A method for increasing the growth rate of a walleye by artificial gynogenesis

Abstract

The present application belongs to the field of artificial gynogenesis breeding of fish, and discloses a method for improving the growth speed of Amur ide by artificial gynogenesis, comprising the following steps: diluting the sperm of Megalobrama amblycephala, irradiating and inactivating under an ultraviolet lamp, stopping the irradiation until the number of active sperm is reduced to 45-55%, and storing the irradiated sperm under light-shielded condition; mixing the eggs of Amur ide and the sperm obtained after the treatment in normal temperature water to make the eggs fertilized, cold-shocking the fertilized eggs at 3-6 DEG C for 23-25 min, and then hatching the eggs in normal temperature water. The gynogenetic Amur ide obtained is different from the female Amur ide in appearance and biological characteristics, has a faster growth speed, and can shorten the breeding cycle; and a new Amur ide strain can be obtained through backcrossing, which provides important germplasm resources for the purification and rejuvenation of Amur ide and the quality improvement of Amur ide.

Description

Technical Field

[0001] This invention belongs to the field of artificial gynogenesis breeding of fish, and particularly relates to a method for improving the growth rate of black carp through artificial gynogenesis. Background Technology

[0002] Black carp, also known as grass carp, is a variant of carp belonging to the order Cypriniformes, family Cyprinidae, and subfamily Cyprinoideae. Black carp have short, stout bodies, relatively small heads, thin skin, tender flesh, no muddy taste, and a purplish-red body color with fine leaf-shaped scales. In some cases, the internal organs are faintly visible due to the thin skin on the abdomen. They grow quickly and have delicious, meaty, and low-bone flesh, making them popular among fish farmers. However, long-term self-pollination and inbreeding have led to a continuous deterioration in their quality, most notably a decrease in growth rate and resistance. Currently, artificial gynogenesis technology is mainly used in the aquaculture industry for the rapid establishment of pure lines, the utilization of asexual populations, and the determination of sex inheritance mechanisms. In traditional breeding techniques, obtaining pure lines generally requires multiple generations of inbreeding, and one mitosis is equivalent to 8-10 generations of inbreeding. Moreover, multiple generations of inbreeding can lead to the degeneration of certain advantageous traits. Therefore, the application of artificial gynogenesis technology can rapidly construct pure lines, greatly shortening the breeding cycle.

[0003] Artificial gynogenesis is a technique that uses heterologous sperm to stimulate egg development and double the genetic material, thereby producing all-female offspring. In the step of doubling the egg's genetic material, researchers achieve this by inhibiting the expulsion of the second polar body or suppressing the first cleavage. This method increases the number of chromosomes in the egg, resulting in offspring with diploid genetic material. Commonly used induction methods include heat shock, cold shock, and hydrostatic pressure. Researching a method to improve the growth rate of black carp using artificial gynogenesis is of great significance in this field. Summary of the Invention

[0004] 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 improving the growth rate of black carp through artificial induction of gynogenesis.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] A method for improving the growth rate of black carp through artificial gynogenesis includes the following steps:

[0007] (1) Dilute the sperm of the blunt snout bream and irradiate it under ultraviolet light to inactivate it until the number of active sperm drops to 45-55%. Then stop the irradiation and store the irradiated sperm under a light-proof condition.

[0008] (2) Mix the black carp eggs and the sperm obtained after step (1) in room temperature water to fertilize them. Then, subject the fertilized eggs to cold shock treatment at 3-6℃ for 23-25 ​​minutes and then place them in room temperature water for incubation.

[0009] The heterologous sperm was selected from blunt-snout bream as the paternal source to ensure that the offspring would be gynogenetic individuals. Cold shock aims to inhibit the release of the second polar body from the fertilized egg, thereby promoting the doubling of the egg's genetic material.

[0010] In the above preparation method, preferably, in step (1), the specific operation of diluting the blunt snout bream sperm is as follows: diluting the blunt snout bream sperm with Hank's solution by a dilution factor of 1-10 times the volume.

[0011] Preferably, in step (1), the specific operation of the inactivation treatment is as follows: the diluted sperm is placed in a culture dish on an ice plate, then placed on a shaker, and irradiated with a 15W ultraviolet lamp. The irradiation distance between the ultraviolet lamp and the sperm is 28-32cm, and the entire ultraviolet irradiation system is covered with a light-shielding cloth.

