CN109874707B - Method for efficiently creating allooctaploid silver crucian carp - Google Patents
Method for efficiently creating allooctaploid silver crucian carp Download PDFInfo
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- CN109874707B CN109874707B CN201910274442.8A CN201910274442A CN109874707B CN 109874707 B CN109874707 B CN 109874707B CN 201910274442 A CN201910274442 A CN 201910274442A CN 109874707 B CN109874707 B CN 109874707B
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Abstract
The invention relates to a method for efficiently creating allooctaploid silver crucian carp. The method comprises an artificial insemination phase comprising: preparing a trypsin solution: adding the trypsin powder into the sperm preservation solution, uniformly mixing to obtain a trypsin solution, and adding 0.9-1.1g of the trypsin powder into every 100mL of the sperm preservation solution; semen treatment: adding mature semen of Carassius auratus into the above trypsin solution, and treating at 22-24 deg.C for 13-17 min; insemination and hatching: and performing dry insemination on the treated semen and mature eggs of the carassius auratus gibelio, and incubating the obtained fertilized eggs to obtain the allooctaploid carassius auratus gibelio. The invention processes the sperms by the trypsin solution dissolved by the sperm preserving fluid, leads the sperms to be decondensed, realizes the successful integration of the allogenic sperms into the carassius auratus gibelio ovum to form the allooctaploid, and further obtains the allooctaploid carassius auratus gibelio with high proportion by setting the proper concentration and processing time of the trypsin.
Description
Technical Field
The invention belongs to the field of fish genetic breeding, and particularly relates to a method for efficiently creating allooctaploid silver crucian carps.
Background
Hybridization and multiplexing can provide the original power and material basis for species evolution and the formation of superior traits. Theoretically, cell size tends to increase with increased genomic ploidy, and polyploids often appear to grow faster and larger in vegetative organs or individuals than diploid relatives. Polyploid breeding (polyploid breeding), a classical and highly effective cell engineering breeding technique, has been widely used in animal and plant breeding, including breeding of breeds for breeding fish and shellfish. In aquaculture animals, examples of success are triploid pacific oysters and homo-or allopolyploids synthesized from natural polyploid fish such as carps and salmonids. Therefore, optimizing or developing a new and efficient ploidy operation technology, creating excellent germplasm, providing a core breeding material for breeding new varieties, and still being one of the important directions of the genetic breeding of aquaculture animals.
Carassius auratus gibelio Bloch is one of the important large fresh water breeding species in China, and the annual total yield of the Carassius auratus gibelio and other crucian carps approaches 300 million tons in recent years. The carassius auratus can carry out natural gynogenesis reproduction, and in an allogenic carassius auratus population artificially reproduced by sperms provided by Xingguo red carps, a small number (about 0.2%) of artificially synthesized allooctaploid carassius auratus which not only maintains the whole set of chromosome set of the carassius auratus but also integrates a set of chromosome set of red carps is discovered, so that the capacity of the carassius auratus to integrate an exogenous genome is displayed. The unique ability can create excellent germplasm for breeding novel carassius auratus gibelio variety. For example, the 'Zhongke No. 5' carassius auratus gibelio is obtained by continuous ten-generation gynogenesis breeding from a breeding core group which is infiltrated with megalobrama amblycephala sperm tiny chromosome fragments and has obviously changed characters; the Changfeng crucian carp is formed by fusing the ovum of the D series of silver crucian carps into a chromosome group of a set of Xingguo red carp sperms. However, allogenic sperms do not disaggregate and are in a solid state after entering into the carpus crassipes egg matter, so the number rate of the allooctaploid filial generation formed spontaneously through the reproduction of the allogenic gynogenesis is only 0.2-0.33%, and the workload of screening and identifying the allooctaploid in breeding is very large.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for efficiently creating allooctaploid silver crucian carps.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for efficiently creating allooctaploid silver crucian carp, comprising an artificial insemination phase, wherein the artificial insemination phase comprises the following operation steps:
step 1, preparing a trypsin solution: adding trypsin powder into the sperm preservation solution, uniformly mixing to obtain a trypsin solution, and adding 0.9-1.1g of the trypsin powder into every 100mL of the sperm preservation solution;
step 2, semen treatment: adding mature semen of Carassius auratus into the above trypsin solution, and treating at 22-24 deg.C for 13-17 min;
step 3, insemination and hatching: and (3) carrying out dry insemination on the semen treated in the step (2) and the mature eggs of the carassius auratus gibelio, and hatching the obtained fertilized eggs to obtain the allooctaploid carassius auratus gibelio.
