Method for creating rapidly-growing gynogenetic grass carp

Abstract

The invention belongs to the technical field of fish variety improvement, and discloses a method for creating rapidly growing gynogenetic grass carps, which comprises the following steps of: screening sexual maturity male mirror carps and sexual maturity female grass carps with relatively obvious growth advantages and stress resistance; the method comprises the following steps: collecting mirror carp sperms, diluting the mirror carp sperms with Hank's liquid, inactivating the sperms by ultraviolet radiation, mixing the obtained inactivated sperms with middle-section ova of grass carps, then carrying out cold shock doubling treatment, and then adding normal-temperature water in batches for hatching to obtain gynogenetic grass carps. The gynogenesis grass carps induced by the mirror carp sperms are excellent in growth speed and disease resistance, a new method and thought are provided for heritage improvement of the grass carps, more possibilities are provided for screening good varieties, and greater economic benefits are brought to the breeding industry of the grass carps.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish variety improvement, and particularly relates to cultivating an improved grass carp variety with fast growth characteristics by inducing gynogenesis of grass carp with sperm of mirror carp ( Cyprinus carpiovar.specularis ). Background Art

[0002] Grass carp ( Ctenopharyngodon Idella ) is one of the important freshwater cultured fish in China, with the advantages of herbivory, fast growth, and delicious meat. However, the overwintering syndrome causes huge economic losses to the grass carp aquaculture industry. At present, there is no effective treatment method, resulting in a low survival rate of grass carp, which seriously restricts the development of the grass carp aquaculture industry. In order to improve the germplasm quality of grass carp and increase the survival rate of grass carp overwintering, using gynogenesis technology to cultivate an excellent new grass carp variety with fast growth characteristics is an effective way.

[0003] Gynogenesis is a genetic breeding technology that uses heterologous sperm to stimulate egg development but does not transmit paternal genetic material. It can produce all-female offspring and improve aquaculture efficiency and quality. Although these heterologous sperm do not participate in inheritance, they may introduce or create some excellent traits through the integration or recombination of chromosomes or DNA fragments. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and defects in the background art, and provide a method for creating gynogenetic grass carp with fast growth characteristics, which significantly improves the overwintering survival rate of the pure line population of gynogenetic grass carp.

[0005] To solve the above technical problem, the technical solution proposed by the present invention is as follows: A method for creating gynogenetic grass carp with fast growth, comprising the following steps: (1) Select sexually mature male mirror carp and sexually mature female grass carp with relatively obvious growth advantages and stress resistance; (2) Collect mirror carp sperm, dilute it with Hank's solution, and inactivate the sperm by ultraviolet irradiation; (3) Mix the obtained inactivated sperm with grass carp mid-section eggs, then perform cold shock doubling treatment, and then add normal temperature water in batches for hatching to obtain gynogenetic grass carp.

[0006] Mirror carp is an excellent carp variety selected through artificial breeding, with characteristics such as fast growth, strong stress resistance, strong cold resistance, and high disease resistance. Using mirror carp sperm as the stimulation source for grass carp gynogenesis will transfer or create excellent traits such as fast growth, low temperature tolerance, and low oxygen tolerance of mirror carp into gynogenetic grass carp, so as to obtain gynogenetic grass carp with fast growth and stress resistance characteristics and cultivate an excellent new grass carp variety.

[0007] Preferably, in step (1), to obtain a gynogenetic grass carp with fast growth characteristics, healthy mirror carp with high specific growth rate and good sperm motility are selected as the male parent, and healthy grass carp with high specific growth rate are selected as the female parent; specifically, by measuring the weight change of mirror carp of the same age, the growth rate is used as the direct criterion to judge the growth advantage of mirror carp, and sexually mature healthy male mirror carp individuals with obvious growth advantage and stress resistance are screened out from the mirror carp population; by measuring the weight change of grass carp of the same age, the growth rate is used as the direct criterion to judge the growth advantage of grass carp, and sexually mature healthy female grass carp individuals with obvious growth advantage are screened out from the grass carp population.

