Efficient cultivation method of triploid mandarin fish

Abstract

The invention discloses an efficient cultivation method for triploid mandarin fish and belongs to the technical field of mandarin fish breeding. In order to solve the problems that the growth and meat quality of the mandarin fish can be influenced by gonad development, the commodity quality of the mandarin fish is reduced and the like, the proper starting time, the optimal hydrostatic pressure, the optimal treatment time, the optimal temperature and the optimal chemical conditions for inducing the triploid mandarin fish are determined by combining the triploid proportion of the triploid mandarin fish; the cultivation method for producing the triploid mandarin fish through combined induction of hydrostatic pressure, temperature shock and chemicals is established. The triploid mandarin fish breeding method established by the invention is clear in operation process and high in practicability, triploid mandarin fish can be efficiently induced and generated, and the induction efficiency of the triploid mandarin fish can be up to 93%. The triploid mandarin fish breeding method provided by the invention opens up a new technical approach for mandarin fish polyploidy breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of mandarin fish breeding, and particularly relates to a high-efficiency breeding method for triploid mandarin fish. Background Art

[0002] Mandarin fish is one of the four famous freshwater fishes in China. Its meat is white, tender, delicious, and has high nutritional value. It is in short supply in the market. Gonadal development will affect the growth and meat quality of mandarin fish and reduce its commercial quality. Moreover, with the expansion of mandarin fish farming area, the probability of mandarin fish escaping into nature has greatly increased, which may cause pollution to fish germplasm resources. It is generally believed that triploid fish cannot mature due to unbalanced chromosome composition, which greatly reduces the adverse effects of growth stagnation and decreased fish meat quality during gonadal development, and can increase the growth rate of fish and improve meat quality. Therefore, conducting research on triploid mandarin fish and cultivating infertile triploid populations will help increase the aquaculture output and commercial value of mandarin fish, and is also crucial for protecting my country's fishery germplasm resources. It can also provide a reference for the breeding of other bream subfamily fish.

[0003] Most fish in nature are diploid. The mature eggs of diploid fish are in the metaphase of the second meiotic division. There are two sets of chromosomes in the eggs. After fertilization, when the sperm (one set of chromosomes) enters the eggs, it stimulates one set of chromosomes in the eggs to be discharged from the eggs as the second polar body, completing the second meiotic division. At this time, the one set of chromosomes in the fertilized egg combines with the one set of chromosomes brought in by the sperm and develops into a normal diploid. If the fertilized egg is treated with high temperature, low temperature, high pressure or chemical drugs when the sperm enters the egg and the second polar body has not yet been discharged, the second meiotic division is prevented, and the second polar body cannot be discharged from the egg. The induced individual with three sets of chromosomes is called artificially induced triploid. Most fish complete the second meiotic division and discharge the second polar body 8-10 minutes after the sperm enters the egg. If you want to obtain triploids, you must perform induction treatment before completing the second meiotic division. No matter which method is used for induction, the starting time and duration of the treatment must be strictly controlled. For example, 15 minutes after the eggs of American trout are fertilized, heat shock treatment with 28℃ high water temperature for 10 minutes can induce triploids, with an induction rate of 100% and a hatching rate of 42%. For catfish, 5 minutes after the eggs are fertilized at 25±1℃, they are placed in 5℃ seawater for cold shock treatment for 1 hour, and then placed in aerated seawater for cultivation. The induction rate of triploids can reach 100%, but if the cold shock treatment time is more than 1.5 hours, it will cause 100% mortality. Hydrostatic induction also requires strict control of the treatment time. Taking crystal crucian carp as an example, the pressure-sensitive period of fertilized eggs is only 4 to 5 minutes after fertilization. Before this, it is the egg initiation period. Applying pressure will destroy the excitement and trimming process of the egg, causing developmental retardation; followed by the pressure refractory period, when the second meiosis tends to be clear, and pressure has no effect on retaining the second polar body. Therefore, in order to obtain triploid crystal crucian carp, hydrostatic induction can only be performed 4 to 5 minutes after fertilization.

