Method for improving nutritional components of oncorhynchus mykiss by artificial gynogenesis

Abstract

The present application belongs to the field of artificial gynogenesis breeding of fish, and discloses a method for improving nutritional components of Oncorhynchus mykiss by artificial gynogenesis, comprising the following steps: diluting Oncorhynchus mykiss sperm, irradiating the sperm under an ultraviolet lamp to inactivate the sperm, and stopping the irradiation until the number of active sperm is reduced to 45-55%, and then storing the irradiated sperm under light-shielded condition; (2) mixing Oncorhynchus mykiss eggs and the sperm obtained in step (1) in normal temperature water to fertilize the eggs, cold-shocking the fertilized eggs at 4-5 DEG C for 19-21 min, and then incubating the eggs in normal temperature water. The improvement method is helpful to accelerate genome separation and fixation of hybrid offspring, makes it possible to identify and screen excellent lines according to economic traits, thereby improving the efficiency of selection of quality traits and quantitative traits, and finally obtaining gynogenesis Oncorhynchus mykiss offspring.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of artificial gynogenesis breeding of fish, and particularly relates to a method for improving the nutritional components of Oncorhynchus mykiss by artificial gynogenesis technology. BACKGROUND

[0002] Artificial gynogenesis technology is an important technique in aquaculture breeding, which uses heterologous sperm to stimulate the development of eggs and the doubling of genetic material, thereby producing all-female offspring. The core of this technology lies in two main steps: first, the activation of heterologous sperm, and second, the doubling of the genetic material of the egg. In the step of activating the egg with heterologous sperm, scientists use inactivated heterologous sperm to activate or initiate the embryonic development process of the egg. Since the heterologous sperm are inactivated, they cannot participate in the normal fertilization process, but they can still activate the development of the egg, causing it to begin to divide and grow.

[0003] Oncorhynchus mykiss is a variety of carp, belonging to the order Cypriniformes, the family Cyprinidae, and the subfamily Cyprininae. It has a short and stout body, a small head, thin skin, tender meat, no muddy smell, and a purple-red body color. It has fine leaf scales, and the internal organs are barely visible due to the thin skin on its abdomen. It grows quickly and has delicious, meaty, and small-spine meat, so it is favored by many breeders. However, due to long-term self-crossing and inbreeding, its quality has been deteriorating, with the most direct feeling being a decrease in growth rate and resistance. In addition, the contents of crude fat, amino acids, fatty acids, and other nutritional components are also decreasing. Therefore, it is of great significance to the field to breed an Oncorhynchus mykiss with improved nutritional components. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies and shortcomings mentioned in the background art and to provide a method for improving the nutritional components of Oncorhynchus mykiss by artificial gynogenesis technology.

[0005] To solve the above technical problems, the technical solution proposed by the present application is as follows:

[0006] A method for improving the nutritional components of Oncorhynchus mykiss using artificial gynogenesis, comprising the following steps:

[0007] (1) Dilute the red crucian carp sperm and irradiate it under a UV lamp for inactivation treatment until the number of active sperm decreases to 45-55%, then stop the irradiation and store the irradiated sperm in a dark condition for cold preservation;

[0008] (2) Mix the Oncorhynchus mykiss eggs with the sperm obtained after step (1) in normal temperature water to allow fertilization, and then perform cold shock treatment on the fertilized eggs at 4-5℃ for 19-21min, and then place them in normal temperature water for incubation.

[0009] The selection of heterologous sperm is to use red sea bream as the male parent to provide heterologous sperm, so as to ensure that the offspring obtained is a gynogenetic individual.

[0010] In the above preparation method, preferably, in step (1), the specific operation of diluting the red sea bream sperm is as follows: the red sea bream sperm is diluted with Hank's solution, and the dilution ratio is 1-10 times the volume.

[0011] In the above preparation method, preferably, in step (1), the specific operation of diluting the red sea bream sperm is as follows: the red sea bream sperm is diluted with Hank's solution, and the dilution ratio is 1-10 times the volume.