[0008] Preferably, the specific growth rate (SGR) calculation formula is used to judge the growth rate, and the calculation formula is , where WB is the weight (g) at the starting time, WE is the weight (g) at the ending time, and t is the time period length (days).

[0009] Preferably, in step (2), the motility of mirror carp sperm is detected under a microscope, and semen of mirror carp with long survival time and good motility is screened out, diluted and mixed evenly with 4 - 4 times the volume of Hank's solution, and the diluted semen is spread on a culture dish according to the standard of 0.1 - 0.2 mm thickness. The culture dish containing the semen is placed on a shaker with an ice bag, and the sperm is inactivated with 2 ultraviolet lamps with a power of 15 watts in a dark environment. The vertical distance between the sperm and the ultraviolet lamp is 30 - 35 cm, and the irradiation time is 43 - 52 min. The above method is used to ensure uniform irradiation of sperm and reduce the rate of sperm inactivation, reducing the over-inactivation of sperm caused by uneven sampling or too fast sperm inactivation by the operator (generally, microscopic examination shows that the tail swing of sperm in the water droplet is weakened by 70 - 80% compared with that before inactivation), thus preventing the activation of grass carp eggs.

[0010] Preferably, in step (2), the Hank's solution comprises the following components in parts by mass: 8 - 8.2 parts of NaCl, 0.4 - 0.45 parts of KCl, 0.14 - 0.15 parts of CaCl2, 0.2 - 0.25 parts of MgSO4·7H2O, 0.06 - 0.065 parts of Na2HPO4·H2O, 0.06 - 0.065 parts of KH2PO4, 0.35 - 0.36 parts of NaHCO3, 12 1.0 - 1.5 parts of C6H

[0011] Preferably, the rotation speed of the shaker is 80 - 120 r / min, and the sperm is maintained at 4 - 6 °C.

[0012] Preferably, the standard for sperm inactivation is as follows: under microscopic examination, the sperm activated by water has a vitality of 20-30% of normal sperm.

[0013] Preferably, in step (3), the inactivated sperm obtained is mixed with the mid-section eggs of grass carp, normal temperature water is added, and the mixture is stirred thoroughly with a feather. Then, it is treated with an ice-water mixture at 4-6 °C for 10-12 min, and the fertilized eggs after cold treatment are transferred to warm water at 24-26 °C for 4-6 min to obtain the fertilized eggs with doubling treatment.

[0014] During the natural or artificial reproduction process of grass carp, the spawning behavior of female fish may be divided into multiple stages (such as the initial stage, the middle stage, and the late stage). The eggs discharged from the mid-section specifically refer to the eggs discharged during the peak spawning period (i.e., the middle stage). The eggs at this stage usually have a relatively high maturity and fertilization potential, which are the key parts for successful reproduction. Preferably, the fertilized eggs with doubling treatment are added to normal temperature water in batches. When the water temperature is consistent with the water temperature in the hatching device, they are transferred into the hatching device for normal hatching. The fry obtained after hatching are transferred to a pre-fertilized cement pond for preliminary feeding, and thus gynogenetic grass carp are obtained.

[0015] Preferably, the hatching is carried out by flowing water hatching. The water flow should be such that it can stir up the fertilized eggs and make them roll over. The water temperature for hatching in the hatching barrel is 22-26 °C.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The mirror carp selected in the present invention is an excellent carp variety through artificial breeding, which has the characteristics of fast growth, strong stress resistance, low temperature tolerance, high disease resistance, etc. At the same time, the specific growth rate is used to screen the parents, and the gynogenesis process is optimized and innovated, and gynogenetic grass carp with fast growth rate and strong stress resistance are successfully developed.