[0004] At present, there are many reports at home and abroad on the use of temperature shock, hydrostatic pressure, chemical treatment and other methods to produce triploid fish. The international community also attaches great importance to the research on triploid induction technology of fish and other aquaculture animals. After the unremitting efforts of scientists from various countries, triploids of many fish have been obtained, such as grass carp, carp, crucian carp, rainbow trout, sweetfish, tilapia, salmon, black silk, beard gill, loach, and flounder. The triploids of some fish such as grass carp, rainbow trout, and sweetfish have reached the level of mass production. Triploid fish have great commercial value and development potential, which can not only improve economic benefits, but also avoid interference with natural resources. This is one of the main factors that triploid breeding still has strong vitality today when genetic engineering technology continues to penetrate into the field of fish breeding.

[0005] So far, no research on the production method of triploid mandarin fish fry has been reported. Due to species differences, when the technical parameters for the reported triploid induction of marine fish and most freshwater fish are used for the induction of triploid mandarin fish, the induction rate of triploid mandarin fish is not high, which is difficult to meet the needs of industrial production. Therefore, it is necessary to establish a breeding method suitable for triploid mandarin fish. Summary of the invention

[0006] In order to solve the problems that gonad development affects the growth and meat quality of mandarin fish and reduces the commercial quality of mandarin fish, the present invention combines hydrostatic pressure treatment with temperature shock and colchicine aqueous solution combined induction treatment, determines the appropriate starting time for inducing triploid mandarin fish, the optimal hydrostatic pressure and treatment time, the optimal temperature and the optimal chemical conditions according to the triploidization ratio of triploid mandarin fish, and establishes a breeding method for producing triploid mandarin fish by combining hydrostatic pressure with temperature shock and chemical induction.

[0007] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0008] The object of the present invention is to provide a method for cultivating triploid mandarin fish, characterized in that it comprises the following steps:

[0009] (1) Artificial insemination of mandarin fish: collect mature eggs and semen of mandarin fish, place the eggs in a dry container, slowly add semen, slowly operate to mix the sperm and eggs, then add breeding water, slowly shake to complete the sperm and egg fertilization process, and obtain fertilized eggs;

[0010] (2) Induction of triploid mandarin fish: The fertilized eggs obtained in step (1) are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment, and the fertilized eggs after hydrostatic pressure treatment are subjected to a combined induction treatment of temperature shock and colchicine aqueous solution, and the fertilized eggs are taken out and transferred to aquaculture water for hatching.

[0011] In one embodiment of the present invention, the volume ratio of semen to egg in step (1) is 2000 μL:500 mL.

[0012] In one embodiment of the present invention, the temperature of the drying container in step (1) is 23-25°C.

[0013] In one embodiment of the present invention, the temperature of the aquaculture water in step (1) is 23-25°C, and the amount added is 20 times the volume of the eggs.

[0014] In one embodiment of the present invention, the hydrostatic pressure treatment in step (2) is performed 10 minutes after insemination.

[0015] In one embodiment of the present invention, the pressure of the hydrostatic pressure treatment in step (2) is 7500 PSI.

[0016] In one embodiment of the present invention, the time of the hydrostatic pressure treatment in step (2) is 6-7 minutes.

[0017] In one embodiment of the present invention, the combined induction treatment of temperature shock and colchicine aqueous solution in step (2) refers to immersing the fertilized eggs in a colchicine aqueous solution with a temperature of 15° C. and a concentration of 400 μg / mL.

[0018] In one embodiment of the present invention, the combined induction treatment time of temperature shock and colchicine aqueous solution in step (2) is 30 minutes.

[0019] In one embodiment of the present invention, the incubation in step (2) is carried out in aquaculture water, and the temperature of the aquaculture water is kept constant at 23-25°C.

