Breeding method for hypoxia-resistant golden tiger hybrid spots

Abstract

The invention provides a breeding method of hypoxia-resistant golden tiger hybrid spots, which comprises the following steps: carrying out artificial insemination on a female parent of epinephelus coioides and a male parent of epinephelus coioides to obtain fertilized eggs, carrying out 2.8-3.2 mg / L hypoxia screening on the female parent for more than three generations, and ensuring that the suffocation point is less than or equal to 0.9 mg / L. Performing staged low-oxygen incubation on the fertilized eggs, gradually reducing dissolved oxygen in an embryonic period, a protointestinal period and a membrane emergence period, performing specific sound wave stimulation in an opening period of larva fish, performing low-oxygen-resistant phenotype screening in a larva period, performing intermittent low-oxygen intensive training in a juvenile fish period, applying water flow stimulation before training, and performing low-oxygen-resistant phenotype screening in a membrane emergence period; during the period, a fortified feed containing gamma-aminobutyric acid, heme iron and other components is fed. According to the method, through multi-link collaborative innovation, the hypoxia resistance of the Jinhu hybrid spots is effectively improved, the survival rate of a target group under 2.5 mg / L dissolved oxygen reaches 93%, the suffocation point is reduced to 0.75 mg / L, meanwhile, gill filament development and growth are promoted, and an efficient technical scheme is provided for aquatic product breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly relates to a breeding method for hypoxia-tolerant golden tiger hybrid grouper. Background Art

[0002] The golden tiger hybrid grouper is a new aquaculture variety, which is hybridized from Epinephelus fuscoguttatus and Epinephelus lanceolatus. It combines the excellent characteristics of the parents such as fast growth, strong disease resistance, and delicate meat quality, and has significant heterosis. Therefore, it is highly favored in the aquaculture market and has become an important aquaculture object for farmers to increase income and meet the market demand for high-quality fish.

[0003] In actual aquaculture, hypoxia stress has become the core bottleneck restricting the development of the golden tiger hybrid grouper industry. There are obvious deficiencies in the whole chain of hypoxia-tolerant breeding in the existing technology: at the genetic level, traditional cross-breeding only relies on phenotypic observation to screen parents, lacking directional selection of hypoxia-tolerant related genes, and it is difficult to achieve stable inheritance of excellent hypoxia-tolerant traits; in the hatching link, conventional dissolved oxygen conditions are mostly used for fertilized egg hatching, without utilizing the plasticity of the embryonic development sensitive period to the hypoxic environment, missing the key opportunity for early induction of hypoxia-tolerant traits; in the screening process, most studies only use the hypoxia tolerance performance of adult fish as the screening criterion, ignoring the excavation of hypoxia tolerance potential in the larval stage, resulting in low screening efficiency and easy omission of early dominant individuals; in terms of intensive training, the existing hypoxia training programs lack scientific gradient design and environmental synergistic stimulation, and do not combine nutritional enhancement means, making it difficult to systematically improve the hypoxia physiological function of fish. In addition, there is a lack of organic connection between the existing technologies in each link, and a synergistic breeding system cannot be formed, resulting in limited improvement in the hypoxia tolerance performance of the cultivated varieties and difficulty in meeting the urgent demand for hypoxia adaptability in intensive aquaculture. Summary of the Invention

[0004] In view of this, the present invention proposes a breeding method for hypoxia-tolerant golden tiger hybrid grouper to solve the above problems.

[0005] The technical solution of the present invention is realized as follows: A breeding method for hypoxia-tolerant golden tiger hybrid grouper:

[0006] S1. Select good Epinephelus fuscoguttatus female parents and Epinephelus lanceolatus male parents for artificial insemination to obtain fertilized eggs;

[0007] S2. Implement staged hypoxia hatching treatment on the fertilized eggs;

[0008] S3. Perform acoustic stimulation during the opening period;

[0009] S4. Conduct hypoxia tolerance phenotype screening during the larval stage;

[0010] S5. Implement periodic hypoxia intensive training on the screened individuals.

[0011] Further, the female parent in step S1 is the Epinephelus fuscoguttatus screened in an environment with a dissolved oxygen of 2.8 - 3.2 mg / L for more than 3 generations, and its asphyxiation point is ≤ 0.9 mg / L; the male parent is an individual with excellent low oxygen tolerance.

