CN109362616B - Breeding method of triploid carp - Google Patents
Breeding method of triploid carp Download PDFInfo
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- CN109362616B CN109362616B CN201811541945.9A CN201811541945A CN109362616B CN 109362616 B CN109362616 B CN 109362616B CN 201811541945 A CN201811541945 A CN 201811541945A CN 109362616 B CN109362616 B CN 109362616B
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- A—HUMAN NECESSITIES
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- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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Abstract
The invention discloses a breeding method of triploid carp, relating to the technical field of carp breeding, and the technical scheme comprises the following steps: taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs; performing hydrostatic pressure treatment and then immersing the product in cold water for treatment; transferring to water for hatching to obtain triploid carp. The method provided by the invention is convenient for field operation, safe, reliable, simple and feasible, has high triploid conversion rate, and has important application value in triploid induction and large-scale culture of carps.
Description
Technical Field
The invention relates to the technical field of carp breeding, in particular to a breeding method of triploid carps.
Background
The vast majority of fishes in nature exist in diploid form and breed offspring in a hermaphroditic fusion mode, the gonad development of diploid fishes consumes a large amount of energy in the sexual maturity stage, the quality of fish flesh is reduced and the time of sale is prolonged due to the gonad development stage and the egg laying season, meanwhile, the growth of individuals is stopped and the death rate is increased in the gonad development stage, and the individual body types are greatly limited. Natural diploid fish need artificial induction to produce triploid. The method for artificially inducing triploid fish mainly comprises physical methods such as cold shock, heat shock, hydrostatic pressure, etc., and chemical methods for treating with chemical substances (such as 6-dimethylaminopurine (6-DMAP), colchicine, etc.). At present, methods such as temperature shock, hydrostatic pressure, drug treatment and the like are used for inhibiting the second polar body discharge of fertilized eggs, so that triploid fish produced by chromosome set triploid production are reported at home and abroad, but most of the triploid fish are still in the stages of exploration and test, a certain distance is left from practical application, most of results still do not completely search out the optimal induction parameters, the phenomena of high fertilized egg necrosis rate, low emergence rate, high juvenile fish teratogenesis rate, low triploid rate and the like influence the progress of triploid industrialization.
From the current development situation of the aquaculture industry, if the processing conditions of triploid induction are further optimized and the triploid induction method and the technical process are perfected, more triploid fishes can be applied to the aquaculture industry, and the healthy development of the fish aquaculture industry is promoted.
Disclosure of Invention
In order to solve the problems, the invention provides a breeding method of triploid carps, which adopts the technical scheme that the breeding method comprises the following steps:
1) taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs;
2) putting the fertilized eggs obtained in the step 1) into a hydrostatic pressure device bin for hydrostatic pressure treatment;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and immersing the fertilized eggs into cold water for treatment;
4) transferring the fertilized eggs obtained in the step 3) into water for hatching.
The carp in the step 1) is preferably mirror carp, and more preferably Songpu mirror carp.
The volume ratio of the semen and the eggs in the step 1) is 500 mu L (50-100) mL.
The water temperature in step 1) is 23-25 ℃.
After the insemination process in the step 1) is finished, the fertilized eggs continue to develop in water for 3-4.5 min.
Step 2) the storehouse of the hydrostatic pressure device is filled with colchicine aqueous solution of 500 mug/ml, and the fertilized eggs obtained in the step 1) are immersed in the colchicine aqueous solution.
Step 2) hydrostatic pressure treatment, wherein the pressure is 600kg/cm2The treatment time was 3 min.
In the hydrostatic pressure treatment process of the step 2), the temperature of the colchicine aqueous solution is 23-25 ℃.
And 3) the temperature of the cold water is 0-2 ℃, and the treatment time is 10-15 min.
The water temperature in the step 4) is 23-25 ℃.