[0020] Beneficial effects of the present invention:

[0021] The present invention designs typical induction condition factors of the triploid mandarin fish breeding method, including hydrostatic pressure, temperature, chemical substances, etc., combines the triploidization ratio of the triploid mandarin fish, determines the appropriate starting time for inducing the triploid mandarin fish, the optimal hydrostatic pressure and treatment time, the optimal temperature and the optimal chemical conditions, and establishes a method for producing triploid mandarin fish by using hydrostatic pressure combined with temperature shock and chemical combined induction. The triploid mandarin fish breeding method established by the present invention has the advantages of clear operation flow and strong practicality, and the method can efficiently induce the triploid mandarin fish, and the induction efficiency of the triploid mandarin fish obtained by the method can be as high as 93%. Therefore, the breeding method provided by the present invention is suitable for artificial and efficient production of triploid mandarin fish. In addition, the triploid mandarin fish breeding method provided by the present invention opens up a new technical approach for mandarin fish polyploid breeding.

[0022] At the same time, the present invention has established a complete identification method for triploid mandarin fish, including red blood cell volume identification method, DNA content identification method, chromosome counting analysis method, and determined the range of red blood cell volume and red blood cell nucleus volume of triploid mandarin fish under the same breeding conditions; determined the ratio of the relative content of DNA of diploid mandarin fish and triploid mandarin fish, determined the absolute content of DNA of triploid mandarin fish; and determined the chromosome number of triploid mandarin fish. The identification method for triploid mandarin fish established by the present invention can quickly identify triploid mandarin fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the test result of DNA content of diploid mandarin fish;

[0024] Figure 2 This is the test result of DNA content of triploid mandarin fish;

[0025] Figure 3 This is the observation diagram of the metaphase division of diploid mandarin fish chromosomes;

[0026] Figure 4 This is an observation diagram of the metaphase division phase of the chromosomes of triploid mandarin fish. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0028] The present invention compares the proportions of triploid mandarin fish obtained under different induction conditions, and optimizes the cultivation method suitable for triploid mandarin fish. Among all the cultivation methods, the method of artificial insemination of mandarin fish is as follows:

[0029] Selectively mature mandarin fish, collect mature eggs and sperm, place the eggs in a dry container at 23-25℃, slowly add semen, the fertilization volume ratio of semen to eggs is 2000μL:500mL, slowly operate to mix the sperm and eggs, then add 20 times the volume of the eggs and the temperature of the aquaculture water at 23-25℃, the aquaculture water meets the fishery water quality standards (GB 11607-1989), and slowly shake to complete the sperm and egg fertilization process.

[0030] 1. Determination of the induction method of triploid mandarin fish

[0031] (1) Determination of the starting time of triplicate

[0032] Comparative Example 1: 4 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0033] Comparative Example 2: 5 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0034] Comparative Example 3: 6 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0035] Comparative Example 4: 7 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0036] Comparative Example 5: 8 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0037] Comparative Example 6: 9 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0038] Comparative Example 7: 10 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0039] The triploid ratios obtained in the above comparison examples were statistically analyzed. The triploid ratio of comparison example 1 was 52%, the triploid ratio of comparison example 2 was 57%, the triploid ratio of comparison example 3 was 59%, the triploid ratio of comparison example 4 was 65%, the triploid ratio of comparison example 5 was 67%, the triploid ratio of comparison example 6 was 69%, and the triploid ratio of comparison example 7 was 76%. Among them, the triploid ratio of comparison example 7 was the highest. Therefore, the starting time of inducing triploidization was determined to be 10 minutes after fertilization.

[0040] (2) Determination of hydrostatic pressure treatment time

[0041] Comparative Example 8: 10 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 4 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0042] Comparative Example 9: 10 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 5 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0043] Comparative Example 7: 10 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0044] Comparative Example 10: 10 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 7 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0045] Comparative Example 11: 10 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 8 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0046] The triploid ratios obtained in the above comparative examples were statistically analyzed, and it was found that the triploid ratios of comparative examples 7 and 10 were 76% and 77%, respectively. The triploid ratios were relatively high among the above comparative examples, so the time for the hydrostatic pressure treatment was determined to be 6-7 minutes.

