CN109964858B - Breeding method for inducing pelteobagrus fulvidraco facultative fish to generate super-male fish and full-female matched line - Google Patents
Breeding method for inducing pelteobagrus fulvidraco facultative fish to generate super-male fish and full-female matched line Download PDFInfo
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Abstract
The invention provides a breeding method for generating a super-male fish and a full-female mating line by inducing pelteobagrus fulvidraco facultative fish, which comprises the following steps: the method comprises the steps of determining the induction and type of XY genotype and XX genotype facultative fishes, utilizing the backcross of the XY genotype facultative fishes to generate candidate super-male fish groups, utilizing a test cross screening method of the super-male fishes, and utilizing the backcross of the XX genotype facultative fishes to generate the technology of the all-female mating line. According to the technology, a plurality of geographic populations introduced according to the technical requirements are respectively constructed into a super-male fish and a full-female matched line, and a full-male pelteobagrus fulvidraco production combination with heterosis is screened out by using a double-row hybridization test. The invention establishes an electronic digital chip label marking management method for preventing population hybridization and accuracy of test cross identification, induced facultative fishes do not need genetic identification, screened supermale families have the characteristic of high male rate, the requirements of instruments and equipment are simple, the technology is easy to operate, and the invention is also suitable for breeding all-male fishes and all-female fishes of other male heterotypic fishes.
Description
Technical Field
The invention belongs to the technical field of sex control of fish genetic breeding, in particular to a breeding method for inducing pelteobagrus fulvidraco facultative fish to generate a super-male fish and full-female fish complete set line, which is also suitable for breeding full-male fish and full-female fish of other male special-shaped fishes.
Background
The biological traits of many fishes such as growth speed, maturation age, breeding mode, body shape and body color are different between female and male, and human beings can obtain the specific traits expected by a single sex through sex control. At present, methods for controlling the sex of fishes comprise manual selection, hormone induction, gynogenesis, androgenesis, interspecific hybridization, polyploidy induction, trilinear matching, GMT technology, sex molecular markers and the like. The male and female individuals are difficult to accurately distinguish in the juvenile fish stage by manual selection, and the labor intensity is high; residual hidden troubles exist in hormone induction, so that the food safety is endangered; interspecific crosses are only suitable for crosses between ZZ and XX types of fish; the three-line matching is similar to the GMT technology, and although a reliable and stable all-male breeding method is established on tilapia, generation-by-generation test cross is required, the work is complicated, and the period is long; the gynogenesis, the androgenesis, the polyploidy induction and the sex molecular marking method have advanced technology and reliable method, but have high requirements on instrument configuration and technology, and the induction rates of the gynogenesis, the androgenesis and the polyploidy are lower, so the technology is difficult to be matched with and matched with the neck and back of a common seedling enterprise.
The pelteobagrus fulvidraco is an important freshwater fish for pond culture in China, is a typical fish with large male and female growth difference, and has the growth speed of about 30 percent faster than that of female fish and over 100 percent faster than that of second-instar fish. The male growth advantage is obtained by sex control and full male breeding, the culture yield of the pelteobagrus fulvidraco can be improved, the culture period is shortened, and the culture cost is reduced. Meanwhile, the sex determination mechanism of the pelteobagrus fulvidraco can be clarified. In 2007, Chinese scholars report a method for producing YY super-male fish from the gynogenesis of XY female fish for the first time, and the YY super-male fish is mated with an original line female fish to produce full-male yellow catfish; however, under the influence of the ordinary chromosome modification genes, the male rate of the super-male fish test cross offspring generated by gynogenesis is 75.9-100%, and the average is 90.30%. The supermale family obtained by the technology can not completely generate a full-male offspring, and the problem of male rate can be solved only by screening the supermale family with high male rate through test crossing; likewise, supermale families bred by sex-specific molecular markers cannot exclude the influence of sex-modifying genes on male rate.
The method for producing the all-male fish through the YY super-male fish has the advantages that another problem to be solved is that female parent fish needing batch breeding are bred through seedlings, the best scheme is to establish an all-female matched line to meet the requirements of the female parent fish in batch, and the method needs to be completed through an all-female breeding method. Meanwhile, the all-female breeding has important significance for the fish with fast female growth. In addition to the above methods of sex control, all-female breeding can also be produced by the method of pseudo-male fish, which can be obtained by: the sex of gynogenesis female fish is reversed, or the gynogenesis female fish is induced by hormone or aromatizing enzyme, but the induced population needs sex molecular marker or other genetic methods for identification, and the defects are obvious. Currently, these methods, whether all-male or all-female breeding, are beneficial for the rapid establishment of pure lines or purification and rejuvenation, but due to high degree of purity, there is also a risk of germplasm degradation due to single population structure, low genetic diversity and possible inbreeding. So far, the induction and physiological characteristics of facultative fish have been reported, but the research of producing super-male fish and full-female fish by utilizing facultative fish has not been reported. Therefore, a simple and efficient super-male fish and full-female matched line breeding method is established by inducing the facultative fish, and the method has important significance for the production of the full-male pelteobagrus fulvidraco.
