Artificial breeding method of epinephelus trifoliatus and epinephelus lanceolatus hybrid

Abstract

The invention discloses an artificial cultivation method of a hybrid of epinephelus trifoliatus and epinephelus lanceolatus, which comprises the following steps: taking healthy epinephelus trifoliatus as female parent fish, and carrying out cultivation strengthening and artificial ripening to obtain strengthened female parent fish; healthy epinephelus lanceolatus is used as male parent fish, and sexual mature male parent fish is obtained through natural sexual maturity; the ovulation time and the spermiation time are consistent; collecting egg liquid and seminal fluid, inseminating to obtain fertilized eggs, and performing embryo incubation and illumination regulation and control cultivation after opening to obtain hybrid spots; carrying out authenticity identification on the hybrid spots by utilizing a primer pair, and determining that the hybrid spots inherit genetic information of the female parent fish and the male parent fish; the primer pair is a sequence pair composed of SEQ ID NO. 1 and SEQ ID NO. 2, and a sequence pair composed of SEQ ID NO. 3 and SEQ ID NO. 4. The technical blank of crossbreeding by taking the epinephelus trifoliatus as the parent epinephelus is filled, and the authenticity of the hybridized spots is reliable.

Description

Technical Field

[0001] This invention relates to the fields of aquaculture and genetic breeding technology, specifically to a method for artificially cultivating a hybrid of black-spotted grouper and saddle-tailed grouper. More particularly, it relates to a method for improving the hybrid fertilization rate, embryonic development stability, and early larval survival rate under non-hormonal conditions through the synergistic effects of artificial maturation regulation, dry artificial insemination, and staged light regulation, and for identifying the authenticity of the hybrid using molecular biology techniques. Background Technology

[0002] In grouper ( Epinephelus spp. The grouper is a diverse and economically valuable fish resource in my country's tropical and subtropical marine aquaculture industry. With its tender flesh and high nutritional value, grouper enjoys stable market demand, leading to a continuous expansion of its aquaculture scale in coastal areas. Integrating the advantages of different grouper varieties in terms of growth rate, environmental adaptability, and stress resistance through interspecific hybridization is considered an important breeding approach for developing new high-performance grouper strains.

[0003] Black-spotted grouper ( Epinephelus corallicola The black-spotted grouper (Salmonella spp.) belongs to the order Perciformes, family Serranidae, and genus Salmonella. It is mainly distributed in the tropical coral reef waters of the Indian Ocean and the western Pacific Ocean, typically inhabiting coral reefs and rocky reefs at depths of 5-80 meters. This medium-sized fish has distinctive mottled patterns on its body, exhibits strong environmental adaptability and good meat quality, and has a certain resource base in some sea areas. While the black-spotted grouper grows relatively quickly and adapts well to artificial aquaculture conditions, its artificial breeding and large-scale seedling production techniques still need further improvement.

[0004] Saddleback grouper ( Epinephelus lanceolatus This fish belongs to the order Perciformes, family Serranidae, and genus Lepidoptera. It is widely distributed in the tropical and subtropical waters of the Indian and Pacific Oceans, primarily inhabiting nearshore coral reefs and rocky reefs. This fish is relatively large, highly resilient, and exhibits good tolerance to changes in the aquatic environment. During aquaculture, it demonstrates high survival rates and stability, making it highly valuable for aquaculture.

[0005] Currently, artificial hybridization breeding methods for some grouper species have been publicly disclosed, and certain artificial propagation and seedling raising procedures have been established. However, systematic research and technical solutions for artificial hybridization breeding using black bream and saddleback grouper as parents have not yet been reported in a mature and stable manner. Significant technical challenges remain, particularly regarding differences in the gonadal development rhythms of the parents, control of artificial maturation, hybridization fertilization efficiency, and the stability of early hybrid embryo development.

[0006] Therefore, in the artificial hybridization breeding of black-spotted grouper and saddle-banded grouper as parents, the existing technology cannot provide an effective and reproducible solution, and there are still obvious technological gaps. Further research and technological breakthroughs are needed to promote the development of grouper hybridization breeding and industrial application. Summary of the Invention

[0007] The purpose of this invention is to provide an artificial breeding method for a hybrid of brown-spotted grouper and blue-spotted sea bass, to fill the technical gap in hybrid breeding of grouper with blue-spotted sea bass as parent, so that the hybrid obtained by crossing blue-spotted sea bass and brown-spotted grouper is authentic and reliable, and inherits the genetic information of the parents at the molecular level.

[0008] To solve this technical problem: This patent provides a method for artificially cultivating a hybrid of black-striped grouper and saddle-banded grouper, comprising the following steps: Step A: Using healthy black-spotted grouper as female parent fish, they are fortified with nutrients and artificially matured to obtain fortified female parent fish; using healthy saddle-banded grouper as male parent fish, they are allowed to reach sexual maturity naturally to obtain sexually mature male parent fish; so that the ovulation time of the fortified female parent fish is consistent with the sperm release time of the sexually mature male parent fish. Artificial ripening was achieved by lowering the temperature from 26±0.5℃ to 22.5±0.5℃ at a rate of 0.5℃ per day for 7 to 10 days, followed by raising the temperature to 28.5±0.5℃ at a rate of 0.5℃ per day for water temperature regulation. Step B: Collect the egg fluid of the enhanced female parent fish and the sperm of the sexually mature male parent fish, fertilize them to obtain fertilized eggs, and then cultivate them under controlled light after embryo hatching and opening to obtain hybrid spots; Step C: Use primer pairs to verify the authenticity of the hybrid spots and determine that the hybrid spots inherited the genetic information of both the female and male parent fish. The primer pairs are sequence pairs consisting of SEQ ID NO.1 and SEQ ID NO.2 and sequence pairs consisting of SEQ ID NO.3 and SEQ ID NO.4.

[0009] Furthermore, step B includes: Step B-1: Collect the egg fluid of the enhanced female parent fish and the sperm of the sexually mature male parent fish, and obtain fertilized eggs after dry insemination and activation; Step B-2: The fertilized eggs are incubated as embryos and cultured under staged light regulation after opening to obtain hybrid spots.

[0010] Further, in step B-1, the dry fertilization method specifically involves: mixing the egg fluid of the female parent fish with the sperm of the sexually mature male parent fish under anhydrous conditions, and then allowing it to stand or gently stirring to ensure full contact. The anhydrous mixing and contact time is 30-90 s, preferably 45-60 s. The activation step involves: adding water once within 1-3 minutes after the egg fluid of the female parent fish and the sperm of the sexually mature male parent fish have been fully mixed. The volume of water added at one time is 1-5 times the volume of the eggs, preferably 2.0-3.5 times the volume.

[0011] Furthermore, the activation temperature of the seawater is 26-28℃, the salinity is 33-35‰, and after adding water, it is gently stirred for 10-30 seconds, and then left to stand for 1-3 minutes to complete the uniform activation and fertilization.

