CN114793957B - Method for artificially inducing gynogenesis Hemibarbus maculatus on a large scale and application - Google Patents
Method for artificially inducing gynogenesis Hemibarbus maculatus on a large scale and application Download PDFInfo
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Classifications
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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
-
- 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
- A01K61/17—Hatching, e.g. incubators
-
- 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 artificially inducing gynogenesis flowers on a large scaleComprises the following steps: first, selecting sexually mature male koi and female flowersArtificially induced spawning, and then ultraviolet inactivating sperm and flower of koiMixing ovum, stirring with feather to activate ovum, placing activated ovum in yellow mud at 0-1deg.C for cold shock treatment for 20-22min, sequentially placing in water at 4-6deg.C, 8-10deg.C and 15-18deg.C for 1-2min, incubating in incubation tank at 22-25deg.C, and identifying and screening to obtain diploid gynogenesis flowerThe cultivation method of the invention can reduce the damage of the temperature rise to the fertilized eggs, improve the hatching rate and the offspring survival rate of the fertilized eggs, and effectively overcome the defect that the conventional method is difficult to obtain the viable gynogenesis flowersIs successful in obtaining gynogenesis flowersGroup, fill up flowersBlank in gynogenesis technology.
Description
Technical Field
The invention belongs to the technical field of fish cultivation, in particular to a flower for artificially inducing gynogenesis in a large scaleIs a method of (2) and gynogenesis flower->Is used in the application of (a).
Background
Gynogenesis is an important genetic breeding method, is an effective way for quickly establishing a pure line, and is equivalent to continuous breeding of 8-10 generations of traditional inbreeding. Through gene purification, individuals carrying harmful genes are naturally eliminated in the gynogenesis process, so that the gynogenesis offspring often show excellent characters such as hypoxia tolerance, strong stress resistance, high growth speed and the like.
Flower patternCommonly known as Ma carp, ma-Chao fish, dachong eye, etc. Flower->Tender meat, high meat yield, less thorn, and protein and polyunsaturated fatThe acid content is higher than that of other famous and excellent cultured fishes in the carp family, so that the fish has a better health care function and is deeply favored by consumers. Flower->The fish is praised as a domestic four-large delicious freshwater fish together with black carp and weever (fresh water) and mandarin fish. Flower->The body of the utility model is long, the front section is round bar-shaped, the rear section is slightly flat and the abdomen is round; the eyes protrude. The head length is smaller than the height and 4.5 times of the eye diameter, and 1 row of mucinous glands are arranged along the edges of the anterior orbital bone, the inferior orbital bone and the anterior branchia cover bone of the head; longer kissing length, smaller than or equal to the big posterior head of the eye, lower mouth, horseshoe shape, undeveloped lower lip and narrow two sides of leaf; the center of the jaw part is provided with a small triangle protuberance, and 1 pair of mouth hair is arranged; the body side and the back are blue brown, the back side is provided with a row of 10-12 black spots above the lateral line scales, the back and the tail fin are also provided with a plurality of irregular small black spots, and the starting point of the tail fin is obviously raised. The abdomen is white, and the pectoral fin, ventral fin, and gluteal fin are off-white.
