A method for inducing gynogenesis of psetta lacustris by sperm of psetta mudia

Abstract

The present application belongs to the field of fish germplasm resource improvement, and discloses a method for inducing gynogenesis of large yellow croaker by sperm of Nibea albiflora, comprising the following steps: selecting female large yellow croakers and male Nibea albiflora as parent fish, artificially inducing parturition to obtain mature eggs of female large yellow croakers and sperm of male Nibea albiflora; after dilution, the sperm of male Nibea albiflora is inactivated, then mixed with the mature eggs of large yellow croakers for insemination, cold shock treatment, and then transferred to seawater at 22-24 DEG C for continuous aeration and oxygenation floating incubation; when the hatched fry can swim, artificial feeding is performed, and gynogenetic large yellow croakers are obtained. After successfully breeding gynogenetic large yellow croaker offspring by inducing gynogenesis of large yellow croakers by sperm of Nibea albiflora, the growth rate, stress resistance and meat quality of the offspring are obviously stronger than those of common large yellow croakers in the breeding process, which not only lays a good foundation for improving the germplasm resource of large yellow croakers, but also has important significance in fish genetic breeding and biological evolution.

Description

Technical Field

[0001] This invention belongs to the field of fish germplasm resource improvement, specifically relating to a method for inducing gynogenesis in large yellow croaker using yellow croaker sperm. Background Technology

[0002] Large yellow croaker (Larimichthys crocea) belongs to the genus Larimichthys in the family Sciaenidae of the order Perciformes. It is also known as yellow croaker, yellow fish, and cucumber. Large yellow croaker has a wide diet, mainly feeding on crustaceans such as shrimp and crabs, as well as small fish. It is a major economic fish species in my country's coastal waters and is one of the traditional "four major economic fish species of the sea" (large yellow croaker, small yellow croaker, ribbonfish, and squid). However, due to overfishing, its population has declined, and it is currently listed as a critically endangered species on the IUCN Red List of Threatened Species. Despite the increasing production of large yellow croaker in my country due to breakthroughs in artificial breeding technology, diseases such as visceral white spot disease and body surface white spot disease, as well as hypoxia stress caused by high summer temperatures, have continued to affect the quantity and quality of farmed large yellow croaker. Furthermore, the low coverage rate of improved varieties and the fact that the number of seedlings produced far exceeds the demand for farming are prominent issues that greatly limit people's demand for high-quality large yellow croaker. Therefore, there is an urgent need for improved breeding and variety improvement to meet the needs of industrial development.

[0003] Gynogenesis technology plays a crucial role in obtaining superior varieties, accelerating population selection, and sex control. Generally, the cultivation of artificially gynogenic fish involves using genetically inactivated heterologous sperm to activate the development of haploid eggs. The eggs then undergo chromosome doubling treatment to form diploid offspring that primarily develop using the egg's genetic material. Although the heterologous sperm undergoes genetic inactivation, some of its genetic material can be integrated into the egg's genetic material through the "heterospermia effect," resulting in genetically improved gynogenic offspring. Large yellow croaker has a very delicious flavor and rich nutritional value, making it very popular. Consumer demand is increasing, and the cultivation of superior varieties is essential to meet this demand. Currently, large yellow croaker germplasm improvement mainly focuses on population selection, with few reports on using gynogenesis for germplasm improvement. The main challenges of artificially inducing gynogenesis in large yellow croaker are as follows: First, large yellow croaker belongs to the order Perciformes, family Sciaenidae, and genus Croaker, making it difficult to find a suitable heterologous sperm to induce gynogenesis, ensuring successful activation of egg development without confusion in hybrid offspring; second, the effect time of artificial spawning in large yellow croaker females is not fixed, with spawning occurring within 30-54 hours; third, large yellow croaker eggs develop asynchronously, being a batch-spawning fish, with only a few individuals able to spawn normally after spawning induction, and the eggs are very prone to over-maturation.

[0004] Therefore, successfully inducing gynogenesis in large yellow croaker by selecting suitable heterologous sperm and breeding gynogenic large yellow croaker with significant improvements in growth rate, stress resistance, and meat quality is of great significance in fish genetics and breeding and biological evolution. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a method for inducing the development of gynogenetic cells in large yellow croaker using yellow croaker sperm, which can be used to cultivate large yellow croaker with faster growth rate, stronger resistance and better meat quality.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A method for inducing gynogenesis in large yellow croaker using yellow croaker sperm includes the following steps:

[0008] (1) Select female large yellow croaker and male yellow croaker as parent fish for intensive breeding, and then artificially induce spawning to obtain mature eggs of female large yellow croaker and sperm of male yellow croaker.