[0012] The purpose of covering the sample with a light-blocking cloth is twofold: firstly, to protect the experimenters from excessive UV exposure, and secondly, to prevent the restoration of genetic activity due to photorepair during irradiation, thus ensuring inactivation efficiency. During UV inactivation, the culture dish is manually shaken every 1-3 minutes, and a microscopic examination is performed. During microscopic examination, a small sample of the culture is placed on a slide using a toothpick, and the microscope is adjusted to a suitable focus to clearly observe the sperm morphology. Subsequently, a small amount of water is added and gently stirred with a toothpick to mix the sperm with the water and activate them for observation of their motility. This process continues until the proportion of active sperm in the field of view decreases to approximately 50%, at which point UV irradiation is stopped.

[0013] Preferably, the culture dish is shaken every 1-3 minutes during the irradiation process, and sperm motility is observed periodically.

[0014] Preferably, the refrigerated storage temperature is 4-5℃.

[0015] Preferably, in step (2), the temperature of the room temperature water is 23-25℃.

[0016] Preferably, in step (2), the fertilization time is 2-3 minutes, and during the incubation process, sufficient space is ensured between each fertilized egg to prevent them from contacting each other. This arrangement helps the fertilized eggs obtain sufficient oxygen during incubation, thus providing a suitable environment for the fertilized eggs to promote successful hatching.

[0017] Preferably, in step (2), the fertilized egg is subjected to cold shock treatment at 4°C for 23 minutes.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The method of the present invention is the first to obtain female-developing black carp offspring through heterologous sperm stimulation from blunt snout bream. These offspring differ from the parent black carp in appearance and biological characteristics, grow faster, and can shorten the breeding cycle. Furthermore, new black carp strains can be obtained through backcrossing, providing important germplasm resources for the purification, rejuvenation, and quality improvement of black carp.

[0020] 2. The method of the present invention has different cold shock temperature, cold shock time, fertilization rate, hatching rate, and morphology, resulting in higher cultivation efficiency. Attached Figure Description

[0021] 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.

[0022] Figure 1 It is a black carp;

[0023] Figure 2 It is the sperm of the blunt snout bream that stimulates the development of the female nucleus in the offspring of the black carp;

[0024] Figure 3 It is the average DNA content of gynogenetic black carp;

[0025] Figure 4 It refers to the number of chromosomes in the female-developed black carp;

[0026] Figure 5 This is a diagram showing the results of fluorescent in situ hybridization of female-developed black carp;

[0027] Figure 6 The results are from the comparison of the 5S rDNA sequences of the female-developed black carp and its parents. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] COC represents the female carp, BSB represents the male bream, and GCB represents artificially gynogenetic carp.

[0032] Example:

[0033] I. Methods to improve the growth rate of black carp through artificial gynogenesis

[0034] 1. The method of cold shock is used to inhibit the expulsion of the second polar body of the egg cell and induce gynogenesis in black carp. The specific operation steps and procedures are as follows:

[0035] 1) The male blunt snout bream was selected as the paternal parent to provide heterologous sperm, ensuring that the offspring obtained are gynogenetic individuals.

[0036] 2) Collect heterologous sperm and dilute them appropriately with Hank's solution, with a dilution factor ranging from 1 to 10 times. Take about 3 mL of the diluted sperm and place it in a 25 mL diameter petri dish, evenly covering the bottom of the dish. Store the remaining diluted sperm in a refrigerator at 4°C for later use.

[0037] 3) Place the culture dish containing sperm on an ice plate wrapped with a dry towel. Next, inactivate the sperm using a 15W UV lamp. To achieve this, place the culture dish and ice plate on a shaker approximately 30cm away from the UV lamp. The entire UV irradiation system is covered with a light-shielding cloth. This serves two purposes: firstly, to protect the experimenter from excessive UV exposure, and secondly, to prevent the restoration of genetic activity due to photorepair during irradiation, thus ensuring inactivation efficiency. During the UV inactivation process, manually shake the culture dish every 1-3 minutes and perform a microscopic examination. During microscopic examination, use a toothpick to take a small sample of the culture and place it on a slide. Then, adjust the microscope to a suitable focus to clearly observe the sperm morphology. Subsequently, add a small amount of water and gently stir with a toothpick to mix the sperm with the water and activate them for observation of their motility. This process continues until the proportion of active sperm in the field of view decreases to approximately 50%, at which point UV irradiation is stopped.

