CN107996468A - A kind of ussuriensis Genetic male reverses physiology female fish to educate screen method - Google Patents
A kind of ussuriensis Genetic male reverses physiology female fish to educate screen method Download PDFInfo
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- BFPYWIDHMRZLRN-SLHNCBLASA-N Ethinyl estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 BFPYWIDHMRZLRN-SLHNCBLASA-N 0.000 description 2
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- 229910052794 bromium Inorganic materials 0.000 description 1
- 210000001136 chorion Anatomy 0.000 description 1
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
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- 239000004467 fishmeal Substances 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/90—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination
- A01K61/95—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination specially adapted for fish
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
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Abstract
Reversed the invention discloses a kind of ussuriensis Genetic male and educate screen method for physiology female fish, including fry hatching, the selection of experiment fry, artemia hatching, estradiol solution immersion artemia, artemia are observed as the physiology gender of hormone carrier feeding fry, fingerling and adult fish, genetic sex identification step.Pass through the suitable feeding initial time of program screening of optimization, the estradiol concentration for soaking artemia and time, the lasting processing time of feeding, Genetic male individual sex reversal is made a living and manages female individuals, use the special molecular markers for identification Genetic male physiology female individuals of male, point pool raise, according to the breeding technique route of milter sex reversal → artificial gynogenesis → supermale fish → all-male fish, the present invention provides a large amount of high-quality basic materials in a short time for ussuriensis all-male fish breeding.
Description
Technical field
The present invention relates to fish biotechnology breeding field, and in particular to a kind of ussuriensis Genetic male is reversed and made a living
The female fish of reason educates screen method.
Background technology
In fish culture production practices, it has been found that have a variety of fish between male and female individual existing forms, the speed of growth,
The male and female dimorphism phenomenon of sexal maturity weight etc., if the marketable fish of neat specification can not only be obtained by carrying out single file cultivation,
The utilization rate of feed is improved, increases the income of raiser, and Modern Fishery industrialization can be promoted and formed to have and independently known
Know the new varieties of property right, there is unique advantage and application.Therefore, it is aquatic products to carry out fish sex control breeding and monosex cultivation
One important directions of industry development.
The fish sex control most common way of breeding is the parent population reversed with sex hormone induction of genetic gender, with heredity
The fish of not identical another gender mates to obtain supermale or superfemale fish, is handed over supermale or superfemale fish with normal raun or milter
With all-male or complete female fry can be obtained, achieve the purpose that monosex cultivation.This is because fish gonadal sex not only by it is hereditary because
Son control, is also easy to be influenced be subject to internal and external environment, particularly the effect of exogenous hormones, at ontogeny early application hormone
Reason can make it that the genetic sex of fry and physiology gender are inconsistent, produce gender reversal.Ussuriensis just has allusion quotation
The build of type, growth gender dimorphism phenomenon.In identical growth cycle, the milter speed of growth is faster than raun, adult fish male
Weight is even more up to two times of female.If all-male monosex cultivation can be carried out, it will yield is substantially improved, improves the conversion of feed
Coefficient, reduces aquaculture cost, greatly improves the social and economic benefit of its cultivation.According to milter sex reversal → artificial gynogenesis
The technology path of → supermale fish → all-male fish, obtains Genetic male and physiology female fish is a breeding successfully crucial ring.But no
Same fish its inhereditary material composition is different, Sex Determination Mechanism is different, gonad development sequential is different from process, in a kind of fish
Hormone-inducible reverses successful method to be not directly applicable another fish with step, it is necessary to is developed again.
The fry sex reversal of sex hormone induction at present mainly has following three kinds of methods：（1）Hormone solution directly soaks.By fry
Taken out after soaking a period of time directly in certain density hormone solution, continue to that gonad development is formed.Infusion method is for crow
Intend in Soviet Union there are sizable risk for this alepidotes of Chang, processing time is long, big to fry stimulation, juvenile fish constitution is poor, right
Fry, which causes damage, influences its survival.（2）Feed, which is mixed, feeds.Hormone is directly admixed in commodity fish meal, feeds and is eaten to fry.
