrate and freeze dry it under vacuum to make powder, and dilute it with isothermal water at a ratio of 1:5 before use.

[0062] (III) Artificial induction of labor and insemination

[0063] During the breeding season care phase, add a reproductive care agent to the water daily. After adding the agent, let the water stand for 2 hours to ensure that the active ingredients fully act on the surface of the sperm and eggs. The reproductive care agent formula is as follows: per liter of water, add: 29mg Astragalus polysaccharide, 25mg Vitamin E, 20mg Vitellin, 10mg Hyaluronic Acid, and 0.75mg Chitosan.

[0064] Artificial spawning was induced using luteinizing hormone-releasing hormone (LRH-A3). The injection was administered intramuscularly at the base of the dorsal fin of the parent fish, in two separate injections 12 hours apart. The dosage was 7 μg / kg body weight for females and half that of males (3.5 μg / kg body weight). During spawning, the parent fish was slowly moved along the path towards the genital opening while pressure was applied to the abdomen to expel the sperm and eggs.

[0065] The dry insemination method was used to complete the fertilization of sperm and eggs. The sperm and eggs were mixed at a volume ratio of 7:1, gently stirred for 1.5 minutes, and then left to stand for 6.5 minutes. If the eggs were stuck together after insemination, they were gently dispersed in 23°C water with a sterile soft brush to avoid the formation of an anaerobic egg mass. Direct sunlight was avoided throughout the insemination and dispersion process.

[0066] (iv) Hatching and Seedling Cultivation

[0067] After fertilization, the fish eggs are placed in a temperature-controlled environment for incubation. The incubation water temperature is controlled at 23℃. Aeration is carried out using micro-nano aeration discs (aeration intensity 0.4m³ / (h·m³)) and a low-speed magnetic stirrer (speed 40r / min). Oxygen is supplied in an intermittent mode of "stirring for 10 minutes and letting stand for 20 minutes".

[0068] Water quality indicators during the entire incubation and seedling cultivation process are controlled as follows: ammonia nitrogen content ≤0.1mg / L, nitrite content ≤0.02mg / L; water is changed once a day, with the water volume being 1 / 2 of the water body. During water change, the aeration intensity is reduced to 0.1m³ / (h·m³) and stirring is suspended.

[0069] After hatching, the fish are raised in three stages: larvae, juveniles, and young fish. During the larval stage, the water temperature is kept stable at 23℃, and the fish are fed rotifers and brine shrimp larvae. During the juvenile and young fish stages, the fish are fed crushed stress-resistant feed with an appropriate amount of copepods. At the same time, the fish are subjected to intensive acclimatization training. In the later stage of acclimatization, the water temperature fluctuation range is gradually expanded to 18℃~28℃. Specifically, the water temperature is lowered to 18℃ at midnight and raised to 28℃ at noon. The increase and decrease of temperature are carried out linearly.

[0070] Comparative Example 1

[0071] This comparative example is based on Example 3, except that the stress-resistant feed is removed and replaced with a conventional marine fish basic feed. All other steps and parameters are completely identical to Example 3. The conventional marine fish basic feed formula, by weight, is: 490 parts fishmeal, 190 parts soybean meal, 110 parts wheat flour, 70 parts fish oil, and 2 parts betaine. It contains no functional additives, and the feeding method and amount are the same as in Example 3.

[0072] Comparative Example 2

[0073] This comparative example is based on Example 3. The shad egg peptide and Haematococcus pluvialis astaxanthin ester were removed from the stress-resistant feed. The remaining feed components and ratios, feeding methods, and feeding amounts were the same as in Example 3. The remaining steps (temperature-sensitive acclimatization, induced spawning and insemination, hatching and cultivation, etc.) and parameters were exactly the same as in Example 3.

[0074] Comparative Example 3

[0075] This comparative example is based on Example 3. The astaxanthin ester of Haematococcus pluvialis (0.55 parts) was removed from the stress-resistant feed. The other feed components and ratios, feeding methods and feeding amounts are the same as in Example 3. The remaining steps (temperature-sensitive acclimatization, induced fertilization, incubation and cultivation, etc.) and parameters are exactly the same as in Example 3.