Due to flowersThe individual is small, the growth speed is relatively slow, and the flower is severely restricted>Is an industrial development of (a). Flower of China>The breeding of the variety has the outstanding problems of chaotic sources of the seedlings, autotrophy and germplasm degradation, single culture mode, reduced culture benefit and the like, and a lot of new varieties need to be created. Fish are the species with the largest species number in vertebrates, and the living habits and propagation characteristics of different fishes are greatly different. Therefore, gynogenesis difficulties vary from fish to fish. In some fish, such as crucian, a large number of gynogenesis offspring can be easily obtained by using a traditional gynogenesis method. Flowers (L.) and (L.) of>As special fishes in China, the fish is sensitive to oxygen content, has relatively poor hypoxia tolerance in the cultivation process, frequently causes a large number of deaths caused by insufficient oxygen supply in the transportation process, has large gynogenesis difficulty and has no gynogenesis flowers yet>Successful report that the excellent variety with strong hypoxia tolerance is still lacking in the whole country at present, so that innovative breeding technology is developed for flowers ∈>The creation of new varieties has very important significance.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects and the shortcomings in the prior art and providing a method for artificially inducing gynogenesis flowers on a large scaleIs a method of (2) and gynogenesis flower->Is used in the application of (a). The method effectively overcomes the difficulty of obtaining viable gynogenesis flower by conventional method>Is successful in obtaining gynogenesis of the flower>Group, fill up flowers->Blank in gynogenesis technology. Gynogenesis also has a "heterospermic effect". Inducing floral +.f. in sperm of heterologous koi utilizing genetic inactivation>In fish, DNA hybridization may occur during induction. Through the micro-satellite map experiments,we demonstrate that gynogenesis flowers +.>The genome contains the segment of koi. DNA fragment of sperm of koi and flower->DNA fragment hybridization of genome can affect biological properties such as growth, stress resistance, body color and the like to a certain extent.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
artificial large-scale induction gynogenesis flowerComprises the following steps: first selecting sexually mature male koi and female flower +.>Artificially inducing parturition, and then adding ultraviolet inactivated sperm and flower of koi>Mixing ovum, stirring with feather to activate ovum, placing activated ovum in yellow mud at 0-1deg.C for cold shock treatment for 20-22min, sequentially placing in water at 4-6deg.C, 8-10deg.C and 15-18deg.C for 1-2min, incubating in incubation tank at 22-25deg.C, and identifying and screening to obtain diploid gynogenesis flower and Japanese ferox>。
During the hatching process of fish fertilized eggs, the rapid temperature change can lead to massive death of fish fertilized eggs. In the previous multiple experiments, the activated ovum after cold shock treatment is directly transferred into water with the water temperature of 22-24 ℃ and is not subjected to gradient heating treatment, and a large amount of gynogenesis flowers are not obtainedOffspring, and the activated ovum is subjected to gradient heating after cold shock treatment,can obviously improve the hatching rate and the survival rate of fertilized eggs. At the same time, the invention discovers flower->The ovum has certain viscosity, and cold shock in yellow mud can remove the viscosity between ovum and ovum, thereby enlarging the use of gynogenesis flower ∈>The number of ova is increased by subsequent running water hatching.
In the above cultivation method, preferably, the artificial induced spawning specifically includes the following steps: the female flowers are subjected to one-time injection methodInjecting with the dosage of 400-500IU/Kg of chorionic gonadotrophin per tail, 6-8ug/Kg of lutein-releasing hormone analogue and 1mg/Kg of diurone; simultaneously injecting 3-4 male flowers->Fish, the dosage is halved for female; the injection dose of male koi is 800-1000IU/Kg of chorionic gonadotrophin per tail, and 6-8ug/Kg of lutein-releasing hormone analogue. Flower->Preferably, the individual with good pathological development and obvious sexual maturity characteristics is selected from the individual with good pathological development and obvious sexual maturity characteristics with more than 2 years old, and the individual with more than two years old is selected from the fancy carp; flower->And the koi is put into a parent fish pond for artificial intensive culture for 3-4 months before artificial parturition.
Preferably, after the injection of the artificial induced spawning is finished, the male flowers and the female flowers are mixedPlacing in the same spawning pond, every 10 female flowers2 male flowers are put in>The method comprises the steps of carrying out a first treatment on the surface of the Putting the male koi into another spawning pond, injecting for 6-10h by artificial induced spawning, fishing the male koi, extruding sperm, diluting the sperm by Hank's liquid, inactivating by ultraviolet, and performing artificial dry insemination.
Preferably, flowersInjecting fresh running water into parent fish pond every day 20-30 days before induced spawning>Ripening is carried out.
Preferably, the water temperature of the pond is maintained at 22-25 ℃ when artificial induced spawning is carried out.
Preferably, the ultraviolet inactivated koi sperm is obtained by the following method: extruding sperm from cloaca by lightly pressing male koi belly, taking the sperm by a dry and clean container, quickly diluting the sperm by 4 ℃ precooled Hank's liquid according to the volume ratio of 1:2-3, then spreading the sperm in a thin layer form in a 0-4 ℃ precooled culture dish, placing the culture dish on a shaking table with an ice plate, and irradiating the culture dish by an ultraviolet lamp until the ultraviolet inactivated koi sperm with the inactivation degree of 70-80% is obtained.