[0009] (2) Dilute the male yellow croaker semen obtained by artificial spawning in step (1) with pre-cooled sperm diluent. Spread the diluted semen evenly in a pre-cooled culture dish and place it on a shaker covered with ice plates for inactivation treatment until 50%-60% of the sperm lose motility.

[0010] (3) Mix the yellow croaker semen obtained after inactivation in step (2) with the mature large yellow croaker eggs obtained by artificial spawning in step (1), fertilize them in seawater at 23-25℃, then perform cold shock treatment, and then transfer the fertilized eggs to seawater at 22-24℃ for continuous aeration and floating incubation; During the breeding season of large yellow croaker, the normal seawater temperature is only a dozen degrees. In this invention patent, the seawater used is heated seawater, which can achieve better breeding results.

[0011] (4) When the fish fry hatched from the floating incubation in step (3) can swim horizontally, they are artificially raised to obtain large yellow croaker with gynogenesis.

[0012] Selecting suitable exogenous semen for induction is crucial for successful gynogenesis. Yellow croaker (Nibea albiflora), belonging to the family Sciaenidae and genus Nibea in the order Perciformes, is an excellent species for marine aquaculture due to its ease of cultivation, rapid growth, strong disease resistance, and rich nutritional content. This invention demonstrates the significant advantages of using yellow croaker semen to induce gynogenesis in large yellow croaker. Yellow croaker exhibits rapid growth, strong disease resistance, and produces a large volume of high-quality semen with good sperm motility, making it an excellent donor for gynogenesis. Previous distant hybridization experiments confirmed that fry produced from crosses between large yellow croaker and yellow croaker all died within 8 days of age due to their inability to feed. Offspring surviving beyond 8 days of age obtained by inducing gynogenesis in large yellow croaker using yellow croaker semen are all gynogenic offspring and require no further differentiation.

[0013] In the above method, preferably, in step (1), the broodstock are two to three years old; the specific operation steps of the intensive rearing are as follows: 1 to 2 months before artificial spawning, select two to three-year-old female large yellow croaker and male yellow croaker with obvious sexual maturity characteristics for intensive rearing in separate ponds. During the intensive rearing period, feed the broodstock with fresh oyster meat twice a day to strengthen their cultivation. Change the water and clean the rearing pond every day to remove residual oyster meat and broodstock feces and maintain good water quality. Half a month before artificial spawning, stimulate the broodstock with flowing water every 2 to 3 days to promote the maturation of the broodstock's gonads.

[0014] Preferably, in step (1), the specific steps of artificial spawning induction are as follows: After one month of intensive cultivation of broodstock, spawning is induced by injecting a luteinizing hormone-releasing hormone analog (LRH-A3). Female large yellow croaker broodstock are injected with two doses: the first dose of LRH-A3 is 0.5-1.0 μg / kg, and the second dose is injected 24-26 hours later, with a dosage of 4.0-4.5 μg / kg. Male large yellow croaker and yellow croaker are injected with one dose of LRH-A3, with a dosage of 0.5-1.0 μg / kg. For gynogenesis breeding of large yellow croaker, ensuring egg quality is crucial, and optimizing the injection dosage for female large yellow croaker selection and spawning induction is essential. These technical improvements are vital for increasing the hatching and survival rates of gynogenic fry and improving the germplasm resources of large yellow croaker.

[0015] Large yellow croakers are batch-spawning fish, with only a few individuals able to spawn normally after induced spawning. Each female lays around several thousand eggs at a time, and the eggs are highly prone to over-maturation, rendering them unfertilizable. Furthermore, the hatching and survival rates of offspring developed through gynogenesis are inherently low. The small egg production and the high rate of over-maturation significantly increase the difficulty of implementing gynogenesis techniques. In addition, the timing of the induced spawning effect in large yellow croakers is difficult to control; spawning can occur anywhere from 30 to 54 hours after induction. The inability to accurately control the timing of induced spawning further complicates the implementation of gynogenesis techniques. Long-term induced spawning experiments using this invention have shown that while a single injection (LRH-A3 dosage of 3-5 μg / kg) can induce spawning in female large yellow croakers, the egg production and the timing of the induced spawning effect are difficult to control, exhibiting significant individual variations and resulting in unsatisfactory induced spawning effects. However, artificial spawning induction using two injections in female large yellow croakers yields ideal results. The specific implementation steps of the two injections are as follows: The dosage of the first injection, LRH-A3, is 0.5 μg / kg. 24-26 hours later, the female large yellow croaker parent fish are injected with the second injection, LRH-A3, at a dosage of 4.5 μg / kg. After the above artificial spawning induction, more than 80% of the female large yellow croakers that have been enhanced and cultivated can be spawned normally, with each female fish laying 80,000 to 120,000 eggs each time. The effect time after spawning induction (starting from the second injection) can be controlled within 33-37 hours.

