CN109548712B - Method for breeding female triploid zebra fish by using heat shock method - Google Patents
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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
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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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
Abstract
The invention discloses a method for breeding female triploid zebra fish by using a heat shock method, which comprises the following steps: taking wild AB strain zebra fish as parent fish, carrying out artificial spawning induction and artificial insemination, placing the obtained fertilized eggs in a culture dish, carrying out still water culture at the temperature of 28-29 ℃, then transferring the fertilized eggs into water at the temperature of 40.5-41.5 ℃ for heat shock treatment for 2min, then placing the fertilized eggs in water at the temperature of 28-29 ℃ for still water incubation, transferring the fish fries into water at the temperature of 22-26 ℃ for constant temperature culture after the fish fries are all incubated out, starting to feed paramecium within 3-5 days after the fish fries are incubated out, feeding fairy shrimp within 15-20 days, and carrying out detection and screening by a biological method after the fish fries are incubated out for 2 months to obtain a female triploid zebra fish individual. The method breaks through the cognition that the triploid zebra fish obtained by the heat shock method is male, has important scientific value, and provides a good material for the sex determination research of the zebra fish.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for breeding female triploid zebra fish by using a heat shock method.
Background
In the field of fish technology, the heat shock technique is a technique of inducing triploid by inhibiting the discharge of second polar body and inducing tetraploid by inhibiting the first cleavage, which is a method widely used for artificially inducing polyploid fish and has the advantages of low cost, simplicity and practicality.
Among zebrafish, Kavumpurath and Pandian reported for the first time that heat shock induced triploid zebrafish was 1990, however, triploid individuals obtained by this method were all male. Scientists reported induction of triploid zebrafish in 2004 and 2007, but both were male individuals. TADelomas, 3 months 2018, published in magazines Molecular Reproduction and Development "what is three zebrafish all? "further confirmed in the review report that triploid zebrafish induced by second polar induction, which is currently inhibited by heat shock, were bred by conventional temperature (28.5 ℃) until the sexually mature triploid zebrafish was substantially male. Therefore, how to induce and breed the female triploid zebra fish by using a heat shock method is a problem to be solved urgently in the technical field.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings in the background technology and provide a method for breeding female triploid zebra fish by using a heat shock method.
In order to solve the technical problems, the technical scheme provided by the invention is to provide a method for breeding female triploid zebra fish by using a heat shock method, which comprises the following steps: taking wild AB strain zebra fish as parent fish, carrying out artificial spawning induction and artificial insemination on the parent fish, placing the obtained fertilized eggs in a culture dish, carrying out still water culture for 2-3min under the condition of 28-29 ℃ (the optimal temperature for the development of zebra fish embryos), then transferring the fertilized eggs into water with the temperature of 40.5-41.5 ℃ (the temperature with the best heat shock effect) for heat shock treatment for 2min, then placing the fertilized eggs into water with the temperature of 28-29 ℃ (the optimal temperature for the development of the zebra fish embryos) for still water incubation, transferring the fish fries into water with the temperature of 22-26 ℃ for constant temperature culture (the low temperature culture is utilized to influence the sexual differentiation of triploid zebra fish) after the fish fries are incubated for 3-5 days, then starting to feed paramecium vernalidius, changing to feed fairy worms after the fish fries are incubated for 15-20 days, and carrying out biological detection and screening after the fish fries are incubated for 2 months, obtaining female triploid zebra fish individuals.
The adopted wild AB strain zebra fish comes from the zebra fish resource center of the institute of aquatic organisms of Chinese academy of sciences. Zebra fish is a tropical fish, and its optimum culture temperature is 28.5 deg.C, and heat shock temperature is 40.5-41.5 deg.C. The heat shock treatment is to destroy the second division of embryo by high temperature to inhibit the discharge of second diode, so as to achieve the effect of chromosome doubling, and the heat shock treatment is carried out at 40.5-41.5 ℃ for 2min, and the tripling efficiency can reach 90-95%.
