Method for breeding interspecific hybrid of pteria penguin and pinctada martensii

Abstract

This invention discloses a method for interspecific hybridization and seedling cultivation of *Pterocarya spp.* and *Pterocarya martensii*, comprising: selecting mature, live male *Pterocarya spp.*, cleaning and drying them, placing them in seawater, continuously aerating and artificially circulating the water to induce natural sperm release; dissecting mature female *Pterocarya martensii* to extract gonads, collecting mature eggs, cleaning them, and allowing them to settle to obtain an egg mixture; adding ammonia to the egg mixture to activate the eggs; adding the activated egg mixture to a sperm-containing water body for fertilization in the dark; after successful fertilization, removing the male *Pterocarya spp.* and transferring the fertilized eggs to a seedling pond; and cultivating the fertilized eggs in seawater to develop into D-shaped larvae. This invention is the first of its kind for distant hybridization and seedling cultivation of *Pterocarya spp.* and *Pterocarya martensii*, breaking through the reproductive isolation between the two species, achieving stable and efficient hybridization and fertilization. The process is simple and highly stable, and can cultivate new hybrid pearl oyster seedlings that combine the excellent traits of both parents. It has significant implications and promotional value for the innovation of marine pearl oyster germplasm and industrial upgrading.

Description

Technical Field

[0001] This invention belongs to the field of marine shellfish breeding technology, specifically relating to a method for interspecific hybridization and seedling raising of the white-lipped oyster and the Pinctada martensii. Background Technology

[0002] White butterfly shell ( Pinctada maxima As a large marine pearl oyster, the oyster is the exclusive mother oyster for cultivating high-quality South Sea pearls. It has the core advantages of large shell, thick nacre, good pearl luster, and large pearl size. However, its natural reproduction conditions are harsh, the artificial seedling release rate is low, and the seedling survival rate is extremely low. Large-scale seedling cultivation has always been a technical challenge for the industry. The Pinctada martensii is the dominant species in my country's marine pearl farming. It has the characteristics of rapid gonadal development, strong reproductive capacity, mature artificial seedling technology, and strong environmental adaptability. However, it has the defects of small individuals and limited pearl quality and size.

[0003] Currently, marine pearl oyster breeding technology is limited to artificial propagation within the same species. Interspecific distant hybridization faces significant technical barriers, including strict reproductive isolation, poor sperm-egg recognition compatibility, difficulty in combining distant sperm and eggs, high rates of embryonic malformation, and inability to survive to the larval stage. Through domestic and international patent searches, academic literature reviews, and industry technical data verification, no expert, scholar, or technician has yet successfully synthesized male sperm from the white-lipped pearl oyster (Pterocarpus spp.) with that of the Pinctada martensii (Pterocarpus natans). Pinctada martensii Artificial induction of hybridization and fertilization of female eggs to obtain surviving hybrid larvae is generally considered in the industry to be unfeasible for distant hybridization of the two pearl oyster species, which greatly restricts the improvement of pearl oyster germplasm, the cultivation of superior hybrid seedlings and the upgrading and development of the marine pearl industry. Summary of the Invention