Disclosure of Invention
In order to overcome the problems, the invention aims to solve the problems of super-male fish, batch female parent fish and male rate required by large-scale artificial propagation of the full-male pelteobagrus fulvidraco by establishing a super-male fish and full-female matched line breeding method which is simple in equipment configuration and easy in technical operation, and prevent germplasm degradation risk caused by simple population structure, low genetic diversity and inbreeding.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the method comprises five aspects: 1 induction of two types of facultative fish; 2, generating, identifying and establishing a super-male fish breeding line; 3 methods for the production of hologynic mating lines; 4, construction and evaluation of a super-male fish breeding line of multiple geographical populations; 5, electronic digital chip label marking technology. The method comprises the following specific steps:
1.1 Artificial reproduction
Selecting female and male pelteobagrus fulvidraco fish with sexual maturity, artificially hastening parturition according to a conventional method, collecting mature ova and sperms, artificially inseminating by adopting a dry method or a hypotonic solution, uniformly spraying fertilized ova on a 40-mesh net, incubating in a cement pond, and obtaining the fry which is the XX-XY gender mixed group after incubation.
Wherein, the conventional methodThe artificial induction of labor is to inject mixed hormone into the body at the temperature of 22-31 ℃ to induce estrus, and the dosage of the mixed hormone is luteinizing releasing hormone analogue (LRH-A)2) 25-30 mg/kg, 6-8 mg/kg of Domperidone (DOM) and 1300-1500 IU/kg of chorionic gonadotropin (HCG);
the dry artificial insemination is to uniformly mix the collected sperms and the ova, then add clean pond water with the volume 2-4 times of the ova, stir for 30-60 seconds, and finish the fertilization process;
the hypotonic solution artificial insemination is to add the collected sperms into 0.3 percent of physiological saline, stir for 2-3 seconds to enable the sperms to be evenly mixed, then pour the sperms into the collected ova together, stir for 30-60 seconds, and finish the fertilization process. The dosage of the physiological saline is 1-2 times of the volume of the egg.
1.2 sex hormone Induction
Sex hormone induction is classified into estradiol valerate (estrogen) induction and 17 α -methyltestosterone (androgen) induction. Estradiol valerate induction aims at producing XY genotype physiological phenotype facultative fish, and 17 alpha-methyltestosterone induction aims at producing XX genotype physiological phenotype facultative fish.
1.2.1 estradiol valerate Induction
Taking artificially propagated XX-XY sex mixed group fish fries, feeding rotifer, fairy shrimp and small cladocera after advection, continuously feeding for 5-7 days (7-10 days old), then feeding estradiol valerate feed with the concentration of 15-20 mg/kg for 25-40 days, and inducing XY genotype fish into physiological phenotype facultative fish.
Wherein, the micro-flowing water is used for feeding in the induction process, the dissolved oxygen is not less than 5mg/L, the feeding is carried out in a separate culture container, and after the induction is finished, the pelteobagrus fulvidraco feed is fed until the sexual maturity. The fish body can be transferred to a cement pond with larger water body for breeding along with the growth of the fish body in the breeding process, but the mixing is prevented.
The purity of the used estradiol valerate reaches the biochemical level, the estradiol valerate is completely dissolved by 100-500 mL of 75-95% alcohol according to the concentration proportion of 15-20 mg/kg, the mixture is uniformly mixed into the yellow catfish powdery feed, and the yellow catfish powdery feed is dried in the shade under the condition of shade and cool and stored in a refrigerator at 4 ℃ for later use.
1.2.217 alpha-methyl testosterone induction
The induction method, feed preparation and feeding process of inducing XX genotype fishes into physiological phenotype facultative fishes by 17 alpha-methyl testosterone are similar to those of estradiol valerate induction, the concentration of the used 17 alpha-methyl testosterone is 30-50 mg/kg, and the feeding time is 45-60 days.
1.3 type determination
The XX genotype fish has female genetic phenotype, theoretically, estradiol valerate induces or does not induce, and the physiological phenotype of the XX genotype fish is female fish; the XY genotype fish has male genetic phenotype, and the induction is successful, and the result is that the fish is a physiological phenotype female fish or a physiological phenotype facultative fish, and the fish is still a physiological phenotype male fish if the induction is unsuccessful. Therefore, the type of the facultative fish induced by the estradiol valerate is determined to be XY genotype.
On the contrary, the genetic phenotype of the XY genotype fish is male, theoretically, the 17 alpha-methyltestosterone is induced or not induced, and the physiological phenotype of the XY genotype fish is male fish; the XX genotype fish has female genetic phenotype and is induced successfully, and the result is physiological phenotype male fish or physiological phenotype facultative fish. Therefore, the type of the facultative fish induced by 17 alpha-methyltestosterone is determined to be XX genotype.