[0038] 4) Place the photographed sperm, along with the culture dish, upright in a dark chamber, allowing the sperm to collect on one side of the dish. The dark chamber is used to prevent genetically inactivated sperm from being exposed to light and regenerating. Aspirate the collected sperm using a syringe and transfer them to prepared light-protected centrifuge tubes, then store them at 4°C.

[0039] 5) After the female fish comes into heat, squeeze the eggs into a ceramic basin, and pour in the inactivated foreign sperm. Use a goose feather to stir the inactivated foreign sperm and eggs to mix them thoroughly. Distribute the fertilized eggs evenly in a pre-set constant temperature water culture dish at 23-25℃, ensuring sufficient space between each egg and avoiding contact. This arrangement helps the fertilized eggs receive adequate oxygen during incubation. The fertilization process is expected to last approximately 2 minutes. This method provides a suitable environment for the fertilized eggs, promoting successful hatching.

[0040] 6) After the heterologous sperm activates the egg and initiates embryonic development, the water in the culture dish needs to be poured out and replaced with ice water. The purpose of this step is to inhibit the release of the second polar body from the fertilized egg, thereby promoting the doubling of the egg's genetic material. Gradient experiments were conducted on this process to obtain the best results, as shown in Tables 1 and 2.

[0041] 7) After the second polar body is inhibited from escaping by ice water, the culture dish containing the fertilized eggs is quickly removed from the 4°C freezer, and the 4°C ice water in the culture dish is discarded and replaced with room temperature water. The inhibition on the fertilized eggs gradually dissipates, and normal development begins. The female carp has the following appearance: Figure 1 As shown, the offspring of the female-developed black carp have the following appearance. Figure 2 As shown, their morphological characteristics (countable and measurable traits) were compared. The data were processed by SPSS software, and the results are shown in Tables 3 and 4. The morphological characteristics of the female development of the black carp stimulated by the sperm of the blunt snout bream were significantly different from those of the parents. The reason for this phenomenon may be due to the large difference in appearance between the two parents.

[0042] Table 1. Statistics on fertilization rate and hatching rate of gynogenetic black carp under different cold shock temperatures.

[0043]

[0044] Table 2. Statistics on fertilization rate and hatching rate of gynogenetic black carp under different cold shock durations.

[0045]

[0046] Table 3 Comparison of countable traits of black carp and their parents in gynogenetic stimulation by bream semen.

[0047]

[0048] Note: Uppercase Roman numerals represent hard fins, and Arabic numerals represent soft fins.

[0049] Note: The superscripts a, b, and c represent different significance levels. If the superscripts are the same, there is no significant difference.

[0050] Table 4. Comparison of measurable traits of black carp and their parents stimulating gynogenesis with sperm from blunt snout bream.

[0051]

[0052] Note: Superscripts a, b, c indicate significant differences; no superscript indicates no significant difference.

[0053] II. Comparison of growth rates between gynogenetic black carp and common black carp

[0054] Common black carp and female-developed black carp were raised under the same conditions. The weights of 10 common black carp and 10 female-developed black carp aged 3 and 6 months were compared. The results are shown in Table 5.

[0055] Table 5. Weight statistics of female-developed black carp and common black carp at 3 and 6 months of age (unit: g)

[0056]

[0057] It is evident that gynogenetic black carp stimulated by blunt snout bream sperm exhibit faster growth rates.

[0058] III. Methods for determining the ploidy of offspring from gynogenetic black carp

[0059] 1. To determine the ploidy of the hybrid offspring, this method will utilize flow cytometry to detect their DNA content. Ten gynogenetic offspring were selected for the experiment. The specific method is as follows:

[0060] 1) The captured gynogenetic offspring were meticulously classified and labeled in the tank to facilitate subsequent experimental procedures and data recording. This step was crucial to ensuring the accuracy and reproducibility of the experiment.

[0061] 2) Prepare sterile syringes and anticoagulant. Each syringe should be pre-filled with 0.2 mL of anticoagulant. The use of anticoagulant is to prevent blood clotting and ensure that the blood sample remains liquid during the experiment for easy subsequent analysis.