[0076] Comparative Example 4

[0077] This comparative example is based on Example 3. No temperature-sensitive conditioning agent is added during the temperature-sensitive acclimatization period. Only the basic water quality indicators are maintained. The remaining steps (parent selection, feed feeding, induced spawning and fertilization, hatching and cultivation, etc.) and parameters are completely consistent with Example 3.

[0078] Test results

[0079] I. Elasticity and pressure test of fish eggs

[0080] (I) Test Methods

[0081] 1. Sample pretreatment: Fresh fish eggs from each group of parent fish within 1 hour of induced spawning in Examples 1 to 3 and Comparative Examples 1 to 4 were selected. Deformed, damaged and adhered individuals were removed, and 20 samples with complete shape and uniform size were retained from each group.

[0082] 2. Instruments and parameters: A high-precision micro-force tester (range 0~100kPa, accuracy 0.1kPa) is used, equipped with a planar probe larger than 5mm, and the detection rate is set to 0.05mm / s; the elasticity test compression is 20% of the initial diameter of the fish egg, and the crack resistance test is performed by continuously applying pressure until the egg wall ruptures.

[0083] 3. Index detection and data processing: Elastic recovery rate = (recovered diameter after compression / initial diameter before compression) × 100%; record the corresponding compression amount when the egg wall ruptures, and calculate the proportion of fracturing compression amount = (fracturing compression amount / initial diameter) × 100%. The average value of the test results within the same group is taken.

[0084] (II) Test Results

[0085]

[0086] The experimental results showed a significant stratification in the performance of fish eggs between the example group and the comparative group. The fish eggs from Examples 1 to 3 all exhibited excellent performance, with elastic recovery rates exceeding 96% and compression ratios ranging from 44.8% to 48.2%, showing minimal differences between groups and demonstrating the stability of the technical solution. Example 3 performed best, with an elastic recovery rate of 97.5% and a compression ratio of 48.2%, indicating extremely high egg toughness. These eggs not only required compression deformation of nearly half their initial diameter to rupture but were also less susceptible to damage during artificial egg release, maximizing the preservation of intact eggs for fertilization. Furthermore, during incubation, they effectively resisted mechanical damage from aeration and agitation, providing double protection for a high hatching rate. Examples 2 and 1 had parameters within their upper and lower limits, with slightly inferior performance compared to Example 3, but both indicators still significantly exceeded those of the comparative group. This demonstrates that within the set range of process parameters and component dosages, fish eggs with excellent toughness can be cultivated, suitable for the requirements of artificial egg release operations.

[0087] This advantage stems from the synergistic effect of stress-resistant feed and reproductive care agents: the precise ratio of 0.75 parts of shad egg-derived peptide and 0.55 parts of Haematococcus pluvialis astaxanthin ester in the stress-resistant feed can work synergistically with the phosphoprotein and hyaluronic acid in the reproductive care agents to thicken the surface of the fish eggs and improve the integrity and toughness of the cell membranes; combined with the synergistic effect of gradient temperature-sensitive acclimatization and temperature-sensitive modifiers, it can reduce the stress response of the parent fish and provide a stable physiological basis for the development of fish eggs.

[0088] The fish eggs in the comparative groups generally exhibited poor performance, with an elastic recovery rate ≤65.4% and a fracturing compression ratio ≤27.3%. The performance was ranked from best to worst as follows: Comparative Example 3, Comparative Example 1, Comparative Example 4, and Comparative Example 2, reflecting the irreplaceable nature of each key component and process. Comparative Example 3 (lacking only Haematococcus pluvialis astaxanthin ester) performed poorly, indicating that the absence of a single active component affects the toughness of the fish eggs. The absence of this component leads to a decrease in the antioxidant capacity of the fish eggs (its main function is to assist in enhancing the antioxidant capacity of the fish eggs), weakening their resistance to compression. Comparative Example 1 (replacing conventional feed) showed a decline in performance, confirming the core supporting role of functional components in stress-resistant feed for the toughness of fish eggs. Conventional feed could not provide sufficient nutrition to strengthen the structure of the fish eggs, resulting in a significant decrease in the fracturing compression ratio. Comparative Example 4 (without temperature-sensitive modifier) ​​performed worse than Comparative Example 1. This study highlights the key value of the modifier in alleviating acclimatization stress and promoting temperature-sensitive adaptation. Stress response can affect the development quality of fish eggs and weaken their resilience. Comparative Example 2 (lacking both active components) performed the worst, with an elasticity recovery rate of only 51.3% and a compression ratio of only 16.5%, which is significantly lower than Example 3. This demonstrates that the synergistic effect of the two active components is the core of optimizing fish egg resilience. The double deficiency leads to a fragile surface structure of fish eggs, which is easily damaged during artificial squeezing and release, resulting in a large loss before fertilization. At the same time, the eggs are also susceptible to mechanical damage during the incubation period, ultimately significantly reducing the hatching success rate.