[0012] Furthermore, in step B-2, during the cleavage stage of the embryo hatching into a fertilized egg and the embryonic development stage, light-proof or low-light conditions are used, so that the light intensity is less than or equal to 100 lx.

[0013] Furthermore, the specific steps of staged light regulation cultivation after opening are as follows: 0-3 days old: Use dark or low light conditions, so that the light intensity is less than or equal to 100 lx; 4-8 days old: Increase the light intensity gradient to 1600-1800 lx; 9-20 days old: Reduce light intensity from 1600-1800 lx to 800-1200 lx in a gradual manner; 21-37 days old: Reduce light intensity from 800-1200 lx to 600-800 lx; 38-45 days old: Gradually increase the light intensity from 600-800 lx to 800-1000 lx; The photoperiod for the 4-45 day old animals was 14 hours of light and 10 hours of darkness; the gradient decrease was to reduce the light intensity by 100-200 lx per hour, and the gradient increase was to increase the light intensity by 100-200 lx per hour.

[0014] Furthermore, the water temperature during the incubation of fertilized embryos and the staged light-controlled cultivation after opening is 25-27℃, the salinity is 33-35‰, the dissolved oxygen concentration is above 6.5 mg / L, and the pH value is 7.9-8.4.

[0015] Further, step C specifically involves extracting DNA from the female parent fish, male parent fish, and hybrid spot to obtain the genomic DNA of the female parent fish, the genomic DNA of the male parent fish, and the genomic DNA of the hybrid spot, respectively, and then using primer pairs for PCR identification; confirming that the hybrid spot inherited the genetic information of the female parent fish and the male parent fish.

[0016] Furthermore, the PCR identification system was a 25 μL amplification system; the 25 μL amplification system included: 1 μL of genomic DNA at a concentration of 100 ng / μL; 1 μL of forward primer Fw at a concentration of 10 μM; 1 μL of reverse primer Rw at a concentration of 10 μM; 12.5 μL of 2×Taq Plus Master Mix II; and 9.5 μL of enzyme-free sterile water.

[0017] Furthermore, the PCR identification procedure includes the following steps: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 56℃ for 20 s, extension at 72℃ for 40 s, for a total of 32 cycles from denaturation to extension, followed by a full extension at 72℃ for 10 min.

[0018] Furthermore, in step A, the standard for health is: no disease, no injury, no deformity, robust physique, and no hormone-induced spawning or ovarian induction treatment; the weight of female parent fish is ≥1.0 kg, and the weight of male parent fish is ≥3.0 kg.

[0019] Furthermore, female and male parent fish are raised separately in an indoor recirculating aquaculture system, with an adaptation period of 2-4 months, preferably no less than 3 months.

[0020] Furthermore, the fortification period is 30-60 days, preferably 40-50 days; fortification involves feeding the fortified feed after the animal has had full feed. The fortified feed includes a basic feed and a nutrient supplement. The nutrient supplement is added at 3.93%-6.37% of the weight of the basic feed. The nutrient supplement includes 0.8%-1.2% deep-sea fish oil, 0.4%-0.6% L-arginine, 0.6%-0.9% coated vitamin E, 0.2%-0.5% taurine, 1.0%-1.5% aquatic-specific lecithin, 0.15%-0.25% microencapsulated folic acid, 0.3%-0.5% vitamin C, 0.2%-0.5% arachidonic acid, 0.08%-0.12% zinc methionine, and 0.2%-0.3% inositol.

[0021] Furthermore, the fertilization rate of the fertilized eggs is ≥95%, and the hatching rate of the fertilized eggs is ≥88%.

[0022] Furthermore, the survival rate of hybrid spots at 35 days of age was ≥50%, and the survival rate of hybrid spots at 65 days of age was ≥40%.

[0023] Compared with existing technologies, this patent has the following beneficial technical effects: This patent provides a method for artificially breeding a hybrid of black bream and saddleback grouper. This application is not a simple superposition of existing breeding measures, but rather achieves this through: (1) using artificially domesticated wild black bream as female parent fish and artificially cultured saddleback grouper as male parent fish. Under non-hormonal conditions, the female parent fish undergoes 45 days of nutritional enhancement and temperature slow-change to artificially promote ovarian maturation, while the male parent fish matures under natural conditions, thus achieving an effective match between the ovarian development process of female black bream and the sperm ejaculation rhythm of male saddleback grouper; (2) further utilizing the sperm activation timing control in dry artificial insemination to improve the combination efficiency of heterologous sperm and eggs and enhance the stability of early hybrid embryo development; (3) from embryo to larvae The synergistic effect of staged lighting regulation matching the developmental stage forms a complete and controllable hybrid breeding regulation system, resulting in hybrid spots with a fertilization rate of over 95%, a hatching rate of over 88% for fertilized eggs, a survival rate of 50% for 35-day-old hybrid spots, and a survival rate of 40% for 65-day-old hybrid spots. This fills the current technological gap in hybrid breeding using black-spotted grouper as female parent fish. Furthermore, the authenticity of the hybrid spots obtained by hybridizing black-spotted grouper and saddle-banded grouper was verified using two pairs of primers. The authenticity is reliable, and the hybrid spots inherit the genetic information of both female and male parent fish at the molecular level, laying the foundation for obtaining superior grouper breeds and also increasing the economic value of black-spotted grouper and saddle-banded grouper. Attached Figure Description

[0024] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0025] Figure 1 This is a diagram illustrating the process of collecting female broodstock eggs in the example. Figure 2 This is a diagram illustrating the process of collecting semen from male parent fish in this embodiment; Figure 3 This is a microscopic observation of the fertilized eggs in the embryo sac stage after hybridization between female and male parent fish in the example (scale bar: 200 μm). Figure 4 This is a microscopic observation of the gastrula stage of fertilized eggs after hybridization between female and male parent fish in the example (scale bar: 200 μm). Figure 5 This is a microscopic observation of the somite stage of fertilized eggs after hybridization between female and male parent fish in the example (scale bar: 200 μm). Figure 6 The image shown is of a 35-day-old hybrid spot obtained after hybridization of female and male parent fish in the example (scale bar is 1cm). Figure 7The image shown is of a 65-day-old hybrid spot obtained after hybridization of female and male parent fish in the example (scale bar is 1cm). Figure 8 The image shown is an agarose gel electrophoresis image used to identify the authenticity of hybrid spots and female and male parent fish in the examples (where M is the marker, 1 and 4 are the PCR product results of female parent fish, single band; 2 and 5 are the PCR product results of male parent fish, single band; 3 and 6 are the PCR product results of hybrid spots, double band, representing hybrid species; lanes 1-3 are for primer sequence detection SEQ ID NO.1-SEQ ID NO.2, and lanes 4-6 are for primer sequence detection SEQ ID NO.3-SEQ ID NO.4). Detailed Implementation

[0026] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0027] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings: As used in this patent, "no disease, no injury, no deformity" refers to the broodstock having an intact body surface, no mechanical damage or infectious lesions, no ulcers on the skin, no broken fins, no obvious parasites, normal swimming posture, and active feeding response.