[0004] The purpose of this invention is to address the problems in the existing technology of reproductive isolation between the white-lipped oyster and the pinnatifida oyster, the inability to fertilize interspecific hybridization, the inability to obtain surviving larvae, and the lack of distant hybridization seedling technology. This invention provides a method for interspecific hybridization seedling production of the white-lipped oyster and the pinnatifida oyster that breaks through the reproductive isolation between species and achieves high fertilization rate, high larval survival rate, and high stability.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A method for interspecific hybridization and seedling cultivation of *Pterocarya stenoptera* and *Pterocarya martensii* includes the following steps: (1) Treatment of male white-lipped oysters to induce sperm release: Select live male white-lipped oysters with mature gonads, clean and dry them, and place them in disinfected seawater with a water temperature of 26-28℃ and a salinity of 28-30‰. Continuously oxygenate and artificially circulate the water for 1-2 hours to induce natural sperm release in the male oysters, and control the sperm density to 1.2×10 8 -1.5×10 8 cells / mL; (2) Collection and purification of female eggs of Pinctada martensii: The gonads of mature live Pinctada martensii oysters were dissected and the gonads were removed. The gonads were cleaned to remove the mucus, and the mature eggs were extracted, cleaned, allowed to settle, and the upper layer of seawater was removed, leaving 500 mL of seawater and egg mixture. (3) Chemical activation induction of oocytes: Add 0.15-0.2 mL of analytical grade ammonia to the oocyte mixture, stir gently, and activate for 10-20 minutes until the oocytes are regular and round; (4) Hybrid fertilization: The activated egg mixture is added in portions to the water containing the white butterfly oyster sperm from step (1), and fertilization is carried out in a static, light-protected environment at a water temperature of 26-28℃. (5) Fertilization detection and transfer of fertilized eggs: After hybridization fertilization, the male white butterfly oysters were removed and the fertilized eggs were left to settle for 30 minutes. Half of the seawater in the upper layer was removed and the remaining fertilized eggs and seawater were transferred to the nursery pond. (6) Larval hatching and development: Fertilized eggs are cultivated at a water temperature of 26-28℃ and a salinity of 28-30‰. After 8-10 hours, they develop into D-shaped larvae.

[0006] Preferably, in step (1), the shell length of the male parent white butterfly oyster is 18-22 cm.

[0007] Preferably, in step (1), the dissolved oxygen content in the water is ≥5 mg / L.

[0008] Preferably, in step (1), the water flow velocity of the artificial water diversion stimulation is 0.8-1.2 m / s.

[0009] Preferably, in step (2), the shell length of the Pinctada martensii mother oyster is 6-8 cm.

[0010] Preferably, in step (2), the eggs are washed using seawater filtered through a 300-mesh sieve, and the egg density is controlled at 8 × 10⁻⁶. 5 -1×10 6 per mL.

[0011] Preferably, in step (4), when adding the egg mixture in several batches, the amount added each time is 150-200 mL, with an interval of 5 minutes between each batch. During this time, the water is gently stirred to avoid excessively high local sperm concentration.

[0012] Preferably, in step (5), fertilization is confirmed to be successful 60 minutes after hybridization fertilization.

[0013] Preferably, in step (5), the hybridization fertilization rate is ≥85%.

[0014] Preferably, in step (6), the survival rate of D-shaped larvae is ≥70%.

[0015] The present invention has the following beneficial effects: (1) Outstanding technological innovation: This invention is the result of 50 years of practice and experimentation in the pearl oyster industry. It is the first in the world to achieve interspecific hybridization and fertilization between male white butterfly oysters and female Pinctada martensii, and successfully obtain surviving D-shaped larvae. It completely breaks the reproductive isolation between the two types of oysters, fills the gap in pearl oyster distant hybridization and seedling breeding technology at home and abroad, and subverts the traditional technical understanding of the industry.

[0016] (2) Stable and efficient fertilization and survival rate: Through precise parental stimulation, chemical activation of eggs and step-by-step fertilization process, the hybridization fertilization rate is ≥85%, and the survival rate of D-shaped larvae in 8-10 hours is ≥70%, which is far higher than the conventional technology level of distant hybridization. The technology is highly stable, repeatable, and suitable for large-scale seedling production.

[0017] (3) Simple and controllable process: The whole seedling process does not require complicated equipment, the reagent cost is low, the operation process is standardized, and ordinary seedling technicians can operate it after simple training, which is suitable for the actual production conditions of seawater pearl seedling farms.

[0018] (4) Significant value in germplasm improvement: The hybrid offspring combine the advantages of large size and excellent pearl quality of the white butterfly oyster and strong reproductive capacity and easy seedling of the Pinctada martensii, providing core technical support for cultivating new high-quality pearl oyster germplasm and improving the yield and quality of seawater pearls, thus promoting the leapfrog development of the seawater pearl industry.

[0019] (5) Thorough experimental verification: This technology has been repeatedly tested hundreds of times and can stably achieve hybridization fertilization and larval development. It has a high degree of technical maturity, strong practicality, and extremely high industrial promotion value. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] 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.

[0022] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0023] The analytical grade ammonia water used in the embodiments of the present invention is a common commercially available reagent with a concentration of 25%-28% (w / w).