Wherein, the physiological phenotype judgment of the sex can be distinguished according to the gonad morphology after sexual maturity. The physiological phenotype of the male fish is that the gonad only has the spermary; physiological phenotype female fish, gonad only ovary; the physiological phenotype facultative fish has the sex glands with the spermary and the ovaries and has the characteristics of hermaphrodite and hermaphrodite.
2 Generation, identification and establishment of super-male fish breeding line
The generation, identification and establishment of the super-male fish breeding line comprise the following steps: generating a super-male fish candidate group, carrying out test cross identification and screening on the super-male fish, and establishing a super-male breeding line.
2.1 super-Male Fish candidate population Generation
A candidate population of super-male fish can be generated in two ways: backcrossing between the XY genotype facultative fish and the original line XY genotype male fish; crossing between a facultative fish of XY genotype with another facultative fish of XY genotype.
2.1.1 backcross between facultative fish of XY genotype and male fish of XY genotype of original line to produce super-male fish candidate population
Selecting a sex-mature estradiol valerate induced XX-XY sex mixed group and an original series XY genotype female fish, dissecting individuals of the sex-mature estradiol valerate induced XX-XY sex mixed group one by one according to an artificial propagation mode, only keeping individuals with an ovary and a spermary, wherein the individuals are XY genotype facultative fishes, taking out mature ova of the individuals, taking out mature sperms of the original series XY genotype male fishes, carrying out artificial insemination on the ova and the sperms, and hatching fries which are the XX, XY and YY genotype super male fish candidate groups.
2.1.2 hybridization between a facultative fish of an XY genotype and another facultative fish of another XY genotype to produce a candidate population of super-male fish
Selecting a sex-mature estradiol valerate induced XX-XY sex mixed group, dissecting individuals of the XX-XY sex mixed group one by one according to an artificial propagation mode, only keeping individuals with both spermary and ovary, separating mature ova and sperms of the individuals, carrying out artificial insemination according to the ova and sperms among different individuals, and hatching fries which are also super-male fish candidate groups with XX, XY and YY genotypes.
2.2 Cross-testing identification and screening of super-male fish
The genetic phenotype of the YY genotype super-male fish is male, and in order to establish a super-male breeding line, the YY genotype physiological phenotype male fish and female fish must be identified and separated from XX, XY and YY genotype super-male fish candidate groups respectively.
2.2.1 identification and isolation of physiological phenotype male fish of YY genotype
Taking XX, XY and YY genotype super-male fish candidate group fry, feeding the fry separately until sexual maturity, rejecting morphological female fish, namely XY-YY genotype mixed group, implanting electronic digital chip labels into test cross individuals one by one, carrying out test cross with original series XX genotype female fish, feeding test cross offspring of each fish separately for 60 days, injecting glacial acetic acid into abdominal cavity for fixation, taking out gonad tabletting and dyeing, identifying sex according to gonad cell morphology, and obtaining candidate male fish with the male ratio of the test cross offspring exceeding 95%, namely the physiological phenotype male fish of the YY genotype super-male fish.
2.2.2 identification and isolation of YY genotype female fish with physiological phenotype
Taking XX, XY and YY genotype super-male fish candidate group fries, feeding estradiol valerate feed with the concentration of 30-60 mg/kg for 45-60 days, carrying out sex reversion to obtain physiological phenotype female fish, then feeding until the female fish is sexually mature, implanting electronic digital chip labels into test cross individuals one by one, and carrying out test cross with XX genotype facultative fish. The test cross identification method is the same as the test cross identification method of the physiological phenotype male fish of the YY genotype super male fish, and the candidate female fish with the test cross offspring male ratio of more than 95 percent is the physiological phenotype female fish of the YY genotype super male fish. When the male fish roe and the XX genotype facultative fish are crossed, only female fish roes of a super-male fish candidate group are selected to be crossed with XX genotype facultative fish sperm.
2.3 establishment of supermale clones
In order to obtain the super-male fish in batches, the physiological phenotype female fish and the physiological phenotype male fish of the YY genotype super-male fish identified by test cross are taken, the batch of YY genotype super-male fish can be obtained according to the breeding mode of artificial estrus promotion and natural spawning, the obtained super-male fish is independently bred to the specification of 5 g/tail, the electronic digital chip tags are implanted one by one to prevent confusion, and the super-male fish is bred to sexual maturity.
Wherein the breeding mode of artificial estrus promotion and natural spawning is that the physiological phenotype female fish and the physiological phenotype male fish of the sexually mature YY genotype super male fish are mixed according to the proportion of 1: 1-1.5, and the female fish is injected with LRH-A215-20 mg/kg, DOM 5-8 mg/kg, HCG 1300-1500 IU/kg, halving the dosage of the male fish, putting the male fish into a cement pond, controlling the water depth to be 40-60 cm, and feeding the male fish in micro-flow water; then a plastic clay tile bucket with the diameter of 30cm and the height of 20cm is placed in the cement pond, a piece of palm slice is placed at the bottom of the bucket to serve as a spawning fish nest, and the number of the fish nests is the same as that of the placed male fish. After the heat and spawning of the parent fish are finished, the fish nests are collected, and the fish nests stuck with the fertilized eggs are placed into another cement pond for microflow water incubation.