[0012] In the above preparation method, preferably, in step (1), the specific operation of diluting the red sea bream sperm is as follows: the red sea bream sperm is diluted with Hank's solution, and the dilution ratio is 1-10 times the volume.

[0013] The purpose of covering the black cloth is twofold: on the one hand, it protects the experimental personnel from excessive ultraviolet radiation, and on the other hand, it avoids the recovery of genetic activity due to photoreactivation during irradiation, thereby ensuring the inactivation efficiency. During the ultraviolet inactivation process, the culture dish is manually shaken every 1-3 minutes, and a microscope examination is performed once. During the microscope examination, a small amount of culture sample is taken with a toothpick and placed on a glass slide, and then the microscope is adjusted to the appropriate focus to clearly observe the morphology of the sperm. Subsequently, a small amount of water is added and gently stirred with a toothpick to mix and activate the sperm, so as to observe their activity. This process will continue until the proportion of active sperm in the field of view decreases to about 50%, at which point the ultraviolet irradiation will be stopped.

[0014] In the above preparation method, preferably, in step (1), the specific operation of diluting the red sea bream sperm is as follows: the red sea bream sperm is diluted with Hank's solution, and the dilution ratio is 1-10 times the volume.

[0015] In the above preparation method, preferably, in step (1), the specific operation of diluting the red sea bream sperm is as follows: the red sea bream sperm is diluted with Hank's solution, and the dilution ratio is 1-10 times the volume.

[0016] In the above preparation method, preferably, in step (2), the temperature of the normal temperature water is 23-25℃.

[0017] In the above preparation method, preferably, in step (2), the fertilization time is 2-3 minutes, and during the incubation process, enough space is ensured between each fertilized egg to avoid contact with each other. Such a layout is conducive to the fertilized eggs obtaining sufficient oxygen supply during the incubation process, and in this way, a suitable environment can be provided for the fertilized eggs to facilitate their smooth incubation.

[0018] Compared with the prior art, the present application has the following advantages:

[0019] 1. The improved method of the present application uses red crucian carp as the heterologous sperm father, and combines the method of cold shock to inhibit the second polar body discharge of the egg cell, inducing gynogenesis in the Amur ide, which helps to accelerate the genomic separation and fixation of hybrid offspring, making it possible to identify and screen excellent lines according to economic traits, thereby improving the efficiency of selection of quality traits and quantitative traits, and ultimately obtaining gynogenetic Amur ide offspring.

[0020] 2. The gynogenetic Amur ide obtained by the improved method is similar to the Amur ide in appearance and biological characteristics, and the nutritional components have changed significantly. The gynogenetic Amur ide offspring have significant differences in crude fat, amino acids, and fatty acids from the mother Amur ide, and therefore have important significance in genetic breeding and production practice. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.

[0022] Figure 1 is the mother Amur ide;

[0023] Figure 2 is the gynogenetic Amur ide;

[0024] Figure 3 is the average DNA content of the gynogenetic Amur ide;

[0025] Figure 4 is the number of chromosomes of the gynogenetic Amur ide. DETAILED DESCRIPTION

[0026] In order to facilitate the understanding of the present application, the following will combine the drawings of the specification and the preferred embodiments to make a more comprehensive and detailed description of the present application, but the protection scope of the present application is not limited to the following specific embodiments.

[0027] Unless otherwise defined, all the professional terms used in the following have the same meaning as generally understood by those skilled in the art. The professional terms used in this paper are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present application.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0029] COC represents the mother Amur ide; RCC represents the father red crucian carp; GCR represents the gynogenetic Amur ide stimulated by red crucian carp sperm.

[0030] Embodiment:

[0031] A method for improving the nutritional components of Oncorhynchus mykiss by artificial gynogenesis, comprising the following steps:

[0032] 1. Using cold shock method to inhibit the second polar body discharge of egg cell, inducing Oncorhynchus mykiss to carry out gynogenesis, the specific operation steps and process are as follows:

[0033] 1) Selection of heterologous sperm Red crucian carp is used as the father to provide heterologous sperm to ensure that the offspring obtained is a gynogenetic individual.

