CN111543390A - Preparation method and application of disease-resistant grass carp - Google Patents
Preparation method and application of disease-resistant grass carp Download PDFInfo
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- CN111543390A CN111543390A CN202010481989.8A CN202010481989A CN111543390A CN 111543390 A CN111543390 A CN 111543390A CN 202010481989 A CN202010481989 A CN 202010481989A CN 111543390 A CN111543390 A CN 111543390A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2227/40—Fish
Abstract
The invention relates to the technical field of grass carp breeding, in particular to a preparation method and application of disease-resistant grass carp. The preparation method of the disease-resistant grass carp comprises the following steps: firstly, selecting seeds; ② parent infection culture; preparing sperms and ova; fourthly, raising the seedlings of the first-generation disease-resistant grass carp fries; fifthly, first-generation disease-resistant grass carp fry infection breeding; sixthly, breeding the second-generation disease-resistant grass carp fry; and seventhly, screening seedlings, and performing the identification of pure lines. The disease-resistant grass carp obtained by the preparation method has excellent disease resistance and survival ability, can be used for the production of large-scale disease-resistant grass carp fries, has the characteristics of low cost and short breeding period, and has good market application prospect.
Description
Technical Field
The invention relates to the technical field of grass carp breeding, in particular to a preparation method and application of disease-resistant grass carp.
Background
The grass carp belongs to the carp herbivorous fish, is one of the main freshwater cultured fishes in China, plays an important role in the aquaculture industry and the national economy of China, and has the characteristics of delicious taste and quick growth. However, in the artificial culture of grass carps, the diseases of the grass carps of the first age and the second age are serious, the average survival rate of the commercial fish culture is less than 30 percent, and the economic benefit of the culture is seriously reduced.
The diseases related to grass carp include grass carp hemorrhage, enteritis, gill rot disease and the like, and the grass carp hemorrhage is one of the most serious diseases. Grass carp hemorrhage is an acute infectious disease caused by Reovirus (Reovirus), and mainly causes harm to grass carp species of first age and second age. The disease has rapid onset, wide prevalence and high mortality, and the histopathological changes of the disease are mainly manifested by systemic small blood vessel injury and bleeding. The main characteristic of reovirus propagation is the overall process of replication, biosynthesis and assembly in the cytoplasm. Unlike other viruses, any stage of such virus propagation does not occur in the nucleus. Therefore, the research on the disease-resistant breeding of the grass carps, in particular to the breeding research on the improvement of the virus infectivity of grass carp species, has great significance.
At present, the establishment of pure lines of biological species by traditional genetic breeding methods requires several generations of continuous inbred breeding. Because the grass carp is a large-scale fish, the sexual maturity period is as long as 5 years, the traditional genetic breeding method for establishing the disease-resistant grass carp variety pure line requires decades or even hundreds of years in the actual production, and a large amount of manpower and material resources are consumed. Moreover, since the scale of the breeding engineering is difficult to include all possible gene combinations, the bred strains are likely to disappear due to inbreeding depression, and the breeding goal cannot be achieved.
The research of cell engineering applied to fish starts in the late 60 s of the 50 s of the 20 th century, and has been greatly developed in the 70 and 80 s. Mainly comprises fish nucleus transplantation, chromosome engineering, cell fusion and cell culture. At present, mainly focusing on nuclear transplantation and cell culture in the aspect of breeding disease-resistant grass carp strains. The disadvantages are that the survival fry after transplantation or fusion of the fish nucleus is quite low, and the chromosome non-diploid changes may occur after several passages of the nucleus before transplantation or fusion. Meanwhile, genetic operation on in vitro cultured cells is difficult, and a disease-resistant grass carp fingerling strain is not formed so far.
The establishment of eukaryotic gene molecular cloning technology provides a means for understanding fish gene structure from nucleotide level. The transgenic fish is successfully made by adopting the recombination technology, and convenience is provided for directed genetic breeding. So far, the obtained transgenic fish is not a line with stable unit site heredity, and the fixed-site integration of transgenes and the establishment of pure transgenic fish lines are still the key for solving the problems, which are also the problems faced by grass carp breeding for disease resistance.