The further technical scheme is as follows: the formula of the sperm preserving fluid is as follows: NaCl 7.5-8.5g/L, KCl0.3-0.5g/L, CaCl20.12-0.16g/L、MgSO4·7H2O 0.1-0.3g/L、Na2HPO4·H2O0.04-0.08g/L、KH2PO40.04-0.08g/L、NaHCO30.33-0.37g/L and 0.9-1.1g/L of glucose; the pH value of the sperm preservation solution is 7.0-7.2.
The further technical scheme is as follows: the hatching mode is that the fertilized eggs are placed in a white porcelain plate for hatching and half water is changed every 12 hours, or the fertilized eggs are placed on a net piece and placed in a water tank for oxygen charging for hatching.
The further technical scheme is as follows: the method also comprises a carassius auratus gibelio parent breeding and reproducing stage, wherein the carassius auratus gibelio parent breeding and reproducing stage comprises the following steps:
step 1, preparing for discharging: in winter, before the parents are put into the pond, the parent culture pond is disinfected in a dry way to kill trash fish, then fresh water is poured into the parent culture pond again, and meanwhile, an isolation net sleeve is arranged at the water inlet of the parent culture pond to prevent the trash fish from mixing into the culture pond;
step 2, parent breeding: male and female fish are screened before the parents are put into the pond, and male fish are eliminated; after beginning spring, the water depth of the parent culture pond is increased to 1.5-2.0 m;
the feeding amount of the feed in the parent breeding process is calculated according to 2-4% of the total weight of the parent;
parent breeding3000-4500kg/hm of decomposed organic fertilizer is splashed into the parent culture pond every time in the process2Or the fermented EM solution is 1200 ml/mu/7-15 days, and the dissolved oxygen of the water body is reduced to 2-4 mg/L;
when the water temperature of the parent culture pond is 16-22 ℃, the parent is promoted to lay eggs in a net pulling mode, the parent is prevented from being damaged in the net pulling process, the parent captured by the net pulling process is identified through the maturity, the parent whose ovary is developed to the middle stage of the fourth stage and later is induced to spawn immediately, and the parent whose ovary is developed to the initial stage of the fourth stage is transferred to the indoor for the preparation of induced spawning propagation;
step 3, hastening parturition: the mixed solution of 1ug/kg of diutanone, 1mg/kg of pituitary gland, 500 international units/kg of human chorionic gonadotropin and 5ug/kg of ovulation-promoting hormone is adopted to inject the parents for induced spawning, and the parents after the injection induced spawning are placed in a flowing water cylinder with indoor constant temperature of 21 ℃ for waiting for spawning.
The further technical scheme is as follows: the method also comprises a ploidy detection mode of the hatched offspring, wherein the ploidy detection mode comprises the following steps:
step 1, blood sampling: cutting tail of fry with length of 1.5cm to obtain blood 0.1 μ L, and cutting fin of fry with age of six months to obtain blood 0.1 μ L;
step 2, detection: the collected fresh blood is put into 200 mu L of CyStain DNA1Step solution pre-cooled at 4 ℃ and mixed evenly, then the mixture is put on ice, and DNA content detection is carried out by utilizing a flow cytometer.
The further technical scheme is as follows: and injecting the white crucian carp male parent according to the dosage of one third of the mixed solution for hastening parturition, wherein the white crucian carp male parent is a robust individual with the age of more than 2 years, and the weight of the white crucian carp male parent is 500 g.
The further technical scheme is as follows: in the parent breeding process, aiming at the area with sudden temperature rise in 3 months, the parents are temporarily bred in 18-22 ℃ indoor circulating water in different jars at the air temperature of 16-20 ℃, and then the subsequent induced spawning is carried out.