[0016] Preferably, in step (2), the 1L sperm diluent comprises the following components in parts by weight: KCl 0.39g, NaCl 8.22g, NaHCO3 0.20g, NaH2PO4·2H2O 0.28g, MgCl2·6H2O 0.23g, CaCl2·2H2O 0.72g, and finally deionized water is added to 1L; the sperm diluent is diluted with the semen at a volume ratio of 20-25:1, the temperature of the pre-cooled culture dish and the sperm diluent is 3-5℃, and the rotation speed of the shaker is 100-110r / min.

[0017] Preferably, in step (2), the specific operation steps of the inactivation treatment are as follows: irradiate with a 20W ultraviolet lamp for 60-120s for inactivation treatment, observe the sperm motility with an optical microscope during ultraviolet inactivation treatment, observe once every 20-30s, and stop inactivation when 50%-60% of the sperm lose their motility, and store the obtained diluted semen in a light-protected environment at 3-5℃.

[0018] Preferably, in step (3), the cold shock treatment is performed at 3-4°C for 9-12 minutes.

[0019] Preferably, in step (3), the fertilization is performed in a basin containing seawater at 23-25°C, and the aeration rate of the continuous aeration and oxygenation floating incubation is adjusted to ensure that all fertilized eggs can float.

[0020] Preferably, in step (3), under the spawning-inducing dosage set by the present invention, the water temperature is 23-25℃, the female fish effect time (starting from the second injection) is 33-37h, and the number of eggs laid by each female fish is 80,000-120,000; that is, the collection time of the mature eggs of the large yellow croaker is 33-37h after the female large yellow croaker parent fish are injected with the second injection of LRH-A3.

[0021] Preferably, in step (4), the fish fry are cultured in seawater at 22-24℃ for 40-50 days.

[0022] Preferably, the specific steps of artificial rearing are as follows: feeding rotifers, brine shrimp, copepods, and artificially formulated micro-particle feed in sequence until the fry completely consume the artificially formulated micro-particle feed. More preferably, the specific steps of rearing gynogenetic large yellow croaker fry are as follows: starting from 3 days of age, feed rotifers for 3-5 days (i.e., 3-7 days of age); when the fry are 6-7 days of age, feed rotifers and brine shrimp simultaneously; when the fry are 8-15 days of age, feed only brine shrimp; during this period, starting from 11 days of age, feed copepods simultaneously with brine shrimp for 5 days; when the fry are 25 days of age and their body color begins to turn black, begin to acclimate them to No. 0 artificially formulated micro-particle feed, gradually increasing the amount of artificially formulated micro-particle feed until it becomes the main feed.

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

[0024] 1. The method of the present invention uses genetically inactivated croaker sperm to induce gynogenesis in large yellow croaker and successfully selects offspring of gynogenic large yellow croaker. During the breeding process, the gynogenic large yellow croaker is significantly stronger than ordinary large yellow croaker in terms of growth rate, stress resistance and meat quality. This not only lays a good foundation for improving the germplasm resources of large yellow croaker, but also has important significance in fish genetic breeding and biological evolution.

[0025] 2. In this invention, female large yellow croaker that has been enhanced and cultured are used as parents to induce gynogenesis. Two injections are administered and the dosage of the spawning-inducing drug is optimized. This ensures that more than 80% of the female large yellow croakers can spawn within the predetermined time and that a large number of high-quality eggs can be obtained. This lays a solid foundation for the successful implementation of gynogenesis technology.