More preferably, the specific operation of the ultraviolet lamp irradiation comprises the following steps: and (3) carrying out irradiation treatment at a position 10-12cm away from sperms by using 2 ultraviolet lamps with the energy of 15W capable of emitting obvious ozone smell, after the irradiation for 15min, dipping semen at intervals of 1-2min, coating the semen on a glass slide, activating the semen by using water, and then judging the activity by microscopic examination, and stopping the irradiation when the sperm motility of 70% -80% of the sperms is obviously weakened, thus obtaining the ultraviolet inactivated koi sperms. During gynogenesis, the degree of sperm inactivation is closely related to the gynogenesis power. Incomplete inactivation will produce filial generation, and excessive inactivation will not activate the ovum to start development. Because sperm from different individual sources may vary in viability, the inactivation time is different. The invention adopts about 70% of sperm motility obviously weakened as a standard, and can improve the success rate of gynogenesis.
Preferably, the activation time of the ovum is 1-2min.
Preferably, the fish fries hatched in the hatching tank are transferred into a pond for cultivation, and then are identified and screened by adopting a flow cytometry method, a hemocyte DNA content detection method and a chromosome detection method, so that the DNA content and the chromosome number of the fish fries can be accurately obtained on the premise of not killing the sample.
Based on a general inventive concept, the invention also provides a gynogenesis flower obtained by the methodUses of said gynogenesis flower +.>Is similar to common male flower->Hybridization to obtain improved flower->And (5) offspring. The improved flower->Has the advantages of fast growth, strong disease resistance, good stress resistance and the like, is suitable for single-culture and intercropping technology, and can generate obvious economic and social benefits when being used for cultivating fishes such as grass carp, white armor and the like.
Flower patternIs poor in hypoxia tolerance, complicated in induced spawning process and is->In the breeding process of parents, oxygen content and food are ensured to be sufficient to ensure that gonad development is good. And the current related flowers->Less research on breeding and its artificial workGynogenesis studies have not been reported. We found in the study that the flower +.>Fertilized eggs are sensitive to temperature changes during hatching. Development of gynogenesis flowers Using the traditional methods of gynogenesis>Namely, after cold shock treatment, fertilized eggs are directly transferred into a hatching pond for hatching, the survival rate of offspring is 0 in multiple experiments, and gynogenesis flowers are difficult to obtain>And (5) offspring. Using the gradient heating method provided in the present invention, the flower +.>After cold shock treatment, the fertilized eggs gradually rise to the incubation temperature of 22-24 ℃ from the environment of 0 ℃ so as to reduce the damage of the temperature rise to the fertilized eggs, thereby breaking through the flowers->Artificial gynogenesis technology bottleneck, successfully obtain gynogenesis flower ++>And (5) offspring. Meanwhile, in the subsequent repeated experiments, the effectiveness of the method is verified.
Compared with the prior art, the invention has the beneficial effects that:
1. the cultivation method of the invention adopts the sperm of the male koi as the stimulus source to lead the flowers to beAfter the fertilized eggs are subjected to cold shock treatment, the temperature of the fertilized eggs is gradually increased to the hatching temperature of 22-24 ℃ from the environment of 0 ℃, and the damage of the temperature rise to the fertilized eggs can be reduced by combining the gradient temperature increasing method, so that the hatching rate and the offspring survival rate of the fertilized eggs are improved, and the problem that the conventional method is difficult to obtain the survivable fertilized eggs is effectively overcomeLiving gynogenesis flower->Is successful in obtaining gynogenesis of the flower>Group, fill up flowers->Blank in gynogenesis technology.
2. The cultivation method adopts flowers in the aspect of selecting the heterologous spermsHybridization does not form viable offspring of koi sperm, which is genetically inactivated to stimulate the flower +.>The ovum development avoids the occurrence of hybridization offspring possibly existing in the gynogenesis offspring due to the incomplete inactivation of the heterologous sperms, and greatly simplifies the detection difficulty of the gynogenesis offspring.