[0024] Example 1 A method for interspecific hybridization and seedling cultivation of *Pearl Oyster* and *Pearl Oyster*, with the specific operating steps as follows: 1. Treatment of male broodstock white butterfly oysters to induce sperm release Four live male white-lipped oysters, each with a shell length of 20cm, full and mature gonads, and strong vitality, were selected. The shells were repeatedly rinsed several times with sterile sand-filtered seawater to thoroughly remove mud, algae, and other attached organisms. After drying the oysters in the sun for 30-60 minutes, they were placed in a 1-ton plastic bucket. Clean seawater that had undergone sedimentation, sand filtration, and ultraviolet disinfection was added. The water temperature was strictly controlled at 27℃, and the salinity at 29‰. Continuous oxygenation was maintained throughout the process to ensure dissolved oxygen ≥5mg / L (e.g., 6mg / L). A small electric water pump was used to increase the water temperature by approximately 2 degrees Celsius, and a continuous water flow of 1.0m / s was applied for 1.5 hours to induce synchronous natural sperm release in the male oysters. The process continued until the water turned a uniform milky white color and the sperm density reached 1.3×10⁻⁶. 8 When the concentration is 100 cells / mL, it is ready for use.

[0025] 2. Collection and purification of female eggs from Pinctada martensii. Ten live female Pinctada martensii oysters, each with a shell length of 7 cm and mature gonads (orange-red and plump), were selected. After aseptic dissection, the gonadal tissue was quickly removed. The gonads were rinsed with seawater filtered through a 300-mesh screen to thoroughly remove mucus, blood, and tissue debris. Mature eggs were extracted using a sterile pipette and transferred to a 1L sterile container. The container was rinsed three times with filtered seawater, allowing it to stand for 10 minutes each time to remove impurities and damaged eggs. The egg mixture was allowed to stand for 30 minutes until the mature eggs had completely settled to the bottom. Excess seawater was then siphoned off, leaving a final 500mL mixture of seawater and eggs, with the egg density controlled at 9 × 10⁻⁶. 5 per mL.

[0026] 3. Chemical activation and induction treatment of oocytes Analytical grade ammonia was used as the oocyte activation reagent. Ammonia was precisely drawn up with a sterile dropper and slowly added to the oocyte mixture with 3 drops (approximately 0.05 mL per drop). The mixture was then gently and evenly stirred with a sterile glass rod for 1 minute to ensure full contact between the ammonia and the oocytes. After 15 minutes of activation treatment, microscopic observation revealed that the oocytes had shrunk to a regular round shape and the oocyte membrane had expanded, indicating that oocyte maturation and activation were complete, significantly enhancing the ability of the oocytes to combine with heterologous sperm.

[0027] 4. Controlled hybridization fertilization procedure After the male white butterfly oysters naturally release sperm and the sperm density reaches the required level, slowly add 500mL of activated egg mixture to a 1-ton water tank containing sperm in three separate additions, each adding 150-200mL at 5-minute intervals, while gently stirring the water to prevent excessively high local sperm concentration. Maintain a water temperature of 27℃ and a still, dark environment throughout the process to allow the sperm and eggs to naturally combine and complete hybridization fertilization.

[0028] 5. Fertilization detection and transfer of fertilized eggs Sixty minutes after hybridization and fertilization, a small amount of egg fluid was aspirated with a sterile dropper and observed under a biological microscope. The appearance of the first polar body on the outer layer of the egg indicated successful fertilization, with a fertilization rate of 88%. After confirming successful fertilization and normal division and development of the fertilized eggs, all male white-lipped oysters were immediately removed from the water to prevent the parent oysters' metabolic products and feces from polluting the water and affecting the development of the fertilized eggs. The fertilized eggs were then left to stand for another 30 minutes until they completely sank to the bottom of the bucket. The top 50% of the seawater was then siphoned off, and the remaining fertilized eggs and seawater were transferred together to a 20-ton nursery tank containing pre-sterilized water.