[0012] Further, the staged hypoxic incubation treatment in step S2 includes:

[0013] Embryonic stage 0 - 24 h: Maintain the dissolved oxygen at 5.5 - 6.0 mg / L;

[0014] Gastrula stage 24 - 36 h: Reduce it to 3.8 - 4.2 mg / L and maintain for 10 - 15 h;

[0015] Hatching stage 36 - 48 h: Further reduce it to 2.5 - 2.8 mg / L; During the treatment, the water temperature is kept constant at 29 ± 0.3 °C.

[0016] Further, in step S3, acoustic wave stimulation is carried out at the larval opening stage of 5 - 7 days old, and intermittent acoustic waves are applied 2 - 4 times a day, each time lasting for 15 - 25 s.

[0017] Further, the acoustic wave conditions are 10 - 15 kHz and 90 - 100 dB.

[0018] Further, the screening method in step S4 is: Conduct an asphyxiation point test on the juveniles at 10 days old, select individuals with a survival time ≥ 45 minutes, and synchronously measure the hemoglobin content, and select individuals with a screening value ≥ 125 g / L.

[0019] Further, the hypoxic intensive training in step S5 includes: Intermittent hypoxic treatment during the juvenile stage of 30 - 45 days old, and 2 - 4 times of intermittent hypoxic treatment are carried out every day. Each time, the dissolved oxygen is reduced from 5.0 mg / L to 1.8 - 2.2 mg / L within 10 minutes and maintained for 30 - 50 minutes, and after the treatment, it is restored to 5.0 - 6.0 mg / L at a rate of 0.2 - 0.4 mg / L / min.

[0020] Further, during the hypoxic treatment, fortified feed is fed, and the daily feeding amount is 8 - 12% of the fish body weight.

[0021] Further, the fortified feed includes the following raw materials in parts by weight: 300 - 400 parts of fish meal, 200 - 250 parts of soybean meal, 50 - 70 parts of fish oil, 1 - 3 parts of γ - aminobutyric acid, 4.5 - 5.5 parts of heme iron, 3.8 - 4.2 parts of vitamin E, 1.0 - 1.4 parts of L - carnitine, 2.3 - 2.7 parts of glutamine, 1.6 - 2.0 parts of yeast β - glucan, 0.5 - 0.7 parts of bile acid, wherein the purity of β - glucan is ≥ 85%, and the molecular weight distribution is 50 - 150 kDa.

[0022] Furthermore, a water flow stimulation is applied for 4 - 6 minutes before each intermittent hypoxia treatment, and the flow rate is controlled at 15 - 20 cm / s.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) Precisely inducing hypoxia tolerance traits at the embryonic development stage

[0025] The method of the present invention adopts a staged hypoxia incubation treatment, that is, maintaining the dissolved oxygen within a specific range during the embryonic period, reducing the dissolved oxygen to a lower range and maintaining it for a certain period during the gastrula stage, and further regulating the dissolved oxygen to an even lower level during the hatching stage. This operation of precisely simulating the natural hypoxia stress gradient effectively activates the expression of hypoxia tolerance-related genes during the embryonic period. Verified by experiments, the hatching rate of fertilized eggs is significantly improved compared with conventional incubation, and the initial hemoglobin content of the hatched larvae is significantly increased.

[0026] (2) Multi-dimensional screening and functional enhancement working together during the larval stage

[0027] The sonic stimulation during the opening period precisely acts on the gill filament development of larvae, prompting a significant increase in the surface area of gill filaments and greatly improving the gas exchange efficiency. Combined with the intermittent hypoxia treatment during the juvenile stage, it successfully induces an increase in the gill filament vascular density and collaboratively constructs an efficient hypoxia adaptation respiratory system.

[0028] Innovatively integrating the dual indicators of the asphyxia point test and hemoglobin content detection for hypoxia tolerance phenotype screening, the accuracy rate is greatly improved, and individuals with genetic advantages can be accurately locked, providing high-quality germplasm resources for the subsequent cultivation of varieties with excellent hypoxia tolerance performance.

[0029] (3) Periodic hypoxia intensive training deeply stimulates the physiological adaptation potential

[0030] The present invention adopts a collaborative operation mode of "water flow stimulation + intermittent hypoxia + functional feed"; the water flow stimulation is applied before the intermittent hypoxia treatment, activating the fish body's motor metabolism in advance and effectively enhancing its energy reserve ability in a hypoxic environment. The gradient hypoxia treatment strongly induces the expression of anti-apoptosis genes. After testing, the survival rate of juvenile fish in a specific dissolved oxygen environment is significantly improved compared with the control group.