3. The cultivation method of the invention is that the female parent flowersIn the artificial induced spawning process, the same species of male flowers are subjected to the same time>Artificial parturition is induced, and the female is stimulated by using the artificial parturition to promote female flowers +.>Improves ovum quality, and provides a subsequent gynogenesis of flower +.>Provides an important guarantee for successful acquisition of the (E) product.
4. The method for cultivating the gynogenesis flower of the inventionHas the characteristics of excellent property, high survival rate, stable heredity and the like, and is the subsequent flower +.>The breeding of good varieties provides important germplasm resources; gynogenesis flower with excellent utilization character>The secondary gynogenesis is hopeful to obtain gynogenesis flower with various excellent characters>The strain is developed, and the disease-resistant fine variety is developed, which has important significance in aquaculture; and 20-30 ten thousand ova can be simultaneously induced to develop the gynogenesis on a large scale, and the production efficiency is high.
5. The application of the invention utilizes gynogenesis flowersIs similar to common male flower->Mating, improved flowers can be obtained on a large scaleThe method comprises the steps of carrying out a first treatment on the surface of the The improved flower->Has the advantages of fast growth, strong disease resistance, good stress resistance and the like, is suitable for single-culture and intercropping technologies, can co-culture grass carp, white beetle and the like, and can generate obvious economic benefit and social benefit.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows gynogenesis flowers according to the inventionA technical roadmap;
FIG. 2 shows a common flower according to the present inventionA DNA content map;
FIG. 3 shows gynogenesis flowers according to the inventionA DNA content map;
FIG. 4 shows gynogenesis flowers according to the inventionCell mitotic metaphase picture (2n=50);
FIG. 5 shows a common flower according to the present inventionMetaphase pictures of cell chromosomes;
FIG. 6 shows gynogenesis flowersMicrosatellite map.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms 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 be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Example 1:
artificial large-scale induction gynogenesis flowerIs a method of (2) gynogenesis flower->The technical route is shown in fig. 1, and comprises the following steps:
1. artificial induced spawning: flowers with age of more than 2 years, no pathological condition, good development and obvious sexual maturity characteristics are selected respectively in 1-2 months each yearPlacing male koi with obvious sexual maturity characteristics, which is more than two years old, has no pathological condition, has good development, and is placed into parent fish pond for artificial intensive culture, and is about flowers->The proportion of natural baits fed by the intensive culture pond every day is not lower than 70% of the total amount of the baits; in middle and late 4 months, when the pond water temperature is stabilized between 22-24 ℃, the flower is added>Carrying out artificial induced spawning on the fancy carp; 10 to 15 days before the induced spawning, every flower of->Carrying out running water ripening; in oxytocic, female flowers->The artificial oxytocic is carried out by adopting an injection method, and the injection dosage is 400-500IU/Kg of chorionic gonadotrophin, 6-8ug/Kg of lutein-releasing hormone analogue and 1mg/Kg of diolone. Simultaneously injecting 3-4 male flowers->The fish dose is halved for female doses. The injection dose of male koi is 800-1000IU/Kg of chorionic gonadotrophin per tail, and 6-8ug/Kg of lutein-releasing hormone analogue. After injection, the female flowers and the male flowers are treated by->Placing into the same spawning pond stimulated by running water for producing, wherein every 10 female flowers are +.>2 male flowers are put in>Is beneficial to stimulating female flowers->Estrus; the male koi is put in another spawning pond stimulated by running water. After injection time 16 hours, the female flowers are salvaged>And male koi.
2. Sperm inactivation: in the prediction of flowers1-2 hours before spawning, starting to take koi sperms for ultraviolet inactivation treatment; selecting male koi with good induced spawning effect, lightly pressing abdomen to extrude sperms from cloaca, rapidly diluting with Hank's liquid precooled at 4 ℃ according to the volume ratio of 1:2-3 after receiving by a dry and clean 50mL cone bottom centrifugal tube, then spreading the sperms in a thin layer form in a culture dish precooled at 4 ℃, then placing the culture dish on a shaking table filled with ice plates, and carrying out irradiation treatment at a position with a distance of 10cm by using an ultraviolet lamp; after 15min of irradiation, a small amount of semen is dipped and coated on a glass slide every 1min, activated by water, examined under a microscope to judge activity, and the irradiation is stopped when about 70% of sperm motility is obviously weakened, thus obtaining ultraviolet inactivated koi sperm, collecting the koi sperm, then placing the koi sperm in an environment of 4 ℃ for light-shielding preservation, and waiting for flower->Spawning.