[0017] 2. The present invention finds that the specific growth rate (one and a half years) of the gynogenetic grass carp induced by mirror carp sperm is 0.104 and 0.034 higher than that of the gynogenetic grass carp induced by the sperm of improved red crucian carp and koi carp respectively, and 0.114 higher than that of common grass carp. Moreover, in terms of survival rate, it is significantly better than the gynogenetic grass carp induced by the sperm of improved red crucian carp and common grass carp. This provides a new method and idea for the genetic improvement of grass carp, offers more possibilities for screening excellent varieties, and brings greater economic benefits to the grass carp aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0019] Figure 1 It is a flow chart of gynogenesis induced by sperm of mirror carp; Figure 2 It is a flow chart of gynogenesis induced by sperm of red crucian carp; Figure 3 It is a flow chart of gynogenesis induced by sperm of common carp; Figure 4 It is a comparison chart of the appearance of three kinds of gynogenetic grass carp and common grass carp; Figure 5 It is a metaphase chromosome map of mitosis of kidney cells of three kinds of grass carp; Figure 6 It is the result of male sex-specific molecular marker of grass carp; Figure 7 It is the result of 5s rDNA molecular marker. Detailed implementation manners

[0020] For the convenience of understanding the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0021] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0022] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0023] Grass carp ( Ctenopharyngodon Idella ), belonging to the genus Ctenopharyngodon in the family Cyprinidae. Mirror carp ( Cyprinus carpiovar.specularis ), belonging to the genus Cyprinus in the family Cyprinidae. Long-term distant hybridization experiments of the present invention show that in most cases, viable fry cannot be obtained in hybridization combinations where the chromosome number of the female parent is greater than that of the male parent. Experiments show that grass carp-mirror carp hybrid fry are often significantly different from grass carp embryos in terms of embryonic development, such as various degrees of embryo deformity, and the yolk sac of the embryo always remains spherical, and they cannot develop to the feeding stage. At the same time, eggs activated by ultraviolet-inactivated sperm of mirror carp cannot obtain normal fry without chromosome doubling treatment. The reasons for the present invention to select mirror carp as the heterologous sperm are as follows: 1. In terms of growth rate, compared with common carp, under the same density of breeding conditions, the growth rate of mirror carp far exceeds that of common carp; 2. The feeding survival rate of mirror carp reaches about 98%, and the overwintering maturation rate reaches 96%, with strong stress resistance; 3. The meat quality of mirror carp is also better than that of common carp.

[0024] Studies have shown that in the experiment of inducing gynogenesis with heterologous sperm, a small amount of paternal genetic material is integrated into the gynogenetic offspring, and the gynogenetic offspring show stronger stress resistance and faster growth rate compared to the offspring of ordinary reproduction.

[0025] Compared with conventional carp varieties, mirror carp has the advantages of a good body shape, fast growth rate, high survival rate, cold tolerance and strong disease resistance. At the same time, the sperm quantity is sufficient and easy to obtain, and the sperm motility is strong and not easily completely inactivated, so the appropriate ultraviolet irradiation time (the inactivation time is generally 43 - 52 min, and the specific time is subject to the microscopic examination result) can be more accurately grasped, and gynogenetic grass carp can be better mass-produced.

[0026] The present invention also uses modified red crucian carp sperm and koi sperm as heterologous sperm to induce gynogenesis of grass carp to obtain another two kinds of gynogenetic grass carp, and at the same time sets ordinary grass carp as a control group to be fed under the same conditions.

[0027] The feeding time from seedling emergence to the first measurement is 180 days, the feeding time for the second measurement is 360 days, and the feeding time for the third measurement is 540 days. It is found that the gynogenetic grass carp induced by mirror carp sperm is superior to the gynogenetic grass carp induced by modified red crucian carp sperm in terms of growth rate and disease resistance, and is superior to the gynogenetic grass carp induced by koi sperm in terms of growth rate.