Ussuriensis belongs to omnivorousness, and in early days using planktonic organism as bait, commercial feed palatability is poor, mating period water temperature is high easily makes
Water body is muddy, growth miscellaneous bacteria influences fry health, hormone polluted-water, and fry survival rate is low.（3）Living bait immersion feeding.Halogen
Worm nauplius is the high-quality bait of fishes and shrimps seedling, full of nutrition and comprehensive, is the outstanding agreeable to the taste bait of ussuriensis prelarva.
In hormone immersion process, hormone is set to enter in polypide by breathing and swallowing, polypide body surface hormone residues amount after filtering cleaning
Seldom, will not polluted-water, fry it is stimulated it is small, survival rate is high.But because fry individual is tender and lovely, when start to process,
How the concentration of hormone selects during processing, and the duration of processing, how long effect was best, slightly will just cause the big of fry accidentally
Amount is dead so that breeding work fails.
Whether sexual hormone induction fry sex reversal is successful, it is necessary to which the verification of being determined property of test cross, generally requires consuming again
The 4-5 years, it is difficult to obtain a large amount of breeding materials in a short time.
The content of the invention
Reverse the technical problem to be solved in the present invention is to provide a kind of ussuriensis Genetic male and educated for physiology female fish
Screen method, for sex reversal rate up to more than 95%, 6 month survival rate up to 60% or so, hormone induction sex reversal combines special point of male
Sub- Marker Identification genetic sex, can obtain a large amount of breeding materials in a short time then in experiment.
The present invention is achieved through the following technical solutions：
A kind of ussuriensis Genetic male reverses physiology female fish to educate screen method, comprises the following steps：
（1）Parent population is hastened parturition；
（2）Fry hatching；
（3）Sex hormone induces fry sex reversal
Hormone soaks artemia live bait：With ethanol it is mother liquor that solvent is configured to 100 mg/mls by estradiol, 4 DEG C of preservations, are pressed
According to 150-250 micrograms per litres dosage immersion artemia nauplii 1-1.5 it is small when, then filter and clean artemia；
Live bait is fed：Untreated artemia living is fed during fry opening, according to it is early, middle and late three times per day, eaten up every time with half an hour
Subject to, 8-9 days after opening, hormone immersion artemia, continuous feeding 37-45 days are fed three times a day, and hormone feeding terminates, continues
After feeding untreated artemia for a period of time, commercial feed feeding is gradually used instead；
（4）Genetic male reverses physiology female fish choosing sieve
Physiology steroid test：Fry through artemia feeding living passes through the presence or absence of gonapophysis, gonopore location determination physiology in 6 months
Gender, and computability reversion rate；
Genetic sex identification：If taking the female fish dry doubling mark that physiology gender is consistent, clip tail fin is a little, extracts genomic DNA,
PCR reactions, agarose electrophoresis are carried out with the special primer of male, ultraviolet light is detected and taken pictures, according to the special amplification piece of male
Duan Youwu judges its genetic sex, and the successful individual of sex reversal is sorted out individually raising as breeding material.
Further improvement project of the invention is that the nucleotides sequence of the primer is classified as：
It is positive：5’→3’(GACAGCAGAACGGAANNNNNN)
Reversely：5’→3’(GGAAGAGGGAGTGTGANNNNNN).
Further improvement project of the invention is that estradiol is α types.
Compared with prior art, the present invention there is following obvious advantage：
First, hormone induction sex reversal, which combines the special molecular markers for identification genetic sex of male, following advantage：（1）Precise Identification
The genetic sex of sex reversal fish, it is not necessary to which whether test cross, that is, confirmability reverses succeeds.（2）Then can be by sex reversal fish in experiment
A detection point pool is raised, this step can save breeding time 3-4.（3）It is high to obtain sex reversal fish success rate, can obtain in a short time
A large amount of breeding materials.
2nd, the present invention uses estradiol as derivant, with Pelteobagrus fulvidraco revulsion（ZL 200610166518.8）Compared to having
Advantages below：（1）Ethinyl estradiol is artificial synthetic drug, unknown applied to fish security.（2）Ethinyl estradiol is under
Thalamus has positive-negative feedback effect.Low dose of use can stimulate gonadotrophin secretion；Heavy dose is using its secretion is suppressed, continuously
Using adenohypophysis gonadotropin-releasing hormone receptor can be caused to lower, sex hormone secretion is reduced, suppresses gonad development.Actually should
Dosage is accurately held in, is not easy to grasp in production.（3）Estradiol is the natural estrogen of ovarian secretion, can be promoted
With the normal development for adjusting female sexual organ and secondary sex character.（4）Commercial goods estradiol is α, β type mixture, wherein α types physiology
Effect is strong, also has anti-androgenic effect, and sex reversal success rate is high.（5）Estradiol dosage scope is wide, easy to produce above
With.