Method for producing complete female fish complete set line
Since 17 α -methyltestosterone was used to induce facultative fish, type XX, a complete female fish complement can be produced in two ways: backcross between the female fish with XX genotype and the facultative fish with XX genotype of the original line, and cross between the facultative fish with XX genotype and another facultative fish with XX genotype.
3.1 backcross between the XX genotype female fish and the XX genotype facultative fish of the original line to generate a complete female fish mating line
Selecting a sex-mature primary XX genotype female fish and an XX-XY sex mixed group induced by 17 alpha-methyl testosterone, extruding mature ova of the primary XX genotype female fish according to an artificial propagation mode, dissecting individuals of the XX-XY sex mixed group one by one, only keeping individuals with both spermary and ovary, taking out mature sperms of the individuals, carrying out artificial insemination on the ova and the sperms, and hatching fry to obtain 100% of XX genotype female fish.
3.2 hybridization between a facultative fish of the XX genotype and another to produce a complete female fish complement
Selecting sexually mature XX-XY sex mixed population induced by 17 alpha-methyltestosterone, dissecting individuals of the XX-XY sex mixed population one by one according to an artificial propagation mode, only keeping individuals with both a spermary and an ovary, separating mature ova and sperms, carrying out artificial insemination on the ova and sperms among different individuals, and obtaining hatched fries which are 100% of XX genotype female fishes.
Construction and evaluation of super-male fish breeding line with multiple geographical populations
The construction and evaluation of the super male fish breeding line of the multi-geographic population comprises the following steps: introduction and selection of different wild geographic populations, construction of super-male fish breeding lines of different geographic populations, and evaluation of super-male fish of different geographic populations.
4.1 introduction and selection of different wild geographical populations
In order to improve the genetic diversity and breeding potential of the populations and prevent inbreeding, at least 4 geographical populations are selected, the populations are required to be isolated from each other or have long distance, and each population is selected from wild pelteobagrus fulvidraco in natural water areas such as large rivers, lakes or reservoirs without artificial release; the introduction population requires large individuals, strong body types, no diseases and no injuries, the germplasm accords with the specification of an industrial standard SC 1070, and the number of female and male individuals of each population is more than 300; the introduced population is disinfected in an isolation area, killed and carried with parasites, and is independently raised and observed for one month, and the population without major epidemic diseases is used as a basic population for constructing the super-male fish. If serious epidemic diseases are found in the introduced population in the isolation stage, harmless destruction treatment is immediately carried out.
4.2 construction of super-male fish breeding lines in different geographical populations
Respectively constructing and culturing the super-male fish breeding lines of different geographical populations according to the contents of the steps 1 and 2 in the technical scheme, and implanting an electronic digital chip label into each super-male fish in order to prevent the super-male fish breeding lines from being mixed among the populations.
4.3 evaluation of super-male fishes in different geographical populations
Carrying out NxN double-row hybridization test on the original line XX genotype female fish and the YY genotype super-male fish among the populations, separately breeding the obtained XY genotype filial generations, taking the fish seeds with the same number of each filial generation, implanting electronic digital chip labels one by one, and carrying out the comparison feeding test with the same pool. And performing data statistical analysis and growth sequencing according to the same-pond feeding test result, thereby screening out the full-male pelteobagrus fulvidraco hybridization combination with the optimal growth population.
5 electronic digital chip label marking technology
The label of electronic digital chip is used to prevent the confusion or mixing of species or test cross individuals and to establish precise management file of species, parent fish or individual data and germplasm, and the technology includes: an electronic digital chip label implanting method, a population and individual electronic digital chip label filing.
5.1 electronic digital chip label implanting method
The specification of the electronic digital chip label is selected according to the size specification of the fish body, and the smaller the specification of the electronic digital chip label is, the smaller the damage to the fish body is, and the specification of 1.25 multiplied by 7.0mm is generally preferred as the electronic digital chip label needs to be implanted into the tissues or organs of the fish body. The specific implantation method comprises the following steps: the electronic digital chip label is placed into a needle head of a special syringe, then the needle head is inserted into the muscle at the dorsal fin base part or the abdominal cavity at the pectoral fin base part at an angle of 45 degrees, a syringe handle is pushed, the label is pushed into the muscle or the abdominal cavity, and the implantation depth is preferably 0.3-0.5 cm. After the fish is implanted, the scanner is connected with a computer, the scanning function is started, and the scanned digital code of each fish is automatically recorded in the computer.
5.2 population and Individual electronic digital chip tag profiling
In the processes of establishing management files of the population and the parent fish, testing, crossing and screening and pond-feeding contrast test, electronic digital chip tags are implanted into corresponding population, parent fish and individuals, and each tag has a specific digital code, so that the tags can be accurately identified after being marked, the population, the parent fish and the testing and crossing individuals are prevented from being confused, and accurate management is realized.