The invention has the beneficial effects that:
(1) according to the invention, the sperm is treated by trypsin through the sperm preserving fluid, the sperm is decondensed, the allogenic sperm (white crucian) is successfully integrated into the carassius auratus gibelio to form allooctaploid, and the high-proportion allooctaploid carassius auratus gibelio is further obtained by setting the proper concentration and treatment time of the trypsin, the allooctaploid formation rate of the invention is up to 16.8%, and the survival rate of the allooctaploid carassius auratus gibelio is 2.4 +/-0.7%.
(2) Sterilization H for sperm preservation solution of the present invention2And O, the formula of the sperm preservation solution can be used for preserving sperm and enabling trypsin to exert activity after being prepared.
(3) Regarding the hatching mode:
the net sheets are stainless steel frames, dense-hole net sheets are arranged on the front surface and the back surface of the net sheets, and the net sheets are made of polyethylene generally. After the roe is adhered to the net sheet, the net sheet is put into a water tank and is oxygenated for incubation. The operation has the advantages of large hatching water body and no need of water change in the hatching process.
The white porcelain plate is a medical experiment plate, roe is laid in the white porcelain plate, water is added for incubation, and half water is replaced every 12 hours. The hatching method has the advantages that: the development form of the roe can be observed at any time, and the dead roe is removed at regular time so as to prevent spoiled water quality from influencing the incubation of normal roe.
(4) The carassius auratus gibelio parent breeding and reproducing stage is the basis for obtaining allooctaploid carassius auratus gibelio with high proportion, wherein the advantages of each step are as follows:
an isolation net sleeve is arranged at the water inlet of the culture pond, and is mainly used for preventing trash fish from mixing into the culture pond, so that the abortion caused by the stimulation of trash fish sperms on carassius auratus gibelio parents in a breeding season is avoided.
The male and female fish are rejected before the parents are put into the pond, so that the male fish is prevented from stimulating the female fish in spring to cause the abortion of the female fish. The function of improving the water depth of the parent culture pond to 1.5-2.0 m after spring beginning is to avoid parent abortion caused by over-quick water temperature rise.
Spraying decomposed organic fertilizer or EM (effective microorganisms) solution into the parent culture pond can ensure that the culture pond has abundant living baits in water. The control on reducing the dissolved oxygen in the water body ensures the need of parent gonad development and does not cause the parents to be aborted early.
The net pulling can promote the parent to lay eggs, but the mode of 'upper-layer net pulling and ignoring bottom line' is adopted during net pulling, so that the abortion caused by stimulation to the parent is avoided.
In the middle and downstream areas of the Yangtze river, sudden temperature increases of several consecutive days often occur in the middle and last 3 months, which may lead to abortion of parents. Therefore, in order to ensure the full maturity of the parents and avoid the abortion phenomenon, the parents are temporarily cultured in indoor constant-temperature circulating water at 18-22 ℃ in different jars at the temperature of 16-20 ℃. When the parents are cultivated, enough nutrition is provided by abundant live baits, and the low oxygen content is not enough to be over mature, so that the parents can still perform induced spawning propagation after temporarily cultivated indoors for one and a half months.
The method realizes the synchronization of parent spawning by performing induced spawning on the parent, thereby being beneficial to developing large-scale seedling breeding. The effect period of putting the injected parent into a room constant-temperature 21 ℃ running water cylinder is about 10 hours, so that the subsequent breeding operation is conveniently arranged. The oxytocic dose and the oxytocic temperature of the invention have the advantages that: the influence of water temperature on the effect period is avoided, the spawning time is easy to control, and the subsequent breeding operation is reasonably arranged; the oxytocic drug is compatible, so that the maturity of parent ova is uniform, and high-quality ova are provided for propagation.
(5) The ploidy detection method has the advantages of less blood taking amount, convenient operation of the method for cutting the fin ray to take blood and no killing of the detected octaploid fish. The mixed detection sample is placed on ice, can be stored for six hours, provides sufficient time support for sampling detection, and can improve the inspection success rate of the instrument by being placed on the ice, so that the detection efficiency is greatly improved.
Drawings
In fig. 1, part a is a morphological diagram of a female parent carassius auratus gibelio a + line, a male parent white carassius auratus gibelio and a new carassius auratus gibelio octaploid.
In fig. 1, the part B is a DNA content histogram of allogenic octaploid peripheral blood of female parent carassius auratus gibelio a + line, male parent carassius auratus gibelio and carassius auratus gibelio.