3rd, the present invention starts to feed hormone immersion artemia live bait for 8-9 days after fry opening, chooses the right moment appropriate, fry
Body is good for, good to drug tolerance, can be directed to induction sexual gland to the development of ovary, sex reversal rate, 6 months survival rates, which reach, significantly to be carried
It is high.
Brief description of the drawings
Fig. 1 is flow chart of the present invention.
Embodiment
1 detailed description ussuriensis Genetic male of the present invention reverses below in conjunction with the accompanying drawings educates screen method for physiology female fish, has
Body is implemented according to the following steps：
1st, parent population selection is with hastening parturition：Select that gonad development is good, the undamaged parent population of healthy skin, female parent population is injected at twice
Human chorionic gonadotrophin and luteinising hormone-releasing hormo mixture（2000 IU/kg HCG, 20 μg/kg LHRH-
A）, male parent population dosage halves and only injects once.
2nd, artificial insemination：When Effect time occurs, fish-egg is squeezed into plastic tub, while dissects milter and takes spermary to add less
Perhaps physiological saline is ground, and takes semidry method artificial insemination.
3rd, incubating oosperm：Embryonated egg is layed on the nylon wire got ready, and nylon wire is hung in the hatchery of inflation,
Keep hatching water to have certain mobility, observe hatching situation three times a day.
4th, fry selects：After fry hatches, the fry for selecting form normal and vivaciously swimming is put into the experiment water marked
Pond, keeps inflation good.
5th, artemia hatching and processing：The hatching of worm's ovum is carried out according to artemia incubation condition, removes chorion, collects nauplius
For being eaten after fry opening.The mother liquor of 100 mg/ml of estradiol mother liquor is prepared, is soaked according to the dosage of 150-250 micrograms per litres
When bubble artemia nauplii 1-1.5 is small, then filters and clean artemia.
6th, fry hormone is fed：Untreated artemia living is fed during fry opening, according to it is early, middle and late three times per day, every time with
Half an hour feeds the artemia of hormone immersion, continuous feeding 37-45 days, terminates to swash subject to eating up after fry opening 8-9 days
Element processing, continues to feed a period of time with artemia, progressively uses commercial feed feeding instead with cost-effective.
7th, physiology steroid test：Test fish raises 6 months i.e. by the presence or absence of gonapophysis, gonopore location determination its physiology
Gender, and computability reversion rate.
8th, genetic sex identification：Simultaneously tail is marked to the individual for needing the physiology gender for carrying out genetic sex identification consistent
Fin samples, and extracts genomic DNA according to standard method, DNA concentration is diluted to 50-100ng/ μ l；Drawn with sex-specific marker
Thing carries out PCR（PCR）, i.e., using genomic DNA as template, special using PCR primer amplification genetic sex
DNA fragmentation, 25 μ l of PCR system, special forward primer 5 ' → 3 ' of 1 μ l of test individual DNA profiling, male and female
(GACAGCAGAACGGAANNNNNN) 1 μ l, reverse primer 5 ' → 3 ' (GGAAGAGGGAGTGTGANNNNNN) 1 μ l, PCR Mix
12μl、ddH2O 10μl；PCR reaction conditions：94 DEG C of 30S pre-degenerations, 94 DEG C of 30S, 55-59 DEG C of 30S, 72 DEG C of 30S, altogether
30-35 circulation, 72 DEG C of extension 6min. PCR products, 4 DEG C of preservations；Agarose gel electrophoresis, that is, the agarose for preparing 1-2% coagulate
Glue, when loading, take 5 μ l of DNA Marker, take each 5 μ l PCR product electrophoresis detections of sample to be tested, deposition condition：Voltage 120-
180V, electric current 300-600mA, time 15-20min；Ultraviolet light is detected and taken pictures, i.e., by Ago-Gel through bromine after electrophoresis
Gel analysis instrument is put into after changing the dyeing of second ingot, appropriate parameter post-exposure is set and takes pictures；Genetic sex judge, i.e., to photo into
Row is understood, if only occurring a 300bp band on corresponding swimming lane, which is genetically XX females, if in phase
Occur a 300bp band and a 471bp band on the swimming lane answered, then the individual is genetically XY males.Determine each
The genetic sex of fish, Genetic male, the individual of physiology female is sorted out, and is raised in single pond, you can be made for unisexuality breeding
Material.