[0028] Example: In early May of the present invention, mirror carp (2n = 100) is used as heterologous sperm to conduct artificial gynogenesis research on grass carp, and a method for creating fast-growing gynogenetic grass carp is explored, such as Figure 1 , including the following steps: (1) Collection and inactivation of mirror carp sperm: Under the same feeding conditions, with a feeding period of half a year, two screenings are carried out. Select sexually mature mirror carp with a high specific growth rate and good health as sperm donors, and use the specific growth rate (SGR) calculation formula to judge the growth rate. The calculation formula is , where WB is the weight of the start time (g), WE is the weight of the end time (g), and t is the time period length (days). The mirror carp's abdomen was squeezed artificially to collect its semen in a disposable plastic cup away from light. The mirror carp sperm motility was detected under a microscope. A small amount of semen was dipped on a glass slide with a toothpick. A small amount of pure water was added to activate the sperm and the sperm activity time was recorded. The mirror carp semen with long survival time and good motility was screened out, diluted and mixed with 4 times the volume of Hank's solution, and stored in a 4 ℃ refrigerator as "mother liquor". The diluted semen was spread in a culture dish according to the standard of 0.1-0.2 mm thickness, and placed on a shaking table with a rotation speed of 90-100 r / min and a frozen plate (a thin towel can be placed on the ice plate to increase friction), and two 15-watt ultraviolet lamps (model: Cnlight ZW20S15W-Z589) were placed 32-35 cm above the culture dish, and the whole set of equipment was covered with a black curtain to avoid light. After irradiation for 48 minutes, the inactivated semen was collected in a light-proof plastic tube, marked and stored at 4 ℃. Hank's solution formula: NaCl 8-8.2 parts, KCl 0.4-0.45 parts, CaCl2 0.14-0.15 parts, MgSO4·7H2O 0.2-0.25 parts, Na2HPO4·H2O 0.06-0.065 parts, KH2PO4 0.06-0.065 parts, NaHCO3 0.35-0.36 parts, C6H 12 O6 1.0-1.5 parts, MgCl2·6H2O 0.1-0.15 parts and water 1000 parts.

[0029] (2) Egg activation: Collect the mid-section eggs of sexually mature grass carp and mix them with the sperm inactivated in step (1). Stir thoroughly with a feather. Then add 26 ℃ warm water and stir for 2 min. Immediately transfer the fertilized eggs to a 4 ℃ ice water mixture and continue stirring for 10-12 min.

[0030] (3) Incubation of fertilized eggs: Transfer the fertilized eggs that have been cold treated for 10-12 min in step (2) in batches into 26°C water and gently stir for 5 min. Then, when the water temperature is basically the same as the room temperature water in the incubation bucket, pour all of them into the incubation bucket.

[0031] (4) Preparation of the seedling pond: Prepare the seedling pond in advance. Disinfect the seedling pond with quicklime on the 15th day before stocking. Add lake water that meets the standards and aerate the pond on the 10th day before stocking. Use tea bran to remove impurities and continue aeration on the 8th day before stocking. Add peanut bran or soy milk along the edge of the seedling pond every morning on the second day before stocking to fertilize the water.

[0032] (5) Feeding of fry: Place the fry into a pre-prepared fry pond for early feeding, and transfer to an earthen pond for feeding after 3-4 months.

[0033] The present invention uses red crucian carp sperm and common carp sperm, and obtains two other gynogenetic grass carps by the same method. Breeding experiments are carried out under the same density and feeding conditions, such as Figure 2 , Figure 3 . The breeding data at different time periods are shown in Table 1.

[0034] The present invention uses three kinds of inactivated sperm to induce gynogenesis of grass carp eggs, and successfully obtains three kinds of gynogenetic grass carps. Their external views are as shown in Figure 4 . Figure 4 -A is common grass carp, Figure 4 -B is the gynogenetic grass carp induced by red crucian carp sperm, Figure 4 -C is the gynogenetic grass carp induced by mirror carp sperm, Figure 4 -D is the gynogenetic grass carp induced by common carp sperm.

[0035] The present invention makes slides and observes the renal cell chromosomes of two kinds of gynogenetic grass carps and common grass carp. The steps of the renal cell chromosome slide-making method are as follows: inject phytohemagglutinin (PHA) into the experimental fish twice at a dose of 1 mg / mL. The injection interval is 12 - 24 h. 2 - 6 h before dissection, inject colchicine into each sample at a dose of 4 - 6 μg / g. Grind the renal tissue with 0.8% NaCl, perform hypotonic treatment with 0.075 M KCl at 37°C for 40 - 60 min, fix it three times in methanol - acetic acid with a ratio of 3:1, stain it with Giemsa stain for 1 hour after dropping the sample on the slide, and observe and photograph the metaphase chromosomes with Pixera Pro 600ES (US). The results are as shown in Figure 4 . The results show that the chromosome number of the renal cells of common grass carp is 48 ( Figure 5 -A), the chromosome number of the renal cells of the gynogenetic grass carp induced by mirror carp sperm is 48 ( Figure 5 -B), the chromosome number of the renal cells of the gynogenetic grass carp induced by red crucian carp sperm is 48 ( Figure 5 -C), the chromosome number of the renal cells of the gynogenetic grass carp induced by common carp sperm is 48 ( Figure 5 -D), and all three kinds of gynogenetic grass carps are diploids.