[0029] 6. Larval Hatching and Development The seawater conditions in the nursery pond are the same as those in the fertilization water: water temperature 27℃, salinity 29‰, dissolved oxygen 6mg / L. The fertilized eggs hatch naturally in the pond. During the cultivation process, the development of the fertilized eggs is observed. After 4.5 hours, trochophore larvae are formed and float to the upper layer of the water. After 8 hours of continuous cultivation, microscopic examination shows that the larvae have successfully metamorphosed into D-shaped larvae with complete morphology and normal vitality. The larval survival rate is stable at 73%, completing the initial cultivation of hybrid seedlings, and can enter the subsequent juvenile shell cultivation stage.

[0030] Example 2 A method for interspecific hybridization and seedling cultivation of *Pearl Oyster* and *Pearl Oyster*, with the specific operating steps as follows: 1. Treatment of male broodstock white butterfly oysters to induce sperm release Three live male white-lipped oysters, each with a shell length of 22cm, full and mature gonads, and strong vitality, were selected. The shells were repeatedly rinsed several times with sterile sand-filtered seawater to thoroughly remove mud, algae, and other attached organisms. After drying the male oysters in the sun for 30-60 minutes, they were placed in a 2-ton plastic tank with clean seawater that had undergone sedimentation, sand filtration, and ultraviolet disinfection. The water temperature was strictly controlled at 26℃, and the salinity at 30‰. Continuous oxygenation was maintained throughout the process to ensure dissolved oxygen ≥5mg / L (e.g., 6mg / L). A small electric water pump was used to increase the water temperature by approximately 3 degrees Celsius, and a continuous water flow of 0.8m / s was applied for 2 hours to induce synchronous natural sperm release in the male oysters. The process continued until the water turned a uniform milky white and the sperm density reached 1.5×10⁻⁶. 8 When the concentration is 100 cells / mL, it is ready for use.

[0031] 2. Collection and purification of female eggs from Pinctada martensii. Ten female Pinctada martensii oysters with a shell length of 8 cm and mature gonads (orange-red and plump) were selected. After aseptic dissection, the gonadal tissue was quickly removed. The gonads were rinsed with seawater filtered through a 300-mesh screen to thoroughly remove mucus, blood, and tissue debris. Mature eggs were extracted using a sterile pipette and transferred to a 1L sterile container. The container was rinsed three times with filtered seawater, allowing it to stand for 10 minutes each time to remove impurities and damaged eggs. The egg mixture was allowed to stand for 30 minutes until the mature eggs had completely settled to the bottom. Excess seawater was then siphoned off, leaving a final 500mL mixture of seawater and eggs, with the egg density controlled at 1×10⁻⁶. 6 per mL.

[0032] 3. Chemical activation and induction treatment of oocytes Analytical grade ammonia was used as the oocyte activation reagent. Ammonia was precisely drawn up with a sterile dropper and slowly added to the oocyte mixture with 4 drops (each drop approximately 0.05 mL). The mixture was then gently and evenly stirred with a sterile glass rod for 1 minute to ensure full contact between the ammonia and the oocytes. After 10 minutes of activation treatment, microscopic observation revealed that the oocytes had shrunk to a regular round shape and the oocyte membrane had expanded, indicating that oocyte maturation and activation were complete, significantly enhancing the ability of the oocytes to combine with heterologous sperm.

[0033] 4. Controlled hybridization fertilization procedure After the male white butterfly oysters naturally release sperm and the sperm density reaches the required level, slowly add 500mL of activated egg mixture to a 1-ton water tank containing sperm in three separate additions, each adding 150-200mL at 5-minute intervals, while gently stirring the water to prevent excessively high local sperm concentration. Maintain a water temperature of 26℃ and a still, dark environment throughout the process to allow the sperm and eggs to naturally combine and complete hybridization fertilization.

[0034] 5. Fertilization detection and transfer of fertilized eggs Sixty minutes after hybridization and fertilization, a small amount of egg fluid was aspirated with a sterile dropper and observed under a biological microscope. The appearance of the first polar body on the outer layer of the egg indicated successful fertilization, with a fertilization rate of 86%. After confirming successful fertilization and normal division and development of the fertilized eggs, all male white-lipped oysters were immediately removed from the water to prevent the parent oysters' metabolic products and feces from polluting the water and affecting the development of the fertilized eggs. The fertilized eggs were then left to stand for another 30 minutes until they completely sank to the bottom of the bucket. The top 50% of the seawater was then siphoned off, and the remaining fertilized eggs and seawater were transferred together to a 20-ton seedling tank containing pre-sterilized and prepared water.