3. Fertilization: when female and male flowersBeginning to chase and exposing the tail part to the water surfaceDuring beating, check flowers by pulling net>Under the egg laying condition, female parent fish with smooth egg laying and high ovum quality is selected, the ovum is extruded into a dry and clean porcelain basin, a proper amount of genetically inactivated koi sperm is immediately added, the clean feather is rapidly and uniformly stirred, a proper amount of water is added, and the stirring is continued for 1-2min to activate the ovum to start development.
4. Cold shock treatment: after the ovum is activated, placing the ovum in pre-adjusted cold yellow mud at 0 ℃ for cold shock treatment for 20-22min, then sequentially placing the ovum in water at 4-6 ℃, 8-10 ℃ and 15-18 ℃ for treatment for 1-2min, and then placing the ovum in a hatching tank for running water hatching at the water temperature of 22-24 ℃; meanwhile, a comparative test is set, fertilized eggs are directly placed in running water at the water temperature of 22-24 ℃ for hatching after cold shock treatment, and test results are shown in table 1.
Table 1: different treatment methods are used for gynogenesis and development of flowersEffects of (3)
The invention also explores the influence of different shock temperatures and shock times on the fertilization rate, the hatching rate and the survival rate, and the results are shown in Table 2. From the following data, it can be seen that the fertilization rate, hatchability and survival rate are highest when the shock temperature is 4℃and the shock time is 22 minutes.
Table 2: influence of different shock temperatures and shock times on fertility, hatching rate and survival rate
Table 3: induction of different spermFlower patternGynogenesis fertility rate, hatchability and survival rate comparative analysis
| Stimulus source | Fertilization rate | Hatchability rate | Survival rate |
| Red crucian sperm | 39.4％ | 30.6％ | 6.1％ |
| Sperm of carp | 32.7％ | 30.2％ | 6.7％ |
| Sperm of fancy carp | 39.8％ | 30.5％ | 8.5％ |
As shown in Table 3, through different sperm stimulation experiments, the survival rate of the sperm stimulation of the koi is highest under the same condition.
5. Feeding: gynogenesis flowersAfter the fish fry hatch, after the fish fry starts to swim at the waist point, transferring the fish fry into a hatching tank for pre-fertilization (flower +.>Is benthic fish, which can be bred by adopting microorganism flora such as green algae, etc., soybean milk is sprayed every day for 2-3 times per day, and waterweed is sprayed along the pool, and meanwhile, the waterweed is added into the pool until the fries grow into cun-form seeds, and then the cun-form seeds are transferred into a soil pool for breeding.
6. And (3) detection: when gynogenesis is carried out on flowersWhen it is up to 4-5 months of age, its appearance is detected (gynogenesis flower +.>The appearance of the obtained product is shown in figure 1), and ploidy detection is carried out by adopting a DNA content detection method and a tail fin cell chromosome number detection method to obtain the gynogenesis flower +.>。
Randomly selecting 15 gynogenesis flowers with ages of 4-5 monthsTheir external morphological characteristics were examined, including dorsal fin, gluteal fin, lateral line scale up and lateral line scale down, and compared with flowers already known in the literature->The profile data were compared and the results are shown in table 4 below.
Table 4: gynogenesis flowersIs combined with common flower->Comparison of countable traits
The results showed that gynogenesis flowersOffspring were scaled on the lateral line, under the lateral line and in the hip fin, and these were less variable in the countable trait, indicating that they were genetically stable and superior.
In addition, experiments prove that the method can improve the hatching rate and the offspring survival rate of fertilized eggs.