[0034] 2) Collect the heterologous sperm and dilute it with Hank's solution, the dilution ratio is from 1 to 10 times. Take about 3mL of the diluted sperm and evenly cover the bottom of the culture dish with a diameter of 25mL. The remaining diluted sperm is placed in a 4℃ refrigerator for standby.

[0035] 3) Place the culture dish containing sperm on an ice plate wrapped with a dry towel. Next, use a 15W ultraviolet lamp for inactivation treatment. In order to achieve this process, place the culture dish and ice plate on a shaker about 30cm away from the ultraviolet lamp tube. The entire ultraviolet irradiation system is covered with a light shield cloth, which serves a dual purpose: on the one hand, it protects the experimental personnel from excessive ultraviolet radiation, and on the other hand, it avoids the recovery of genetic activity due to photoreactivation during irradiation, thereby ensuring the inactivation efficiency. During the ultraviolet inactivation treatment, manually shake the culture dish every 1-3 minutes and perform a microscope examination. When examining under the microscope, use a toothpick to take a small amount of culture sample and place it on a glass slide, then adjust the microscope to the appropriate focus to clearly observe the morphology of the sperm. Then, add a small amount of water and gently stir with a toothpick to mix and activate the sperm, so as to observe their activity. This process will continue until the proportion of active sperm in the field of view decreases to about 50%, at which point the ultraviolet irradiation will be stopped.

[0036] 4) Place the irradiated sperm together with the culture dish in a dark box to allow the sperm to concentrate on one side of the culture dish. The dark box is to prevent the genetically inactivated sperm from photorepair. The concentrated sperm is sucked with a syringe and transferred to a prepared light-proof centrifuge tube and stored in a 4℃ refrigerator.

[0037] 5) After the female fish is in estrus, the eggs are squeezed into a ceramic basin, and the inactivated heterologous sperm is poured in as well, and the inactivated heterologous sperm and the eggs are stirred with a goose feather to mix them thoroughly, and the fertilized eggs are evenly dispersed and placed in a pre-set constant temperature water culture dish at 23-25°C, ensuring that there is enough space between each fertilized egg to avoid contact with each other. Such a layout is conducive to the fertilized eggs to obtain sufficient oxygen supply during the hatching process. The fertilization process is expected to last about 2 min. In this way, a suitable environment can be provided for the fertilized eggs to facilitate their smooth hatching.

[0038] 6) After the heterologous sperm activates the eggs and initiates embryonic development, the water in the culture dish needs to be poured out and replaced with ice water at 4°C, and then the culture dish is transferred to a refrigerator at 4°C for cold storage. The purpose of this step is to inhibit the release of the second polar body by the fertilized eggs, thereby facilitating the completion of the doubling of the genetic material of the eggs. This process stimulated by red carp sperm requires 20 min (GCR).

[0039] 7) After the ice water inhibits the discharge of the second polar body, the culture dish containing the fertilized eggs is quickly removed from the 4°C refrigerator, and the 4°C ice water in the culture dish is poured out and replaced with normal temperature water. The inhibition of the fertilized eggs is gradually lifted, and normal development begins. The appearance of the gynogenetic Koi carp offspring is as shown in Figure 1 The morphological characteristics (countable and quantifiable traits) were compared, and the data obtained were processed by SPSS software, and the results are shown in Tables 1 and 2. The morphological characteristics of the gynogenetic Koi carp stimulated by red carp sperm are closer to those of the female parent Koi carp.

[0040] Table 1 Comparison of countable traits of gynogenetic Koi carp stimulated by red carp sperm and parent

[0041]

[0042] Note: The capital Roman numerals represent hard fin rays, and the Arabic numerals represent soft fin rays.

[0043] Note: The upper superscripts a, b, and c represent different significant differences, and the same superscript indicates no significant difference.

[0044] Table 2 Comparison of quantifiable traits of gynogenetic Koi carp stimulated by red carp sperm and parent

[0045]

[0046]

[0047] Note: The upper superscript a represents significant difference.