When the population and the parent fish are established, the countable characters and the quantifiable characters of the population or the parent fish such as the body length, the body weight, the fin type, the number of branchial rakes outside a first branchial arch, the body length/the body height, the body length/the head length, the body length/the tail stalk length, the body length/the fat fin base length, the head length/the pectoral fin length and the like need to be measured, recorded and filed to serve as the germplasm file of each population or parent fish.
Compared with the prior art, the technical scheme of the invention can realize the following beneficial effects: 1, the induced XY genotype or XX genotype facultative fish only needs morphological identification and does not need genetic methods such as test cross or molecular markers. By the method for generating the YY genotype super-male fish and the XX genotype full-female matched line by the XY genotype and XX genotype facultative fish, the XY transgenic test cross identification step is omitted, and compared with a three-line matched line or GMT technology, one generation is saved; instruments, control conditions and operation techniques required for gynogenesis, androgenesis and sex specific molecular marker identification are not required to be configured, and the method is simpler and more efficient. 2, the XX genotype double cross test line established by identifying and separating the super-male fish, namely the YY genotype physiological phenotype male fish is identified by using the XX genotype female fish test cross of the original line and the YY genotype physiological phenotype female fish is identified by using the induced XX genotype facultative fish test cross, which is more accurate than the method for identifying the original line XY genotype male fish by using the three-line matching or GMT technology. The method can screen the super-male family with high male rate characteristic, solves the problem of the super-male family with low male rate which cannot be identified by gynogenesis or molecular sex markers, and ensures that the bred full-male yellow catfish has high and stable male rate. 3, by constructing the super-male fish in multiple geographic populations, the genetic diversity is improved, the problem of simple structure of the super-male fish in a single population is solved, the germplasm degradation risk possibly generated by inbreeding is prevented, and the full-male pelteobagrus fulvidraco hybridization combination with the best production performance can be screened out. The method for marking by the electronic digital chip label ensures that the filing management of the population and the parent fish germplasm is more accurate, prevents the population, the parent fish or the individual test cross from being mixed or confused, and has traceability.
Drawings
The invention is further described below with reference to the following figures and examples:
FIG. 1 is a technical route diagram of a breeding method for inducing pelteobagrus fulvidraco facultative fish to produce a super-male fish and full-female mating line
In the figure 1. artificial propagation; 2. inducing facultative fish; 3. backcrossing; 4. generating a candidate population of super male fish or a hologynic mating line; 5. test cross screening; 6. building a supermale breeding line; 7. constructing a multi-geographic population super-male fish and full-female mating line; n × N two-line hybridization assay.
Detailed Description
In order to further illustrate the technical scheme of the invention, a super-male fish breeding line and a full-female mating line are constructed for introduced geographic populations of 4 wild pelteobagrus fulvidraco in the inner Mongolia city west temple reservoir, the Hongze lake, the eastern lake attached to the Dongting lake and the Poyang lake through specific embodiments, the technical scheme and the flow shown in the attached drawings are described in detail, and the specific implementation modes and the steps are as follows:
1 selection, introduction and profiling of geographic populations
Selecting 4 wild geographical populations of inner Mongolia city west temple reservoir (number DXM), Hongze lake (number HZL), Dongting lake auxiliary lake east lake (number DHL) and yang lake (number PYL), wherein each population is introduced with more than 300 tails of female and male fishes, male individuals are more than or equal to 200 g/tail, female individuals are more than or equal to 100 g/tail, physique is strong, body side stripes are clear, diseases and injuries do not exist, injured individuals are removed after being transported to a destination, pathogens are detected, the wild geographical populations are disinfected by 3% saline, and 12m in an isolation area room2Feeding and observing in a cement poolOne month.
Each group selects 200 male and female fishes with large individual, good body type, no disease and no injury respectively, and the male and female fishes are used as basic breeding groups of a super-male fish and a full-female matched line. A1.25 multiplied by 7.0mm electronic digital chip label is implanted into the muscle at the base of dorsal fin of each fish by a special syringe, and the digital code of each fish is scanned and recorded, so that the mutual mixing of populations is prevented.
Taking 30 fish from each population, measuring countable characters and quantifiable characters such as body length, body weight, fin type, branchial rake number outside the first gill arch, body length/body height, body length/head length, body length/tail stalk length, body length/fat fin base length, head length/pectoral fin thorn length, recording and filing as parent fish files of each population,
2 Induction of two types of facultative Fish
2.1 Artificial reproduction
Selecting 30 sexually mature female fishes and male fishes of yellow catfish from each population, and under the condition that the water temperature is 22-31 ℃, the injection dosage of the female fishes is luteinizing releasing hormone analogue (LRH-A)2) 25-30 mg/kg, 6-8 mg/kg of Domperidone (DOM), 1300-1500 IU/kg of chorionic gonadotropin (HCG), and halving the dosage of the male fish. Collecting mature ova after estrus, dissecting male fish, taking out a spermary, grinding the spermary into slurry by using a mortar, putting 0.5mL of semen into 100mL of 0.2-0.4% physiological saline, stirring for 2-3 seconds to uniformly mix the sperms, then pouring the sperms into the collected ova together, and stirring for 30-60 seconds to finish the fertilization process. And uniformly spraying fertilized eggs on a 40-mesh net, hatching in a cement pond, and obtaining the hatched fry as the XX-XY gender mixed population.