[0026] 3. This invention uses *Croton tigrinosa* (Yellow Croaker) of the Sciaenidae family (Perciformes) as the sperm source. Yellow Croaker is easy to cultivate, grows rapidly, has strong disease resistance, and is rich in nutrients, making it an excellent species for marine aquaculture. This invention demonstrates significant advantages in using Yellow Croaker sperm to induce gynogenesis in large yellow croaker. Yellow Croaker has a fast growth rate, is rich in nutrients, and its sperm has good activity, high quality, and large volume, making it a good donor for heterologous sperm in gynogenesis. Furthermore, previous distant hybridization experiments confirmed that fry produced by hybridization between large yellow croaker and Yellow Croaker all died within 8 days of age due to their inability to feed. Using genetically inactivated Yellow Croaker sperm to induce gynogenesis in large yellow croaker... Offspring that survive for more than 8 days after development are considered gynogenetic offspring and do not require further screening. Since yellow croaker can continuously provide high-quality sperm, each male yellow croaker can provide 3-6 mL of sperm, which can meet the needs of 3-5 gynogenetic experiments. Moreover, collecting sperm will not affect its survival, avoiding unnecessary losses. Yellow croaker and large yellow croaker both belong to the Sciaenidae family of the Perciformes order. Therefore, when using genetically inactivated yellow croaker sperm to activate large yellow croaker eggs for gynogenetic development, the efficiency is very high, and a "heterospermic effect" is formed in the gynogenetic large yellow croaker, thereby achieving genetic improvement in growth rate, stress resistance, and meat quality. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is an image of the common large yellow croaker (scale bar = 1cm);

[0029] Figure 2 An illustration of the external appearance of a large yellow croaker undergoing gynogenesis (scale bar = 1 cm);

[0030] Figure 3 The image shows the detection of microsatellite markers in gynogenetic large yellow croaker (M represents pBR322DNA / Mspl, the red box is the electrophoretic lane of yellow croaker, the green box is the electrophoretic lane of common large yellow croaker, and the blue box is the electrophoretic lane of gynogenetic large yellow croaker).

[0031] Figure 4 Image showing the homozygosity of microsatellite markers for gynogenetic large yellow croaker (black lines mark the electrophoretic lanes of common large yellow croaker, and red lines mark the electrophoretic lanes of gynogenetic large yellow croaker);

[0032] Figure 5 Flow cytometry plot of common large yellow croaker;

[0033] Figure 6 Flow cytometry diagram of gynogenesis in large yellow croaker;

[0034] Figure 7 Chromosome diagram of common large yellow croaker (scale bar = 3μm);

[0035] Figure 8 Chromosome diagram of gynogenetic large yellow croaker (scale bar = 3 μm). Detailed Implementation

[0036] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0037] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0039] Example:

[0040] A method for inducing gynogenesis in large yellow croaker using croaker sperm according to the present invention includes the following steps:

[0041] (1) Selection and rearing of broodstock: One to two months before artificial spawning, select female large yellow croakers that are two to three years old, free from injury or disease, and exhibit obvious sexual maturity characteristics. Figure 1 The male and female yellow croakers were separated and intensively cultured in separate ponds. During the intensive culture period, the parent fish were fed fresh oyster meat twice a day to strengthen their growth. The water in the culture pond was changed and cleaned every morning to remove residual oyster meat and parent fish feces to maintain good water quality. Half a month before artificial spawning, the parent fish were stimulated with flowing water every 2-3 days to promote the maturation of their gonads.

[0042] (2) Artificial spawning induction: After one month of intensive rearing of broodstock, spawning was induced by injecting luteinizing hormone-releasing hormone analogue (LRH-A3). Female large yellow croaker broodstock were injected with two doses. The first dose of LRH-A3 was 0.5 μg / kg. 24-26 hours later, the female large yellow croaker broodstock were injected with a second dose of LRH-A3 at a dose of 4.5 μg / kg. Male large yellow croaker and yellow croaker were injected with one dose of LRH-A3 at a dose of 0.5 μg / kg.

[0043] (3) Semen collection and inactivation: Observe and record the state of the fish to be induced to spawn at all times. When the large yellow croaker makes a "cooing" sound before spawning, use a fine-mesh scoop to gently scoop up whether there are any floating eggs on the surface of the spawning pond. When a large number of floating eggs are found, the gynogenesis breeding experiment can begin. Collect croaker semen carefully using a 5mL sterile syringe. Dilute the semen 20-25 times with pre-cooled sperm diluent. Spread the diluted semen evenly in a pre-cooled culture dish, with a thickness of about 0.1-0.2mm. Place the culture dish on a shaker with ice plates at 100-110 rpm for UV irradiation. The distance between the UV lamp and the shaker should be 20cm. Inactivate the sperm with a 20W UV lamp for 60-120s. Observe under a microscope every 20-30s. Stop UV irradiation when only about 40%-50% of the sperm are motile. Store the irradiated semen in a light-proof tube at 4℃. Perform artificial insemination with the irradiated semen on eggs promptly.