[0048] II. Method for detecting ploidy of gynogenetic Koi carp offspring

[0049] 1. For the ploidy of hybrid offspring, this method will use flow cytometry to detect the DNA content. Ten female offspring of the experiment were selected for the experiment. The specific method is as follows:

[0050] 1) The captured gynogenesis offspring were carefully classified and labeled in the tank to facilitate subsequent experimental operation and data recording. This step is crucial to ensure the accuracy and repeatability of the experiment.

[0051] 2) Prepare sterile syringes and anticoagulants, each syringe pre-absorbed with 0.2 mL of anticoagulant. The use of anticoagulants is to prevent blood clotting and ensure that the blood sample remains liquid during the experiment, facilitating subsequent analysis.

[0052] 3) When drawing blood samples, collect from the fish's tail vein to minimize damage to the fish. After collection, the blood sample is properly stored in an ice box to maintain its freshness and activity.

[0053] 4) Perform nuclear staining, prepare sterile EP tubes, and add 0.3 mL of DAPI staining solution and 1 mL of 8% physiological saline. DAPI is a fluorescent dye that specifically binds to DNA, allowing us to observe the morphology of the cell nucleus under a microscope.

[0054] 5) Use a 1 μL pipette to gradually add the drawn blood to the EP tube containing the DAPI staining solution until the liquid color turns slightly red. This step is to ensure that the blood sample is fully mixed with the staining solution.

[0055] 6) After sample preparation, place them in the dark for 10-15 min to allow the DAPI dye to fully penetrate the cell nucleus. After the light avoidance treatment is complete, use a 20 μm aperture filter to filter the sample to remove unbound dye and cell debris, then dilute it for detection on the flow cytometer, as shown in Figure 3 .

[0056] 2. In this method, 10 gynogenesis offspring with a body weight of about 50 g were selected and labeled for each experimental fish to ensure the accuracy of the experiment. Before the experiment, prepare frozen slides, sterilize 1.5 mL EP tubes, sterilize glass culture dishes, sterilize 15 mL EP tubes, sterilize surgical scissors, sterilize pipettes, physiological saline (4 g / 500 mL), KCL (2.8 g / mL), methanol, glacial acetic acid, Giemsa staining solution, Na2HPO4 (0.7098 g / 500 mL), NaH2PO4 (0.78005 g / mL), and other reagents.

[0057] 1) The first night at 8:30 PM, inject PHA with a concentration of 4 mg / mL at the fish tail direction at a 45-degree angle to the fish chest fin (PHA is phytohemagglutinin, the PHA dosage calculation formula is body weight * dosage / concentration, the PHA dosage is 10 μg / g, the calculation result is 0.0025*body weight); the next day at 8:30 AM, inject the same concentration of PHA at the same position of the experimental fish (the dosage is 15 μg / g, the calculation result is 0.00375*body weight); 3 hours later, inject the same concentration of PHA at the same position of the experimental fish (the dosage is 6 μg / g, the calculation result is 0.0015*body weight), and inject colchicine with a concentration of 2.5 mg / ml at the same position on the other side of the experimental fish (the colchicine dosage is 4 μg / g, the calculation result is 0.00075*body weight). After the third injection, place the experimental fish in the tank for 1 hour before the experiment.

[0058] 2) First, the selected experimental fish is subjected to bloodletting treatment. After completing the bloodletting, the fish body is carefully cut along the direction of the urogenital opening of the fish body to take out the kidney as completely as possible from the back. The removed kidney is immediately thoroughly washed with physiological saline to remove possible residual blood and other impurities. After washing, the kidney is gently placed in a pre-prepared sterile culture dish.

[0059] 3) An appropriate amount of physiological saline is added to the culture dish to facilitate the processing of the kidney tissue. Then, the culture dish is tilted to about 45 degrees to facilitate operation. Using surgical scissors, the kidney tissue is carefully cut into small pieces until it is completely dispersed into a uniform aqueous solution. This step is crucial to ensure that the kidney cells are fully dispersed and the subsequent experiment proceeds smoothly.