2.2 preparation of the feed
Weighing 15-20 mg of biochemical-grade estradiol valerate or 30-60 mg of biochemical-grade 17 alpha-methyltestosterone by using an electronic balance, putting the weighed materials into 300mL of 95% alcohol, stirring until the materials are completely dissolved, uniformly mixing the materials into 1kg of yellow catfish powdery feed, drying the materials in the shade under the condition of shade, and storing the materials in a refrigerator at 4 ℃ for later use.
The feed containing the estradiol valerate is used for inducing the XY genotype physiological phenotype facultative fishes, and the feed containing the 17 alpha-methyltestosterone is used for inducing the XX genotype physiological phenotype facultative fishes.
2.3 hormone Induction
2.3.1 estradiol valerate Induction
And taking the XX-XY sex mixed group fry of which the 5-7 days or 7-10 days old are artificially bred from 500 fish, independently breeding in a 300L round glass fiber reinforced plastic barrel, feeding estradiol valerate feed with the concentration of 15-20 mg/kg for 25-40 days, and inducing the XY genotype fish into physiological phenotype facultative fish.
2.3.217 alpha-methyl testosterone induction
Separately breeding the XX-XY sex mixed group fry of which the 5-7 days or 7-10 days old are opened at 500 tails of each group in a 300L round glass fiber reinforced plastic barrel, feeding estradiol valerate feed with the concentration of 30-60 mg/kg for 45-60 days, and inducing the XY genotype fish into physiological phenotype facultative fish.
2.4 Breeding management
Feeding in micro-flowing water in the induction process, sucking dirt once in the morning and afternoon every day, increasing oxygen by using an air pump, wherein the dissolved oxygen is not less than 5mg/L, and feeding the pelteobagrus fulvidraco commercial feed to reach sexual maturity after induction is finished. The fish body grows during the feeding process and is transferred to 10m2The cement pond, but the transferring and breeding process needs to prevent the mixing of various populations.
2.5 type determination
The XX genotype fish has female genetic phenotype, theoretically, estradiol valerate induces or does not induce, and the physiological phenotype of the XX genotype fish is female fish; the XY genotype fish has male genetic phenotype, and the induction is successful, and the result is that the fish is a physiological phenotype female fish or a physiological phenotype facultative fish, and the fish is still a physiological phenotype male fish if the induction is unsuccessful. Therefore, the type of the facultative fish induced by the estradiol valerate is determined to be XY genotype.
On the contrary, the genetic phenotype of the XY genotype fish is male, theoretically, the 17 alpha-methyltestosterone is induced or not induced, and the physiological phenotype of the XY genotype fish is male fish; the XX genotype fish has female genetic phenotype and is induced successfully, and the result is physiological phenotype male fish or physiological phenotype facultative fish. Therefore, the type of the facultative fish induced by 17 alpha-methyltestosterone is determined to be XX genotype.
Wherein, the physiological phenotype judgment of the sex can be distinguished according to the gonad morphology after sexual maturity. The physiological phenotype of the male fish is that the gonad only has the spermary; physiological phenotype female fish, gonad only ovary; the physiological phenotype facultative fish has the sex glands with the spermary and the ovaries and has the characteristics of hermaphrodite and hermaphrodite.
3 Generation, identification and establishment of super-male fish breeding line
3.1 Generation of a super Male Fish candidate population
Selecting a sex-mature estradiol valerate induced XX-XY sex mixed group and an original series XY genotype female fish, injecting an aphrodisiac hormone according to an artificial propagation mode, dissecting individuals of the sex-sex XX-XY mixed group one by one after estrus, only keeping individuals with ovaries and sperms, namely the XY genotype facultative fish, taking out mature ova and sperms, utilizing the facultative ova and the original series XY genotype male fish sperms or the facultative sperms to artificially inseminate, hatching fries, namely a XX, XY and YY genotype super-male fish candidate group, wherein the theoretical proportion of the XX genotype in the super-male fish candidate group is 25%, the proportion of the XY genotype is 50%, and the proportion of the YY genotype is 25%.
3.2 test-crossing identification and screening of super-male fish
The genetic phenotype of the YY genotype super-male fish is male, and in order to establish a super-male breeding line, the YY genotype physiological phenotype male fish and female fish must be identified and separated from XX, XY and YY genotype super-male fish candidate groups respectively.
3.2.1 identification and isolation of Male Fish with YY genotype physiological phenotype
Taking XX, XY and YY genotype super-male fish candidate group fry, feeding the fry separately until sexual maturity, rejecting morphological female fish, namely XY-YY genotype mixed group, implanting electronic digital chip labels into test cross individuals one by one, testing cross with original series XX genotype female fish one by one, feeding test cross offspring of each fish separately for 60 days, injecting glacial acetic acid into abdominal cavity for fixation, taking out gonad tabletting and dyeing, identifying sex according to gonad cell morphology, and obtaining candidate male fish with test cross offspring male ratio exceeding 95%, namely physiological phenotype male fish of YY genotype super-male fish.