The triploid zebra fish induced by heat shock cultured by the conventional method cannot obtain females, mainly because the zebra fish is a model organism influenced by the sex by the environment, the sex differentiation can be influenced by the culture temperature after hatching, and the triploid zebra fish cultured to be sexually mature by the conventional temperature (28.5 ℃) is basically male. The female triploid zebra fish is obtained by culturing the triploid zebra fish obtained by the method through the constant temperature of 22-26 ℃ for two months under the condition of inhibiting the second polar body from discharging and inducing the heat shock, and influencing the sex differentiation of the triploid zebra fish by utilizing the temperature, wherein the proportion of the female triploid zebra fish is more than 10%. The temperature of 22-26 ℃ is selected because zebra fish cannot survive due to too low temperature, the sexual differentiation of the triploid zebra fish cannot be obviously influenced if the temperature is too close to the optimum temperature, and the temperature of 22-26 ℃ is the optimum temperature for influencing the sexual differentiation of the triploid zebra fish into female. Through identification, the gonad of the female triploid zebra fish obtained by the method is good in development, the sexual maturity can be reached after 3 months of age, and normal eggs can be produced.
Preferably, the selection standard of the parent fish is the wild AB strain zebra fish which is selectively mature, disease-free, injury-free, good in physical sign and strong in body.
Preferably, the specific operation of artificial induced spawning comprises the following steps: the specific operation of the artificial induced spawning comprises the following steps: putting the parent fish into an incubation box according to the quantity ratio of male to female being 2:1, carrying out dark treatment for 10-14h, and then carrying out illumination for 20-30min to finish artificial spawning induction. The zebra fish individual is small, the injection amount of the medicine is difficult to control, and the wound of the zebra fish is relatively large due to injection, so that the wound of the zebra fish is easy to be inflamed, and the zebra fish can die due to carelessness; the induced spawning method adopted by the invention is the most suitable induced spawning method for the zebra fish, and the optical treatment can stimulate the zebra fish to produce sperms and spawn, so that the method is not only efficient, but also does not damage the zebra fish, and is efficient and safe.
Preferably, the specific operation of artificial insemination comprises the following steps: taking the male zebra fish obtained after artificial spawning induction, extruding semen, diluting with Hank's solution according to the volume ratio of 1:100 (the volume ratio of the semen to the Hank's solution), and placing on ice for later use; and (3) squeezing out the eggs of the female zebra fish which successfully spawns after artificial spawning induction, covering the eggs with Hank's solution containing semen, stirring and uniformly mixing, and adding water to complete artificial insemination. When artificial fertilization is carried out, a sufficient volume of semen diluent is needed to cover all ova, if the content of Hank's solution is too low, more original semen is needed, and more male fishes are needed; too high Hank's liquid content causes too small sperm concentration, which affects fertilization rate; and the volume ratio of the semen to the Hank's liquid is 1:100, so that the required individual number of the male fish is relatively proper, and the normal fertilization rate is not influenced.
Preferably, the specific operation of performing detection screening by a biological method comprises the following steps: after the fry is hatched for 2 months, shearing tail fins of the fry to disperse into cell suspension, and screening triploid zebra fish through detection of a flow cytometer; and then placing the screened triploid zebra fish into water at 28-29 ℃ to be continuously cultured until sexual maturity, screening female triploid zebra fish according to the appearance characteristics, and taking the gonad of the female triploid zebra fish for histological identification.
Preferably, the specific operation of detecting by the flow cytometer comprises the following steps: adding 400-500uL of DNA fluorescent dye 4', 6-diamidino-2-phenylindole into the cell suspension, dyeing for 8-10min in a dark environment, filtering by using a sieve with the aperture of 30 mu m, detecting by using a flow cytometer, and screening the triploid zebra fish according to the detection result.
Preferably, the screening standard of the female triploid zebra fish is to screen triploid zebra fish with hypertrophic body and obvious abdominal distension.
Preferably, the specific operation of performing histological identification comprises the following steps: and (3) fixing the gonad of the screened female triploid zebra fish by using Bohne's fluid, carrying out paraffin section, carrying out HE dyeing on the section, observing the gonad structure by using an optical microscope, taking a picture and identifying.