Advantageous effects
The method provided by the invention is convenient for field operation, safe, reliable, simple and feasible, the triploid carp cultivated by the method provided by the invention is tested to have a triploid conversion rate of 91%, and the triploid carp cultivated by the method has a red cell volume ratio of 1.63:1 and a red cell nucleus volume ratio of 1.56:1 compared with a common diploid, and has extremely obvious differences in red cell nucleus long diameter, red cell area, nuclear area and nuclear volume compared with the diploid; the gonad of the triploid carp does not develop, and the observation of the spermary section shows large-area vacuolation; and in comparison with 3-year fish, the weight ratio of the triploid carp to the diploid carp is 1.7 +/-0.21. The triploid carp cultivated by the method has no problems that a large amount of energy is consumed by gonad development in the sexual maturity stage of diploid fish, the fish quality is reduced and the time to market is prolonged due to the gonad development stage and the laying season, the growth of individuals is stopped and the death rate is increased in the gonad development stage, and the like, and has important application value in triploid induction and large-scale cultivation of cyprinid fish.
Drawings
Figure 1 diploid versus triploid carp size comparison (3-age group);
FIG. 23 age diploid vs triploid gonad differential; wherein 2n represents diploid and 3n represents triploid;
FIG. 33 is a 40 Xphotograph taken by an optical microscope of a spermary tissue section of a diploid carp aged;
FIG. 43 is a 40-fold photograph taken by means of an optical microscope of a spermary tissue section of a triploid carp aged;
FIG. 5 diploid pine Pumiri red cells (HE staining);
FIG. 6 triploid Songpu mirror carp red blood cells (HE staining);
FIG. 7 is the metaphase mitosis phase of diploid pine pur mirror carp;
FIG. 8 mid-split phase of triploid Songpu mirror carp;
FIG. 9 is the DNA content of diplodia Songpu mirror carp;
FIG. 10 is the DNA content of triploid Songpu mirror carp.
Detailed Description
Example 1
A breeding method of triploid carp comprises the following steps:
1) taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs;
2) rapidly folding the screen with the fertilized eggs obtained in the step 1), putting the screen into a hydrostatic pressure device bin, and performing hydrostatic pressure treatment;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and immersing the fertilized eggs into cold water for treatment;
4) transferring the fertilized eggs obtained in the step 3) into water for hatching.
The carp in the step 1) is Songpu mirror carp.
The volume ratio of the semen and the eggs in the step 1) is 500 muL to 100 mL.
The water temperature in step 1) was 23 ℃.
And 1) after the insemination process is finished, the fertilized eggs continue to develop in water for 3 min.
Step 2) the storehouse of the hydrostatic pressure device is filled with colchicine aqueous solution of 500 mug/ml, and the fertilized eggs obtained in the step 1) are immersed in the colchicine aqueous solution.
Step 2) hydrostatic pressure treatment, wherein the pressure is 600kg/cm2The treatment time was 3 min.
In the hydrostatic pressure treatment process of the step 2), the temperature of the colchicine aqueous solution is 23 ℃.
And 3) the temperature of the cold water is 0 ℃, and the treatment time is 10 min.
The water temperature in step 4) was 23 ℃.
And (3) operating the fertilized eggs under the load of a screen mesh.
Example 2
A breeding method of triploid carp comprises the following steps:
1) taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs;
2) rapidly folding the screen with the fertilized eggs obtained in the step 1), putting the screen into a hydrostatic pressure device bin, and performing hydrostatic pressure treatment;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and immersing the fertilized eggs into cold water for treatment;
4) transferring the fertilized eggs obtained in the step 3) into water for hatching.
The carp in the step 1) is Songpu mirror carp.
The volume ratio of the semen and the eggs in the step 1) is 500 muL to 50 mL.
The water temperature in step 1) was 25 ℃.
After the insemination process in the step 1) is finished, the fertilized eggs continue to develop in the water for 4.5 min.