Wherein, the test cross process adopts the mode of artificial estrus promotion and natural spawning, namely, female fish of XX genotype of original system and male fish of XY-YY genotype mixed population are paired one to one at the water temperature of 25-28 ℃, and the female fish is injected with LRH-A dosage215-18 mg/kg + DOM 5-6 mg/kg + HCG 1300-1500 IU/kg mixed hormone, the dosage of male fish is halved, the male fish is placed into a 300L round glass fiber reinforced plastic barrel, a palm slice is placed at the bottom of the barrel to serve as a spawning fish nest, after spawning, the fish nest is collected, and the fish nest stuck with fertilized eggs is placed into another 300L round glass fiber reinforced plastic barrel for microflow water incubation.
3.2.2 identification and isolation of YY genotype female fish with physiological phenotype
Taking XX, XY and YY genotype super-male fish candidate group fries, feeding estradiol valerate feed with the concentration of 30-60 mg/kg for 45-60 days, carrying out sex reversion to obtain physiological phenotype female fish, then feeding until the female fish is sexually mature, implanting electronic digital chip labels into test cross individuals one by one, and carrying out test cross with XX genotype facultative fish. The test cross identification method is the same as the test cross identification method of the physiological phenotype male fish of the YY genotype super male fish, and the candidate female fish with the test cross offspring male ratio of more than 95 percent is the physiological phenotype female fish of the YY genotype super male fish.
Wherein, when testing and crossing with XX gene type facultative fish one-to-one, adopting artificial estrus promotion and artificial insemination modes, namely taking super male fish candidate population to reverse female fish and XX gene type facultative fish, female fish injecting dose LRH-A215-18 mg/kg + DOM 5-6 mg/kg + HCG 1300-1500 IU/kg mixed hormone, the dosage of the male fish is reduced by half, the female fish eggs are taken out after estrus and are artificially inseminated with XX genotype facultative sperms, the fertilized eggs are uniformly sprinkled on a 40-mesh net, and the net is put into a 300L round glass fiber reinforced plastic barrel for microflow water incubation.
3.3 establishment of supermale clones
Obtaining the super-male fish in batches, taking the physiological phenotype female fish and the physiological phenotype male fish of the YY genotype super-male fish identified by test cross, obtaining the batches of the YY genotype super-male fish according to the breeding mode of artificial estrus promotion and natural spawning, independently breeding the obtained super-male fish to the specification of 5 g/tail, implanting electronic digital chip tags one by one to prevent confusion, and breeding the super-male fish to sexual maturity. Wherein the breeding mode of artificial estrus promotion and natural spawning is that the physiological phenotype female fish and the physiological phenotype male fish of the sexually mature YY genotype super male fish are mixed according to the proportion of 1: 1-1.5, and the female fish is injected with LRH-A215-20 mg/kg, DOM 5-8 mg/kg, HCG 1300-1500 IU/kg, halving the dosage of the male fish, putting the male fish into a cement pond, controlling the water depth to be 40-60 cm, and feeding the male fish in micro-flow water; then a plastic clay tile bucket with the diameter of 30cm and the height of 20cm is placed in the cement pond, a piece of palm slice is placed at the bottom of the bucket to serve as a spawning fish nest, and the number of the fish nests is the same as that of the placed male fish. After the heat and spawning of the parent fish are finished, the fish nests are collected, and the fish nests stuck with the fertilized eggs are placed into another cement pond for microflow water incubation.
The physiological phenotype female fish and the physiological phenotype male fish of each group of YY genotype super male fish are mated independently, batch YY genotype super male fish obtained after mating are fed independently, and electronic digital chip labels are implanted one by one to prevent YY genotype super male fish from mixing among groups.
Generation of 4 complete female fish lines
Taking each population of sex-matured primary XX genotype female fish and an XX-XY sex mixed population induced by 17 alpha-methyl testosterone, injecting an aphrodisiac hormone under the condition of water temperature of 22-31 ℃ according to an artificial propagation mode, wherein the dosage is the same as that of the step 2.1, extruding mature ova of the primary XX genotype female fish for later use, dissecting individuals of the XX-XY sex mixed population one by one, only keeping individuals with both spermary and ovaries, namely the XX genotype facultative fish, taking out the mature ova and sperms of the XX genotype facultative fish, carrying out artificial insemination on the ova of the primary XX genotype female fish or the ova of the XX genotype facultative fish and the sperms of the XX genotype facultative fish, wherein the hatched fries are 100% of the XX genotype female fish, and breeding the fries to sexual maturity, namely the mature female fish in batches. Wherein, each population is mated only in the population, and the obtained fry is raised independently and can not be confused. The number of female fish required for each population depends on the number of artificial breedings.