[0044] 1L of sperm diluent comprises the following components in parts by weight: KCl 0.39g, NaCl 8.22g, NaHCO3 0.20g, NaH2PO4·2H2O 0.28g, MgCl2·6H2O 0.23g, CaCl2·2H2O 0.72g, and finally deionized water is added to 1L.

[0045] (4) Egg activation and cold treatment: Squeeze the large yellow croaker eggs into a clean sterilized culture dish, add the irradiated yellow croaker semen, and gently stir with a feather to mix them evenly. Then add seawater at about 24℃ to fertilize them. 2-3 minutes after fertilization, transfer the fertilized eggs to a basin containing seawater at 3-4℃ for cold shock treatment for 9-12 minutes. Then transfer the fertilized eggs after cold shock treatment to a black bucket containing seawater at 22-24℃ for continuous aeration and floating hatching. Adjust the aeration rate to just ensure that all fertilized eggs can float. After 12 hours, remove the fertilized eggs that have stopped developing (the fertilized eggs will sink after aeration stops). Replace half the volume of fresh seawater in the black bucket every 6 hours. When the water temperature is 22-24℃, the fry will hatch in about 2 days and can swim horizontally in 3-5 days.

[0046] (5) Feeding: Starting at 3 days old, feed rotifers for 3-5 days (i.e., 3-7 days old). When the fry are 6-7 days old, feed both rotifers and brine shrimp. When the fry are 8-15 days old, feed only brine shrimp. During this period, at 11 days old, start feeding copepods, feeding them simultaneously with brine shrimp for 5 days. When the fry are 25 days old and their body color begins to turn black, start acclimatizing them to No. 0 artificial micro-particle feed, gradually increasing the amount of artificial micro-particle feed until it becomes the main feed. After 40-50 days of rearing in black buckets, transfer them to fish rafts for further rearing, thus obtaining gynogenetic large yellow croaker ( Figure 2 ).

[0047] During incubation, the fertilization rate and hatching rate of the embryos were recorded. In the process of artificially inducing gynogenesis in large yellow croaker using genetically inactivated yellow croaker sperm, the fertilization rate and hatching rate of the embryos both exceeded 60%, which was significantly improved compared with similar gynogenesis breeding experiments.

[0048] (6) Detection of gynogenetic large yellow croaker: The offspring types of gynogenetic large yellow croaker were detected using the microsatellite marker method. The detection results of microsatellite primer LYC0033 showed that the bands of gynogenetic large yellow croaker were all consistent with those of ordinary large yellow croaker, and there were no common bands with yellow croaker, indicating that all the gynogenetic large yellow croakers detected were gynogenetic offspring. Figure 3 (As shown). Simultaneously, the homozygosity of genomic DNA in the offspring of gynogenetic large yellow croaker was detected using microsatellite marker primer Lcr-01. The results showed that the homozygosity of gynogenetic large yellow croaker was higher than that of ordinary large yellow croaker. Figure 4 (As shown), it was further identified as a gynogenetic offspring.

[0049] The method for detecting offspring types using microsatellite markers was as follows: The caudal fins of gynogenetic large yellow croaker, common female large yellow croaker, and male yellow croaker were cut, and DNA was extracted using a DNA extraction kit (Sangon Biotech, Shanghai). Using the extracted DNA as a template, PCR amplification was performed using microsatellite primers (LYC0033: F-GGATGGAGGAGTGATGATGG, R-GCACTGAGACCTGAATGCTCC, as shown in SEQ ID Nos. 1-2, respectively). The PCR reaction system included 5 μL of 2×Tap PCR Mix (Tiangen Biotech Co., Ltd.), 1 μL of 50 ng genomic DNA template, 0.5 μL each of forward and reverse primers, and finally, ddH2O was added to a final volume of 20 μL. The PCR reaction conditions were: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 55℃ annealing for 30 s, 72℃ extension for 45 s, for 35 cycles; and a final extension at 72℃ for 10 min. Finally, the PCR products were subjected to electrophoresis using denaturing polyacrylamide gel. After electrophoresis, the denaturing polyacrylamide gel was stained with silver nitrate to show the bands. According to the banding results, if the band of the gynogenetic large yellow croaker is consistent with that of the common large yellow croaker and there is no common band with that of the yellow croaker, it indicates that all the gynogenetic large yellow croakers tested are gynogenetic offspring.