Compared with the prior art, the invention has the beneficial effects that:
the method successfully obtains the female triploid zebra fish by using the low-temperature (22-26 ℃) continuous induction method on the basis of obtaining the triploid zebra fish by the heat shock technology, breaks through the cognition that the triploid zebra fish obtained by the heat shock method is all male, has important scientific value, and provides a good material for the sex determination research of the zebra fish.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph of DNA content of diploid zebra fish;
FIG. 2 is a graph of the (female) DNA content of triploid zebra fish;
FIG. 3 is a chromosome map of a triploid zebrafish;
FIG. 4 is a photograph of a section of triploid zebrafish (female) ovarian paraffin;
FIG. 5 is a male egg drawing of a diploid zebrafish;
FIG. 6 is an egg (female) subgraph of a triploid zebra fish;
figure 7 is a (female) profile of triploid zebra fish;
FIG. 8 is a diagram of the diploid zebrafish (female) anatomy;
figure 9 is a (female) anatomical view of a triploid zebra fish.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example (b):
the invention relates to a method for breeding female triploid zebra fish by using a heat shock method, which comprises the following steps:
(1) selecting parent fishes: selectively mature, disease-free and injury-free, good-sign and healthy wild AB strain zebra fish is used as parent fish; the wild AB strain zebra fish comes from the national zebra fish resource center of aquatic organism institute of Chinese academy of sciences;
(2) spawning induction of parent fish: putting the selected parent fish into an incubation box according to the quantity ratio of male to female being 2:1, performing dark treatment for 12h, and then illuminating for 25min to finish artificial spawning induction;
(3) artificial insemination: taking male zebra fish obtained after artificial spawning induction, extruding semen, diluting the semen according to the volume ratio of 1:100 of the semen to Hank's liquid, and placing the diluted semen on ice for later use; taking the female zebra fish which successfully spawns after artificial spawning induction, extruding out eggs, covering the eggs with Hank's solution containing semen, adding water, stirring and mixing uniformly to complete artificial insemination;
(4) induction of triploid zebra fish by heat shock: placing the obtained fertilized eggs in a culture dish, culturing for 2min in still water at 28.5 ℃, then transferring the fertilized eggs into water at 41 ℃ for heat shock treatment for 2min, taking out the obtained embryos, and then placing the embryos in the still water at 28.5 ℃ for hatching to obtain fish fries;
(5) continuous low-temperature induction: after all the fish fries are hatched out after the films are removed, the obtained fish fries are transferred into water with the temperature of 24 ℃ for constant-temperature culture;
(6) fry rearing: feeding paramecium for 4 times a day after 3 days after the fry is hatched; feeding fairy shrimp for 3 times every day after the fry is hatched for 15 days;
(7) flow cytometry screening: obtaining 2-month-old small fish after the fry is hatched for 2 months, taking diploid zebra fish as a control, shearing tail fins of the small fish, placing the small fish in an EP tube, adding 75ul of physiological saline into the EP tube, and shearing the tail fins by using scissors which are cleaned by alcohol with the volume concentration of 70% to prepare cell suspension; adding 450ul of DNA fluorescent dye 4', 6-diamidino-2-phenylindole (DAPI) into the cell suspension, staining for 9min in dark environment, filtering with a sieve with aperture of 30 μm, detecting with a flow cytometer (see figure 1-3), and screening triploid zebra fish according to the detection result;
(8) feeding at normal temperature: transferring the 2-month-old triploid zebra fish into water at 28.5 ℃ for continuous culture until sexual maturity;
(9) gonad paraffin section: and (3) screening the female triploid zebra fish with the external shape like the external shape according to the external shape characteristics, wherein the female triploid zebra fish is fat in body and obviously expanded in abdomen, otherwise, the triploid zebra fish with a slender body, large fins and yellow body is male, taking the gonad of the female triploid zebra fish as a paraffin section, fixing the paraffin section by using Bonn's fluid, carrying out HE (high-intensity emission) staining on the section, observing the histological structure of the gonad, and taking a picture (see fig. 4-6).
The sex glands of the obtained female triploid zebra fish are well developed after identification, sexual maturity can be reached after 3 months of age, and normal ova can be generated, namely 10-15% of female triploid zebra fish individuals are obtained (see figures 7-9).