Step 2) the storehouse of the hydrostatic pressure device is filled with colchicine aqueous solution of 500 mug/ml, and the fertilized eggs obtained in the step 1) are immersed in the colchicine aqueous solution.
Step 2) hydrostatic pressure treatment, wherein the pressure is 600kg/cm2The treatment time was 3 min.
In the hydrostatic pressure treatment process of the step 2), the temperature of the colchicine aqueous solution is 25 ℃.
And 3) the temperature of the cold water is 2 ℃, and the treatment time is 15 min.
The water temperature in step 4) was 25 ℃.
And (3) operating the fertilized eggs under the load of a screen mesh.
Example 3
A breeding method of triploid carp comprises the following steps:
1) taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs;
2) rapidly folding the screen with the fertilized eggs obtained in the step 1), putting the screen into a hydrostatic pressure device bin, and performing hydrostatic pressure treatment;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and immersing the fertilized eggs into cold water for treatment;
4) transferring the fertilized eggs obtained in the step 3) into water for hatching.
The carp in the step 1) is Songpu mirror carp.
The volume ratio of the semen and the eggs in the step 1) is 500 muL to 80 mL.
The water temperature in step 1) was 24 ℃.
And 1), after the insemination process is finished, the fertilized eggs continue to develop in the water for 4 min.
Step 2) the storehouse of the hydrostatic pressure device is filled with colchicine aqueous solution of 500 mug/ml, and the fertilized eggs obtained in the step 1) are immersed in the colchicine aqueous solution.
Step 2) hydrostatic pressure treatment, wherein the pressure is 600kg/cm2The treatment time was 3 min.
In the hydrostatic pressure treatment process of the step 2), the temperature of the colchicine aqueous solution is 24 ℃.
And 3) the temperature of the cold water is 1 ℃, and the treatment time is 13 min.
The water temperature in step 4) was 24 ℃.
And (3) operating the fertilized eggs under the load of a screen mesh.
Comparative example
Experimental group 1
1) Taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs;
2) rapidly folding the screen with the fertilized eggs obtained in the step 1), putting the screen into a hydrostatic pressure device bin, and performing hydrostatic pressure treatment;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and transferring the fertilized eggs into water for hatching;
the carp in the step 1) is Songpu mirror carp.
The volume ratio of the semen and the eggs in the step 1) is 500 muL to 80 mL.
The water temperature in step 1) was 24 ℃.
And 1), after the insemination process is finished, the fertilized eggs continue to develop in the water for 4 min.
Step 2), water is filled in the still water pressure device bin, and the fertilized eggs obtained in the step 1) are immersed in the water.
Step 2) hydrostatic pressure treatment, wherein the pressure is 600kg/cm2The treatment time was 3 min.
The water temperature in step 3) was 24 ℃.
And (3) operating the fertilized eggs in the steps 1) to 2) under the load of a screen mesh.
1) Taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs;
2) rapidly folding the screen with the fertilized eggs obtained in the step 1), putting the screen into a hydrostatic pressure device bin, and performing hydrostatic pressure treatment;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and immersing the fertilized eggs into cold water for treatment;
4) transferring the fertilized eggs obtained in the step 3) into water for hatching.
The carp in the step 1) is Songpu mirror carp.
The volume ratio of the semen and the eggs in the step 1) is 500 muL to 80 mL.
The water temperature in step 1) was 24 ℃.
And 1), after the insemination process is finished, the fertilized eggs continue to develop in the water for 4 min.
Step 2), water is filled in the still water pressure device bin, and the fertilized eggs obtained in the step 1) are immersed in the water.
Step 2) hydrostatic pressure treatment, wherein the pressure is 600kg/cm2The treatment time was 3 min.
In the hydrostatic pressure treatment process of the step 2), the temperature of water is 24 ℃.
And 3) the temperature of the cold water is 1 ℃, and the treatment time is 13 min.
The water temperature in step 4) was 24 ℃.
And (3) operating the fertilized eggs under the load of a screen mesh.