[0036] The present invention uses grass carp male sex-specific molecular markers to identify the genders of common grass carp (1-6), gynogenetic offspring (7-12), and the female parents of gynogenetic grass carp (13-18). The caudal fins of common grass carp, gynogenetic grass carp, and the female parents of gynogenetic grass carp were respectively cut, and genomic DNA was extracted using a kit. PCR amplification was performed using grass carp male-specific sex marker primers. The reaction conditions were pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 10 min, with 35 cycles. The PCR products were separated by 1.5% agarose gel electrophoresis, and the electrophoresis results were compared as Figure 6 shown. It was found that the grass carp male sex-specific molecular marker sequences were cloned from sample 3 and sample 6 in common grass carp, and this result was consistent with the observation results of gonad sections; the grass carp male sex-specific molecular marker sequences were not cloned from either the gynogenetic offspring or the female parents of gynogenetic grass carp. The results indicate that all the grass carp obtained in this experiment are gynogenetic offspring.

[0037] The present invention uses 5s rDNA molecular specific markers to identify the germplasm of gynogenetic grass carp. It was found that the 5s rDNA electrophoresis bands of gynogenetic grass carp were consistent with those of the parental common grass carp.

[0038] 5s rDNA molecular specific marker method: The caudal fins of common grass carp (1-6), gynogenetic offspring (7-12), and the female parents of gynogenetic grass carp (13-18) were respectively cut, and genomic DNA was extracted using a kit. PCR amplification reaction was performed using 5s rDNA specific primers (5s-F: 5’-CAGGTTGGTATGGCCGTAAGC-3’ and 5S-R: 5’-GCTATGCCCGATCTCGTCTGA-3’). The reaction conditions were pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 59.2 °C for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 10 min, with 35 cycles. The PCR products were separated by 1.5% agarose gel electrophoresis, purified and then ligated to the Pmd18-T vector, and single clone colonies were selected for Sanger sequencing. The electrophoresis results were compared as Figure 7 shown. Two identical types of 5S rDNA bands were obtained from common grass carp, gynogenetic grass carp, and the female parents of gynogenetic grass carp, and the 5s rDNA molecular specific marker results of the three types of fish were consistent.

[0039] The present invention used common grass carp as a control and conducted a breeding experiment on two kinds of gynogenetic grass carp. Under the condition of keeping the pond size, water source, feed, light and other conditions consistent, the average weight of the three kinds of fish was statistically counted every six months (three times in total), and the survival number after overwintering was statistically counted, as shown in Table 1. The statistical data show that the gynogenetic grass carp induced by mirror carp sperm is superior to the gynogenetic grass carp induced by improved red crucian carp sperm in terms of growth rate and disease resistance, and is superior to the gynogenetic grass carp induced by common carp sperm in terms of growth rate. At the same time, the measurable traits of the three kinds of grass carp were statistically counted, and the results are shown in Table 2, and the countable traits are shown in Table 3.

[0040] Table 1 Statistical data of comparative breeding experiments of three kinds of grass carp

[0041] In the same column, different lowercase letters a, b, c represent significant differences among different fish species ( P <0.05).