[0060] 4) The kidney tissue treated into an aqueous solution is transferred to a 15 mL EP tube. To dilute and uniformly mix the kidney cell suspension, we add physiological saline to the EP tube until the total amount reaches 4 mL. Then, using a pipette, the suspension is vigorously blown for about 200 times to promote cell dispersion. After blowing, we continue to add physiological saline until the total amount of liquid in the EP tube reaches 12 mL, and perform another 200 times of blowing to ensure the uniformity of the cell suspension and reduce cell aggregation.

[0061] 5) After completing the blowing step, we let the EP tube stand for 10 min to allow the solid particles in the cell suspension to settle. Then, we carefully transfer the supernatant in the upper layer to a new 15 mL EP tube, and then supplement physiological saline to make the total volume reach 11 mL. Use the centrifuge to centrifuge the EP tube, set the speed to 1500 revolutions per minute (rpm), and the centrifugation time to 5 min. After centrifugation, carefully pour off the supernatant, and retain the precipitate at the bottom of the tube.

[0062] 6) Add 4 mL KCL, gently blow the EP tube with a pipette to mix, then add to 10 mL, stand for 60 min, every 10 min during the interval, suck the bottom of the precipitate and discard. After standing, centrifuge the test tube in the centrifuge for 5 min at 1500 rpm, discard the supernatant after centrifugation. After the removal of the precipitate is completed, 4 mL KCL solution is added to the EP tube, which is a solution for cell lysis and chromosome preparation. We use a pipette to gently blow to ensure that the KCL solution is fully mixed with the precipitate. Then, continue to add physiological saline to make the total volume reach 10 mL to dilute the suspension and promote the further separation of cell components. The EP tube is left to stand for 60 min. During this process, the bottom precipitate is carefully sucked and discarded every 10 min, and after the standing time is over, the EP tube is placed in the centrifuge again at a speed of 1500 rpm for 5 min. After centrifugation is completed, the supernatant is poured off, and only the precipitate at the bottom of the tube is retained.

[0063] 7) After the completion of the precipitation, 2 mL of Carnoy's fixative (methanol: glacial acetic acid = 1:3) is added to the EP tube, and the EP tube is gently blown to ensure that the fixative is fully mixed with the precipitate. To further ensure the fixing effect, continue to add fixative to 6 mL, and then let the mixture stand for 15 min. After fixing, the EP tube is centrifuged in the centrifuge at a speed of 1500 rpm for 5 min. After centrifugation, the supernatant is removed, and this fixing and centrifugation step is repeated three times. After the three fixing and centrifugation processes are completed, an additional 2 mL of fixative is added to the EP tube, and then the EP tube is sealed. In order to maintain the stability of the cell nucleus sample, the sealed EP tube is stored in a refrigerator at 4°C.

[0064] 8) Take out a frozen slide that has been stored at -20°C for 24 h, and use a pipette to add the fixative stored at 4°C to the frozen slide from the vertical top, and gently pass the slide over the outer flame of the alcohol lamp a few times.

[0065] 9) Use the staining solution (5 ml Na2HPO4+5 ml NaH2PO4+20 drops of Giemsa staining solution) to stain the baked slide, and the staining time is 45 min. After staining, wash the slide under running water to remove the staining agent, and the running water is gentle during this period. After natural air drying, observe and take pictures under an electron microscope, and the results are shown in Figure 4 , the chromosome number of both gynogenetic offspring is 100.

[0066] III. Comparison of fatty acids in gynogenetic Misgurnus anguillicaudatus and common Misgurnus anguillicaudatus

[0067] In the present invention, the fatty acid changes of the Atlantic salmon and the gynogenetic Atlantic salmon were explored. Under the same cultivation conditions, 3 fish of each species were randomly selected as experimental samples. Before the experiment, the experimental fish were first disinfected with 70% alcohol to ensure aseptic operation during the experiment. The specific experimental content is as follows:

[0068] 1) Using high-temperature sterilized scissors, carefully cut the abdomen along the cloaca of the fish, and completely remove all internal organs. After cleaning the internal organs, carefully clean the abdomen to remove residual blood and ensure the cleanliness of the sample.