[0035] 6. Larval Hatching and Development The seawater conditions in the nursery pond are the same as those in the fertilization water: water temperature 26℃, salinity 30‰, dissolved oxygen ≥5mg / L (e.g., 6mg / L). The fertilized eggs hatch naturally in the pond. During the cultivation process, the development of the fertilized eggs is observed. After 4 hours, trochophore larvae are formed and float to the upper layer of the water. After 8 hours of continuous cultivation, microscopic examination shows that the larvae have successfully metamorphosed into D-shaped larvae with complete morphology and normal vitality. The larval survival rate is stable at 71%, completing the initial cultivation of hybrid seedlings, and can enter the subsequent juvenile shell cultivation stage.

[0036] Example 3 1. Treatment of male broodstock white butterfly oysters to induce sperm release Three live male white-lipped oysters, each 18cm in shell length, with full, mature gonads and strong vitality, were selected. The shells were repeatedly rinsed several times with sterile sand-filtered seawater to thoroughly remove mud, algae, and other attached organisms. After drying in the sun for 30-60 minutes, the treated oysters were placed in a 1-ton plastic bucket. Clean seawater, which had undergone sedimentation, sand filtration, and ultraviolet disinfection, was added. The water temperature was strictly controlled at 28℃, and the salinity at 28‰. Continuous oxygenation was maintained throughout the process to ensure dissolved oxygen ≥5mg / L (e.g., 6mg / L). A small electric water pump was used to increase the water temperature by approximately 2 degrees Celsius, and a continuous water flow of 1.2m / s was applied for 1 hour to induce synchronous natural sperm release in the male oysters. The process continued until the water turned a uniform milky white and the sperm density reached 1.2×10⁻⁶. 8 When the concentration is 100 cells / mL, it is ready for use.

[0037] 2. Collection and purification of female eggs from Pinctada martensii. Ten female Pinctada martensii oysters with a shell length of 6 cm and mature gonads (orange-red and plump) were selected. After aseptic dissection, the gonadal tissue was quickly removed. The gonads were rinsed with seawater filtered through a 300-mesh screen to thoroughly remove mucus, blood, and tissue debris. Mature eggs were extracted using a sterile pipette and transferred to a 1L sterile container. The container was rinsed three times with filtered seawater, allowing it to stand for 10 minutes each time to remove impurities and damaged eggs. The egg mixture was allowed to stand for 30 minutes until the mature eggs had completely settled to the bottom. Excess seawater was then siphoned off, leaving a final 500mL mixture of seawater and eggs, with the egg density controlled at 8 × 10⁻⁶. 5 per mL.

[0038] 3. Chemical activation and induction treatment of oocytes Analytical grade ammonia was used as the oocyte activation reagent. Ammonia was precisely drawn up with a sterile dropper and slowly added to the oocyte mixture with 3 drops (each drop approximately 0.05 mL). The mixture was then gently and evenly stirred with a sterile glass rod for 1 minute to ensure that the ammonia and oocytes were in full contact. After 20 minutes of activation treatment, microscopic observation showed that the oocytes had shrunk to a regular round shape and the oocyte membrane had expanded, indicating that the oocytes had matured and been activated, which significantly improved their ability to combine with heterologous sperm.

[0039] 4. Controlled hybridization fertilization procedure After the male white butterfly oysters naturally release sperm and the sperm density reaches the required level, slowly add 500mL of activated egg mixture to a 1-ton water tank containing sperm in three separate additions, each adding 150-200mL at 5-minute intervals, while gently stirring the water to prevent excessively high local sperm concentration. Maintain a water temperature of 28℃ and a still, dark environment throughout the process to allow the sperm and eggs to naturally combine and complete hybridization fertilization.