Intend Chang sex hormone induction fry sex reversal situation in group experiment Soviet Union, embodiment experimental condition and result of the test are shown in
Following table.
Claims (3)
1. a kind of ussuriensis Genetic male reverses physiology female fish to educate screen method, it is characterised in that comprises the following steps：
（1）Parent population is hastened parturition；
（2）Fry hatching；
（3）Sex hormone induces fry sex reversal
Hormone soaks artemia live bait：With ethanol it is mother liquor that solvent is configured to 100 mg/mls by estradiol, 4 DEG C of preservations, are pressed
According to 150-250 micrograms per litres dosage immersion artemia nauplii 1-1.5 it is small when, then filter and clean artemia；
Live bait is fed：Untreated artemia living is fed during fry opening, according to it is early, middle and late three times per day, eaten up every time with half an hour
Subject to, 8-9 days after opening, hormone immersion artemia, continuous feeding 37-45 days are fed three times a day, and hormone feeding terminates, continues
After feeding untreated artemia for a period of time, commercial feed feeding is gradually used instead；
（4）Genetic male reverses physiology female fish choosing sieve
Physiology steroid test：Fry through artemia feeding living passes through the presence or absence of gonapophysis, gonopore location determination physiology in 6 months
Gender, and computability reversion rate；
Genetic sex identification：If taking the female fish dry doubling mark that physiology gender is consistent, clip tail fin is a little, extracts genomic DNA,
PCR reactions, agarose electrophoresis are carried out with the special primer of male, ultraviolet light is detected and taken pictures, according to the special amplification piece of male
Duan Youwu judges its genetic sex, and the successful individual of sex reversal is sorted out individually raising as breeding material.
2. a kind of ussuriensis Genetic male according to claim 1 reverses physiology female fish breeding method, its feature
It is：The nucleotides sequence of the primer is classified as：
It is positive：5’→3’(GACAGCAGAACGGAANNNNNN)
Reversely：5’→3’(GGAAGAGGGAGTGTGANNNNNN).
3. a kind of ussuriensis Genetic male according to claim 1 reverses physiology female fish breeding method, its feature
It is：Estradiol is α types.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110250052A (en) * | 2019-07-04 | 2019-09-20 | 淮阴师范学院 | A kind of selection of ussuriensis lower oxygen concentration resistance family |
| CN112195252A (en) * | 2020-10-27 | 2021-01-08 | 淮阴师范学院 | Multiple PCR primers for pseudobagrus ussuriensis gender detection and detection method |
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| CN110250052B (en) * | 2019-07-04 | 2022-04-26 | 淮阴师范学院 | Breeding method for pseudobagrus ussuriensis hypoxia-resistant family |
| CN112195252A (en) * | 2020-10-27 | 2021-01-08 | 淮阴师范学院 | Multiple PCR primers for pseudobagrus ussuriensis gender detection and detection method |
| CN112195252B (en) * | 2020-10-27 | 2024-04-30 | 淮阴师范学院 | Multiplex PCR primer and detection method for detecting pseudobagrus ussuriensis gender |
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| CN114058713A (en) * | 2021-11-30 | 2022-02-18 | 淮阴师范学院 | Acquisition method of Y chromosome sequence of Pseudobagrus ussuriensis |
| CN114058713B (en) * | 2021-11-30 | 2023-09-01 | 淮阴师范学院 | Method for obtaining Y chromosome sequence of Pseudobagrus ussuriensis |
| CN115024251A (en) * | 2022-07-04 | 2022-09-09 | 淮阴师范学院 | Rapid acquisition method for full-male Pseudobagrus ussuriensis |
| CN115428752A (en) * | 2022-10-12 | 2022-12-06 | 华中农业大学 | Method for producing all-female grass carp in large scale and application |
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