At the same time, by common flowersFor reference, the flow cytometer was used to provide a flower for gynogenesis>The DNA content of the offspring erythrocytes was determined. The flow cytometry DNA content assay generally comprises the steps of: blood was collected from the venous blood vessels of the fish tail by a disposable syringe wetted with heparin sodium at about 0.lmL, 10ul was poured into an Eppendorf tube containing 0.49mL of 0.8% physiological saline, 0.5ml of DNA staining solution (DAPI, supplied by Partec GmbH, germany) was added to the mixture of blood and physiological saline, and the stained sample was left to stand in the dark for about 5 to 10min: the samples were filtered through a 20 μm nylon filter (supplied by Partec GmbH, germany) and run on a machine. The measurement results are shown in fig. 2 and 3, respectively. Analysis of the data in the data plot shows that the common flowers +.>DNA average fluorescence intensity of 65.80, control gynogenesis flower->The average fluorescence intensity of DNA is 64.78, and the ratio of the average fluorescence intensity of the two DNA is 1.02:1, the DNA content of the two is not obviously different, so that the gynogenesis flower can be primarily judged>Is combined with common flower->The fish are diploid.
Finally, detecting gynogenesis flowers by a fish tail fin cell methodThe specific operation is as follows:
(1) taking materials, preparing fish for taking materials outside a cell room, taking tissues needing to be made into a centrifuge tube, spraying alcohol and bringing the tissues into the cell room (the cell room needs to be signed for a period of time in advance, ultraviolet is started before the materials are taken, the objects needing to be used are placed into an ultraviolet table for at least 15 minutes, if scissors tweezers need to be used, the objects needing to be used are packaged by tinfoil and placed into a 180 sterilization box for sterilization, and then brought into the cell room), rinsing the tissues for 6-7 times by using PBS with double antibodies, replacing an EP tube for rinsing for 6-7 times, and finally leaving 300-500ul PBS for clipping (the clipping amount of PBS can be determined according to the amount of materials);
(2) centrifuging the chopped tissue in a centrifuge for 1000 min (this is the time for tail fin, the muscle should rotate at a higher speed, and the number of times of centrifugation may be more), discarding supernatant, adding new PBS300-500ul, and centrifuging for 2 times;
(3) after centrifugation, the supernatant was discarded, and 300ul of fetal bovine serum FBS was added to the centrifuge tube and allowed to stand;
(4) taking a small dish, spreading a layer of gelatin which can be added with 700ul, spreading the bottom of the dish fully, sucking the gelatin, and spreading tissues and fetal bovine serum on the small dish uniformly for repeated use, and sucking FBS;
(5) the mixture was placed in an incubator for 1 hour, and then the medium was added.
Chromosome suspension preparation:
1. taking the cultured cells into an EP tube, taking out the cell house, transferring the cells into a 15ml centrifuge tube, centrifuging for 5-10min, discarding the supernatant, adding hypotonic solution, and properly adding the amount of cell sediment.
2. Hypotonic for 1h-2h (different hypotonic time of different fishes), adding a few drops of fixing solution, centrifuging for 5-10min, discarding supernatant, adding fixed solution (methanol: glacial acetic acid=3:1) according to cell precipitation amount, placing into refrigerator for 1h at 4deg.C, centrifuging, discarding supernatant, and adding fixing solution for fixing for about 30min to obtain dripping tablet.
As shown in FIG. 4 and FIG. 5, gynogenesis flowers are shownAnd common flower->There were 50 chromosomes of the same number.
As shown in FIG. 6, gynogenesis flowersMicrosatellite map (1-4 is koi, 5 represents pBR322DNA/Mspl Marker,6-11 is gynogenesis flower->12-15 is flower->Red arrow indicates gynogenesis flower +.>The genetic material is from fancy carp, and blue arrow indicates that the genetic material is from female parent flower +.>) The heterospermic effect means that in the gynogenesis breeding process, the heterosperms not only can stimulate the development of ova, but also can influence certain properties of offspring, such as the growth, reversibility and the like of the offspring. From this figure, it can be seen that: gynogenesis flower->Has obvious heterospermia effect.
In conclusion, both the DNA content detection and the tail fin cell culture chromosome ploidy detection methods show that the offspring obtained by the cultivation of the invention is diploid gynogenesis flowers。
Example 2:
the present invention also provides a gynogenesis flower obtained in example 1Comprises the following steps:
gynogenesis flowers obtained in example 1Is similar to common male flower->Hybridization to obtain improved flower->And (5) offspring.