[0062] 3) When drawing blood samples, collect them from the tail vein to minimize harm to the fish. After collection, the blood samples are properly stored in an icebox to maintain their freshness and activity.

[0063] 4) For nuclear staining, sterile EP tubes were prepared and 0.3 mL of DAPI staining solution and 1 mL of 8% physiological saline were added. DAPI is a fluorescent dye that specifically binds to DNA, allowing us to observe the morphology of the cell nucleus under a microscope.

[0064] 5) Using a 1μL pipette, gradually add the drawn blood to the EP tube containing DAPI staining solution until the liquid turns slightly pink. This step is to ensure that the blood sample is thoroughly mixed with the staining solution.

[0065] 6) After sample preparation, place them in the dark for 10-15 minutes to allow the DAPI dye to fully penetrate the cell nuclei. After the dark treatment, filter the samples using a 20μm pore size filter to remove unbound dye and cell debris, then dilute them for flow cytometry analysis. The results are as follows: Figure 3 As shown.

[0066] 2. In this method, 10 female offspring weighing approximately 50g were selected and each fish was marked to ensure the accuracy of the experiment. Before the experiment, frozen slides, sterile 1.5mL EP tubes, sterile glass petri dishes, sterile 15mL EP tubes, sterile surgical scissors, sterile pipettes, physiological saline (4g / 500mL), KCl (2.8g / mL), methanol, glacial acetic acid, Giemsa stain, Na2HPO4 (0.7098g / 500mL), and NaH2PO4 (0.78005g / mL) were prepared in advance.

[0067] 1) At 8:30 PM on the first day, a 4 mg / mL concentration of PHA (phytohemagglutinin) was injected into the pectoral fin of the experimental fish at a 45-degree angle towards the tail. The PHA dosage was calculated as body weight * dose / concentration, with a PHA dose of 10 μg / g, resulting in a PHA dose of 0.0025 * body weight. At 8:30 AM on the second day, the same concentration of PHA was injected into the same location on the experimental fish (dose of 15 μg / g, resulting in a PHA dose of 0.00375 * body weight). Three hours later, the same concentration of PHA was injected into the same location on the experimental fish (dose of 6 μg / g, resulting in a PHA dose of 0.0015 * body weight). On the other side of the experimental fish, a 2.5 mg / mL concentration of colchicine was injected into the same location (colchicine dose of 4 μg / g, resulting in a colchicine dose of 0.00075 * body weight). After the third injection, the experimental fish were placed in a tank for 1 hour before the experiment.

[0068] 2) First, bleed the selected experimental fish. After bleeding, carefully cut open the fish along the direction of the vent to remove the kidneys as intact as possible from the back. Immediately rinse the removed kidneys thoroughly with physiological saline to remove any remaining blood and other impurities. After rinsing, gently place the kidneys into a pre-prepared sterile culture dish.

[0069] 3) Add an appropriate amount of physiological saline to the culture dish to facilitate the handling of the kidney tissue. Then, tilt the culture dish to approximately a 45-degree angle for easier handling. Using surgical scissors, carefully cut the kidney tissue into small pieces until it is completely dispersed into a homogeneous aqueous solution. This step is crucial for ensuring adequate dispersion of the kidney cells and the smooth progress of subsequent experiments.

[0070] 4) Transfer the processed kidney tissue, now in an aqueous solution, to a 15 mL EP tube. To dilute and homogenize the kidney cell suspension, add physiological saline to the EP tube until the total volume reaches 4 mL. Then, vigorously pipette the suspension approximately 200 times to promote cell dispersion. After pipetting, continue adding physiological saline until the total volume in the EP tube reaches 12 mL, and pipette again 200 times to ensure homogeneity of the cell suspension and reduce cell aggregation.

[0071] 5) After completing the pipetting step, we let the EP tube stand for 10 minutes to allow solid particles in the cell suspension to settle. Then, we carefully transferred the supernatant to a new 15 mL EP tube, added physiological saline to bring the total volume to 11 mL, and centrifuged the EP tube at 1500 rpm for 5 minutes. After centrifugation, we carefully discarded the supernatant, retaining the precipitate at the bottom of the tube.