[0089] II. Fish fry rearing and adult fish farming experiment

[0090] (I) Test Methods

[0091] 1. Randomly select fish eggs (all products of the combination of maternal freshwater grouper eggs and paternal brown-spotted grouper sperm) after fertilization in Examples 1 to 3 and Comparative Examples 1 to 4, with 1000 eggs per group. Complete hatching and fry rearing according to the corresponding group's process parameters. Calculate the hatching rate (hatching rate = number of successfully hatched fry / total number of fertilized fish eggs per group × 100%) and fry survival rate (fry survival rate = number of hatched fry / total number of fish eggs per group × 100%). Set up 3 parallel experiments for each group and take the mean to eliminate random errors.

[0092] 2. Select fry from each group cultivated in Examples 1 to 3 and Comparative Examples 1 to 4, and place them in three water temperature environments of 18℃, 23℃, and 28℃ at a rate of 500 fry / group. Maintain uniform breeding and management conditions (feeding amount, water change frequency, water quality indicators, etc.). Raise them to adulthood, and count the survival rate of adult fish (survival rate of adult fish = number of adult fish / number of fry × 100%) and the average weight of a single fish (average weight of a single fish = total weight of a single group of fish / number of adult fish). Also, evaluate whether the average weight of a single fish is greater than the weight of the parent fish.

[0093] (II) Test Results

[0094]

[0095] The following analysis can be drawn from the above experimental results.

[0096] 1. Hatching and fry rearing stage (no temperature zone variable)

[0097] The core assessment at this stage is the hatching efficiency of hybrid sperm and eggs and the initial vitality of the seedlings, which directly determines the basic quality of subsequent adult fish farming.

[0098] (1) Example group: The overall performance was excellent and extremely stable. The hatching rate of fish eggs remained stable at 94.5%~96.5%, and the survival rate of fry remained at 93.9%~95.3%. The fluctuation of indicators between groups was less than 2%, which reflects the reliability of the technical solution of this invention. This advantage comes from the synergistic effect of shad egg peptides and Haematococcus pluvialis astaxanthin ester in the stress-resistant feed, together with temperature-sensitive modifiers and reproductive care agents. It not only optimized the quality of parent sperm and eggs and reduced egg wall damage during hatching, but also alleviated temperature-sensitive acclimatization stress and improved the stress resistance of fry after hatching, laying a high-quality fry foundation for subsequent cross-temperature zone adult fish farming.

[0099] (2) Comparative example group: All indicators were significantly lower than those of the example group, and showed obvious shortcomings, which confirmed the irreplaceability of key components and processes. Comparative Example 2 (lacking shad egg-derived peptides and Haematococcus pluvialis astaxanthin esters) performed the worst, with an egg hatching rate of only 50.6%~55.1% and a fry survival rate of 49.3%~53.8%, a difference of more than 40 percentage points compared to Example 3. This indicates that the dual active components are the core for strengthening the structure of sperm and eggs and improving hatching and fry rearing efficiency. Comparative Example 3 (lacking only Haematococcus pluvialis astaxanthin esters) had better indicators than Comparative Example 2, but its hatching rate (68.2%~71.5%) and fry survival rate (66.9%~70.1%) were still much lower than the Example 3 group, reflecting that the lack of a single active component weakens the antioxidant and structural strengthening effects, indirectly reducing fry vitality. Comparative Example 1 (conventional feed replacing stress-resistant feed) and Comparative Example 4 (without temperature-sensitive modifiers) had similar and low indicators, further proving that the functional nutrition of stress-resistant feed and the environmental adaptation of temperature-sensitive modifiers are key to overcoming the bottleneck of hybrid grouper hatching in different temperature zones.