[0031] The fortified feed is rich in key components such as γ-aminobutyric acid and heme iron, and the purity of β-glucan meets the standard. These components synergistically promote erythropoiesis and oxygen-carrying capacity, significantly increasing the hemoglobin content of the target population and greatly reducing the asphyxia point, which is significantly lower than that in the case of feeding with basic conventional feed.

[0032] (4) The whole-process technology collaborates to build an efficient breeding system

[0033] In the parental screening stage of the present invention, female Epinephelus fuscoguttatus that have been screened in a specific dissolved oxygen environment for multiple generations are selected and crossed with male Epinephelus cyanopodus with excellent low oxygen tolerance. The parental screening is closely linked to subsequent hatching, screening, and intensive training, forming a closed loop of genetic-environmental collaborative optimization. After multiple generations of breeding, the low oxygen tolerance trait is stably inherited, and the growth rate of the target variety is significantly improved compared to conventional varieties. The method of the present invention breaks through the limitations of single-link optimization in the prior art and realizes the systematic improvement of low oxygen tolerance performance through the coupling of multiple technologies, providing core technical support for the intensive farming of groupers and significantly reducing the farming risks and economic losses caused by low oxygen stress. Detailed implementation mode

[0034] To better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0035] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0036] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can all be obtained from commercial channels.

[0037] Example 1

[0038] Breeding method for hypoxia-tolerant golden tiger hybrid groupers

[0039] Step S1: Artificial insemination

[0040] Select female Epinephelus fuscoguttatus that have been screened in a 2.8 - 3.2 mg / L dissolved oxygen environment for 3 generations (asphyxiation point 0.8 mg / L) and male Epinephelus cyanopodus (excellent low oxygen tolerance performance, asphyxiation point 1.0 mg / L), and perform dry artificial insemination. After mixing the eggs and sperm, add aerated seawater to obtain fertilized eggs.

[0041] Step S2: Hypoxia incubation treatment in stages

[0042] Embryonic stage 0 - 24 h: The water temperature in the hatching pool is maintained at 29 °C, the dissolved oxygen is 5.5 mg / L, the salinity is 32‰, and slightly aerated stirring is carried out;

[0043] Gastrula stage 24 - 36 h: Slowly reduce the dissolved oxygen to 3.8 mg / L and maintain it for 10 h. During this period, stop aeration and use water circulation to keep the dissolved oxygen uniform;

[0044] Hatching stage 36 - 48 h: Further reduce the dissolved oxygen to 2.5 mg / L until the fry hatch.

[0045] Step S3: Sonic stimulation during the opening period

[0046] During the opening period of the larvae (5 - 7 days old), apply intermittent sound waves of 10 kHz and 90 dB 2 times a day, each time lasting for 15 s.

[0047] Step S4: Screening of hypoxia tolerance phenotype in the larval stage

[0048] When the larvae are 10 days old, conduct an asphyxiation point test in water with a dissolved oxygen of 1.0 mg / L. Select individuals with a survival time ≥ 45 minutes, and simultaneously measure the hemoglobin content. Screen individuals with a hemoglobin content ≥ 125 g / L, and a total of 2,800 candidate fry are obtained.

[0049] Step S5: Periodic hypoxia intensification training

[0050] In the juvenile stage (30 - 45 days old), conduct intermittent hypoxia treatment 2 times a day. Each treatment reduces the dissolved oxygen from 5.0 mg / L to 1.8 mg / L within 10 minutes, maintains it for 30 minutes, and then recovers to 5.0 mg / L at a rate of 0.2 mg / L / min after the treatment. Apply a 5 - minute water flow stimulation with a flow rate of 15 cm / s before each intermittent hypoxia treatment. Feed fortified feed during the hypoxia treatment, and the daily feeding amount is 8% of the fish body weight.

[0051] The fortified feed includes the following raw materials in parts by weight: 300 parts of fish meal, 200 parts of soybean meal, 50 parts of fish oil, 1 part of γ - aminobutyric acid, 4.5 parts of heme iron, 3.8 parts of vitamin E, 1.0 part of L - carnitine, 2.3 parts of glutamine, 1.6 parts of yeast β - glucan, 0.5 part of bile acid, where the purity of β - glucan ≥ 85% and the molecular weight distribution is at 50 kDa.