[0072] 6) Add 4 mL of KCl, gently pipette to mix, and bring the volume to 10 mL. Let stand for 60 min, discarding the precipitate at the bottom every 10 min. After standing, centrifuge the tube at 1500 rpm for 5 min, discarding the supernatant. After removing the precipitate, add 4 mL of KCl solution to the EP tube. This is a solution used for cell lysis and chromosome preparation. Gently pipette to ensure the KCl solution is thoroughly mixed with the precipitate. Then, continue adding physiological saline to bring the total volume to 10 mL to dilute the suspension and promote further separation of cell components. Let the EP tube stand for 60 min. During this process, carefully aspirate and discard the precipitate at the bottom every 10 min. After standing, return the EP tube to the centrifuge and centrifuge at 1500 rpm for 5 min. After centrifugation, discard the supernatant, retaining only the precipitate at the bottom of the tube.

[0073] 7) After precipitation, add 2 mL of Carnot fixative (methanol:glacial acetic acid = 1:3) to the EP tube and gently pipette to ensure thorough mixing of the fixative and precipitate. To further ensure fixation, add fixative up to 6 mL and allow the mixture to stand for 15 min. After fixation, centrifuge the EP tube at 1500 rpm for 5 min. After centrifugation, remove the supernatant and repeat the fixation and centrifugation steps three times. After three fixation and centrifugation processes, add an additional 2 mL of fixative to the EP tube and seal it. To maintain the stability of the cell nuclear samples, store the sealed EP tube at 4°C.

[0074] 8) Take out a frozen slide that has been stored at -20℃ for 24 hours, and use a pipette to drop the fixative stored at 4℃ onto the frozen slide from a vertical position. Gently sweep the slide over the outer flame of the alcohol lamp a few times.

[0075] 9) Stain the baked slides with a staining solution (5 ml Na₂HPO₄ + 5 ml NaH₂PO₄ + 20 drops Giemsa stain) for 45 minutes. After staining, rinse the back of the slides under running water to remove the stain, ensuring a gentle flow. After air drying, observe and photograph the slides under an electron microscope. The results are shown below. Figure 4 As shown, the offspring of gynogenesis all have 100 chromosomes.

[0076] IV. Genetic Identification Methods for Gynogenetic Offspring

[0077] 1. In this method, the chromosome fixation solution prepared for the ploidy detection experiment is used. After normal slide preparation, the slides are placed in a 70℃ oven for drying, while the water bath is preheated (70℃, 80℃). The probe used in this chapter is the 5S sequence of blunt snout bream. The specific operating steps are as follows:

[0078] 1) When performing fluorescence in situ hybridization (FISH) experiments, the probe hybridization solution needs to be prepared first. This includes mixing purified probe (5 μL), deionized formamide (DDM, 4 μL), 50% dextran sulfate (DS, 3 μL), and 20× sodium citrate hydrochloride buffer (SSC, 2 μL). Next, the slide containing the chromosome is preheated in a 70°C oven for 1-2 hours to facilitate the subsequent hybridization process.

[0079] 2) The slides were immersed in 2×SSC solution for 30 min to reduce background signal. Then, the slides were dehydrated by a gradient of 70% and 100% alcohol for 5 min each time, and then briefly placed in 70% 2×SSC / DDM solution for 2 min to further prepare for hybridization.

[0080] 3) After drying at room temperature, add the denaturing hybridization solution to a glass slide to cover the chromosome, and then mount the slide. Place the mounted slide in a humidified chamber and incubate overnight at 37°C to promote the binding of the probe to the target DNA.

[0081] 4) After incubation, the slides were washed twice in 2×SSC / DDM solution at 43℃ for 5 min each time, and then washed sequentially in 2×SSC and 1×SSC for 5 min each time. Afterward, the slides were dried at room temperature, and 8 μL of fluorescein isothiocyanate (FITC) was added. The slides were then incubated in the dark for 20-30 min.

[0082] 5) The washing step was repeated. The slide was washed in washing solution at 43°C for 15 min, and then dried at room temperature. Finally, 6 μL of DAPI and a fluorescence quencher were added to enhance nuclear staining and reduce fluorescence background. After completing the above steps, the slide could be observed under a fluorescence microscope to evaluate the hybridization effect and the specific binding of the probe. The results of hybridization between the paternal-specific primer and the female nucleosuria carp showed no signal, consistent with the results of the maternal nucleosuria carp. The results are as follows: Figure 5 As shown.