[0069] 2) Next, cut off the head and tail fin of the fish, and scrape off the scales to facilitate subsequent quality analysis. After processing, the remaining tissue samples were placed in a refrigerator at -20℃ for frozen storage. After overnight freezing treatment, the samples were sent to Qingdao Stand Company for professional nutrient component detection.

[0070] 3) Dry lipid samples were obtained from freeze-dried muscle samples by chloroform-methanol method. Then, add 2mL CH4O with KOH to the dry lipid sample, and heat the mixture at 50℃ until the oil droplets disappear due to saponification. After cooling to room temperature, add 2mL 14% BF3-CH3OH, mix in a 50℃ water bath for 30min for FAMEs derivatization. Then, add 1mL HPLC (>95%) to the above solution and shake for 2min. Then, add saturated sodium chloride solution to separate the organic phase, and take the supernatant for gas chromatography analysis. The fatty acid composition was determined by gas chromatography equipped with a 50m x 0.25mm x 0.20μm chromatographic column (Trace1310ISQ). High-purity N2 was used as the carrier gas at a constant flow rate of 7.8mL / min. The temperature of the gas chromatograph was raised from 140℃ to 210℃ at a rate of 5℃ / min, and maintained at 210℃ for 10min. The temperature of the injector and detector was set to 250 and 300℃ respectively. The identification of fatty acids was determined by comparing the retention time with the known standard mixture. By calculating the peak area ratio of each fatty acid, the results were expressed as the relative content of each fatty acid, with the unit of g / 100g.

[0071] The data in the experimental process was analyzed by One Way ANOVA Analysis of SPSS 17.0 software, and Duncan test was used for multiple comparisons. The experimental results were expressed as "mean ± standard error", and the significance level was set as P<0.05. The COC, GCR were cut off the head, tail fin, and the muscle tissue sample of the fish scale was scraped to detect the fatty acid. The gynogenesis of the red gynogenetic crucian carp was stimulated by the sperm of the female parent, and 19 kinds of fatty acids were detected in the female parent and the ordinary crucian carp. The ordinary crucian carp did not detect eicosatrienoic acid n-6, and the red gynogenetic crucian carp did not detect omega-6 octadecatrienoic acid. The gynogenesis of the gynogenetic crucian carp stimulated by the sperm of the common bullhead was detected. Among the 20 kinds of fatty acids detected, there were 6 kinds of saturated fatty acids and 14 kinds of unsaturated fatty acids. Compared with the ordinary crucian carp, the red gynogenetic crucian carp stimulated by the sperm of the red gynogenetic crucian carp had higher contents of palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, sorbic acid, eicosatrienoic acid n-6, eicosatrienoic acid n-3, eicosapentaenoic acid n-3 and docosahexaenoic acid n-3. The results are shown in Table 3.

[0072] Table 3 Statistical results of fatty acids of gynogenesis of the crucian carp and ordinary crucian carp

[0073]

[0074] Note: The superscript * represents significant.

[0075] It can be seen that the nutritional components of the gynogenesis of the crucian carp obtained by the breeding method of the present application have changed obviously, and the contents of crude fat, amino acid and fatty acid have significant differences with those of the female parent crucian carp, so it has important significance in genetic breeding and production practice.

Claims

1. A method for improving the nutritional composition of black carp using artificial gynogenetic development, characterized in that, Includes the following steps: (1) After diluting the red crucian carp sperm, 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. (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 4-5℃ for 19-21 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 red crucian carp sperm is as follows: dilute the red crucian carp 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 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.

4. The method according to claim 3, characterized in that, During the irradiation process, the culture dish should be shaken well every 1-3 minutes, and sperm motility should be observed periodically.

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

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

7. The method according to any one of claims 1-6, characterized in that, In step (2), the fertilization time is 2-3 minutes, and during the incubation process, sufficient space is ensured between each fertilized egg to avoid contact with each other.

Images