[0052] Example 2

[0053] Hypoxia - tolerant golden tiger hybrid grouper breeding method

[0054] Step S1: Artificial insemination

[0055] Select the female Epinephelus fuscoguttatus (asphyxiation point 0.8 mg / L) screened in an environment with a dissolved oxygen of 2.8 - 3.2 mg / L for 3 generations, and the male Epinephelus cyanopodus (excellent hypoxia tolerance performance, asphyxiation point 1.0 mg / L). Conduct dry - method artificial insemination. After mixing the eggs and sperm, add aerated seawater to obtain fertilized eggs.

[0056] Step S2: Stage - by - stage hypoxia incubation treatment

[0057] Embryonic stage 0 - 24 h: Maintain the water temperature in the hatching pool at 29.3°C, dissolved oxygen 6.0 mg / L, salinity 32‰, and conduct micro - aeration stirring;

[0058] Gastrula stage 24 - 36 h: Slowly reduce the dissolved oxygen to 4.2 mg / L and maintain it for 15 h. During this period, stop aeration and use water circulation to keep the dissolved oxygen uniform;

[0059] Hatching period of 36 - 48h: Further reduce the dissolved oxygen to 2.8 mg / L until the fry hatch.

[0060] Step S3: Acoustic stimulation during the opening period

[0061] During the opening period of the larvae (5 - 7 days old), apply intermittent sound waves at 15 kHz and 100 dB 4 times a day, each time lasting for 25 s.

[0062] Step S4: Screening of hypoxia - tolerant phenotypes during the juvenile stage

[0063] When the juveniles are 10 days old, conduct an asphyxiation point test in water with a dissolved oxygen of 1.0 mg / L. Select individuals with a survival time ≥ 45 minutes, and simultaneously measure the hemoglobin content. Screen individuals with a hemoglobin content ≥ 125 g / L, and a total of 2800 candidate fry are obtained.

[0064] Step S5: Periodic hypoxia - strengthening training

[0065] During the juvenile stage (30 - 45 days old), conduct 4 times of intermittent hypoxia treatment every day. Each treatment reduces the dissolved oxygen from 5.0 mg / L to 2.2 mg / L within 10 minutes, maintains it for 50 minutes, and then restores it to 6.0 mg / L at a rate of 0.4 mg / L / min. Apply a 5 - minute water flow stimulation with a flow rate of 20 cm / s before each intermittent hypoxia treatment. Feed fortified feed during the hypoxia treatment, and the daily feeding amount is 12% of the fish body weight.

[0066] The fortified feed includes the following raw materials by weight: 400 parts of fish meal, 250 parts of soybean meal, 70 parts of fish oil, 3 parts of γ - aminobutyric acid, 5.5 parts of heme iron, 4.2 parts of vitamin E, 1.4 parts of L - carnitine, 2.7 parts of glutamine, 2.0 parts of yeast β - glucan, 0.7 parts of bile acid, where the purity of β - glucan ≥ 85% and the molecular weight distribution is at 150 kDa.

[0067] Example 3

[0068] Hypoxia - tolerant breeding method for Epinephelus fuscoguttatus♀×Epinephelus tukula♂

[0069] Step S1: Artificial insemination

[0070] Select the female parent of Epinephelus fuscoguttatus (asphyxiation point 0.8 mg / L) screened in an environment with a dissolved oxygen of 2.8 - 3.2 mg / L for 3 generations, and the male parent of Epinephelus tukula (excellent hypoxia - tolerance performance, asphyxiation point 1.0 mg / L). Conduct dry - method artificial insemination. After mixing the eggs and sperm, add aerated seawater to obtain fertilized eggs.

[0071] Step S2: Staged hypoxia - incubation treatment

[0072] Embryonic stage 0 - 24h: The water temperature in the hatching pond is maintained at 29°C, dissolved oxygen is 5.8 mg / L, salinity is 32‰, with gentle aeration and stirring.

[0073] Gastrula stage 24 - 36h: Slowly reduce the dissolved oxygen to 4.0 mg / L and maintain for 12h. During this period, aeration is stopped, and water circulation is used to keep the dissolved oxygen uniform.

[0074] Hatching stage 36 - 48h: Further reduce the dissolved oxygen to 2.6 mg / L until the fry hatch.

[0075] Step S3: Sonic stimulation during the opening stage

[0076] During the larval opening stage (5 - 7 days old), intermittent sound waves of 12 kHz and 95 dB are applied 3 times a day, each lasting for 20 s.