In the figure 1, part C is a mitosis metaphase map of the allooctaploid of female parent carassius auratus gibelio A + line, male parent carassius auratus gibelio and carassius auratus gibelio.
Detailed Description
The technical scheme of the invention is more specifically explained by combining the following embodiments:
the invention relates to a method for efficiently creating allooctaploid silver crucian carp, which comprises a carassius auratus gibelio parent breeding and reproduction stage, an artificial insemination stage and a ploidy detection mode of hatched offspring.
The method comprises the following steps of breeding and propagating carassius auratus gibelio parents:
example 1
Step 1, preparing for discharging: in winter, before the parents (variety: carassius auratus gibelio A + series) are put into the pond, the parent culture pond is disinfected in a dry pond to kill trash fish, then new water is poured into the parent culture pond again, and meanwhile, an isolation net sleeve is arranged at the water inlet of the parent culture pond to prevent the trash fish from mixing into the culture pond;
step 2, parent breeding: male and female fish are screened before the parents are put into the pond, and male fish are eliminated; after spring beginning, the water depth of the parent culture pond is increased to 1.8 meters;
the feeding amount of the feed in the parent breeding process is calculated according to 3 percent of the total weight of the parent;
the decomposed organic fertilizer is sprinkled into the parent cultivation pond every 4000kg/hm in the parent cultivation process2Reducing the dissolved oxygen of the water body to 3 mg/L;
when the water temperature of the parent culture pond is 20 ℃, the parent is promoted to lay eggs in a net pulling mode, the parent is prevented from being damaged in the net pulling process, the parents captured by the net pulling process are identified through the maturity, the parents after the ovary is developed to the middle stage of the fourth stage are induced to spawn immediately, and the parents after the ovary is developed to the initial stage of the fourth stage are transferred to the room for the preparation of induction breeding;
step 3, hastening parturition: the mixed solution of 1ug/kg of diutanone, 1mg/kg of pituitary gland, 500 international units/kg of human chorionic gonadotropin and 5ug/kg of ovulation-promoting hormone is adopted to inject the parents for induced spawning, and the parents after the injection induced spawning are placed in a flowing water cylinder with indoor constant temperature of 21 ℃ for waiting for spawning. And (3) injecting a white crucian carp male parent (selected from Nanning area) into the mixed solution at a dosage of one third for hastening parturition, wherein the white crucian carp male parent is a healthy individual with the age of more than 2 and the weight of the white crucian carp male parent is about 400 g.
Example 2
Step 1, preparing for discharging: in winter, before the parents (variety: carassius auratus gibelio A + series) are put into the pond, the parent culture pond is disinfected in a dry pond to kill trash fish, then new water is poured into the parent culture pond again, and meanwhile, an isolation net sleeve is arranged at the water inlet of the parent culture pond to prevent the trash fish from mixing into the culture pond;
step 2, parent breeding: male and female fish are screened before the parents are put into the pond, and male fish are eliminated; after spring beginning, the water depth of the parent culture pond is increased to 1.5 meters;
the feeding amount of the feed in the parent breeding process is calculated according to 2 percent of the total weight of the parent;
3000kg/hm of decomposed organic fertilizer is sprinkled into the parent cultivation pond every time in the parent cultivation process2Reducing the dissolved oxygen of the water body to 4 mg/L;
when the water temperature of the parent culture pond is 16 ℃, the parent is promoted to lay eggs in a net pulling mode, the parent is prevented from being damaged in the net pulling process, the parents captured by the net pulling process are identified through the maturity, the parents after the ovary is developed to the middle stage of the fourth stage are induced to spawn immediately, and the parents after the ovary is developed to the initial stage of the fourth stage are transferred to the room for the preparation of induction breeding;
step 3, hastening parturition: the mixed solution of 1ug/kg of diutanone, 1mg/kg of pituitary gland, 500 international units/kg of human chorionic gonadotropin and 5ug/kg of ovulation-promoting hormone is adopted to inject the parents for induced spawning, and the parents after the injection induced spawning are placed in a flowing water cylinder with indoor constant temperature of 21 ℃ for waiting for spawning. And injecting the white crucian carp male parent according to the dosage of one third of the mixed solution for hastening parturition, wherein the white crucian carp male parent is a healthy individual with the age of more than 2 years and the weight of the white crucian carp male parent is about 300 g.