The improved flowerHas the advantages of fast growth, strong disease resistance, good stress resistance and the like, is suitable for single-culture and intercropping technologies, can co-culture with farmed fishes such as grass carp, white beetles and the like, and can generate obvious economic and social benefits.
Claims (7)
1. A method for artificially inducing gynogenesis Hemibarbus maculatus on a large scale, which is characterized by comprising the following steps: firstly, selecting sexually mature male koi and female Hemibarbus maculatus for artificial induced spawning, then mixing koi sperms and Hemibarbus maculatus eggs subjected to ultraviolet inactivation, stirring with feathers to activate the eggs, placing the activated eggs in yellow mud at 4 ℃ for cold shock treatment for 22min, then sequentially placing the eggs in water at 6 ℃, 8-10 ℃ and 15-18 ℃ for treatment for 1-2min, then placing the eggs in a hatching tank with water temperature of 22-25 ℃ for hatching, and obtaining diploid gynogenesis Hemibarbus maculatus after identification and screening;
the artificial induced spawning specifically comprises the following steps: injecting female Hemibarbus maculatus by one-time injection method with dosage of 400-500IU/kg chorionic gonadotrophin, and luteinizing hormone releasing hormone A 2 6-8ug/kg and 1mg/kg Di OuDose injection of ketone; simultaneously injecting 3-4 male Hemibarbus maculatus, and halving the dosage of female; male koi is injected according to dosage of chorionic gonadotrophin of 800-1000IU/kg and lutein releasing hormone analogue of 6-8 ug/kg;
the ultraviolet inactivated koi sperm is obtained by the following method: extruding sperms from cloaca by lightly pressing male koi abdomen, taking the sperms by a dry and clean container, quickly diluting the sperms by 4 ℃ precooled Hank's liquid according to the volume ratio of 1:2-3, then spreading the sperms in a thin layer form in a 0-4 ℃ precooled culture dish, placing the culture dish on a shaking table filled with ice plates, and irradiating the culture dish by an ultraviolet lamp until the ultraviolet inactivated koi sperms with the inactivation degree of 70-80 percent are obtained;
the specific operation of the ultraviolet lamp irradiation comprises the following steps: and (3) carrying out irradiation treatment on the sperm 10-12cm away from the sperm by using 2 ultraviolet lamps with the width of 15W, after the irradiation is carried out for 15min, dipping semen every 1-2min, coating the semen on a glass slide, activating the semen by using water, and carrying out microscopic examination to judge the activity of the semen, and stopping the irradiation when the swimming capacity of 70% -80% of sperms is obviously weakened, thus obtaining the ultraviolet inactivated koi sperm.
2. The method for artificially inducing gynogenesis imperial generation of Hemibarbus maculatus on a large scale according to claim 1, wherein after the injection of the artificial induced spawning is completed, the female Hemibarbus maculatus are placed in the same spawning pond, and 2 male Hemibarbus maculatus are placed in every 10 female Hemibarbus maculatus; putting the male koi into another spawning pond, injecting for 6-10h by artificial induced spawning, fishing the male koi, extruding sperm, diluting the sperm by Hank's liquid, inactivating by ultraviolet, and performing artificial dry insemination.
3. The method of artificially mass inducing gynogenesis Hemibarbus maculatus according to claim 1, wherein Hemibarbus maculatus are matured by injecting fresh running water into the parent fish pond daily 20-30 days prior to the induction of parturition.
4. The method of artificially mass inducing gynogenesis Hemibarbus maculatus according to claim 1, wherein the pond water temperature is maintained at 22-25 ℃ during artificial oxytocic.
5. The method of artificially mass inducing gynogenesis Hemibarbus maculatus according to claim 1, wherein the activation time of the ovum is 1-2min.
6. The method of artificially mass inducing gynogenesis Hemibarbus maculatus according to claim 1, wherein the identifying and screening comprises the following: flow cytometry, blood cell DNA content detection, chromosome detection.
7. Use of a gynogenic Hemibarbus maculatus obtainable by a method according to any of the claims 1 to 6, characterized in that a modified Hemibarbus maculatus progeny is obtained by crossing said gynogenic Hemibarbus maculatus with a normal male Hemibarbus maculatus.
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