[0028] As used in this patent, "satisfying feeding" refers to feeding the parent fish in an amount that is sufficient to satisfy their hunger and prevent them from constantly searching for food due to hunger.

[0029] All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, and in particular, meaning that one of ordinary skill in the art, upon reading the claims, specification and drawings of this patent, can directly and without doubt determine how the technical solution of this patent can be implemented.

[0030] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, specification, and drawings of this patent, a person skilled in the art can still arrive at the only correct understanding by reading the claims, specification, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this patent.

[0031] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0032] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0033] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0034] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0035] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0036] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0037] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0038] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0039] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0040] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0041] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0042] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0044] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0045] (1) Source of sample materials Black-spotted grouper: Purchased from Guangzhou Blue Ocean Marine Technology Co., Ltd. (Liwan District, Guangzhou), from a wild population that has been domesticated.

[0046] Saddleback grouper: Purchased from Lingshui Delin Chengxin Aquaculture Co., Ltd. (Lingshui County, Hainan Province), from an artificially bred group.

[0047] Sequence synthesis: carried out by Sangon Biotech (Shanghai) Co., Ltd., hereinafter referred to as Sangon.

[0048] (2) Sources of reagents and consumables Table 1. Sources and Product Codes of Reagents and Consumables Required for the Experiment ; (3) Source of instruments and equipment Table 2. Sources and Models of Instruments and Equipment Required for the Experiment ;

[0049] Example A method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper includes the following steps: S1. Selection and rearing of broodstock, the specific steps are as follows: 1. Select sexually mature black-spotted grouper with a symmetrical body shape, weight between 1.0-1.5 kg, no obvious abdominal deformities, intact scales, no mechanical damage, no parasitic infections, no obvious disease symptoms, and a sensitive and stable feeding response as female broodstock. A preliminary assessment of their sexual maturity is made through a combination of palpation and visual observation to ensure that individuals entering the artificial maturation stage have the physiological basis for further maturation.

[0050] 2. Select robust, sexually mature saddleback grouper weighing 3.0-5.0 kg with normal swimming ability, smooth and intact body surface, and no obvious damage or disease as male broodstock. Observe the sperm expulsion by gently pressing the abdomen, and select individuals that can naturally expel milky white semen as male broodstock to ensure that the sperm quality and quantity meet the needs of artificial hybridization.

[0051] To avoid potential interference from exogenous hormones on subsequent hybridization fertilization and embryonic development, neither the female nor male broodstock underwent any form of hormone injection, spawning induction, or forced maturation before the experiment. Gonadal development was regulated solely through nutritional and environmental factors. This provides a stable and reproducible physiological basis for subsequent artificial maturation regulation and hybridization fertilization.

[0052] 3. Broodstock rearing: The aforementioned female and male broodstock are separately transferred to an indoor recirculating aquaculture system for individual rearing. Adaptation to the environment is preferably 2-4 months, more preferably at least 3 months, to eliminate the short-term stress caused by transportation or sudden environmental changes. During the acclimatization period, the water temperature in the indoor recirculating aquaculture system is controlled at 25-27℃, the salinity is maintained at 33-35‰, the dissolved oxygen concentration (DO) is kept above 6.5 mg / L, and the pH value is controlled between 7.9 and 8.4 to simulate the natural nearshore ecological environment.

[0053] Meanwhile, a combined ultraviolet and biological filter water treatment device in the recirculating aquaculture system is used to maintain water quality. Grouper feed is given twice daily, supplemented with fresh small fish and squid; the amount of fresh small fish is 10%–15% of the total feed, and the amount of squid is 8%–10%. The feeding behavior, swimming status, and body condition of the parent fish are observed daily, and individuals exhibiting abnormal behavior or health risks are removed.

[0054] S2. Artificial maturation regulation of female parent fish, the specific steps are as follows: Because female and male broodstock exhibit significant differences in gonadal development rhythms under natural conditions, direct artificial hybridization can easily lead to problems such as asynchronous maturation and misaligned ovulation and spermation times. Therefore, artificial maturation regulation of female broodstock is employed, systematically intervening in the ovarian development process to effectively match the ovarian development process of female broodstock with the spermation rhythm of male broodstock under natural conditions. This provides physiologically consistent and highly mature gametes for subsequent artificial insemination. The specific steps are as follows: 1. Nutritional fortification regulation Forty-five days before the start of the artificial maturation stage for the broodstock, the broodstock are transferred to a dedicated maturation incubation tank for intensive nutrition. The maturation incubation tank uses an independent circulating water system with a high water turnover rate to ensure long-term water quality stability.

[0055] The salinity of the aquaculture water in the maturation and rearing ponds should be maintained at 33-35‰, the water temperature at 25-27℃, and the dissolved oxygen at a level above 6.5 mg / L to avoid the inhibitory effect of low dissolved oxygen conditions on gonadal development. Water quality parameters should be tested daily to ensure that ammonia nitrogen and nitrite concentrations are maintained within safe ranges.

[0056] The basic feed mainly consists of frozen triggerfish, with small amounts of squid, oysters, sandworms, etc.

[0057] In addition to the basic feed, add appropriate amounts of feed ingredients rich in high-quality protein and unsaturated fatty acids, and supplement with vitamins, taurine, arginine, and polyunsaturated fatty acids to promote yolk deposition and oocyte growth. Feed twice daily, at fixed times in the morning and afternoon. The amount of feed should be such that the parent animals can finish it within 30 minutes to avoid uneaten feed polluting the water. Change 5-10% of the water daily.

[0058] The basic feed consists of a coarsely ground mixture of triggerfish and squid at a mass ratio of 6:1, serving as the main protein source and flavor enhancer. Scallops, sandworms, oysters, and other shellfish are added at 10% of the total basic feed mass. For example, 100 portions of basic feed would include 77.14 portions of triggerfish, 12.86 portions of squid, 3.33 portions of scallops, 3.33 portions of sandworms, and 3.34 portions of oysters.

[0059] At the same time, additional nutrients are added to the basic feed at a weight percentage of 3.93% to 6.37%, including 0.8% to 1.2% deep-sea fish oil, 0.4% to 0.6% L-arginine, 0.6% to 0.9% coated vitamin E, 0.2% to 0.5% taurine, 1.0% to 1.5% aquatic-specific lecithin, 0.15% to 0.25% microencapsulated folic acid, 0.3% to 0.5% vitamin C, 0.2% to 0.5% arachidonic acid, 0.08% to 0.12% zinc methionine, and 0.2% to 0.3% inositol.

[0060] All feed should be refrigerated after preparation and thawed before each use, and the temperature should be controlled to not exceed room temperature to avoid loss of nutrients or spoilage.