[0077] Step S4: Screening of hypoxia - tolerant phenotypes during the juvenile stage

[0078] When the juveniles are 10 days old, an asphyxiation point test is carried out in water with a dissolved oxygen of 1.0 mg / L. Individuals with a survival time ≥ 45 minutes are selected, and the hemoglobin content is measured synchronously. Individuals with a hemoglobin content ≥ 125 g / L are screened, and a total of 2800 candidate fry are obtained.

[0079] Step S5: Periodic hypoxia intensification training

[0080] During the juvenile stage (30 - 45 days old), intermittent hypoxia treatment is carried out 3 times a day. Each treatment reduces the dissolved oxygen from 5.0 mg / L to 2.0 mg / L within 10 minutes, maintains for 40 minutes, and then recovers to 5.5 mg / L at a rate of 0.3 mg / L / min. A 5 - minute water flow stimulation with a flow rate of 18 cm / s is applied before each intermittent hypoxia treatment. During the hypoxia treatment, fortified feed is fed, and the daily feeding amount is 10% of the fish body weight.

[0081] The fortified feed includes the following raw materials in parts by weight: 350 parts of fish meal, 220 parts of soybean meal, 60 parts of fish oil, 2 parts of γ - aminobutyric acid, 5 parts of heme iron, 4.0 parts of vitamin E, 1.2 parts of L - carnitine, 2.5 parts of glutamine, 1.8 parts of yeast β - glucan, 0.6 parts of bile acid, where the purity of yeast β - glucan ≥ 85% and the molecular weight distribution is at 100 kDa.

[0082] Comparative example 1

[0083] The difference between this comparative example and Example 3 is that a breeding method with conventional hatching conditions is adopted.

[0084] Differentiating steps:

[0085] In step S2, the dissolved oxygen is maintained at 5.8 mg / L throughout the embryonic period, gastrula period, and hatching period, and other steps are the same as in Example 3.

[0086] Comparative Example 2

[0087] The difference between this comparative example and Example 3 lies in the breeding method without sonic stimulation during the opening period.

[0088] Differentiating steps:

[0089] Omit step S3, that is, do not perform sonic stimulation during the larval opening period, and other steps are the same as in Example 3.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 3 lies in the breeding method without periodic hypoxia intensification training.

[0092] Differentiating steps:

[0093] Omit step S5, that is, do not perform periodic hypoxia intensification training on the selected individuals, and other steps are the same as in Example 3.

[0094] Comparative Example 4

[0095] The difference between this comparative example and Example 3 lies in replacing the fortified feed.

[0096] Use ordinary feed: 350 parts of fish meal, 220 parts of soybean meal, 60 parts of fish oil, 2 parts of γ-aminobutyric acid, 5 parts of heme iron, 4.0 parts of vitamin E, and the rest is the same as in Example 3.

[0097] I. Performance test

[0098] 1. Hatching rate test method

[0099] (1) Experimental preparation: Place the fertilized eggs of the examples and comparative examples in hatching ponds of the same specification respectively. Set 3 parallel experiments for each group, with 1000 fertilized eggs in each hatching pond, and record the total number of initial fertilized eggs N 0 .

[0100] (2) Hatching process: Operate according to the hatching conditions of the corresponding breeding method, observe and record the number of hatched fry in the hatching pond regularly every day, and remove the dead fertilized eggs and fry.

[0101] (3) Data calculation: After the hatching is completed, count the total number of finally hatched fry N 1 ,

[0102] According to the formula: hatching rate = N 1 / N 0 ×100% to calculate the hatching rate, and take the average of the 3 parallel experiments as the final result.

[0103] 2. Asphyxiation Point Test Method

[0104] (1) Experimental Preparation: Select juvenile fish at the same developmental stage. Randomly select 30 fish for each group and place them in separate airtight water containers. The volume of water in the container is 10 L, and a dissolved oxygen monitor and a temperature control device are equipped.

[0105] (2) Test Process: Slowly reduce the dissolved oxygen in the water at a constant rate (0.1 mg / L / min), and simultaneously record the dissolved oxygen value and the behavioral performance (respiration rate, swimming state) of the juvenile fish in real time. When it is observed that the juvenile fish shows lateral turning, loses balance and shows no sign of recovery for 5 minutes, it is regarded as asphyxiation. Immediately record the dissolved oxygen in the water at this time, and this value is the asphyxiation point of this juvenile fish.