Experimental group 3
1) Taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs;
2) rapidly folding the screen with the fertilized eggs obtained in the step 1), putting the screen into a hydrostatic pressure device bin, and performing hydrostatic pressure treatment;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and transferring the fertilized eggs into water for hatching;
the carp in the step 1) is Songpu mirror carp.
The volume ratio of the semen and the eggs in the step 1) is 500 muL to 80 mL.
The water temperature in step 1) was 24 ℃.
And 1), after the insemination process is finished, the fertilized eggs continue to develop in the water for 4 min.
Step 2) the storehouse of the hydrostatic pressure device is filled with colchicine aqueous solution of 500 mug/ml, and the fertilized eggs obtained in the step 1) are immersed in the colchicine aqueous solution.
Step 2) hydrostatic pressure treatment, wherein the pressure is 600kg/cm2The treatment time was 3 min.
In the hydrostatic pressure treatment process of the step 2), the temperature of the colchicine aqueous solution is 24 ℃.
The water temperature in step 3 was 24 ℃.
And (3) operating the fertilized eggs in the steps 1) to 2) under the load of a screen mesh.
And (5) result verification:
triploid identification method
1) Method for identifying volume of red blood cell
Collecting blood from vein of fish tail, preparing blood smear, HE staining, sealing with neutral optical gum, measuring length and length of red blood cell and nucleus under oil lens, measuring size of 10 red blood cells and nucleus at random for each blood smear, and making into oral liquid according to formula ab2And/1.91, calculating the volume of the red blood cells and the nucleus by using a long diameter and a short diameter as a part of the red blood cells, and determining a long diameter standard for identifying ploidy through a long diameter range of the two-triploid red blood cells.
1) Karyotyping analysis
Injecting PHA (dissolving calf serum) into pectoral fins in an amount of 5 mu l/g per tail, injecting colchicine into fish bodies in a weight ratio of 1.5 mu g/g after 12-24 hours, cutting off tails and gill cutting for bloodletting after two hours, taking head and kidney, washing 3-5 times in 0.75% physiological saline, centrifuging for 5-7 min at 1000r/min after grinding, taking supernatant, treating with 0.5% KCl at room temperature for 40min, centrifuging to collect cells, fixing with Carnot fixed solution for 3 times, not less than 15min each time, dripping ice slides, standing and airing, staining with Giemsa for 20min, washing with tap water and airing, and observing under a microscope.
3) DNA content detection
Taking blood from fish veins, fully mixing 300-400 mul of whole fish blood with 900 mul of physiological saline (0.75%), slowly dripping the mixed solution on the liquid surface of 6ml of human lymphocyte separation solution, centrifuging at the normal temperature of 2500-3000 rpm for 20min, discarding the supernatant, rinsing with physiological saline (0.75%) for 2-3 times, adding 1ml of physiological saline (0.75%) for resuspension, taking 100 mul of cell suspension, fixing in 6ml of precooled Carnot fixed solution (glacial acetic acid: methanol: 1: 3), centrifuging at 4 ℃ for 2h, 800rpm, 5min, discarding the supernatant, adding 6ml of physiological saline (0.75%), rinsing at 600rpm for 3min, repeating for 3 times, finally 1ml of heavy suspension saline, dyeing PI in dark place, dyeing at 4 ℃ for 30min, filtering with 400 meshes, loading on a machine, and comparing the chicken blood with a standard diploid.
Experimental results (in the following results, triploid is the triploid carp obtained in example 3, and diploid is the common diploid carp raised under the same conditions)
1. Observation of apparent traits
Fig. 1 shows a comparison of the sizes of diploid and triploid carps (example 3), in 3 rd and 2 rd age groups, there were significant differences in body weight and quantitative trait between 3 rd and 2 rd-ploid carps, and in 3 rd age group, the body weight 3n/2n was 1.7 ± 0.21.