[0061] Through continuous nutritional fortification, the energy reserve level of female parent fish gradually increases, providing a sufficient material basis for rapid ovarian maturation in the later stages.

[0062] 2. Regulation by environmental factors While the female parent fish were being nutritionally fortified, the temperature was slowly and controllably adjusted to induce changes in the development process of the female parent fish's ovaries.

[0063] First, the water temperature is slowly reduced from 26℃ to 22-23℃ at a rate of 0.5-1.0℃ per day, preferably 0.5-0.8℃, and maintained for 7-10 days. This stage, by simulating temperature changes in the natural environment, induces gonadal development into a new regulatory phase.

[0064] The water temperature is then gradually increased to 28-29℃ at the same rate, entering the rewarming activation phase. During rewarming, the rate of temperature increase is strictly controlled to avoid stress responses caused by sudden temperature changes. Through this environmental factor regulation method of cooling-rewarming, the synchronization of ovarian maturation can be significantly promoted.

[0065] After artificial maturation regulation, it can be clearly observed that the abdomen of the female parent fish gradually swells and becomes soft to the touch, the maturity of the ovaries is significantly improved, and the ovulation time tends to be concentrated, thus obtaining matured female parent fish, which provides a basis for eggs with consistent maturity for subsequent artificial insemination.

[0066] S3. Artificial insemination, the specific steps are as follows: 1. After completing the artificial maturation control of female black-spotted grouper in step S2 above, the female parent fish will show a bulging upper abdomen and a slightly reddish genital pore, indicating sexual maturity. Once the ovaries have reached maturity, artificial egg collection is performed on the matured female parent fish. Before egg collection, the female parent fish is gently restrained, and her abdomen and the area around the genital pore are wiped with a dry, clean towel to thoroughly remove any surface moisture and prevent premature contact with water, which could lead to unintended activation of the eggs. Then, moderate pressure is applied along the abdomen from front to back to allow the mature eggs to flow out naturally. The egg fluid is collected in a dry, clean container for later use. The entire egg collection process is conducted under waterless conditions. Figure 1 The diagram shows the process of collecting eggs from female parent fish.

[0067] 2. Male broodstock fish did not undergo artificial maturation induction and reached sexual maturity only under natural conditions. The method for collecting sperm from male broodstock fish corresponds to the process for collecting eggs from female broodstock fish. Before collection, the area around the male broodstock's genital opening was wiped clean to remove moisture. The sperm was then allowed to flow out naturally by gently pressing the abdomen and collected in a waterless container. The collected sperm was used for artificial insemination within a short period to ensure sperm motility. Figure 2 The diagram shown illustrates the process of collecting semen from male parent fish.

[0068] 3. Dry insemination: Under anhydrous conditions, the male broodstock semen collected in step (2) is placed in a container containing female broodstock eggs. The male broodstock semen and female broodstock eggs are gently stirred to ensure thorough mixing. Mixing is performed gently for 30-90 seconds, preferably 45-60 seconds, under anhydrous conditions to avoid damage to the egg membrane caused by vigorous stirring. During this process, since there is no contact with water, the sperm remain in an inactive state, thus preventing premature activation and energy consumption, while ensuring that the sperm can fully contact the eggs in an inactive state.

[0069] After thoroughly mixing the male parent fish sperm and the female parent fish egg fluid, a one-time water activation is completed within 1-3 minutes. The volume of water added at one time is 1-5 times the volume of the eggs (based on the apparent volume of the egg fluid), preferably 2.0-3.5 times the volume. The temperature of the added seawater is the same as or no more than 1°C different from the incubation water temperature, and the salinity is 33-35‰. After adding water, gently mix for 10-30 seconds, and then let it stand for 1-3 minutes to complete fertilization and obtain fertilized eggs.

[0070] The fertilized eggs are gently rinsed 1-2 times with seawater of the same salinity and then transferred to an incubation container. This allows the sperm activation, sperm-egg identification, and fertilization processes to be completed in a short time window. This unified activation method avoids the problem of sperm dispersion in the water and premature activation leading to decreased sperm motility.

[0071] By employing the aforementioned dry artificial insemination and sperm activation timing control process, the effective fertilization window of sperm can be effectively extended, the synchronous combination efficiency of sperm and heterologous eggs can be improved, and polyspermy and fertilization failure can be significantly reduced. Hybridized zygotes obtained using this method exhibit high fertilization rates and good early embryonic development, providing stable foundational conditions for subsequent embryo hatching and larval rearing.

[0072] S4. Hatching of fertilized eggs and cultivation of hybridization spots, the specific steps are as follows: 1. After artificial insemination, the fertilized eggs are allowed to settle and gently washed before being transferred to an incubation container for incubation. The incubation water temperature is controlled at 26-28℃, the salinity is maintained at 33-35‰, and the dissolved oxygen is kept above 6.5 mg / L. Continuous and slow aeration is provided to ensure uniform water distribution and prevent the fertilized eggs from settling or accumulating.

[0073] During incubation, the cleavage and embryonic development of the fertilized eggs are observed regularly, and abnormal and unfertilized eggs are removed promptly to maintain a stable incubation environment. During hybridization, the fertilized eggs obtained from the cross between female and male parent fish develop successfully, successively experiencing typical embryonic development stages such as the blastocyst stage, gastrula stage, and somites stage.

[0074] After fertilization, the fertilized egg is like Figures 3-5 As shown: Among them, Figure 3 The fertilized egg is in the blastocyst stage. The fertilized egg has a regular shape, a transparent membrane, and a clearly visible blastocoel. The embryonic cells are evenly distributed at one end of the membrane. The overall development is good, and no deformities or fragmentation are observed. Figure 4 As a fertilized egg in the gastrula stage, the cells gradually invaginate towards the center of the embryo, forming a distinct primitive opening. The cell arrangement gradually changes from scattered to regular, showing the process of early germ layer differentiation, and the embryonic development enters a critical stage. Figure 5The fertilized egg is in the somite stage. The embryo gradually elongates and segments, with obvious somite differentiation bands visible. The embryonic axis is clear, and the head and tail are initially differentiated, indicating that the hybrid fertilized egg can normally enter the pre-organogenesis stage, laying the foundation for subsequent hatching.

[0075] Further fertilization rate statistics showed that the fertilization rate of the fertilized eggs was 95.5%.

[0076] 2. To further improve the developmental stability of hybrid embryos of black-spotted grouper and saddleback grouper and the early survival rate of larvae, in addition to feeding according to the traditional methods used in the artificial domestication and rearing of black-spotted grouper or saddleback grouper during the period from fertilized egg hatching to early larval development, staged light regulation is introduced as a key developmental control technique. Based on the physiological characteristics of different developmental stages from hybrid embryo to larvae, the light intensity and photoperiod are specifically adjusted. The light intensity can be adjusted within ±10-20% according to the stocking density and water transparency. This aims to reduce light stress, optimize developmental rhythms, and promote the establishment of normal feeding behavior in larvae.