5 two-line hybridization assay
Carrying out 4 x 4 double-row hybridization test on the full-female matched line and the YY genotype super-male fish among the populations, separately cultivating the obtained XY genotype hybridized offspring, removing the head fish and the tail fish when the obtained XY genotype hybridized offspring is cultured to the specification of 5.0 g/tail, combining each family to take 100 tails, implanting an electronic digital chip label, recording digital codes, and carrying out the comparative breeding test with the same pool. At the end of the test, the digital code of each tail is scanned out by a scanner, weighed, subjected to data statistical analysis and growth sequencing. According to the invention, the results of 4 x 4 double-row hybridization tests show that the growth sequence of the supermale fish of the four populations is DHL & gt DXM & gt PYL & gt HZL, and the optimal growth hybridization combination of the hologynic pelteobagrus fulvidraco is DXM (XX female) XDHL (YY male).
6. Production of full-male yellow catfish
According to the 4X 4 biserial hybridization test result, mature female fish of the full female mating line of the DXM population and the YY genotype super male fish of the DHL population are taken, and the obtained offspring is 100 percent of the XY genotype full male fish according to the artificial propagation mode, and the growth advantage of the population is most obvious.
Claims (6)
1. The breeding method for inducing the pelteobagrus fulvidraco facultative fish to generate the super-male fish and the full-female matched line is characterized by comprising the following steps of: respectively inducing XY and XX genotype facultative fishes with normal breeding capacity by feeding sex hormone estradiol valerate with the concentration of 10 mg/kg-20 mg/kg for 30-45 days and 17 alpha-methyltestosterone with the concentration of 30 mg/kg-60 mg/kg for 45-60 days, generating a super-male fish candidate group by hybridization between the XY genotype facultative fishes and an original system XY genotype male fish or another XY genotype facultative fish, and screening the Y genotype super-male fish physiological phenotype male fishes from the super-male fish candidate group by using XX genotype female fishes for test cross breeding; feeding a super-male fish candidate group for 40-60 days, and screening YY genotype super-male fish physiological phenotype female fish by using XX genotype facultative sperm test crossing after the sex reversal of a estradiol valerate feed containing 30-45 mg/kg concentration; crossing the XX genotype facultative fish with the original line female fish or another XX genotype facultative fish to generate an XX genotype full female mating line; the method is used for culturing YY genotype super-male fish and XX genotype full-female matched lines of a plurality of geographic populations, and N multiplied by N double-row hybridization tests are carried out on the YY genotype super-male fish and XX genotype full-female matched lines of each population, so that a full-male pelteobagrus fulvidraco production combination with hybridization advantages is screened out.
2. The breeding method for inducing the pelteobagrus fulvidraco facultative fish to produce the super-male fish and the full-female complete-set line according to claim 1, which is characterized in that: the two XY and XX genotype facultative fishes are facultative fishes induced by estradiol valerate, the type I of the facultative fishes is XY genotype, and the type I of the facultative fishes induced by 17 alpha-methyltestosterone is XX genotype; the sexually mature individuals have hermaphroditic characteristics, the spermary and the ovary have normal breeding capability, and the required spermary or ovary is dissected and taken out according to judgment of the gonad morphological characteristics when the sex-mature individuals are used.
3. The breeding method for inducing the pelteobagrus fulvidraco facultative fish to produce the super-male fish and the full-female complete-set line according to claim 1, which is characterized in that: the super-male fish candidate population is generated by crossing mature ova of the XY genotype facultative fish with mature sperms of an original XY genotype male fish or mature sperms of another XY genotype facultative fish and artificial insemination, and the physiological phenotype female fish and the physiological phenotype male fish of the YY genotype super-male fish are screened by test cross.
4. The breeding method for inducing the pelteobagrus fulvidraco facultative fish to produce the super-male fish and the full-female complete-set line according to claim 1, which is characterized in that: the full-female mating line is generated by hybridizing the mature sperm of the XX genotype facultative fish with the mature ovum of the original XX genotype female fish or the mature ovum of another XX genotype facultative fish and artificial insemination.
5. The breeding method for inducing the pelteobagrus fulvidraco facultative fish to produce the super-male fish and the full-female complete-set line according to claim 1, which is characterized in that: the number of the super-male fishes in the plurality of geographical populations is at least more than 4, the original line of each population is introduced, the naturally-growing individuals of large-scale rivers, lakes and reservoir water areas without artificial release are selected, the number of the female fishes and the number of the male fishes are respectively more than 300, only one population is introduced into each water area, and the distance between the populations is more than 100 kilometers.
6. The breeding method for inducing the pelteobagrus fulvidraco facultative fish to produce the super-male fish and the full-female complete-set line according to claim 1, which is characterized in that: the production combination of the all-male yellow catfish is characterized in that an N multiplied by N double-row hybridization test is carried out on the super-male fish and the all-female matched line among different geographical populations, and a combination with hybridization advantages is screened out for production.
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| CN113661948A (en) * | 2021-09-06 | 2021-11-19 | 华中农业大学 | Method for obtaining full-female homozygous pelteobagrus fulvidraco clone line through self fertilization |
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