The invention successfully prepares the female triploid zebra fish for many times through repeated experiments. The method successfully obtains the female triploid zebra fish by using a low-temperature (22-26 ℃) continuous induction method on the basis of obtaining the triploid zebra fish by a heat shock technology, breaks through the cognition that the triploid zebra fish obtained by the heat shock method is all male, has important scientific value, and provides a good material for the sex determination research of the zebra fish.
Claims (3)
1. A method for breeding female triploid zebra fish by using a heat shock method comprises the following steps: taking wild type AB strain zebra fish as parent fish, carrying out artificial spawning induction and artificial insemination on the parent fish, placing the obtained fertilized eggs in a culture dish, carrying out still water culture at the temperature of 28-29 ℃ for 2-3min, then transferring the fertilized eggs into water at the temperature of 40.5-41.5 ℃ for heat shock treatment for 2min, then placing the fertilized eggs in water at the temperature of 28-29 ℃ for still water incubation, after all fish fries are incubated, transferring the fish fries into water at the temperature of 24 ℃ for constant temperature culture for 2 months, starting to feed paramecium as the fish fries after the fish fries are incubated for 3-5 days, feeding fairy shrimp after the fish fries are incubated for 15-20 days, shearing tail fins of the fish fries into cell suspension after the fish fries are incubated for 2 months, and screening the triploid zebra fish by a flow cytometer; then placing the screened triploid zebra fish in water at 28-29 ℃ for continuous culture until sexual maturity, screening out female triploid zebra fish according to appearance characteristics, and taking gonads of the female triploid zebra fish for histological identification to obtain a female triploid zebra fish individual;
the selection standard of the parent fish is the wild AB strain zebra fish which is selectively mature, disease-free, injury-free, good in physical sign and healthy;
the specific operation of the artificial induced spawning comprises the following steps: putting the parent fish into an incubation box according to the quantity ratio of male to female being 2:1, performing dark treatment for 10-14h, and then performing illumination for 20-30min to finish artificial spawning induction;
the specific operation of the artificial insemination comprises the following steps: taking the male zebra fish obtained after artificial spawning induction, diluting extruded semen with Hank's solution according to the volume ratio of 1:100, and placing on ice for later use; taking the female zebra fish which successfully spawns after artificial spawning induction, extruding out eggs, covering the eggs with Hank's solution containing semen, stirring and mixing uniformly, and adding water to complete artificial insemination;
the screening standard of the female triploid zebra fish is to screen triploid zebra fish with a large body type and obvious abdominal distension.
2. The method according to claim 1, characterized in that said specific operation of detection by flow cytometry comprises the following steps: adding 400-500uL of DNA fluorescent dye 4', 6-diamidino-2-phenylindole into the cell suspension, dyeing for 8-10min in a dark environment, filtering by using a sieve with the pore size of 30 mu m, detecting by using a flow cytometer, and screening triploid zebra fish according to the detection result.
3. The method according to claim 1, characterized in that said specific operation of performing histological identification comprises the following steps: and (3) fixing the gonad of the screened female triploid zebra fish by using Bohne's fluid, carrying out paraffin section, carrying out HE dyeing on the section, observing the gonad structure by using an optical microscope, taking a picture and identifying.
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| CN102696515A (en) * | 2012-06-01 | 2012-10-03 | 北京市水产科学研究所 | Preparation method of game fish triploid fries |
| EP2688396B1 (en) * | 2011-03-23 | 2016-01-20 | Scea Les Poissons Du Soleil | Method for obtaining a triploid interspecific hybrid of meagre and red drum |
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| EP2688396B1 (en) * | 2011-03-23 | 2016-01-20 | Scea Les Poissons Du Soleil | Method for obtaining a triploid interspecific hybrid of meagre and red drum |
| CN102696515A (en) * | 2012-06-01 | 2012-10-03 | 北京市水产科学研究所 | Preparation method of game fish triploid fries |
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| 热休克诱导斑马鱼异源三倍体的研究;吴玉萍等;《海洋与湖沼》;20001031;第31卷(第5期);第465-470页 * |
| 虹鳟鱼三倍体生产规模制种技术研究;沈希顺等;《北京水产》;20050228(第1期);第37-41页 * |
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