[0077] (1) Embryonic development period During the period from fertilization to embryonic development, incubation should be managed under low-light or light-protected conditions, and no feeding should be provided in the purified water. The incubation container should be placed in a light-protected environment or the external light source should be blocked with light-blocking materials to keep the light intensity of the incubation water below 100 lx, preferably under weak diffused light conditions.

[0078] During embryonic development, hybrid embryos are highly sensitive to changes in the external environment. Strong light stimulation can easily lead to increased embryonic metabolic activity and disrupted developmental rhythms. By reducing light intensity, the impact of external light stimulation on the embryo can be effectively reduced, minimizing metabolic fluctuations and stabilizing the cleavage process. This leads to a more consistent embryonic developmental rhythm, which is beneficial for the smooth progress of subsequent organ differentiation and somitosis formation.

[0079] By implementing the above-mentioned lighting control measures during embryonic development, abnormalities such as asymmetrical cleavage and developmental arrest can be significantly reduced, thereby improving the synchronicity and overall stability of embryonic development.

[0080] (2) Hybrid larvae's opening period (0-3 days after hatching) During the critical early stage of 0-3 days after hatching of hybrid larvae and before their first feeding, they should be fed nutritionally fortified rotifers (rich in DHA, vitamins and other nutrients), with the daily count density controlled at 10-20 rotifers / mL. Hybrid larvae are quite sensitive to external light stimulation. Strong light or long photoperiods can easily induce stress responses, leading to ineffective swimming and increased energy consumption, thus affecting early survival rates.

[0081] Therefore, during the initial feeding period of hybrid fry, light control management should be carried out under weak light or light-shielding conditions. It is preferable to control the light intensity of the seedling water body at 0-100 lx, and more preferably at 0-50 lx. If necessary, light-shielding materials can be used to block the external light source so that the water body maintains a weakly diffused dark light environment.

[0082] Using dark / light-protected conditions during the first 0-3 days of incubation can effectively reduce the impact of light stress on hybrid larvae, decrease energy consumption due to ineffective activity, and maintain the physiological stability of the larvae. It also helps improve the uniformity of group development, reduces the risk of early mortality caused by asynchronous individual development, and thus improves the survival rate of larvae in the first 0-3 days. After this stage, the larvae can be gradually transitioned to moderate lighting conditions based on their established feeding behavior to promote subsequent feeding recognition and growth.

[0083] Further analysis of the hatching rate revealed that it was 88.5%.

[0084] (3) Growth and development period of hybrid fry (4-8 days) Once the hybrid fry have started feeding for the first time and entered a stable feeding phase, copepods and artichoke larvae are introduced for feeding. The lighting conditions are further adjusted, gradually increasing the light intensity of the hatching water to 1600-1800 lx, and adopting a 14-hour light / 10-hour dark light cycle to establish a relatively stable diurnal light environment.

[0085] During this stage, moderately increasing light intensity can significantly improve the visual recognition ability of hybrid larvae to the external environment and food, enhance their active feeding behavior and feeding enthusiasm, and help them quickly establish a stable feeding pattern. By improving feeding efficiency, the nutritional intake level of hybrid larvae can be effectively improved, promoting their growth and development, and reducing the risk of early mortality caused by insufficient or unstable feeding.

[0086] During the adjustment of lighting conditions, a gradual increase in intensity should be adopted, such as increasing the light intensity by 100 lx per hour, to avoid a large increase in light intensity at one time, so as to prevent stress response of hybrid fry caused by sudden changes in the light environment, thereby affecting their normal growth and behavioral stability.

[0087] (4) Growth and development period of hybrid fry (9-20 days) When the hybrid fry have established a stable feeding habit and entered a stage of continuous growth and development (about 9-20 days after hatching), copepods and artichoke larvae are introduced for feeding. While keeping the established photoperiod conditions in step (3) unchanged (14 h light / 10 h darkness), the light intensity is adjusted in stages. The light intensity of the hatching water is gradually reduced from 1600-1800 lx in step (3) and stably controlled within the range of 800-1200 lx.

[0088] During this stage, the hybrid larvae have developed relatively complete feeding and movement abilities, and their identification of food no longer relies on high-intensity light. By maintaining a stable diurnal rhythm while appropriately reducing light intensity, the chronic photostress caused by continuous strong light to the hybrid larvae can be effectively reduced, the level of ineffective activity can be lowered, and more energy can be allocated to growth and tissue development.

[0089] Meanwhile, a moderate light intensity of 800-1200 lx can maintain the normal feeding behavior and group distribution of hybrid larvae, avoiding collisions caused by excessive light or decreased feeding efficiency caused by insufficient light. This ensures growth rate while improving the overall stability and survival rate of the hybrid larvae population. Furthermore, during the adjustment of light intensity from high to medium levels, a gradual reduction method is still used, for example, reducing the light intensity by 100 lx per hour, to avoid drastic changes in the light environment in a short period, thus preventing stress responses caused by sudden light changes that could affect the normal growth of the hybrid larvae.

[0090] (5) Late growth stage of hybrid fry (21-37 days) When the hybrid fry enter the late growth stage (about 21-37 days after hatching), introduce adult Artemia worms for feeding, and gradually add microparticle artificial compound feed with a protein content of more than 45% after the 30th day of age. On the basis of keeping the established photoperiod conditions in step (4) unchanged (14 h light / 10 h darkness), the light intensity is adjusted in stages, and the light intensity of the hatching water is gradually reduced from 800-1200 lx in step (4) and stably controlled in the range of 600-800 lx.

[0091] During this stage, the hybrid larvae have completed the establishment of their feeding system and basic behavioral patterns, and their dependence on light has further decreased. By using lower but stable light intensity, the chronic stress caused by continuous light stimulation to the hybrid larvae can be effectively reduced, as can ineffective swimming and energy consumption, allowing more energy to be used for weight gain and tissue development.

[0092] Meanwhile, a light intensity of 600-800 lx can maintain the normal feeding activities and group distribution of hybrid fry, avoiding adverse behaviors such as panic and collisions caused by excessive light. This ensures growth stability while improving group uniformity and survival rate. Furthermore, during the adjustment of light intensity from 800-1200 lx to 600-800 lx, a gradual reduction method is still used, for example, reducing the light intensity by 100 lx per hour. This avoids drastic changes in the light environment in a short period, preventing stress reactions in the hybrid fry caused by sudden changes in the light environment, which could affect their normal growth and health.

[0093] like Figure 6 The image shown is of a 35-day-old hybrid spotted fish obtained from the crossbreeding of female and male parent fish. The average body length is approximately 3 cm, and the average weight is 0.8 g (±0.1 g). The body is semi-transparent, with clearly visible muscle segments and vertebrae, indicating a critical stage in the transition from larvae to juveniles. The head is relatively large, the eyes are significantly enlarged and completely blackened, the mouth is fully developed, and the fish possesses the ability to actively feed. The fish is in good health.