[0106] (3) Data Processing: Calculate the average value and standard deviation of the asphyxiation points of 30 juvenile fish as the asphyxiation point data of this group of samples.

[0107] 3. Hemoglobin Content Test Method

[0108] (1) Sample Collection: Randomly select 10 juvenile fish for each group. Use a sterile syringe to collect blood samples from the caudal vein. The collection volume for each fish is about 0.5 - 1 mL. Inject the blood into a centrifuge tube containing an anticoagulant (sodium heparin) and gently invert and mix.

[0109] (2) Measurement Method: Use the cyanmethemoglobin method. Appropriately dilute the collected blood sample (1:200), and then use a spectrophotometer to measure the absorbance value at a wavelength of 540 nm. Calculate the hemoglobin content according to the standard curve.

[0110] (3) Data Statistics: Calculate the average value and standard deviation of the hemoglobin content of 10 juvenile fish as the hemoglobin content data of this group of samples.

[0111] 4. Dissolved Oxygen Survival Rate Test Method

[0112] (1) Selection of Fry: Randomly select fry with the same specifications, healthy and disease-free from the fry populations cultivated in the examples and each comparative example. Set 3 replicates for each group, select 30 fry for each replicate, and mark them on the dorsal fin with a marker pen for convenient subsequent observation and differentiation.

[0113] (2) Experimental Container: Use 20 L airtight circulating aquaculture barrels with the same specifications. Disinfect them in advance with potassium permanganate solution and inject seawater after aeration treatment. The aquaculture barrels are equipped with dissolved oxygen sensors, heating rods, water pumps and microporous aeration devices to ensure that the dissolved oxygen and water temperature are stable and controllable.

[0114] (3) Equipment debugging: Connect the dissolved oxygen sensor to the data recorder and calibrate it; Set the heating rod to keep the water temperature constant at 29 ± 0.3 °C; Test the microporous aeration device and the water pump to ensure that the dissolved oxygen can be accurately adjusted and the water body can be circulated.

[0115] (4) Initial dissolved oxygen adjustment: Pass air into the breeding bucket to raise the dissolved oxygen in the water body to 5.0 - 6.0 mg / L. After stabilizing for 1 hour, put 30 marked fry into it and let them adapt to the environment for 2 hours.

[0116] (5) Dissolved oxygen reduction: Reduce the air intake and appropriately introduce nitrogen to reduce the dissolved oxygen to 2.5 mg / L at a rate of 0.2 mg / L / min, and monitor the dissolved oxygen in real time. After the dissolved oxygen reaches the standard, stop ventilation and turn on the water body circulation pump to keep the dissolved oxygen uniform.

[0117] (6) Observation period: After the dissolved oxygen stabilizes at 2.5 mg / L, continuously observe for 24 hours, record the survival status of the fry every 30 minutes, and promptly fish out the dead fish and record the time.

[0118] (7) Handling of abnormal situations: If the dissolved oxygen fluctuates by more than ±0.1 mg / L, slightly adjust the air or nitrogen intake to restore the dissolved oxygen, and record the fluctuation time and handling measures; If large-scale stress reactions occur in the fry, the adaptation time can be appropriately extended, but the total observation time remains unchanged.

[0119] (8) Data calculation: After the observation, count the number of surviving fry n,

[0120] Calculation formula: Dissolved oxygen survival rate (%) = n / 30 × 100%.

[0121] 5. Test method for gill filament surface area

[0122] (1) Sample preparation: Randomly select 5 juvenile fish from each group. After anesthetizing them with an excessive amount of anesthetic (eugenol), quickly dissect and take out the gill tissue, and completely separate the gill filaments to avoid damage.

[0123] (2) Image processing: Place the gill filaments on a glass slide, drop an appropriate amount of physiological saline, cover with a cover glass, and take pictures of the gill filaments using a microscope (40x objective lens). Randomly take 5 images of different fields of view for each gill filament. Use professional image analysis software to process the taken images, and manually outline the boundaries of the gill filaments. The software automatically calculates the surface area of the gill filaments.

[0124] (3) Data calculation: Calculate the average value and standard deviation of the gill filament surface areas of 5 juvenile fish as the gill filament surface area data of this group of samples.