[0047] (3) Determination of pressure during hydrostatic pressure treatment

[0048] Comparative Example 12: 10 minutes after fertilization, the hydrostatic pressure was 6000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0049] Comparative Example 13: 10 minutes after fertilization, the hydrostatic pressure was 6500PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0050] Comparative Example 7: 10 minutes after fertilization, the hydrostatic pressure was 7000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0051] Comparative Example 14: 10 minutes after fertilization, the hydrostatic pressure was 7500 PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0052] Comparative Example 15: 10 minutes after fertilization, the hydrostatic pressure was 8000PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0053] The triploid ratios obtained in the above comparative examples were statistically analyzed. Among all comparative examples, the comparative example 14 had the highest triploid ratio, which was 86%. Therefore, the induction conditions were determined as follows: 10 minutes after sperm and egg fertilization, the eggs were treated at a hydrostatic pressure of 7500PSI for 6-7 minutes, and then transferred to aquaculture water to complete hatching.

[0054] (4) Determination of whether to perform temperature shock and chemical combination induction treatment

[0055] Comparative Example 14: 10 minutes after fertilization, the hydrostatic pressure was 7500 PSI, the action time was 6 minutes, and the cells were transferred into the breeding water to complete the hatching.

[0056] Comparative Example 16: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 0°C and a concentration of 300 μg / mL for 20 minutes, and then transferred to breeding water to complete hatching.

[0057] Comparative Example 17: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 0°C and a concentration of 400 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0058] Comparative Example 18: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 0°C and a concentration of 500 μg / mL for 40 minutes, and then transferred to the breeding water to complete the hatching.

[0059] Comparative Example 19: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 15°C and a concentration of 300μg / mL for 20 minutes, and then transferred to breeding water to complete hatching.

[0060] Comparative Example 20: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 15°C and a concentration of 400 μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0061] Comparative Example 21: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 15°C and a concentration of 500μg / mL for 40 minutes, and then transferred to breeding water to complete hatching.

[0062] Comparative Example 22: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 25°C and a concentration of 300μg / mL for 20 minutes, and then transferred to breeding water to complete hatching.

[0063] Comparative Example 23: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 25°C and a concentration of 400 μg / mL for 30 minutes, and then transferred to breeding water to complete the hatching.

[0064] Comparative Example 24: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and combined induction treatment with chemical substances. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 25°C and a concentration of 500μg / mL for 40 minutes, and then transferred to breeding water to complete hatching.

[0065] Comparative Example 25: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 35°C and a concentration of 300μg / mL for 20 minutes, and then transferred to breeding water to complete hatching.

[0066] Comparative Example 26: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 35°C and a concentration of 400μg / mL for 30 minutes, and then transferred to breeding water to complete hatching.

[0067] Comparative Example 27: 10 minutes after fertilization, the hydrostatic pressure was 7500PSI, and the action time was 6 minutes. After the hydrostatic pressure treatment, the fertilized eggs were subjected to temperature shock and chemical combination induction treatment. The fertilized eggs were immersed in a colchicine aqueous solution with a temperature of 35°C and a concentration of 500μg / mL for 40 minutes, and then transferred to breeding water to complete hatching.

[0068] The triploid ratios obtained in the above comparative examples were statistically analyzed, as shown in Table 1. Among the comparative examples, comparative example 20 had the highest triploid ratio, which was 93%.

[0069] Table 1 Triploid ratios obtained in comparative examples

[0070]

[0071] Based on the triploid ratios of the above comparisons, the optimal breeding method for producing triploid mandarin fish was finally determined:

[0072] (1) Artificial insemination of mandarin fish

[0073] Selectively mature mandarin fish, collect mature eggs and sperm, place the eggs in a dry container at 23-25℃, slowly add semen, the fertilization volume ratio of semen to eggs is 2000μL:500mL, slowly operate to mix the sperm and eggs, then add 20 times the volume of the eggs and the temperature of the aquaculture water at 23-25℃, the aquaculture water meets the fishery water quality standards (GB 11607-1989), and slowly shake to complete the sperm and egg fertilization process.