Therefore, the method can rapidly cultivate the disease-resistant grass carp with strong disease resistance and high survival rate, and is a technical difficulty which needs to be solved urgently at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the preparation method of the disease-resistant grass carp, which can be used for quickly culturing the disease-resistant grass carp with strong disease resistance and high survival rate.
The invention also aims to provide the application of the preparation method of the disease-resistant grass carp, and the disease-resistant grass carp obtained by the preparation method has the characteristics of strong disease resistance and high survival rate.
The above object of the present invention is achieved by the following technical solutions:
a preparation method of disease-resistant grass carp comprises the following steps:
firstly, seed selection: selecting healthy sexual mature female parent grass carps with uniform body types, marking, and putting the grass carps into a special pond for culture;
secondly, parent infection culture: putting diseased grass carps with hemorrhagic disease into a special pond, wherein the diseased grass carps infect all parental grass carps, and the parental grass carps which are fatigued and the diseased grass carps are eliminated in time;
preparing sperms and ova: selecting carp sperms which are lethal by hybridization with grass carp, diluting the carp sperms with 2-4 times of volume of Hank's solution at 0-4 ℃, placing the diluted carp sperms under two 15W ultraviolet sterilizing lamps for irradiation for 70min, continuously shaking semen by a shaking table in the irradiation process, and maintaining the temperature of the semen at 0-4 ℃ to obtain the genetically inactivated carp sperms; during the period, artificial spawning induction is carried out on the parent grass carp to obtain mature parent grass carp eggs;
activating the eggs of the parent grass carps by using genetically inactivated sperms of the carps, carrying out cold shock on the activated eggs of the grass carps for 15min in cold water at 4-6 ℃ after the eggs of the grass carps are contacted for 2min, controlling the meiosis of the eggs of the parent grass carps, and enabling a second diode not to be separated to obtain a high homozygous gynogenesis diploid grass carp fry with two genomes from homologous chromosomes of a female parent and without a male parent genome, namely a generation of disease-resistant grass carp fry;
fifthly, first-generation disease-resistant grass carp fry infection cultivation: putting diseased grass carps with hemorrhagic disease into the first-generation disease-resistant grass carp fry at the first-age stage of the first-generation disease-resistant grass carp fry, infecting all the first-generation disease-resistant grass carp fries, screening healthy female first-generation disease-resistant grass carp fries, adopting artificial induced spawning to breed the first-generation disease-resistant grass carp with sexual maturity, and then carrying out artificial induced spawning on the first-generation disease-resistant grass carp fries to obtain mature first-generation grass carp ova;
sixthly, second-generation disease-resistant grass carp fry breeding: activating eggs of a first-generation grass carp by using genetically inactivated sperms of the carp, carrying out cold shock on the activated eggs of the grass carp for 15min in cold water at 4-6 ℃ after the eggs of the carp are contacted for 2min, controlling meiosis of the eggs of the first-generation grass carp, and enabling a second polar body not to be separated to obtain a high-homozygous gynogenesis diploid grass carp fry with two genomes from maternal homologous chromosomes and without paternal genomes, namely the second-generation disease-resistant grass carp fry;
and seventhly, screening seedlings: in the first-age stage of the second-generation disease-resistant grass carp fries, the diseased grass carp fries with hemorrhage are thrown again, the second-generation disease-resistant grass carp fries are infected, and healthy second-generation disease-resistant grass carp fries are screened out to obtain the final disease-resistant grass carp pure line;
and eighthly, pure line identification: randomly taking 30 individual tail fin samples from the disease-resistant pure grass carp line population and tail fin samples of the carp and grass carp in comparison, carrying out extensive scanning analysis and comparison on the genomes of the tail fin samples through polymorphic molecular markers, determining the homozygosity and consistency of the disease-resistant pure grass carp line genome, and only after no exogenous grass carp is mixed in the population, the method can be used for large-scale production of disease-resistant grass carp fries.