[0050] The method for detecting the homozygosity of offspring genomic DNA using microsatellite markers was as follows: The caudal fins of gynogenetic large yellow croaker, common female large yellow croaker, and male yellow croaker were cut, and DNA was extracted using a DNA extraction kit (Sangon Biotech, Shanghai). Using the extracted DNA as a template, PCR amplification was performed using microsatellite primers (Lcr-01: F-ATAGCTGTCTCCATGCCCAC, R-AAAATTGACCTCCAGCCAAA, as shown in SEQ ID No. 3-4, respectively). The PCR reaction system included 9 μL of standard 2×Tap PCR Mix (Tiangen Biotech), 1 μL of 50 ng genomic DNA template, 0.5 μL each of forward and reverse primers, and finally, ddH2O was added to a final volume of 20 μL. The PCR reaction conditions were: 96℃ pre-denaturation for 4 min; 95℃ denaturation for 30 s, 53℃ annealing for 40 s, 72℃ extension for 1 min, for 35 cycles; and a final extension at 72℃ for 7 min. Finally, the PCR products were subjected to electrophoresis using denaturing polyacrylamide gel. After electrophoresis, the denaturing polyacrylamide gel was stained with silver nitrate to show banding. According to the banding results, if the number of alleles in the gynogenetic offspring is less than that in the common large yellow croaker, it indicates that the genomic DNA homozygosity of the gynogenetic offspring is higher.

[0051] The silver staining procedure is as follows: the denatured polyacrylamide gel is fixed in 10% glacial acetic acid for 10 min, then transferred to 0.1% silver nitrate for staining for 10 min, rinsed with pure water for about 30 s, and finally transferred to the developing solution (the developing solution consists of 9 g NaOH, 0.25 g anhydrous Na2CO3, 2.5 mL formaldehyde, and double-distilled water to 500 mL) for development. The gel is removed after bands are visible.

[0052] Furthermore, gynogenesis in large yellow croaker with a chromosome number of 48 (2n=48) was further identified using flow cytometry DNA content determination and kidney tissue chromosome ploidy detection. Common large yellow croaker was used as a reference. Figure 5 The DNA content in the erythrocytes of gynogenetic large yellow croaker was detected by flow cytometry, and the results are as follows: Figure 6 As shown in the figure. Analysis of the data shows that the DNA content of common large yellow croaker and gynogenetic large yellow croaker is 50.55 and 48.36, respectively. The DNA content ratio of gynogenetic large yellow croaker to common large yellow croaker is 0.96, which is not significantly different from the expected theoretical ratio of 1:1. Sampling and detection of somatic cell chromosome number in common and gynogenetic large yellow croaker using the kidney tissue chromosome preparation method showed that both common and gynogenetic large yellow croaker have 48 chromosomes, indicating diploidity (2n=48). The chromosome diagram is shown below. Figure 7 , Figure 8As shown in the figure. The above-mentioned flow cytometry DNA content determination and kidney tissue chromosome ploidy detection methods further identified that the ploidy level and chromosome number of the gynogenetic large yellow croaker (2n=48) were consistent with those of the common large yellow croaker, and that it was a diploid fish with 48 chromosomes.

[0053] The procedure for flow cytometry DNA content determination is as follows: Approximately 0.2 mL of blood is collected from the fish tail vein using a heparin-moistened disposable syringe and injected into an Eppendorf tube containing 0.8% physiological saline. 1 mL of nuclear extraction solution DAPI-A (provided by Partec GmbH, Germany) is added to the blood and saline mixture, and the mixture is processed for 10–15 min. The sample is then filtered through a 20 μm nylon filter (provided by Partec GmbH, Germany). DNA staining solution (DAPI-B, provided by Partec GmbH, Germany) is used to stain the sample in the dark for approximately 8–10 min, after which the sample is analyzed by the flow cytometry instrument.