[0074] (2) Induction of triploid mandarin fish

[0075] 10 minutes after sperm and egg fertilization, the fertilized eggs are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment at a pressure of 7500PSI for 6-7 minutes; after hydrostatic pressure treatment, the fertilized eggs are subjected to temperature shock and combined induction treatment with chemical substances, and the fertilized eggs are immersed in a colchicine aqueous solution with a concentration of 400μg / mL and a temperature of 15°C for 30 minutes; finally, the fertilized eggs are transferred to the breeding water, the temperature of which is kept constant at 23-25°C to complete the incubation.

[0076] Identification of triploid mandarin fish

[0077] Whether the individuals induced by triploidization are truly triploid still needs to be strictly identified. The present invention uses the following method to identify triploid mandarin fish:

[0078] (1) Identification of red blood cell volume

[0079] It is generally believed that cell size is related to chromosome and DNA content. In a single cell, because the number of triploid chromosomes is more than that of diploid, in order to maintain a constant nuclear-cytoplasmic ratio, the volume of triploid cells is generally larger than that of diploid cells. There are large differences in the volume of triploid and diploid red blood cells and the volume of red blood cell nuclei in different fish. Theoretically, the red blood cell nucleus volume of diploid fish and triploid fish is 1:1.5. The present invention uses a blood collection needle to collect blood from diploid mandarin fish and triploid mandarin fish obtained by the cultivation method provided by the present invention, prepares blood smears, HE stains, and observes under an optical microscope. The length of the long and short diameters of 10 red blood cell nuclei is measured for each smear, and the volume of red blood cells and nuclei is calculated. The values ​​of red blood cells and red blood cell nuclei obtained for diploid mandarin fish and triploid mandarin fish are shown in Table 2.

[0080] As can be seen from Table 2, all the values ​​of triploid mandarin fish are significantly higher than those of diploid mandarin fish. The long diameter, short diameter, red blood cell area and red blood cell volume of triploid erythrocytes are 1.48, 1.59, 1.58 and 1.42 times that of diploid, respectively, and the long diameter, short diameter, nuclear area and red blood cell nuclear volume of triploid erythrocytes are 1.66, 1.41, 1.71 and 1.66 times that of diploid, respectively. There are significant differences in all parameters between diploid mandarin fish and triploid mandarin fish (P < 0.05).

[0081] The volume range of diploid erythrocytes is 74.21±10.21 μm 3 The range of red blood cell nuclear volume is 53.23±5.17μm 3 The volume range of triploid red blood cells is 105.02±25.62μm 3 The range of red blood cell nuclear volume is 88.47±13.56μm 3 between.

[0082] Table 2 Erythrocyte and erythrocyte nucleus values ​​of diploid and triploid mandarin fish

[0083]

[0084] (2) DNA content identification

[0085] Blood was collected from the tail vein of the diploid mandarin fish and the triploid mandarin fish obtained by the breeding method of the present invention, and the blood was fully mixed with 0.75% saline in a ratio of 1:4. Lymphocytes were separated with lymphocyte separation fluid, stained with PI in the dark at 4°C for 30 minutes, and filtered with a PARTEC ploidy meter with 400 mesh before ploidy identification. 100 samples were tested, and chicken blood with known DNA content was used as a control. Flow cytometry was used to analyze the relative content of DNA in the diploid mandarin fish and the triploid mandarin fish obtained by the breeding method of the present invention to determine the ploidy. The flow cytometry experimental results are shown in Figure 1 and Figure 2 .

[0086] The experimental results show that the relative DNA content of diploid mandarin fish is 73.43±12.57, and the relative DNA content of triploid mandarin fish is 108.22±7.21. The DNA content of triploid mandarin fish is about 1.47 times that of diploid mandarin fish, which is consistent with the results of red blood cell size determination (see Table 3). It is known that the DNA content of chicken red blood cells is 2.3pg / N -1 , then the absolute DNA content of diploid mandarin fish somatic cells is 2.98 pg.N -1 The absolute DNA content of triploid mandarin fish somatic cells is 4.40 pg.N -1 .