By adopting the technical scheme, three times of artificial pathogenicity and artificial induced parthenogenesis are adopted, compared with a traditional breeding mode, the method promotes sexual maturity of grass carp fries in two generations by utilizing artificial induced spawning, and effectively shortens the cultivation period of the grass carp; compared with cell engineering breeding and genetic engineering breeding modes, the disease resistance of the grass carp has good stability.
The disease-resistant grass carp fry breeding method has the advantages that diseased grass carps are directly used for screening the parent grass carps at the beginning of breeding grass carps, so that the growth period from growth of grass carp fries to sexual maturity can be saved, the survival rate of disease-resistant grass carp fries of the first generation can be improved, and the effects of saving breeding cost and improving the disease resistance and survival ability of the disease-resistant grass carp fries are achieved.
The carp has strong adaptability, and the sperm of the carp is subjected to in vitro irradiation of ultraviolet rays for 70min to thoroughly destroy the genetic material of the carp, but the technical condition of activating the developmental capacity of the ovum of the grass carp is kept.
The carp sperms which are hybridized and killed with the grass carp ova are subjected to genetic inactivation treatment, but the capability of activating the development of the grass carp ova is reserved, so that female parthenogenetic homozygous diploid fries of the grass carp generated after artificial doubling of chromosomes can be simply, conveniently and accurately identified, the possibility that the grass carp is hybridized with heterogeneous sperms to generate hybrid offspring is eliminated, and the pure-line progeny grass carp with high survival rate is obtained.
In conclusion, the disease-resistant grass carp obtained by the preparation method has excellent disease resistance and survival capability, can be used for the production of large-scale disease-resistant grass carp fries, has the characteristics of low cost and short breeding period, and has good market application prospect.
The present invention in a preferred example may be further configured to: in the third and fifth steps, the artificial induced spawning method comprises the following steps: injecting oxytocic to grass carp to obtain mature ovum.
By adopting the technical scheme, in the current aquaculture regulation, the oxytocic can effectively accelerate the sexual maturity of the fish and greatly shorten the growth period of the fish, thereby effectively improving the efficiency of fish breeding.
The present invention in a preferred example may be further configured to: the oxytocic is a luteinizing hormone releasing hormone analogue LRH-A2And fish pituitary PG in a weight ratio of 2: 1.
The present invention in a preferred example may be further configured to: the injection amount of the oxytocic is as follows: the female grass carp per kg is injected with 120 μ g oxytocic.
By adopting the above technical means, in the present application, when the oxytocic drug in the above weight ratio and injection amount is used, mature and high-quality ova can be obtained in the shortest time, and therefore, the present application takes this as a preference.
The present invention in a preferred example may be further configured to: in the step I, the weight of the parental grass carps is 10-20% larger than the average weight of grass carps of the same age.
By adopting the technical scheme, the grass carp with the weight of 10-20% has larger weight gain rate and stronger vitality than other grass carps, so that the grass carp can be used as a primary screen to further improve the disease resistance and survival capability of the first-generation disease-resistant grass carp fries.
The present invention in a preferred example may be further configured to: in the second step, the amount of the sick grass carps is 20 percent of the total amount, and the sick grass carps are cultured for 30 days.
The present invention in a preferred example may be further configured to: in the fifth step and the seventh step, the amount of the sick grass carps is 5 percent of the total amount, and the sick grass carps are cultured for 20 days.