[0083] 2. For genetic analysis experiments, 10 parent fish and 10 female-developed offspring were selected. First, the tail fins were thoroughly cleaned with a 70% alcohol solution to remove any potential microorganisms and impurities. After cleaning, the tail fins were cut off using autoclaved scissors. The cut tail fins were immediately placed into pre-labeled EP tubes. To preserve these samples long-term, they were stored in a freezer at -20°C. The specific experimental methods are as follows:

[0084] 1) DNA extraction was performed using the OMG (Omega Tissue DNA Kit). The preserved tail fin sample was removed, shredded with sterile scissors, and placed into a sterile EP tube. 200 μL of TL Buffer and 25 μL of OB Protease Solution were added. After addition, the tube was incubated at 55°C for 5 minutes.

[0085] 2) After completing the water bath step, add 220 μL of BL buffer to the sample and gently shake the EP tube to ensure thorough mixing. After mixing, adjust the water bath temperature to 70°C and return the sample to the water bath for another 10 min.

[0086] 3) After the water bath treatment is completed, add 220 μL of 100% alcohol to the sample, shake well, transfer the sample to the adsorption column using a pipette, and centrifuge for 1 min.

[0087] 4) After centrifugation, carefully remove the supernatant and replace with a new collection tube. Next, add 500 μL of HBC Buffer, centrifuge again for 30 seconds, and then discard the supernatant. Then add 700 μL of DNA Wash Buffer, centrifuge for 30 seconds, and discard the supernatant. This step needs to be repeated twice to ensure DNA purification.

[0088] 5) After completing the washing steps above, replace the tube with a new one and centrifuge for 1 minute. After centrifugation, open the cap and let the tube stand at room temperature for 5 minutes to allow the DNA to precipitate at the bottom. After precipitation, add 35-50 μL of preheated (70°C) Elution Buffer and centrifuge again for 2 minutes. The purified DNA has now been successfully extracted from the sample and can be stored at -20°C for subsequent experiments.

[0089] 6) DNA was extracted from selected fish samples, and PCR amplification was performed using 5S-specific primers. After PCR amplification, the amplified product was gel-cleaved to further purify the target DNA fragment for subsequent cloning and sequencing. The purified DNA fragment was then ligated into an appropriate vector and transformed into host cells for single-clone screening and amplification. After screening, the samples were sent to Sanger sequencing at Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were analyzed in detail using Jaiview software (version 2.11.3.2). No differences were found between the two gynogenetic black carp species and the maternal black carp in this coding region, while a 4-base change was observed in the NTS region, indicating that the offspring's 5S genetic information mainly came from the maternal parent. The results are as follows: Figure 6 As shown, the offspring obtained are female-developed carp.

Claims

1. A method for improving the growth rate of black carp through artificial gynogenesis, characterized in that, Includes the following steps: (1) After diluting the sperm of the blunt snout bream, it was irradiated under a UV lamp for inactivation treatment until the number of active sperm dropped to 45-55% and then the irradiation was stopped. The irradiated sperm was then stored in a dark place under cold storage. The specific operation of the inactivation treatment is as follows: the diluted sperm was placed in a culture dish on an ice plate and then placed on a shaker. It was then irradiated with a 15 W UV lamp. The irradiation distance between the UV lamp and the sperm was 28-32 cm. The entire UV irradiation system was covered with a light-shielding cloth. During the irradiation process, the culture dish was shaken every 1-3 minutes and the sperm motility was observed regularly. (2) Mix the black carp eggs and the sperm obtained after step (1) in room temperature water for 2-3 minutes to fertilize them. Then, subject the fertilized eggs to cold shock treatment at 4°C for 23 minutes and then place them in room temperature water for incubation.

2. The method according to claim 1, characterized in that, In step (1), the specific operation of diluting the blunt snout bream sperm is as follows: dilute the blunt snout bream sperm with Hank's solution, and the dilution factor is 1-10 times the volume.

3. The method according to claim 1, characterized in that, In step (1), the refrigeration temperature is 4-5℃.

4. The method according to claim 1, characterized in that, In step (2), the temperature of the room temperature water is 23-25℃.

5. The method according to claim 1, characterized in that, In step (2), during the incubation process, sufficient space is ensured between each fertilized egg to prevent them from coming into contact with each other.

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