[0054] The procedure for detecting chromosome ploidy in kidney tissue is as follows: Inject 5 μL of hemagglutinin PHA (diluted with 0.75% physiological saline to 5 μg / μL) per gram of fish body weight, once every 12 hours, for a total of two injections; dissolve colchicine in physiological saline and inject 1 μg of colchicine per gram of fish, injecting colchicine into the pectoral fins; 4 hours after colchicine injection, euthanize the fish by cutting off the gill arches and bleeding it out; 10 minutes later, remove the head and mid-kidneys and place them in a culture dish containing 2 mL of physiological saline. Cut the kidneys into small pieces with scissors, repeatedly pipette the mixture, allow it to settle naturally for 5 minutes, collect the supernatant, and centrifuge at 1000 rpm for 8 minutes; discard the supernatant and use 8 mL of 0.075M... Resuspend the cells in KCl solution and let stand at room temperature for 1 hour. Add 2 mL of freshly prepared fixative (methanol: glacial acetic acid = 3:1) at room temperature, let stand for 5 minutes, and then centrifuge at 1000 rpm for 8 minutes. Remove the supernatant, add 2 mL of fixative to resuspend the cells, fix at 4°C for 15 minutes, and then centrifuge at 1000 rpm for 8 minutes. Repeat this step 3 times. Finally, add an appropriate volume of fixative to resuspend the cells to the appropriate concentration. Take a clean glass slide from the -20°C freezer and place it on a support. Use a pipette to draw a certain volume of the above solution and drop it onto the glass slide from a height of about 1.5 meters. Dry the glass slide on a slide oven at 42°C and then store it in a -20°C freezer for later use. Stain with Giemsa stain for 30 minutes, discard the stain, and gently rinse with water. Dry the glass slide on a slide oven at 42°C. Examine under a 100x oil immersion microscope.

[0055] This invention primarily utilizes gynogenesis technology, an important method for fish genetic breeding. Gynogenesis relies mainly on the genetic material of the egg to develop into an individual. Because it requires doubling the chromosome set of the egg, its hatching rate is typically very low. For the gynogenesis breeding experiment with large yellow croaker, which is a batch-spawning fish, only a few individuals can spawn normally after induced spawning. Each female lays only a few thousand eggs at a time, and the eggs are very prone to over-maturation, making fertilization impossible. The hatching and survival rates of offspring induced by gynogenesis are inherently low, and the small egg production and high rate of over-maturation undoubtedly increase the operational difficulty of gynogenesis technology. Furthermore, the timing of the spawning induction effect in large yellow croaker is difficult to control; spawning can occur 30-54 hours after induction. The inability to accurately control the spawning time undoubtedly increases the difficulty of implementing gynogenesis technology. In this invention, through extensive preliminary exploratory experiments, it was found that using pre-cultured female large yellow croaker and artificially inducing spawning with two injections can achieve ideal results. The specific implementation steps of the two injections are as follows: The dosage of the first injection, LRH-A3, is 0.5 μg / kg. 24-26 hours later, the female large yellow croaker parent fish are injected with the second injection, LRH-A3, at a dosage of 4.5 μg / kg. After the above artificial spawning induction, more than 80% of the female large yellow croakers that have been enhanced and cultured can be spawned normally, with each female fish laying 80,000 to 120,000 eggs each time. Moreover, the effect time after spawning induction (starting from the second injection) can be controlled within 33-37 hours, which lays a solid foundation for the successful implementation of gynogenetic technology.

[0056] This invention uses *Cyprinus maculatus* (Yellow Croaker) of the Sciaenidae family (Perciformes) as the sperm source, which is highly significant. Firstly, *Cyprinus maculatus* possesses many excellent traits, such as ease of cultivation, rapid growth, strong disease resistance, and rich nutrition, making it an excellent species for marine aquaculture. Secondly, using *Cyprinus maculatus* sperm as a stimulant is very convenient in terms of semen acquisition; the sperm has good activity, high quality, and a large volume, making it a good donor for gynogenetic heterologous sperm. Furthermore, the effectiveness of sperm inactivation using ultraviolet light is easily assessed. Most importantly, previous distant hybridization experiments confirmed that fry produced by hybridization of large yellow croaker and *Cyprinus maculatus* all died within 8 days of age due to inability to feed. Offspring surviving for more than 8 days after inducing gynogenesis in large yellow croaker using genetically inactivated *Cyprinus maculatus* sperm are considered gynogenetic offspring and require no further screening. Both yellow croaker and large yellow croaker belong to the Sciaenidae family of the order Perciformes. Therefore, using genetically inactivated yellow croaker sperm to activate large yellow croaker eggs for gynogenesis is highly efficient and creates a "heterospermic effect" in the gynogenic large yellow croaker, resulting in genetic improvements in growth rate, stress resistance, and flesh quality. Furthermore, this invention selects an appropriate ultraviolet dose for sperm inactivation and appropriate cold treatment time and temperature when the eggs double, which is highly valuable for reference in the field.