[0040] 5. Fertilization detection and transfer of fertilized eggs Sixty minutes after hybridization and fertilization, a small amount of egg fluid was aspirated with a sterile dropper and observed under a biological microscope. The appearance of the first polar body on the outer layer of the egg indicated successful fertilization, with a fertilization rate of 87%. After confirming successful fertilization and normal division and development of the fertilized eggs, all male white-lipped oysters were immediately removed from the water to prevent the parent oysters' metabolic products and feces from polluting the water and affecting the development of the fertilized eggs. The fertilized eggs were then left to stand for another 30 minutes until they completely sank to the bottom of the bucket. The top 50% of the seawater was then siphoned off, and the remaining fertilized eggs and seawater were transferred together to a 20-ton nursery tank containing pre-sterilized water.

[0041] 6. Larval Hatching and Development The seawater conditions in the nursery pond are the same as those in the fertilization water: water temperature 28℃, salinity 28‰, dissolved oxygen ≥5mg / L (e.g., 6mg / L). The fertilized eggs hatch naturally in the pond. During the cultivation process, the development of the fertilized eggs is observed. After 5 hours, trochophore larvae are formed and float to the upper layer of the water. After 10 hours of continuous cultivation, microscopic examination shows that the larvae have successfully metamorphosed into D-shaped larvae with complete morphology and normal vitality. The larval survival rate is stable at 72%, completing the initial cultivation of hybrid seedlings, and can enter the subsequent juvenile shell cultivation stage.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for interspecific hybridization and seedling cultivation of *Pterocarya stenoptera* and *Pterocarya martensii*, characterized in that, Includes the following steps: (1) Treatment of male white-lipped oysters to induce sperm release: Select live male white-lipped oysters with mature gonads, clean and dry them, and place them in disinfected seawater with a water temperature of 26-28℃ and a salinity of 28-30‰. Continuously oxygenate and artificially circulate the water for 1-2 hours to induce natural sperm release in the male oysters, and control the sperm density to 1.2×10 8 -1.5×10 8 cells / mL; (2) Collection and purification of female eggs of Pinctada martensii: The gonads of mature live Pinctada martensii oysters were dissected and the gonads were removed. The gonads were cleaned to remove the mucus, and the mature eggs were extracted, cleaned, allowed to settle, and the upper layer of seawater was removed, leaving 500 mL of seawater and egg mixture. (3) Chemical activation induction of oocytes: Add 0.15-0.2 mL of analytical grade ammonia to the oocyte mixture, stir gently, and activate for 10-20 minutes until the oocytes are regular and round; (4) Hybrid fertilization: The activated egg mixture is added in portions to the water containing the white butterfly oyster sperm from step (1), and fertilization is carried out in a static, light-protected environment at a water temperature of 26-28℃. (5) Fertilization detection and transfer of fertilized eggs: After hybridization fertilization, the male white butterfly oysters were removed and the fertilized eggs were left to settle for 30 minutes. Half of the seawater in the upper layer was removed and the remaining fertilized eggs and seawater were transferred to the nursery pond. (6) Larval hatching and development: Fertilized eggs are cultivated at a water temperature of 26-28℃ and a salinity of 28-30‰. After 8-10 hours, they develop into D-shaped larvae.

2. The method according to claim 1, characterized in that, In step (1), the shell length of the male parent white butterfly oyster is 18-22cm.

3. The method according to claim 1, characterized in that, In step (1), the dissolved oxygen content in the water body is ≥5 mg / L.

4. The method according to claim 1, characterized in that, In step (1), the water flow velocity stimulated by artificial water transfer is 0.8-1.2 m / s.

5. The method according to claim 1, characterized in that, In step (2), the shell length of the Pinctada martensii mother oyster is 6-8 cm.

6. The method according to claim 1, characterized in that, In step (2), the eggs were washed using seawater filtered through a 300-mesh sieve, and the egg density was controlled at 8 × 10⁻⁶. 5 -1×10 6 per mL.

7. The method according to claim 1, characterized in that, In step (4), when adding the egg mixture in several batches, add 150-200 mL each time, with a 5-minute interval between each addition. Gently stir the water during this time to avoid excessively high local sperm concentration.

8. The method according to claim 1, characterized in that, In step (5), fertilization is confirmed to be successful 60 minutes after hybridization.

9. The method according to claim 1, characterized in that, In step (5), the hybridization fertilization rate is ≥85%.

10. The method according to claim 1, characterized in that, In step (6), the survival rate of D-shaped larvae is ≥70%.