[0100] 2. Adult fish farming stage (three temperature zone variables)

[0101] This stage focuses on evaluating the wide-temperature adaptability of hybrid seedlings. The core objective is to verify the actual effectiveness of the wide-temperature domestication process of this invention by measuring the survival rate and growth performance of adult fish in different temperature zones.

[0102] (1) Example group: It exhibited excellent wide temperature adaptability, and all performance remained stable in the three temperature zones. The survival rate of adult fish was maintained above 92.9%, of which 93.5%~94.5% was achieved at a low temperature of 18℃ and 92.9%~94.1% was achieved at a high temperature of 28℃. The temperature range fluctuated little, and there was no obvious shortcoming due to high and low temperature stress. The average weight of the single fish exceeded 3000g, far exceeding the parental specifications of the maternal clear water grouper (≥2.5kg) and the paternal brown spot grouper (≥3kg). The highest weight reached 3205g at the suitable temperature of 23℃. The weight fluctuation under high and low temperature environments was less than 6%, indicating that the fish fry cultivated in the example had a wide suitable temperature range and the temperature change had little impact on their growth rate.

[0103] (2) Comparative group: Poor wide-temperature adaptability, performance showed a sharp decline with temperature fluctuations, and could not meet the needs of cross-regional aquaculture. The survival rate of adult fish was only maintained at a relatively high level under the suitable temperature of 23℃ (67.8% for Comparative Example 3 and 60.1% for Comparative Example 1), and deteriorated significantly under the low temperature of 18℃ and the high temperature of 28℃. Among them, the survival rate of Comparative Example 2 was only 45.7% at the high temperature of 28℃, which was 47.2 percentage points lower than that of Example 3. The highest average weight of a single fish was 2280g at 23℃ for Comparative Example 3 and the lowest was 1590g at 28℃ for Comparative Example 2. Both of them did not reach or barely approached the parent size, and the temperature fluctuation exceeded 30%. The combined effect of temperature stress and insufficient initial vitality of seedlings led to stagnation of adult fish growth, and could not meet the yield and quality requirements of large-scale aquaculture.

[0104] 3. Conclusion

[0105] The example group, through a synergistic design of "stress-resistant feed + temperature-sensitive modifier + standardized process + wide-temperature acclimatization," not only achieved a high egg hatching rate of over 94.5% and a fry survival rate of over 93.9%, but also endowed the hybrid fry with a wide temperature range adaptability of 18℃~28℃, ensuring stable survival rate and growth performance of adult fish. The comparative group, due to the lack of key components or processes, could not overcome the hatching bottleneck of grouper hybridization in different temperature zones and lacked the wide temperature adaptability of fry, resulting in poor overall performance. This technical solution effectively solves the core problems of low efficiency and narrow temperature range adaptability in traditional grouper hybridization breeding across different temperature zones, providing reliable technical support for the cross-regional industrialization of grouper farming.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for hybrid breeding of grouper, characterized in that, Includes the following steps: Parent breeding: Two types of adult grouper that grow in cold water and hot water were selected as parents, with clear water grouper as the female parent and brown spot grouper as the male parent. The parents had excellent growth performance, were free from diseases, and met the sexual maturity standard. The two types of parents were temporarily raised in separate ponds. Temperature-sensitive acclimatization: The two parent fish were subjected to gradient temperature-sensitive acclimatization, with the water temperature gradually adjusted to the midpoint of the natural growth water temperature of the two parent fish at a rate of 0.5℃~1℃ per day, for a acclimatization period of 7~8 days. During the acclimatization process, stress-resistant feed was used, which, by weight, included: 480~500 parts fish meal, 180~200 parts soybean meal, 100~120 parts wheat flour, 60~80 parts fish oil, 1.5~2.5 parts betaine, 0.8~1 parts sodium hyaluronate, 0.6~0.9 parts shad egg peptide, and 0.4~0.7 parts Haematococcus pluvialis astaxanthin ester. A temperature-sensitive conditioner was added to the aquaculture water daily, with the following formula per liter of water: 45~55 mg vitamin C, 18~24 mg betaine, 13~18 mg glutamine, 8~11 mg licorice extract, and 8~12 mg garlic extract. Artificial spawning and insemination: During the reproductive period care phase, a reproductive care agent is added to the water daily. The formula for the reproductive care agent is: 27-32 mg Astragalus polysaccharide, 23-27 mg Vitamin E, 18-22 mg Vitellin, 8-12 mg Hyaluronic acid, and 0.5-1 mg Chitosan per liter of water. Artificial spawning is induced using luteinizing hormone-releasing hormone, and fertilization is achieved using a dry insemination method. Hatching and seedling cultivation: After fertilization, the fish eggs are placed in a temperature-controlled environment for incubation. During the incubation process, oxygen is supplied by stirring and aeration, and the incubation water temperature is controlled at a moderate temperature. After hatching, seedling cultivation is carried out, and water quality is controlled and water is changed in a standardized manner throughout the process.