Example 3
Step 1, preparing for discharging: in winter, before the parents (variety: carassius auratus gibelio A + series) are put into the pond, the parent culture pond is disinfected in a dry pond to kill trash fish, then new water is poured into the parent culture pond again, and meanwhile, an isolation net sleeve is arranged at the water inlet of the parent culture pond to prevent the trash fish from mixing into the culture pond;
step 2, parent breeding: male and female fish are screened before the parents are put into the pond, and male fish are eliminated; after spring beginning, the water depth of the parent culture pond is increased to 2.0 m;
the feeding amount of the feed in the parent breeding process is calculated according to 4 percent of the total weight of the parent;
spraying 1200 ml/mu/10 days of fermented EM (effective microorganisms) solution into a parent culture pond in the parent culture process, and reducing the dissolved oxygen of a water body to 2 mg/L;
in the embodiment, the temperature rises rapidly in the middle and lower reaches of Yangtze river in 3 months, and the parents are temporarily cultured in indoor circulating water at 18-22 ℃ in different jars at the temperature of about 18 ℃ and then subjected to subsequent spawning induction.
Step 3, hastening parturition: the mixed solution of 1ug/kg of diutanone, 1mg/kg of pituitary gland, 500 international units/kg of human chorionic gonadotropin and 5ug/kg of ovulation-promoting hormone is adopted to inject the parents for induced spawning, and the parents after the injection induced spawning are placed in a flowing water cylinder with indoor constant temperature of 21 ℃ for waiting for spawning. And injecting the white crucian carp male parent according to the dosage of one third of the mixed solution for hastening parturition, wherein the white crucian carp male parent is a healthy individual with the age of more than 2 years and the weight of the white crucian carp male parent is about 500 g.
The criteria for determining the maturity of the parents before induction are as follows:
the carassius auratus gibelio parent (female parent) is soft and swollen in abdomen, clear in ovary outline and reddish in genital pore, and the parent with reddish genital pore is induced by spontaneous abortion and should be removed. Sometimes, the maturity of carassius auratus gibelio parent needs to be identified more accurately, and an egg digging device can be used for egg digging examination. Slowly inserting the ovum fetching device into the genital hole, slightly rotating for several times, slightly extracting, taking out a small amount of ovum, adding a small amount of fixed transparent liquid, soaking for 2-3 min, and observing the position of the ovum core. If the nuclear position of all or most of the eggs is eccentric or polarized, the maturity of the parent is good, and the method is suitable for immediate induced spawning propagation; if the white nucleus is in the central position, the parent is poor in sexual maturity and needs to be further cultivated and matured; if most of the eggs have no white nuclei, the eggs are mostly degenerated and are not suitable for reproduction.
The judgment standard of the female parent maturity is as follows: slightly extruding the abdomen near the genital orifice of the female parent, and hastening parturition if the extruded oocytes in the fourth stage are separated and one granule per granule (indicating that the female parent ovary develops to the middle stage of the fourth stage and later); if the extruded mass is unseparated (indicating maternal ovary development to early stage four), then the feed is continued for an additional 7-15 days.
The male parent of the invention is selected from the white crucian carp of Nanning, is a pure line variety with the age of more than 2 years, the weight of 300-. The mature white crucian carp parent generally has obvious 'chasing stars' on pectoral fins, ventral fins and gill covers, the genital pore is slightly concave, and milky thick seminal fluid flows out when the abdomen is lightly pressed, which indicates that the male fish has better maturity.
The artificial insemination phase comprises the following operation steps:
example 4
Step 1, preparing a trypsin solution: adding trypsin powder into the sperm preservation solution, uniformly mixing to obtain a trypsin solution, and adding 1.0g of the trypsin powder into every 100mL of the sperm preservation solution;
the formula of the sperm preserving fluid is as follows: NaCl 8.0g/L, KCl 0.4.4 g/L, CaCl20.14g/L、MgSO4·7H2O0.2g/L、Na2HPO4·H2O 0.06g/L、KH2PO40.06g/L、NaHCO30.35g/L and 1.0g/L glucose; the sperm preservation solution has a pH value of 7.1.