By adopting the technical scheme, 20 percent of the input amount is enough to ensure that the parental grass carps are completely infected with diseases within one week, and corresponding antibodies are generated in the rest time to resist the diseases of the parental grass carps, so that the parental grass carps which survive and recover in the environment have more excellent disease resistance, and the disease-resistant pure grass carp line with strong disease resistance and high survival rate is obtained. In addition, the first-generation grass carp fries and the second-generation grass carp fries are small in excellent fish age and easy to infect, and a suitable disease-resistant grass carp system can be effectively screened out after 5% of putting amount is cultured for 20 days. From this, this application is through control of the input amount and the breed time of sick grass carp to this quick effective grass carp of screening disease-resistant ability stronger has easy operation, the excellent characteristics of screening effect.
The second aim of the invention is realized by the following technical scheme:
the application of the preparation method of the disease-resistant grass carp is suitable for the disease-resistant grass carp pure line obtained by using the preparation method of the disease-resistant grass carp.
Through adopting above-mentioned technical scheme, the disease-resistant grass carp pure line that the disease-resistant ability that this application can obtain is strong, the survival rate is high to this effectively promotes the development of grass carp aquaculture.
In summary, the invention includes at least one of the following beneficial technical effects:
1. the disease-resistant pure grass carp line prepared by the method has excellent disease resistance and survival ability through three times of artificial pathogenicity and artificial induced parthenogenesis, can be used for the production of large-scale disease-resistant grass carp fries, has the characteristics of low cost and short breeding period, and has good market application prospect;
2. the application aims to select the luteinizing hormone releasing hormone analogue LRH-A2The oxytocic drug mixed with the fish pituitary PG can quickly and effectively promote the sexual maturity of the grass carp to ovulation and/or spermiation, thereby further accelerating the breeding of disease-resistant pure grass carp lines;
3. the method has the advantages that the feeding amount and the breeding time of the sick grass carps are controlled, so that the grass carps with stronger disease resistance can be quickly and effectively screened, and the method is simple to operate and excellent in screening effect.
Detailed Description
The parent female and male grass carps used in the application are derived from a fishfry culture base of a rolling dam in Tokyo county, Fushun, Sichuan province, a special pond is a grass carp culture farm set for culturing other fishes, and all medicaments are commercially available products.
Example 1
A pure line establishment method of disease-resistant grass carp comprises the following steps:
firstly, seed selection: selecting healthy and well-balanced sexually mature female grass carps, marking the female grass carps with the weight of the parent grass carps being more than 10% of the average weight of the grass carps of the same age, and putting the female grass carps into a special pond for culture.
Secondly, parent infection culture: the sick grass carps with hemorrhagic disease are thrown into the special pond, the feeding amount of the sick grass carps accounts for 20 percent of the total amount, the sick grass carps infect the parent grass carps for 30 days, and the parent grass carps which are fatalities and the sick grass carps are cleared away in time.
Preparing sperms and ova: selecting carp sperms which are lethal by hybridization with grass carp, diluting the carp sperms with 2-4 times of volume of Hank's solution at 0-4 ℃, placing the diluted carp sperms under two 15W ultraviolet sterilizing lamps for irradiation for 70min, continuously shaking semen by a shaking table in the irradiation process, and maintaining the temperature of the semen at 0-4 ℃ to obtain the genetically inactivated carp sperms; during the period, artificial spawning induction is carried out on the parent grass carp to obtain mature parent grass carp eggs;
wherein, the artificial spawning-inducing prescriptionThe method comprises the following steps: injecting oxytocic drug into grass carp to obtain mature ovum, wherein the oxytocic drug is luteinizing hormone releasing hormone analogue LRH-A2Mixed with fish pituitary PG according to the weight ratio of 2: 1, and the injection amount of the oxytocic is as follows: injecting 120 mug oxytocic per kg female grass carp;
fourthly, breeding the first-generation disease-resistant grass carp fry: activating ovum of parent grass carp with genetically inactivated sperm of Cyprinus Carpio, contacting the ovum with sperm for 2min, cold shocking the activated ovum of grass carp in 4-6 deg.C cold water for 15min, controlling meiosis of ovum of parent grass carp, and making second polar body unable to separate to obtain high homozygous gynogenesis diploid grass carp fry with two genomes from maternal homologous chromosome and no paternal genome, i.e. first generation disease-resistant grass carp fry;