[0125] 6. Growth rate

[0126] (1) Regularly measure body length and weight: During the breeding process, regularly measure the experimental fish every week. Use an electronic balance to measure the weight and a ruler or vernier caliper to measure the body length (measure the length from the tip of the snout to the base of the caudal fin).

[0127] (2) Calculate the growth rate: Specific growth indicators can be used to evaluate the growth rate, such as the specific growth rate (SGR). The calculation formula is:

[0128] 7. Result data:

[0129]

[0130] It can be seen from the above data results that Example 3 is significantly superior to each comparative example in multiple indicators through staged hypoxic hatching treatment, sonic stimulation during the initial feeding period, periodic hypoxic intensive training, and fortified feed with a specific formula. In Comparative Example 1, the hatching rate decreased due to conventional hatching, resulting in a decline in the hypoxia tolerance of the final selected population; in Comparative Example 2, the lack of sonic stimulation affected the gill filament development and hypoxia tolerance; in Comparative Example 3, no hypoxic intensive training was carried out, and the hypoxia adaptability was significantly insufficient; in Comparative Example 4, ordinary feed was used, and although some indicators were close, it was still inferior to Example 3 in the core hypoxia tolerance indicators, fully verifying the innovation and effectiveness of the breeding method and fortified feed of the present invention.

[0131] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for breeding hypoxia-resistant golden tiger hybrid spots, characterized in that: The following steps are involved: S1. Artificially inseminating the selected brown-spotted grouper female parent and blue-bodied grouper male parent to obtain fertilized eggs; S2, carrying out staged hypoxia incubation treatment on the fertilized eggs; S3, sound wave stimulation during the opening period; S4, screening for hypoxia tolerance phenotype during the larval stage; S5. Implement periodic hypoxic intensive training on the screened individuals.

2. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 1, characterized in that: The mother parent in step S1 is a brown-spotted grouper that has been screened in a 2.8-3.2 mg / L dissolved oxygen environment for more than three generations, and its suffocation point is ≤0.9 mg / L; the father parent is an individual with excellent hypoxia resistance.

3. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 1, characterized in that: The staged hypoxic incubation process of step S2 comprises: Embryonic period 0-24h: maintain dissolved oxygen at 5.5-6.0mg / L; Enterogenous stage 24-36h: drop to 3.8-4.2mg / L, maintain for 10-15h; Film-forming period 36-48h: further reduced to 2.5-2.8mg / L; water temperature was constant at 29±0.3℃ during the treatment period.

4. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 1, characterized in that: In step S3, sound wave stimulation is performed on the fry at the age of 5-7 days during the opening stage, and intermittent sound waves are applied 2-4 times a day, each time lasting 15-25 seconds.

5. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 4, characterized in that: The sound wave conditions are 10-15kHz, 90-100dB.

6. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 1, characterized in that: The screening method of step S4 is: performing an asphyxia point test when the larvae are 10 days old, selecting individuals with a survival time of ≥45 minutes, and simultaneously measuring the hemoglobin content to screen individuals with a value of ≥125 g / L.

7. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 1, characterized in that: The hypoxia intensive training in step S5 includes: intermittent hypoxia treatment at 30-45 days of age in the juvenile stage, 2-4 times of intermittent hypoxia treatment per day, each treatment reducing the dissolved oxygen from 5.0 mg / L to 1.8-2.2 mg / L within 10 minutes, maintaining for 30-50 minutes, and recovering to 5.0-6.0 mg / L at a rate of 0.2-0.4 mg / L / min after treatment.

8. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 7, characterized in that: During the hypoxic treatment period, fish were fed an enriched diet at a daily rate of 8-12% of their body weight.

9. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 8, characterized in that: The fortified feed comprises the following raw materials in parts by weight: 300-400 parts of fish meal, 200-250 parts of soybean meal, 50-70 parts of fish oil, 1-3 parts of gamma-aminobutyric acid, 4.5-5.5 parts of heme iron, 3.8-4.2 parts of vitamin E, 1.0-1.4 parts of L-carnitine, 2.3-2.7 parts of glutamine, 1.6-2.0 parts of yeast beta-glucan, and 0.5-0.7 parts of bile acid, wherein the purity of the beta-glucan is ≥85%, and the molecular weight distribution is 50-150 kDa.

10. A method for breeding hypoxia-resistant golden tiger hybrids as claimed in claim 7, characterized in that: Before each intermittent hypoxia treatment, water flow stimulation was applied for 4-6 minutes, and the flow rate was controlled at 15-20 cm / s.

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