Step 2, semen treatment: adding mature semen of Carassius auratus into the above trypsin solution, and treating at 20 deg.C for 15 min;
step 3, insemination and hatching: and (2) carrying out dry insemination on the semen (derived from the white crucian carp in the example 1) treated in the step (2) and the mature eggs (derived from the carassius auratus gibelio A + line in the example 1) of the carassius auratus gibelio, and hatching the obtained fertilized eggs to obtain the allooctaploid carassius auratus gibelio. The hatching mode is that the fertilized eggs are placed in a white porcelain plate for hatching and half water is changed every 12 hours.
Example 5
Step 1, preparing a trypsin solution: adding trypsin powder into the sperm preservation solution, uniformly mixing to obtain a trypsin solution, and adding 0.9g of the trypsin powder into every 100mL of the sperm preservation solution;
the formula of the sperm preserving fluid is as follows: NaCl 7.5g/L, KCl 0.3.3 g/L, CaCl20.12g/L、MgSO4·7H2O0.1g/L、Na2HPO4·H2O 0.04g/L、KH2PO40.04g/L、NaHCO30.33g/L and glucose 0.9 g/L; the sperm preservation solution has a pH value of 7.0.
Step 2, semen treatment: adding mature semen of Carassius auratus into the above trypsin solution, and treating at 22 deg.C for 13 min;
step 3, insemination and hatching: and (2) carrying out dry insemination on the semen (derived from the white crucian carp in the example 2) treated in the step (2) and the mature eggs (derived from the carassius auratus gibelio A + line in the example 2) of the carassius auratus gibelio, and hatching the obtained fertilized eggs to obtain the allooctaploid carassius auratus gibelio. The hatching mode is that the fertilized eggs are placed on the net sheets and put into a water tank for oxygenation and hatching.
Example 6
Step 1, preparing a trypsin solution: adding trypsin powder into the sperm preservation solution, uniformly mixing to obtain a trypsin solution, and adding 1.1g of the trypsin powder into every 100mL of the sperm preservation solution;
the formula of the sperm preserving fluid is as follows: NaCl 8.5g/L, KCl 0.5.5 g/L, CaCl20.16g/L、MgSO4·7H2O0.3g/L、Na2HPO4·H2O 0.08g/L、KH2PO40.08g/L、NaHCO30.37g/L and 1.1g/L glucose; the sperm preservation solution has a pH value of 7.2.
Step 2, semen treatment: adding mature semen of Carassius auratus into the above trypsin solution, and treating at 24 deg.C for 17 min;
step 3, insemination and hatching: and (3) carrying out dry insemination on the semen (derived from the white crucian carp in the example 3) treated in the step (2) and the mature eggs (derived from the carassius auratus gibelio A + line in the example 3) of the carassius auratus gibelio, and hatching the obtained fertilized eggs to obtain the allooctaploid carassius auratus gibelio. The hatching mode is that the fertilized eggs are placed in a white porcelain plate for hatching and half water is changed every 12 hours.
The above trypsin solution concentrations and semen treatment times were determined by the relevant parallel experiments. Experiments show that overhigh or overlow concentration of the trypsin solution and overlong or overlong semen processing time can bring adverse effects on the fry survival rate and the allopolyoctaploid number rate index, and the concentration of the trypsin solution and the semen processing time provided by the invention are simultaneously beneficial to obtaining ideal fry survival rate and allopolyoctaploid number rate indexes.
Example 7
Regarding the ploidy detection method:
step 1, blood sampling: cutting tail of fry with length of 1.5cm to obtain blood 0.1 μ L, and cutting fin of fry with age of six months to obtain blood 0.1 μ L;
step 2, detection: the collected fresh blood was placed in 200. mu.L of a CyStain DNA1Step (source: Partec, Germany) solution pre-cooled at 4 ℃ and mixed well, and then placed on ice, and DNA content was detected by a flow cytometer.