fifthly, first-generation disease-resistant grass carp fry infection cultivation: in the first-age stage of the first-generation disease-resistant grass carp fries, the diseased grass carps with hemorrhagic disease are thrown, the throwing amount of the diseased grass carps accounts for 5 percent of the total amount, the disease-resistant grass carp fries are cultured for 20 days, all the first-generation disease-resistant grass carp fries are infected, healthy female first-generation disease-resistant grass carp fries are screened out, artificial induced spawning is carried out according to the method in the third step, and ova of the first-generation disease-resistant grass carp fries with sexual maturity are cultured;
sixthly, second-generation disease-resistant grass carp fry breeding: activating eggs of a first-generation grass carp by using genetically inactivated carp sperms, carrying out cold shock on the activated eggs of the grass carp for 15min in cold water at 4 ℃ after the eggs of the grass carp are contacted for 2min, controlling meiosis of the eggs of the first-generation grass carp, and enabling a second polar body not to be separated to obtain a high-homozygous gynogenesis diploid grass carp fry with two genomes from maternal homologous chromosomes and without paternal genomes, namely the second-generation disease-resistant grass carp fry;
and seventhly, screening seedlings: in the first-age stage of the second-generation disease-resistant grass carp fries, the diseased grass carp fries with hemorrhage are thrown again, the feeding amount of the diseased grass carp fries accounts for 5 percent of the total amount, the grass carp fries are cultured for 20 days, the second-generation disease-resistant grass carp fries are infected, and the healthy second-generation disease-resistant grass carp fries are screened out to obtain the final disease-resistant grass carp pure line;
and eighthly, pure line identification: randomly taking 30 individual tail fin samples from the disease-resistant pure grass carp line population and tail fin samples of the carp and grass carp in comparison, carrying out extensive scanning analysis and comparison on the genomes of the tail fin samples through polymorphic molecular markers, determining the homozygosity and consistency of the disease-resistant pure grass carp line genome, and only after no exogenous grass carp is mixed in the population, the method can be used for large-scale production of disease-resistant grass carp fries.
Examples 2 to 3
Examples 2-3 adjustments were made to the preparation parameters based on the procedure of example 1, see table 1 below for specific adjustments.
TABLE 1 preparation parameters of examples 1-4
Comparative example 1
Comparative example 1 on the basis of the method of example 1, the infection operation of step (c) was omitted, and the female parent grass carp selected was directly raised.
Comparative example 2
Comparative example 2 on the basis of the method of example 1, the infection operation of step two was omitted and the female parent grass carp selected for breeding was directly raised. Repeating the operations of the fourth step to the sixth step on the second-generation disease-resistant grass carp fry obtained in the sixth step to obtain a third-generation disease-resistant grass carp fry, and continuing the operations of the seventh step and the eighth step on the third-generation disease-resistant grass carp fry.
Measurement of Performance
The disease-resistant pure grass carp lines obtained in the above examples 1-3 and comparative examples 1-2 were subjected to the following performance measurement, and the detection results are shown in the following table 2.
1. Seedling culture period: correspondingly recording the time for obtaining the disease-resistant grass carp pure line in the examples 1-3 and the comparative examples 1-2.
2. Disease resistance: the disease-resistant grass carp pure lines obtained in the examples 1-3 and the comparative examples 1-2 are used as parents, the grass carp pure lines are cultivated to sexual maturity, artificial insemination is carried out to obtain the first filial generation grass carp, the grass carp is cultivated to the first age stage, diseased grass carp with hemorrhagic disease is added according to the quantity ratio of 1: 1, and the prevalence rate of the first filial generation grass carp is counted after 7 days of infection. According to the same operation, the prevalence rate of the second generation and the third generation is 3, the survival ability is obtained: the disease-resistant grass carp pure line obtained in the embodiment 1-3 and the comparative example 1-2 is cultured to sexual maturity, artificial insemination is carried out to obtain the first filial generation grass carp, the first filial generation grass carp is cultured to the first age stage, and the diseased grass carp with hemorrhagic disease is added according to the quantity ratio of 1: 1 to infect for 40 days, and then the survival rate of the first filial generation grass carp is counted. According to the same operation, the survival rate% of the second generation and the third generation is obtained.