2. Gonadal phenotype
FIG. 2 is a comparison of gonadal differences between 3-year old diploids and triploids (example 3), showing that the 3-fold gonadal abortion can be seen; by comparing the spermary slices (fig. 3 and 4), the triploid carp spermary slice shows large area vacuolation compared with the diploid carp spermary slice.
3. Observation and statistics of erythrocyte nucleus size
TABLE 1 measurement and ratio of diploid and triploid erythrocytes
**Shows very significant difference
The results of the significance test of the sizes of the red blood cells and nuclei and the difference between the red blood cells and nuclei of the diploid fish and the triploid fish (example 3) are shown in Table 1. As can be seen from the table, the values of the triploid fish are higher than those of the diploid fish, and the triploid fish and the diploid fish show very significant difference. The volume ratio of the diploid fish to the triploid fish is 1:1.63, the volume ratio of red cell nuclei is 1:1.56, and the difference between the red cell nucleus length, the red cell area, the nuclear area and the nuclear volume of the triploid fish is extremely obvious compared with that of the diploid fish (P < 0.001).
4. Chromosome detection
The chromosome number of diplodia pine mirror carp is 100 as 2n, and the chromosome number of triploid pine mirror carp (example 3) is 150 as 3n, see fig. 7 and 8, and the chromosome numbers of diplodia pine mirror carp and triploid pine mirror carp are consistent with theoretical expectation.
5. DNA content detection
The relative DNA content of Songpu mirror carp 2n and Songpu mirror carp 3n (example 3) is shown in FIGS. 9 and 10, and the ratio of chromosome number and DNA content of the triple sample to that of the standard diploid is about 1:1.58, which is consistent with the results of red blood cell size measurement. The chromosome number and the DNA content are in proportion relation and are in accordance with the result of the red blood cell size measurement.
The ratio of the nuclear DNA content of the triploid to that of the diploid is about 1:1.58, and is positively correlated with the diameter, volume and chromosome number of the red blood cells.
6. Tripling ratio for different induction methods (comparative example)
According to the method for identifying the triploid, 100 tails are identified for each experimental group, and the triploid ratio of each experimental group is determined. See table 2.
TABLE 2 triploid induction ratio of different experimental groups
The triploid conversion rate of the experimental group 1 is 53%, the triploid conversion rate of the experimental group 2 is 67%, the triploid conversion rate of the experimental group 3 is 62%, the triploid conversion rate of the embodiment 3 is 91%, and the triploid conversion rates of the embodiments 1 and 2 are 83% and 87%, respectively.
Claims (1)
1. A breeding method of triploid carp is characterized in that: the method comprises the following steps:
1) taking mature eggs and semen of carps, uniformly mixing, spreading on a screen, and immersing in water to finish insemination to obtain fertilized eggs; the volume ratio of the semen to the eggs is 500 mu L (50-100) mL; the water temperature of the water is 23-25 ℃; after the insemination process is finished, the fertilized eggs continue to develop in water for 3-4.5 min;
2) putting the fertilized eggs obtained in the step 1) into a hydrostatic pressure device bin for hydrostatic pressure treatment; the bin of the hydrostatic pressure device is filled with colchicine solution of 500 mug/mlLiquid; the hydrostatic pressure treatment is carried out, and the pressure is 600kg/cm2The treatment time is 3 min; in the hydrostatic pressure treatment process, the temperature of colchicine aqueous solution is 23-25 ℃; soaking the fertilized egg in colchicine water solution;
3) taking out the fertilized eggs subjected to the hydrostatic pressure treatment in the step 2), and immersing the fertilized eggs into cold water for treatment; the temperature of the cold water is 0-2 ℃, and the treatment time is 10-15 min;
4) transferring the fertilized eggs obtained in the step 3) into water for hatching; the water temperature is 23-25 ℃.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811541945.9A CN109362616B (en) | 2018-12-17 | 2018-12-17 | Breeding method of triploid carp |
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