[0042] Table 2 Measurable traits of two kinds of gynogenetic grass carp and common grass carp

[0043] Table 3 Countable traits of two kinds of gynogenetic grass carp and common grass carp

[0044] To sum up, the gynogenetic grass carp induced by mirror carp sperm in the present invention is superior to the gynogenetic grass carp induced by improved red crucian carp sperm and common grass carp in terms of growth rate and disease resistance, and is superior to the gynogenetic grass carp induced by common carp sperm in terms of growth rate. The present invention provides an experimental basis for the selection of artificially induced gynogenetic heterologous sperm, and expands the germplasm resources of high-quality grass carp.

Claims

1. A method for creating rapidly growing gynogenetic grass carp, characterized in that, It includes the following steps: (1) Screen sexually mature male mirror carps and sexually mature female grass carps with relatively obvious growth advantages and stress resistance; (2) Collect mirror carp sperm, dilute it with Hank's solution, and inactivate the sperm by ultraviolet irradiation; (3) Mix the obtained inactivated sperm with the middle-section eggs of grass carp, perform cold shock doubling treatment, and then add normal-temperature water in batches for hatching to obtain gynogenetic grass carp.

2. The creation method according to claim 1, wherein In step (1), by measuring the weight change of mirror carps of the same age, using the growth rate as the direct criterion to judge the growth advantage of mirror carps, screen out sexually mature and healthy male mirror carp individuals with obvious growth advantages and stress resistance in the mirror carp population; by measuring the weight change of grass carps of the same age, using the growth rate as the direct criterion to judge the growth advantage of grass carps, screen out sexually mature and healthy female grass carp individuals with obvious growth advantages in the grass carp population.

3. The creation method according to claim 2, wherein Use the specific growth rate (SGR) calculation formula to determine the growth rate. The calculation formula is , where WB is the weight at the starting time (g), WE is the weight at the ending time (g), and t is the time period length (days).

4. The creation method according to claim 1, wherein In step (2), use a microscope to detect the sperm motility of mirror carps, screen out the semen of mirror carps with a long survival time and good motility, dilute and mix it with Hank's solution at a volume ratio of 4-5 times, spread the diluted semen on a culture dish according to the standard of 0.1-0.2 mm thick, place the culture dish containing the semen on a shaker with an ice pack, and inactivate the sperm with 2 15-watt ultraviolet lamps in a dark environment. The vertical distance between the sperm and the ultraviolet lamp is 30-35 cm, and the irradiation time is 43-52 min.

5. The creation method according to claim 4, characterized in that, In step (2), the Hank's solution comprises the following components in parts by mass: 8 - 8.2 parts of NaCl, 0.4 - 0.45 parts of KCl, 0.14 - 0.15 parts of CaCl2, 0.2 - 0.25 parts of MgSO4·7H2O, 0.06 - 0.065 parts of Na2HPO4·H2O, 0.06 - 0.065 parts of KH2PO4, 0.35 - 0.36 parts of NaHCO3, 1.0 - 1.5 parts of C6H 12 O6, 0.1 - 0.15 parts of MgCl2·6H2O and 1000 parts of water.

6. The creation method according to claim 4, characterized in that The rotation speed of the shaker is 80-120 r / min, and the sperm is maintained at 4-6 °C.

7. The creation method according to claim 4, characterized in that, The standard for sperm inactivation is: under microscopic examination, the sperm has 20-30% of the motility of normal sperm when activated by water.

8. The creation method according to claim 1, characterized in that, In step (3), mix the obtained inactivated sperm with the middle-section eggs of grass carp, add normal-temperature water and stir well with a feather, treat with a 4-6 °C ice-water mixture for 10-12 min, and transfer the cold-treated fertilized eggs to warm water at 24-26 °C for 4-6 min to obtain the fertilized eggs after doubling treatment.

9. The creation method according to claim 8, characterized in that, Add the fertilized eggs after doubling treatment to normal-temperature water in batches, transfer them to an incubation device for normal incubation when the water temperature is the same as that in the incubation device, and transfer the hatched fry to a pre-fertilized cement pond for preliminary feeding to obtain gynogenetic grass carp.

10. The creation method according to any one of claims 1-9, characterized in that, The incubation is flow-through incubation, and the water flow should be such that it can stir up and tumble the fertilized eggs. The water temperature for incubation in the incubation barrel is 22-26 °C.

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