[0057] In production, sex reversal technology can be further combined to induce gynogenetic large yellow croakers into physiologically male large yellow croakers, which can then be mated with gynogenetic large yellow croakers to obtain a large number of one-sex large yellow croaker offspring with multiple advantageous traits. One-sex large yellow croakers with multiple advantageous traits can be introduced into many artificial aquaculture areas for cultivation. This will not only yield large yellow croaker populations with fast growth rates and strong resistance, but also provide a large amount of high-quality large yellow croaker protein. Furthermore, it will control the decline in the germplasm quality of large yellow croaker populations caused by over-breeding in artificially controlled aquaculture areas. Therefore, the one-sex large yellow croakers obtained by combining gynogenetic large yellow croakers with sex reversal technology bred in this invention have good market prospects and economic value. This invention not only lays a good foundation for improving large yellow croaker germplasm resources, but also has important significance in fish genetics and breeding and biological evolution.

Claims

1. A method for inducing gynogenesis in large yellow croaker using yellow croaker sperm, characterized in that, Includes the following steps: (1) Select female large yellow croaker and male yellow croaker as broodstock for intensive rearing, and then perform artificial spawning to obtain mature eggs from female large yellow croaker and sperm from male yellow croaker; the specific operation steps of the artificial spawning are as follows: inject LRH-A3 spawning induced agent to induce spawning. Inject two doses into the female large yellow croaker broodstock. To ensure egg quality, the injection dosage for female large yellow croaker selection and spawning induction is optimized. The dosage of the first dose of LRH-A3 is 0.5 µg / kg. 24-26 h later, inject the female large yellow croaker broodstock with the second dose of LRH-A3 at a dosage of 4.5 µg / kg; inject one dose into the male large yellow croaker and yellow croaker at a dosage of 0.5 µg / kg; the collection time of the mature eggs of large yellow croaker: 33-37 h after the second dose of LRH-A3 is injected into the female large yellow croaker broodstock. h; The broodstock are two to three years old; The specific steps of the intensive rearing are as follows: One to two months before artificial spawning, select two to three-year-old female large yellow croaker and male yellow croaker with obvious sexual maturity characteristics for intensive rearing in separate ponds. During the intensive rearing period, feed the broodstock with fresh oyster meat every day to strengthen their cultivation. Change the water and clean the rearing pond every day to remove residual oyster meat and broodstock feces and maintain good water quality. Half a month before artificial spawning, stimulate the broodstock with flowing water every 2 to 3 days to promote the maturation of the broodstock's gonads. (2) Dilute the male yellow croaker semen obtained by artificial spawning in step (1) with pre-cooled sperm diluent. The volume ratio of sperm diluent to semen is 20-25:

1. Spread the diluted semen evenly in a culture dish pre-cooled at 3-5℃ and place it on a shaker covered with ice. The shaker speed is 100-110 r / min. Irradiate with a 20 W ultraviolet lamp for 60-120 s for inactivation treatment. During the ultraviolet inactivation treatment, use an optical microscope to observe the sperm motility. Observe once every 20-30 s until 50%-60% of the sperm lose their motility and then stop the inactivation. Store the inactivated semen in a dark environment at 3-5℃. (3) Mix the yellow croaker semen obtained after inactivation in step (2) with the mature large yellow croaker eggs obtained by artificial spawning in step (1), fertilize them in seawater at 23-25℃, then perform cold shock treatment at 3-4℃ for 9-12 minutes, and then transfer the fertilized eggs to a basin filled with seawater at 22-24℃ for continuous aeration and floating incubation. The aeration rate of the continuous aeration and floating incubation is adjusted to just ensure that all the fertilized eggs can float. (4) When the fish fry hatched from the floating hatching in step (3) can swim horizontally, they are artificially raised. The specific steps of artificial raising are as follows: the fish fry are raised in seawater at 22-24℃ for 40-50 days, and rotifers, brine shrimp, copepods and artificially formulated micro-particle feed are fed in sequence until the fish fry completely eat artificially formulated micro-particle feed, thus obtaining large yellow croaker with gynogenesis.

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