TABLE 2 test results of examples 1 to 3 and comparative examples 1 to 2
As can be seen from Table 2, the disease-resistant pure grass carp line obtained by the preparation method is lower in morbidity and higher in survival rate, and the change of the morbidity and the survival rate from the first generation to the third generation is more gradual, compared with the disease-resistant pure grass carp line obtained by the comparative example 1. Therefore, the disease-resistant pure grass carp line with more excellent disease resistance and survival capability can be obtained by the preparation method of the disease-resistant grass carp, and the disease resistance and survival capability of the disease-resistant grass carp line are more stable.
Examples 4 to 6
Examples 4-6 adjustments were made to the oxytocic based on the method of example 1. Wherein the oxytocin of example 4 is the luteinizing hormone releasing hormone analogue LRH-A2(ii) a The oxytocic of example 5 is fish pituitary PG; the oxytocin of example 6 is an oxytocin and is the luteinizing hormone releasing hormone analogue LRH-A2And fish pituitary PG in the weight ratio of 1 to 1.
Examples 7 to 8
Examples 7-8 adjustments were made to the amount of oxytocin injected based on the method of example 1. Wherein. The injection amount of the oxytocin in example 7 was: 100 mug of oxytocic is injected into every kg of female grass carps. The injection amount of the oxytocin in example 8 is: 150 mug of oxytocic is injected into every kg of female grass carps.
Performance testing
The disease-resistant pure grass carp lines obtained in the above example 1 and examples 4 to 8 are subjected to an induced spawning efficiency test, and the test results are shown in the following table 3.
And (3) spawning induction efficiency testing: the time of maturation of sperm and ovum of grass carp was recorded from day 0 of the injection of oxytocin.
TABLE 3 test results of examples 1, 4 to 8
| The time/day for sperm to induce spawning | Time/day for ovum induced spawning | |
| Example 1 | 10 | 21 |
| Example 4 | 16 | 28 |
| Example 5 | 18 | 30 |
| Example 6 | 13 | 25 |
| Example 7 | 12 | 24 |
| Example 8 | 10 | 20 |
Referring to Table 3, it can be seen that when the oxytocin is the luteinizing hormone releasing hormone analogue LRH-A2Is mixed with the fish pituitary PG according to the weight ratio of 2: 1, and the injection amount of the oxytocic is as follows: the establishment of the disease-resistant pure grass carp line can be effectively promoted by injecting 120 micrograms of oxytocic drugs into every kg of female grass carps.
Examples 9 to 11
Examples 9-11 the weight selection of the parent grass carp was adjusted based on the method of example 1. In example 9, the weight of the parent grass carp is 15% higher than the average weight of grass carps of the same age; in example 10, the weight of the parental grass carp is more than 20% of the average weight of the grass carp of the same age; in example 11, the body weight of the parent grass carp is 25% higher than the average body weight of grass carps of the same age.
Performance testing
The disease-resistant pure grass carp lines obtained in the above example 1 and examples 9 to 11 were tested for weight gain efficiency and survival performance, and the test results are shown in the following table 4.
Weight gain efficiency: the disease-resistant pure grass carp line obtained in the embodiment is used as a sample to be tested, the disease-resistant pure grass carp line obtained by selecting the average weight of grass carps of the same age is used as a blank sample, the first filial generation is cultivated through artificial insemination, and the weight gain rate of the first filial generation sexual maturity of the sample to be tested relative to the blank sample is calculated.