[0094] Survival rate statistics show that the survival rate of hybrid spots at 35 days old is 50%.

[0095] (6) Hybrid juvenile stage (38-45 days) When the hybrid individuals develop to the juvenile stage (approximately 38-45 days after hatching), the juveniles have developed strong swimming abilities and stable feeding behaviors. At this stage, their dependence on live food or transitional feed is gradually reduced, and they are fed formulated feed with a particle size matching their mouth size to meet their needs for rapid growth and balanced nutrition. Simultaneously with the feed transition, the lighting conditions in the rearing water are adjusted accordingly, maintaining a photoperiod of 14 hours of light / 10 hours of darkness, and keeping the light intensity stably controlled within the range of 800-1000 lx. This ensures good recognition and feeding efficiency of the formulated feed for the hybrid juveniles while avoiding panic or stress reactions caused by excessive light.

[0096] By using moderate light intensity that matches the formulated feed during the juvenile stage, stable feeding behavior can be established, feed utilization can be improved, and the risk of growth stagnation or mortality due to feeding maladaptation during the initial feed transition can be reduced. This is beneficial to the healthy growth of juvenile fish and the improvement of group uniformity. The comprehensive effect of phased light control is evident.

[0097] By employing weak or dark conditions during embryonic development, dim or dark conditions during the larval feeding stage, and gradually increasing light intensity and setting a reasonable photocycle after the larvae enter a stable feeding stage, the hybrid embryos and larvae are kept in a relatively stable light environment that matches their physiological needs throughout the early development process.

[0098] Continue training until 65 days old, if Figure 7 As shown, the average body length of the hybrid spotted fish is about 4.5 cm, and the average weight is 4.5 g (±0.5 g). The body is completely opaque, showing the typical dark brown or black base color of an adult fish. The body surface is covered with irregular golden or beige patches and stripes, and there is a distinct ring-shaped color band at the caudal peduncle. All fins are fully developed, with thick fin rays of the dorsal fin, caudal fin, and pectoral fin with light-colored edging. The spines of the anterior part of the dorsal fin have hardened, and the pelvic and anal fins have also formed. The fish is in good health.

[0099] Survival rate statistics show that the survival rate of hybrid spots at 65 days old is 40%.

[0100] S5. Identification of hybridization spots, the specific steps are as follows: To verify the genetic origin and authenticity of the hybrid spots of female black-striped grouper and male saddle-banded grouper, the authenticity of the hybrid spots was determined at the molecular level.

[0101] 1. DNA extraction Muscle tissue was collected from female and male broodstock fish, as well as from hybrid spots, for DNA extraction. The DNA extraction method was performed according to the instructions of the marine animal genome extraction kit. The specific steps for DNA extraction are as follows: (1) DNA was extracted from the above-mentioned female broodstock, male broodstock, and 35-day-old hybrid fish using a marine animal genome extraction kit (Tiangen Biochemical; all reagents and consumables below are from this kit). The DNA extraction steps included: 1) Sample pretreatment: Weigh 30 mg of muscle tissue, add 200 μL of GA buffer to a centrifuge tube, mix gently, and shake for 15 seconds to ensure complete infiltration of muscle tissue.

[0102] 2) Proteinase K digestion: Add 20 mL of Proteinase K solution (20 mg / mL) to a centrifuge tube, mix thoroughly, and incubate at 56°C until the tissue is completely dissolved.

[0103] 3) Pyrolysis and precipitation: Add 200 μL of GB lysis buffer, mix thoroughly, place in a 70℃ water bath for 10 minutes, then add 200 μL of anhydrous ethanol and mix well.

[0104] 4) Adsorption column purification: Transfer the mixture to a CB3 adsorption column, place it in a collection tube, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid. Then add 500 μL of GD buffer and 600 μL of PW wash buffer, centrifuge for 30 seconds each time, and wash the adsorption column to remove impurities.

[0105] 5) Drying the adsorption column: Place the cleaned adsorption column back into a centrifuge tube, centrifuge at 12,000 rpm for 2 minutes, and then let it stand at room temperature for 2-5 minutes to ensure that the ethanol in the adsorption column is completely evaporated.

[0106] 6) DNA elution: Place the adsorption column into a new centrifuge tube, add 50-200 μL of sterile water to the middle of the adsorption column, let it stand at room temperature for 2 minutes, then centrifuge at 12000 rpm for 2 minutes and collect the eluted DNA solution.

[0107] 7) Quality Inspection and Preservation: Use a spectrophotometer to detect the concentration and purity of DNA to ensure OD. 260 / OD 280 Between 1.8 and 2.0, OD 260 / OD 230 Greater than 2.0. Genomic DNA of female broodstock, male broodstock, and hybrid spot was obtained respectively.

[0108] 2. Reference genome alignment Using the genomic DNA of the male parent fish from step 1 above, a third-generation genomic library was constructed and sequenced. The male parent fish genome was used as a reference, and the female parent fish genome (referring to the genomic information recorded in the non-patent literature Zhao B, Jin C, Jiang Y, et al. Chromosome-level genome assembly of the coral grouper, Epinepheluscorallicola and its evolutionary insights into Eupercaria[J]. BMC Genomics, 2025, 2025 (000). DOI:10.1186 / s12864-025-11996-x.) was used for whole-genome alignment. Genetic variation sites with stable differences between male and female parent fish were screened. These genetic variation sites included insertion / deletion sites (InDel), single nucleotide polymorphism sites (SNPs), and their combinations. Genetic variation sites that showed significant differences between male and female parent fish and had unique localization characteristics were selected as candidate sites.

[0109] Two candidate loci were identified between male and female parent fish: (1) The first candidate site is marked as 1656, corresponding to the male parent fish (NC_047009.1:16568637-16568906 on NCBI, a total of 270 bp) and the female parent fish (Chr10_hap1:20039898-20040228 in the female parent fish genome, a total of 331 bp); (2) The second candidate site is marked as 2778, corresponding to the male parent fish (NC_047009.1:23018317-23018586 on NCBI, totaling 270 bp) and the female parent fish (female parent fish genome Chr10_hap1:32426960-32427286, totaling 327 bp).

[0110] 3. Primer design and optimization Primers were designed for the two candidate sites obtained in step 2 above (highly conserved InDel regions (more than 50 qualified fragments before and after each) using Primer3 software. The product length was set to 100~300 bp, and the Tm value, GC content and secondary structure parameters were optimized to screen out candidate primer pairs. The specific primer sequences are shown in Table 3.

[0111] Table 3 Primer sequence listing ;

[0112] 4. Specificity verification Both primer pairs in step 3 above have been BLAST-aligned with the parent genomes to ensure a unique matching site only in the target InDel region. Primer pairs with specificity and no non-specific amplification are used as the final genotyping primers.