[0087] Table 3 DNA content of diploid, triploid mandarin fish and chicken

[0088]

[0089] (3) Chromosome count analysis:

[0090] The metaphase of chromosomes was prepared by in vivo culture of kidney cells, stained with Giemsa, and observed under a microscope. The number of chromosomes in 100 metaphases of diploid and triploid mandarin fish was counted. The counting results showed that the number of chromosomes in diploid mandarin fish was 2n=48, and the number of chromosomes in triploid mandarin fish was 3n=72. Figure 3 and Figure 4 As shown, the chromosome numbers of diploid and triploid mandarin fish conform to the ploidy relationship.

[0091] The above identification methods were combined to find that polyploidy increases the number of chromosomes, the DNA content also increases proportionally, and the cell nucleus volume also increases. There are significant differences in the red blood cells and red blood cell nuclear area and volume, DNA content, chromosome number, etc. between diploid and triploid mandarin fish, and the data differences are consistent with the ploidy relationship.

[0092] In this experiment, the identification of triploid mandarin fish was mainly based on the determination of red blood cells and red blood cell nuclear volume, assisted by the identification methods of chromosome counting and DNA content detection. Red blood cell determination can be conveniently and quickly identified, and the experimental process is relatively simple, which is suitable for large-scale determination at the breeding site. Chromosome karyotype analysis can very intuitively observe the ploidy of cells, but the operation is relatively complicated and time-consuming. The flow cytometer method for identifying DNA content requires professional experimental instruments and is relatively limited. In summary, the red blood cell determination method can be used as the preferred method for large-scale identification of ploidy, and the accuracy of identification can be improved by methods such as chromosome counting and DNA content detection, so that large quantities of mandarin fish can be quickly and effectively identified for ploidy.

[0093] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for cultivating triploid mandarin fish, characterized in that: The following steps are involved: (1) Artificial insemination of mandarin fish: collect mature eggs and semen of mandarin fish, place the eggs in a dry container, slowly add semen, slowly operate to mix the sperm and eggs, then add breeding water, slowly shake to complete the sperm and egg fertilization process, and obtain fertilized eggs; (2) Induction of triploid mandarin fish: The fertilized eggs obtained in step (1) are placed in a hydrostatic pressure chamber for hydrostatic pressure treatment, and the fertilized eggs after hydrostatic pressure treatment are subjected to a combined induction treatment of temperature shock and colchicine aqueous solution, and the fertilized eggs are taken out and transferred to aquaculture water for hatching.

2. The cultivation method according to claim 1, characterized in that The volume ratio of the semen to the egg in step (1) is 2000 μL:500 mL.

3. The cultivation method according to claim 1, characterized in that The temperature of the drying container in step (1) is 23-25°C.

4. The cultivation method according to claim 1, characterized in that The temperature of the aquaculture water in step (1) is 23-25° C., and the amount added is 20 times the volume of the eggs.

5. The cultivation method according to claim 1, characterized in that: The hydrostatic pressure treatment in step (2) is performed 10 minutes after insemination.

6. The cultivation method according to claim 1, characterized in that: The pressure of the hydrostatic pressure treatment in step (2) is 7500PSI.

7. The cultivation method according to claim 1, characterized in that: The time of the hydrostatic pressure treatment in step (2) is 6-7 minutes.

8. The cultivation method according to claim 1, characterized in that: The combined induction treatment of temperature shock and colchicine aqueous solution in step (2) refers to immersing the fertilized eggs in a colchicine aqueous solution with a temperature of 15° C. and a concentration of 400 μg / mL.

9. The cultivation method according to claim 1, characterized in that: The time for the combined induction treatment of temperature shock and colchicine aqueous solution in step (2) is 30 minutes.

10. The cultivation method according to claim 1, characterized in that: In step (2), the incubation is carried out in aquaculture water, and the temperature of the aquaculture water is kept constant at 23-25°C.

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