Survival performance: the disease-resistant grass carp pure line obtained in the example 1 and the examples 9-10 is cultured to sexual maturity, artificial insemination is carried out to obtain the first filial generation grass carp, the first filial generation grass carp is cultured to the first age stage, the diseased grass carp with the hemorrhagic disease is added according to the quantity ratio of 1: 1, and the survival rate of the first filial generation grass carp is counted after 40 days of infection.
TABLE 4 test results of examples 1 and 9 to 11
| Rate of weight gain/%) | Sub-generation survival rate/%) | |
| Example 1 | 10 | 98.0 |
| Example 9 | 14 | 98.6 |
| Example 10 | 20 | 99.1 |
| Example 11 | 20 | 99.0 |
Referring to table 4, the weight gain rates of the parent grass carps in examples 1 and 9-10 are in a positive correlation function with the weight gain of the parent grass carps, but when the weight gain of the parent grass carps reaches 20%, the weight gain rate of the corresponding disease-resistant grass carps tends to be stable, so that the weight of the parent grass carps selected by the application is 10-20% higher than the average weight of grass carps of the same age, and the parent grass carps have a higher weight gain rate and stronger vitality than other grass carps, so that the weight gain rate can be used as a primary screening to further improve the disease resistance and survival ability of the disease-resistant grass carp fries.
Examples 12 to 15
Examples 12-15 all adjusted the amount of diseased grass carp and the time of farming based on the method of example 1. In the example 12, the amount of the diseased grass carps in the step two accounts for 15% of the total amount, and the diseased grass carps are cultured for 40 days. In the example 13, the amount of the diseased grass carps in the step two accounts for 25% of the total amount, and the diseased grass carps are cultured for 30 days. In example 14, the amount of the diseased grass carps in the steps (c) and (c) accounts for 3% of the total amount, and the diseased grass carps are cultured for 30 days. In example 15, the amount of the diseased grass carps in the steps (iv) and (iv) accounts for 10% of the total amount, and the diseased grass carps are cultured for 20 days.
Performance testing
The disease-resistant pure grass carp lines obtained in the above example 1 and examples 12 to 15 were subjected to the same disease resistance and survival ability tests and infection efficiency tests, and the test results are shown in the following table 5.
TABLE 5 test results of examples 1, 12 to 15
Referring to table 5, the infection efficiency (from all infected days), the morbidity and the survival rate of the example 13 and the example 15 are similar to those of the example 1, and the cost is considered, so that the example 1 can effectively reduce the seedling raising cost compared with the examples 13 and 15. In addition, the infection efficiency, the prevalence rate, and the survival rate of example 1 are superior to those of examples 12 and 14, and thus example 1 is preferred. Therefore, the method can be used for rapidly and effectively screening out the grass carps with stronger disease resistance by controlling the putting amount and the breeding time of the diseased grass carps, and has the characteristics of simple operation and excellent screening effect.