[0113] The genomic DNA of the female parent fish, the genomic DNA of the male parent fish, and the hybrid spot obtained in step 1 were amplified by PCR using the two primer pairs in Table 3 above (1656-F and 1656-R are one primer pair, and 2778-F and 2778-R are one primer pair).

[0114] (1) The PCR amplification reaction system is as follows: Genomic DNA (100 ng / μL), 1 μL; Forward primer Fw (10 μM), 1 μL; Reverse primer Rw (10 μM), 1 μL; 2×Taq Plus Master Mix Ⅱ (Plus), 12.5 μL; Enzyme-free sterile water, 9.5 μL.

[0115] (2) The PCR amplification reaction procedure is as follows: Pre-denaturation at 95℃ for 3 min; Denaturation at 95℃ for 15 seconds; Anneal at 56℃ for 20 seconds; The denaturation and extension process was performed at 72°C for 40 seconds, with a total of 32 cycles. Extend the heat at 72℃ for 10 minutes. Store at 4°C.

[0116] After the PCR reaction was completed, PCR products of female parent fish, male parent fish, and hybrid spot PCR products were obtained. The PCR amplification products were detected by 1.5% agarose gel electrophoresis.

[0117] The results are as follows Figure 8 As shown, all hybrid individuals amplified allelic bands exhibiting characteristics of both the father and mother, clearly distinguishing and confirming their hybrid status. This demonstrates that the offspring individuals obtained by this invention inherit the genetic information of their parents at the molecular level, further verifying the reliability of the hybridization breeding results.

[0118] Comparative Example 1 The difference between this comparative example and the previous example is that this comparative example does not involve artificial maturation control of female parent fish.

[0119] The results showed that the female and male parent fish matured at different times, and the ovulation time of the female parent fish was out of sync with the sperm release time of the male parent fish, making it impossible for sperm and egg to combine and obtain fertilized eggs.

[0120] Comparative Example 2 The difference between this comparative example and the previous example is that the female parent fish in this comparative example were not given a 45-day advance nutritional enhancement treatment during artificial maturation.

[0121] The results showed that female parent fish deposited less yolk material, oocytes grew more slowly, and the ovaries could not mature quickly in the later stages. This caused the ovulation time of female parent fish to be out of sync with the sperm release time of male parent fish, making it impossible for sperm and egg to combine and obtain fertilized eggs.

[0122] Therefore, only by subjecting the female parent fish to a 45-day pre-treatment with nutritional enhancement before artificial maturation can the yolk material in the female parent fish be deposited, the ovaries mature rapidly, and the sperm and egg combine normally to obtain fertilized eggs.

[0123] Comparative Example 3 The difference between this comparative example and the previous example is that the female parent fish in this comparative example underwent pre-treatment with enhanced nutrition 30 days earlier during artificial maturation.

[0124] The results showed that the amount of yolk sac deposited and the growth rate of oocytes in female parent fish were higher than those in Comparative Example 2, but there was still a significant difference compared to the Example. The ovarian maturation rate was still slow in the later stage, which caused the ovulation time of female parent fish to be out of sync with the sperm release time of male parent fish, making it impossible for sperm and egg to combine and obtain fertilized eggs.

[0125] Therefore, only by subjecting the female parent fish to a 45-day pre-treatment with nutritional enhancement before artificial maturation can the yolk material in the female parent fish be deposited, the ovaries mature rapidly, and the sperm and egg combine normally to obtain fertilized eggs.

[0126] Comparative Example 4 The difference between this comparative example and the embodiment is that this comparative example uses wet insemination during the artificial insemination process.

[0127] The results showed that the effective fertilization window of male parent fish sperm was shortened, the synchronous binding efficiency of male parent fish sperm and heterologous eggs of female parent fish was significantly lower than in the previous example, and the fertilization rate was significantly reduced.

[0128] Therefore, only by conducting a 45-day pre-treatment with nutritional enhancement before artificially inducing maturation in female parent fish, so that the ovulation time of female parent fish corresponds to the sperm release time of male parent fish, and then performing dry artificial insemination, can the fertilization rate of fertilized eggs reach a technical effect of over 95%.

[0129] Comparative Example 5 The difference between this comparative example and the previous example is that this comparative example uses fixed light conditions for the hatching of fertilized eggs and the cultivation of hybrid spots, adopts a constant cycle throughout the process, and the light intensity does not change with the developmental stage.

[0130] The results showed that embryonic development in this comparative example was not concentrated, the proportion of abnormalities was significantly higher than in the example, the opening time of hybrid spots was not concentrated, and the hatching rate of fertilized eggs and the survival rate of hybrid spots at 35 days of age were significantly lower than in the example.

[0131] Therefore, feeding with nutritional feed alone cannot significantly improve the hatching rate of fertilized eggs and the survival rate of hybrid spots at 35 days old. It is necessary to combine it with staged light control in order to achieve the technical effect of a hatching rate of 88.5% for fertilized eggs and a survival rate of 50% for hybrid spots at 35 days old.

[0132] Comparative Example 6 The difference between this comparative example and the embodiment is that this comparative example uses the traditional method of feeding fish during the artificial domestication and breeding of black barbel grouper or saddle grouper at this stage, and uses the same light control at the entry stage as the key development control technology as in this embodiment. Finally, the survival rate of hybrid groupers at 35 days and 65 days is statistically analyzed.

[0133] The results showed that the survival rates of hybrid spots aged 35 days and 65 days were significantly lower than those in this embodiment. In particular, the survival rate of 65-day-old hybrid spots in this comparative example was only 20%, which was half the survival rate in this embodiment.

[0134] Therefore, using staged lighting control alone cannot significantly improve the survival rate of hybrid spots. It needs to be combined with nutritious feed to achieve the technical effect of a 50% survival rate for 35-day-old hybrid spots and a 40% survival rate for 65-day-old hybrid spots.

[0135] Therefore, it can be concluded that this patent provides an artificial breeding method for a hybrid of black bream and saddleback grouper. This application is not a simple superposition of existing breeding measures, but rather achieves this by: (1) using artificially domesticated wild black bream as female parent fish and artificially cultured saddleback grouper as male parent fish. Under non-hormonal conditions, the female parent fish undergoes 45 days of nutritional enhancement and temperature slow-change to artificially promote ovarian maturation, while the male parent fish matures under natural conditions, thus achieving an effective match between the ovarian development process of female black bream and the sperm ejaculation rhythm of male saddleback grouper; (2) further utilizing the sperm activation timing control in dry artificial insemination to improve the efficiency of heterologous sperm-egg combination and enhance the stability of early hybrid embryo development; (3) The synergistic effect of staged lighting regulation matching the embryonic to larval development stages forms a complete and controllable hybrid breeding regulation system, resulting in hybrid spots with a fertilization rate of over 95%, a hatching rate of over 88% for fertilized eggs, a survival rate of 50% for 35-day-old hybrid spots, and a survival rate of 40% for 65-day-old hybrid spots. This fills the current technological gap in hybrid breeding using black-spotted grouper as female parent fish. Furthermore, the authenticity of the hybrid spots obtained by hybridizing black-spotted grouper and saddle-banded grouper was verified using two pairs of primers. The authenticity is reliable, and the hybrid spots inherit the genetic information of both female and male parent fish at the molecular level, laying the foundation for obtaining superior grouper breeds and also increasing the economic value of black-spotted grouper and saddle-banded grouper.