2. The method for hybrid breeding of grouper according to claim 1, characterized in that, The parent fish were brown-spotted grouper and clear-water grouper. The brown-spotted grouper females weighed ≥2.5kg and the males weighed ≥3kg. The clear-water grouper females weighed ≥2kg and the males weighed ≥2.5kg. The temporary holding density was 1-2 fish per cubic meter of water. The criteria for judging sexual maturity were: the female fish had a swollen and soft abdomen and a slightly open genital pore, and the male fish had milky white semen flowing out when the abdomen was gently pressed.

3. The method for hybrid breeding of grouper according to claim 1, characterized in that, During the temperature-sensitive acclimatization period, the dissolved oxygen in the water should be maintained at ≥6mg / L and the pH value at 7.8~8.5; the stress-resistant feed should be given 1~2 times a day, and the amount of feed should be 2%~3% of the body weight of the parent fish.

4. The method for hybrid breeding of grouper according to claim 1, characterized in that, In the artificial labor induction process, a person slowly moves along the direction close to the genital opening and presses on the parent's abdomen to expel the sperm and egg from the genital opening.

5. The method for hybrid breeding of grouper according to claim 1, characterized in that, During artificial spawning, luteinizing hormone-releasing hormone is injected into the dorsal fin base muscle of the parent fish in two separate injections, 12 hours apart. The injection dosage is 5-8 μg / kg body weight for female fish and half the dosage for male fish.

6. The method for hybrid breeding of grouper according to claim 1, characterized in that, During the dry insemination method, fish eggs and sperm are mixed in a volume ratio of 5:1 to 8:

1. After gently stirring for 1 to 2 minutes, the mixture is left to stand for 5 to 8 minutes to complete the fertilization process. If the fish eggs stick together after insemination, they are gently dispersed in clean water at a constant temperature of 23°C to 24°C with a sterile soft brush to avoid the formation of an egg mass that would lead to oxygen deficiency. Direct sunlight should be avoided during the insemination and dispersion process.

7. The method for hybrid breeding of grouper according to claim 1, characterized in that, During the incubation and seedling cultivation process, the water quality indicators are controlled as follows: ammonia nitrogen content ≤0.1mg / L, nitrite content ≤0.02mg / L; the water is changed once a day, and the water volume is 1 / 3 to 1 / 2 of the water body. When changing the water, the aeration intensity is reduced and stirring is suspended.

8. The method for hybrid breeding of grouper according to claim 1, characterized in that, During the seedling cultivation period, the seedlings are cultivated in three stages: larvae, juveniles, and young fish. During the larval stage, the water temperature is kept stable at 23℃~24℃, and the fish are fed rotifers and brine shrimp larvae. During the juvenile and young fish stages, the fish are fed crushed stress-resistant feed and an appropriate amount of copepods. At the same time, wide-temperature acclimatization is carried out. In the later stage of acclimatization, the water temperature fluctuation range is gradually expanded to 18℃~28℃.

9. The method for hybrid breeding of grouper according to claim 1, characterized in that, The preparation process of the garlic extract is as follows: Select fresh garlic that is free from mold and rot, peel it and crush it into garlic paste, add sterile distilled water at a solid-liquid ratio of 1:8, and place it in a constant temperature environment of 35~40℃ for anaerobic fermentation for 24~36 hours. After fermentation, filter it through an 80-mesh filter to remove residue, take the filtrate and freeze dry it under vacuum to make powder, and dilute it with isothermal water at a ratio of 1:5 before use.

10. A method for hybrid breeding of grouper according to claim 1, characterized in that, The temperature-sensitive conditioning agent must be prepared and used immediately, and added to the aquaculture water within 2 hours after preparation; the reproductive care agent should be added and the water should be left to stand for 2 hours.

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