The application of the preparation method of the disease-resistant grass carp is suitable for the disease-resistant grass carp pure line obtained by using the preparation method of the disease-resistant grass carp, and the disease-resistant grass carp pure line has excellent disease resistance and survival ability, so that the development of grass carp breeding industry is effectively promoted.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (8)
1. A preparation method of disease-resistant grass carp is characterized by comprising the following steps:
firstly, seed selection: selecting healthy sexual mature female parent grass carps with uniform body types, marking, and putting the grass carps into a special pond for culture;
secondly, parent infection culture: putting diseased grass carps with hemorrhagic disease into a special pond, wherein the diseased grass carps infect all parental grass carps, and the parental grass carps which are fatigued and the diseased grass carps are eliminated in time;
preparing sperms and ova: selecting carp sperms which are lethal by hybridization with grass carp, diluting the carp sperms with 2-4 times of volume of Hank's solution at 0-4 ℃, placing the diluted carp sperms under two 15W ultraviolet sterilizing lamps for irradiation for 70min, continuously shaking semen by a shaking table in the irradiation process, and maintaining the temperature of the semen at 0-4 ℃ to obtain the genetically inactivated carp sperms; during the period, artificial spawning induction is carried out on the parent grass carp to obtain mature parent grass carp eggs;
fourthly, breeding the first-generation disease-resistant grass carp fry: activating ovum of parent grass carp with genetically inactivated sperm of Cyprinus Carpio, contacting the ovum with sperm for 2min, cold shocking the activated ovum of grass carp in 4-6 deg.C cold water for 15min, controlling meiosis of ovum of parent grass carp, and making second polar body unable to separate to obtain high homozygous gynogenesis diploid grass carp fry with two genomes from maternal homologous chromosome and no paternal genome, i.e. first generation disease-resistant grass carp fry;
fifthly, first-generation disease-resistant grass carp fry infection cultivation: putting diseased grass carps with hemorrhagic disease into the first-generation disease-resistant grass carp fry at the first-age stage of the first-generation disease-resistant grass carp fry, infecting all the first-generation disease-resistant grass carp fries, screening healthy female first-generation disease-resistant grass carp fries, adopting artificial induced spawning to breed the first-generation disease-resistant grass carp with sexual maturity, and then carrying out artificial induced spawning on the first-generation disease-resistant grass carp fries to obtain mature first-generation grass carp ova;
sixthly, second-generation disease-resistant grass carp fry breeding: activating eggs of a first-generation grass carp by using genetically inactivated sperms of the carp, carrying out cold shock on the activated eggs of the grass carp for 15min in cold water at 4-6 ℃ after the eggs of the carp are contacted for 2min, controlling meiosis of the eggs of the first-generation grass carp, and enabling a second polar body not to be separated to obtain a high-homozygous gynogenesis diploid grass carp fry with two genomes from maternal homologous chromosomes and without paternal genomes, namely the second-generation disease-resistant grass carp fry;
and seventhly, screening seedlings: in the first-age stage of the second-generation disease-resistant grass carp fries, the diseased grass carp fries with hemorrhage are thrown again, the second-generation disease-resistant grass carp fries are infected, and healthy second-generation disease-resistant grass carp fries are screened out to obtain the final disease-resistant grass carp pure line;
and eighthly, pure line identification: randomly taking 30 individual tail fin samples from the disease-resistant pure grass carp line population and tail fin samples of the carp and grass carp in comparison, carrying out extensive scanning analysis and comparison on the genomes of the tail fin samples through polymorphic molecular markers, determining the homozygosity and consistency of the disease-resistant pure grass carp line genome, and only after no exogenous grass carp is mixed in the population, the method can be used for large-scale production of disease-resistant grass carp fries.
2. The method for preparing disease-resistant grass carp according to claim 1, wherein in the third and fifth steps, the artificial spawning method comprises: injecting oxytocic to grass carp to obtain mature ovum.
3. The method for preparing disease-resistant grass carp according to claim 2, wherein said oxytocic is luteinizing hormone-releasing hormone analogue LRH-A2And fish pituitary PG in a weight ratio of 2: 1.
4. The method for preparing disease-resistant grass carp according to claim 2, wherein the injection amount of the oxytocic is as follows: the female grass carp per kg is injected with 120 μ g oxytocic.
5. The method for preparing disease-resistant grass carp according to claim 1, wherein in step (i), the weight of parent grass carp is 10-20% greater than the average weight of grass carp of the same age.
6. The method for preparing disease-resistant grass carp according to claim 1, wherein in the second step, the amount of the diseased grass carp is 20% of the total amount, and the diseased grass carp is cultured for 30 days.
7. The method for preparing disease-resistant grass carp according to claim 1, wherein the amount of diseased grass carp added in steps (c) and (c) is 5% of the total amount, and the disease-resistant grass carp is cultured for 20 days.
8. The use of the method for preparing disease-resistant grass carp of any one of claims 1-7, which is suitable for the disease-resistant grass carp pure line obtained by the method for preparing disease-resistant grass carp.
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