The results of the ploidy test of the offspring hatched in examples 4, 5 and 6 and the survival rate of the obtained octaploid fry are as follows:
| group of | Example 4 | Example 5 | Example 6 |
| Survival rate | 3.i | 2.8 | 2.4 |
| Number of allopolyoctaloids | 16.8 | 16.5 | 16.3 |
Remarking:
survival rate (normal fry tail/total fertilized roe count at the beginning of ingestion) × 100%
The allooctaploid number rate (number of octaploid fry/total fry tail detected) × 100%.
Claims (4)
1. A method for efficiently creating allooctaploid silver crucian carp is characterized by comprising the following steps: the method comprises an artificial insemination stage and a carassius auratus gibelio parent breeding and propagating stage, wherein the artificial insemination stage comprises the following operation steps:
step 1, preparing a trypsin solution: adding trypsin powder into the sperm preservation solution, uniformly mixing to obtain a trypsin solution, and adding 0.9-1.1g of the trypsin powder into every 100mL of the sperm preservation solution;
step 2, semen treatment: adding mature semen of Carassius auratus into the above trypsin solution, and treating at 22-24 deg.C for 13-17 min;
step 3, insemination and hatching: performing dry insemination on the semen treated in the step 2 and mature eggs of the carassius auratus gibelio, and incubating the obtained fertilized eggs to obtain the allooctaploid carassius auratus gibelio;
the carassius auratus gibelio parent breeding and propagating stage comprises the following steps:
step 1, preparing for discharging: in winter, before the parents are put into the pond, the parent culture pond is disinfected in a dry way to kill trash fish, then fresh water is poured into the parent culture pond again, and meanwhile, an isolation net sleeve is arranged at the water inlet of the parent culture pond to prevent the trash fish from mixing into the culture pond;
step 2, parent breeding: thoroughly cleaning the parent pond before the parent pond is put into the pond, and carrying out male and female screening to eliminate all male fishes; after beginning spring, the water depth of the parent culture pond is increased to 1.5-2.0 m;
the feeding amount of the feed in the parent breeding process is calculated according to 2-4% of the total weight of the parent;
3000-4500kg/hm of decomposed organic fertilizer is splashed into a parent cultivation pond in the parent cultivation process each time2Or 1200 ml of fermented EM bacteria solutionReducing the dissolved oxygen of the water body to 2-4mg/L after 7-15 days per mu;
when the water temperature of the parent culture pond is 16 ℃, carrying out high-water-level net pulling, checking the maturity of the parent, carrying out induced spawning on the parent after the ovary grows to the fourth-stage middle stage, and transferring the parent after the ovary grows to the fourth-stage initial stage to the indoor for preparing induced spawning propagation;
step 3, hastening parturition: the mixed solution of 1ug/kg of diutanone, 1mg/kg of pituitary gland, 500 international units/kg of human chorionic gonadotropin and 5ug/kg of ovulation-promoting hormone is adopted to inject the parents for induced spawning, and the parents after the injection induced spawning are placed in a flowing water cylinder with indoor constant temperature of 21 ℃ for waiting for spawning.
2. The method for efficiently creating the allooctaploid silver crucian carp as claimed in claim 1, wherein the method comprises the following steps: the method also comprises a ploidy detection mode of the hatched offspring, wherein the ploidy detection mode comprises the following steps:
step 1, blood sampling: cutting tail of fry with length of 1.5cm to obtain blood 0.1 μ L, and cutting fin of fry with age of six months to obtain blood 0.1 μ L;
step 2, detection: the collected fresh blood is put into 200 mu L of CyStain DNA1Step solution pre-cooled at 4 ℃ and mixed evenly, then the mixture is put on ice, and DNA content detection is carried out by utilizing a flow cytometer.
3. The method for efficiently creating the allooctaploid silver crucian carp as claimed in claim 1, wherein the method comprises the following steps: and injecting the white crucian carp male parent according to the dosage of one third of the mixed solution for hastening parturition, wherein the white crucian carp male parent is a robust individual with the age of more than 2 years, and the weight of the white crucian carp male parent is 500 g.
4. The method for efficiently creating the allooctaploid silver crucian carp as claimed in claim 1, wherein the method comprises the following steps: in the parent breeding process, aiming at the area with sudden temperature rise in 3 months, the parents are temporarily bred in the indoor circulating water body at the temperature of 18-22 ℃ in different jars at the temperature of 16-20 ℃ at constant temperature, and then the subsequent induced spawning is carried out.
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