[0136] In the foregoing description of exemplary embodiments / specific implementations of this patent, various features of this patent are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, unless expressly stated otherwise or in obvious technical contradiction or exclusion, the descriptive method of this patent should not be construed as reflecting an intention that the claimed features of the invention are more than expressly stated in each claim. Rather, the inventive aspect reflected in the claims lies in not all the features of a single foregoing disclosed embodiment / specific implementation. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim existing independently as a separate embodiment / specific implementation of this patent.

[0137] The terms and expressions used in this specification are for illustrative purposes and not for limitation. Their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to facilitate the understanding that various modifications may be possible within the scope of this patent claim. Therefore, it should be understood that while this patent has been specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, variations or modifications of the concepts disclosed herein may be adopted by those skilled in the art, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it will be apparent to those skilled in the art that this patent can be implemented using many variations of the devices, device components, and method steps disclosed herein.

[0138] The foregoing description of specific embodiments fully discloses the general features of this patent, enabling others to easily modify and / or adapt such embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation or deviation from the general concept of this patent. Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0139] Furthermore, the scope of this patent should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents.

Claims

1. A method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper, characterized in that, Includes the following steps: Step A: Using healthy black-spotted grouper as female parent fish, and through nutritional fortification and artificial maturation, fortified female parent fish are obtained; Healthy saddle-tailed grouper were used as male parent fish, and sexually mature male parent fish were obtained through natural sexual maturation; so that the ovulation time of the enhanced female parent fish was consistent with the sperm release time of the sexually mature male parent fish. The artificial ripening process involves lowering the temperature from 26±0.5℃ to 22.5±0.5℃ at a rate of 0.5℃ per day for 7-10 days, followed by raising the temperature to 28.5±0.5℃ at a rate of 0.5℃ per day to regulate the water temperature. Step B: Collect the egg fluid of the enhanced female parent fish and the sperm of the sexually mature male parent fish, fertilize them to obtain fertilized eggs, and then cultivate them under controlled light after embryo hatching and opening to obtain hybrid spots; Step C: Use primer pairs to verify the authenticity of the hybrid spot and determine that the hybrid spot inherits the genetic information of both the female and male parent fish; The primer pairs are sequence pairs consisting of SEQ ID NO.1 and SEQ ID NO.2 and sequence pairs consisting of SEQ ID NO.3 and SEQ ID NO.

4.

2. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 1, characterized in that, Step B includes: Step B-1: Collect the egg fluid of the enhanced female parent fish and the semen of the sexually mature male parent fish, and obtain fertilized eggs after dry insemination and activation; Step B-2: The fertilized eggs are subjected to embryo incubation and staged light-controlled culture after opening to obtain hybrid spots.

3. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 2, characterized in that, In step B-2, The embryo hatching process involves the cleavage stage of the fertilized egg to the embryonic development stage, and is carried out under conditions of avoiding light or weak light, such that the light intensity is less than or equal to 100 lx.

4. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 2, characterized in that, The specific method of staged light-controlled cultivation after opening is as follows: 0-3 days old: Use dark or low light conditions, so that the light intensity is less than or equal to 100 lx; 4-8 days old: Increase the light intensity gradient to 1600-1800 lx; 9-20 days old: Reduce light intensity from 1600-1800 lx to 800-1200 lx in a gradual manner; 21-37 days old: Reduce light intensity from 800-1200 lx to 600-800 lx; 38-45 days old: Gradually increase the light intensity from 600-800 lx to 800-1000 lx; The photoperiod for the 4-45 day old animals was 14 hours of light and 10 hours of darkness; the gradient decrease was to reduce the light intensity by 100-200 lx per hour, and the gradient increase was to increase the light intensity by 100-200 lx per hour.

5. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 2, characterized in that, The water temperature for the fertilized eggs during the staged light-controlled cultivation after embryo hatching and opening was 25-27℃, the salinity was 33-35‰, the dissolved oxygen concentration was above 6.5 mg / L, and the pH value was 7.9-8.

4.

6. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 1, characterized in that, Specifically, step C is as follows: DNA was extracted from the female parent fish, the male parent fish, and the hybrid spot to obtain the genomic DNA of the female parent fish, the genomic DNA of the male parent fish, and the genomic DNA of the hybrid spot, respectively. Then, PCR identification was performed using the primer pairs to confirm that the hybrid spot inherited the genetic information of the female parent fish and the male parent fish.

7. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 6, characterized in that, The PCR identification system was a 25 μL amplification system; The 25 μL amplification system comprises: 1 μL of genomic DNA at a concentration of 100 ng / μL; 1 μL of forward primer Fw at a concentration of 10 μM; 1 μL of reverse primer Rw at a concentration of 10 μM; 12.5 μL of 2×Taq Plus Master Mix II; and 9.5 μL of enzyme-free sterile water. The PCR identification procedure includes the following steps: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 56℃ for 20 s, extension at 72℃ for 40 s, for a total of 32 cycles from denaturation to extension, followed by a full extension at 72℃ for 10 min.

8. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 1, characterized in that, In step A The standard for health is: no disease, no injury, no deformity, robust physique, and no hormone-induced labor or ovarian induction treatment; The weight of the female parent fish is ≥1.0 kg, and the weight of the male parent fish is ≥3.0 kg.

9. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to claim 8, characterized in that, The nutritional fortification involves feeding the patient with fortified feed after full-feeding. The fortified feed includes a basic feed and a nutrient agent. The nutrient agent is added at 3.93% to 6.37% of the weight of the basic feed. The nutrient agent includes 0.8% to 1.2% deep-sea fish oil, 0.4% to 0.6% L-arginine, 0.6% to 0.9% coated vitamin E, 0.2% to 0.5% taurine, 1.0% to 1.5% aquatic-specific lecithin, 0.15% to 0.25% microencapsulated folic acid, 0.3% to 0.5% vitamin C, 0.2% to 0.5% arachidonic acid, 0.08% to 0.12% zinc methionine, and 0.2% to 0.3% inositol.

10. The method for artificially cultivating a hybrid of black-spotted grouper and saddle-banded grouper according to any one of claims 1 to 9, characterized in that, The fertilization rate of the fertilized eggs is ≥95%, and the hatching rate of the fertilized eggs is ≥88%. The survival rate of the hybrid spots is ≥50% at 35 days of age and ≥40% at 65 days of age.

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