Preparation method of rainbow trout tetraploid fry

Abstract

The invention relates to the technical field of aquatic organism breeding, in particular to a rainbow trout tetraploid fry preparation method. The method provided by the invention comprises the following steps: carrying out dry fertilization on naturally mature rainbow trout parents to obtain fertilized eggs, inducing the rainbow trout fertilized eggs after 6.2-6.4 hours to obtain tetraploid rainbow trout, and carrying out induction for 10-18 minutes; after rainbow trout tetraploid offspring seeds are obtained through induction, chromosome ploidy of the rainbow trout body is measured through the technologies of chromosome karyotype analysis, flow cytometry DNA content measurement and the like, and the rainbow trout tetraploid offspring seeds are identified and screened out to be bred. According to the method, a drug induction method is adopted, the induction starting time and the induction lasting time are controlled, and the first mitotic time of fertilized eggs is inhibited, so that the induction rate and the survival rate of the tetraploid rainbow trout are improved, the induction rate of the obtained rainbow trout tetraploid can reach 25-30%, and the survival rate of the obtained rainbow trout tetraploid can reach 90% or above.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic biological breeding, and in particular to a method for preparing tetraploid rainbow trout seedlings. Background Art

[0002] Rainbow trout is an important economic fish, named for the rainbow-colored texture along the lateral line of its adult individuals. It is native to North America and the Pacific coast of the Russian Far East. It belongs to the genus Oncorhynchus of the family Salmonidae of the order Salmoniformes. It mainly lives in springs and streams below 20°C. It has high requirements for the water quality of the breeding waters. During the breeding process, the dissolved oxygen is usually required to be maintained above 6 mg / L and the pH value of the water body is maintained between 6.5 and 8. Rainbow trout meat has the characteristics of high protein, low cholesterol, high content of vitamins and trace elements, etc.

[0003] Polyploid breeding of fish is an important cell engineering breeding method. Infertile triploid fish can use all the energy used for reproduction and breeding for somatic cell growth, avoiding the phenomenon of slow growth in gonadal development and mature reproduction stages, and decreased fish meat quality during sexual maturity. The survival rate and disease resistance of mature triploid individuals are higher than those of diploids, and they have important economic value. Rainbow trout triploids cannot reproduce normally, and each generation of triploid seedlings needs to be artificially induced. However, the technology for directly preparing triploid seedlings is still immature, the survival rate of each batch of seedlings prepared is unstable, and the triploid rate cannot be guaranteed, which restricts the application and promotion of triploid fish. However, since the reproductive cells of tetraploid rainbow trout contain an even number of chromosome sets and are fertile, mature tetraploid parents can be fertilized with normal diploid parents to stably and mass-produce seedlings with a triploid rate of 100%. Therefore, it is necessary to develop efficient and stable preparation technology for rainbow trout tetraploids with high seedling hatching rate and tetraploid rate, and use tetraploid rainbow trout and diploid rainbow trout to breed and achieve stable large-scale production of rainbow trout triploids. Summary of the invention

[0004] The invention provides a method for preparing tetraploid rainbow trout seedlings.

[0005] At present, physical methods such as hydrostatic pressure method and heat shock method are commonly used to induce tetraploid rainbow trout. These methods generally have problems such as low hatching rate of fry, high malformation rate, low and unstable tetraploid rate, and cannot form large-scale production. To solve the above problems existing in the prior art, the present invention provides a method for preparing tetraploid rainbow trout using drug induction technology. This method precisely utilizes water temperature to control the fertilized eggs to develop to the metaphase of the first mitosis. At this stage, the chromosomes have completed division and are located in the center of the equatorial plate. The site for the first division is in place, and the spindle fibers develop to the peak stage and wrap the chromosomes, about to pull the chromosomes to both poles of the egg cell. At this time, the spindle fibers are decomposed using the optimal concentration of the drug, so that it cannot drag the chromosomes to both ends of the egg cell, thereby inhibiting the first mitosis of the fertilized eggs and forming a tetraploid egg cell. The process of preparing tetraploid fry is more complex than the process of preparing triploid fry by inhibiting the discharge of the second polar body of the fertilized eggs out of the egg cell. The selection of the drug concentration needs to be more precise, and the correlation between water temperature control and the starting induction time needs to be more precise.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] The present invention provides a method for preparing tetraploid rainbow trout fry, the method comprising: after fertilizing rainbow trout eggs, placing them at a water temperature of 6-10 °C for 6.2-6.4 h, and then subjecting the fertilized eggs obtained from the fertilized eggs to induction treatment with 6-dimethylaminopurine; In the induction treatment, the concentration of 6-dimethylaminopurine is 120-130 mg / L; The duration of the induction treatment is 10-18 min.

[0008] Induction treatment with 6-dimethylaminopurine can inhibit the first mitosis of fertilized eggs and achieve the preparation of tetraploid rainbow trout fry. The present invention finds that compared with physical methods such as hydrostatic pressure method and heat shock method for preparing tetraploid fry, using 6-dimethylaminopurine as an induction drug and combining with optimizing the drug concentration, induction starting time and induction duration can significantly improve the eye emergence rate of fertilized eggs and the hatching rate of fry, and can also effectively reduce the malformation rate of fry and increase the tetraploid rate. The selection of the above induction drug and the drug action concentration, induction starting time and induction duration cooperate with each other and act synergistically to jointly promote the improvement of the tetraploid induction rate and survival rate of fertilized eggs. Changing the drug action concentration, induction starting time and duration will significantly reduce the eye emergence rate of fertilized eggs, the hatching rate of fry and / or the tetraploid rate.

[0009] Preferably, 6.25-6.30 h after fertilizing rainbow trout eggs, the fertilized eggs obtained from the fertilized eggs are subjected to induction treatment with 6-dimethylaminopurine.

[0010] Preferably, the duration of the induction treatment is 10 - 15 min.

[0011] Preferably, the temperature of the induction treatment is 6 - 10 °C.

[0012] In the above method, the induction treatment is: soaking the fertilized eggs in a 6-dimethylaminopurine solution with a concentration of 120 - 130 mg / L.

[0013] During the above soaking process, the temperature of the 6-dimethylaminopurine solution is 7 - 9 °C.

[0014] The above method further includes: after the induction treatment, hatching the fertilized eggs in water at 6 - 10 °C for 40 - 45 days.

[0015] Preferably, the water body is hatching water that meets the national fishery water quality standards.

[0016] Preferably, after the induction treatment, the fertilized eggs are hatched in water at 7 - 9 °C for 40 - 45 days. After hatching, fry of rainbow trout are obtained.

[0017] In the above method, the fertilization of the eggs is in vitro fertilization.

[0018] Preferably, the in vitro fertilization adopts dry fertilization.

[0019] Preferably, well-developed rainbow trout parents are selected for egg collection and fertilization.

[0020] In some embodiments of the present invention, a method for preparing tetraploid fry of rainbow trout is provided, which includes the following steps: selecting well-developed rainbow trout parents for egg collection and fertilization; after the eggs are fertilized and placed at a water temperature of 7 - 9 °C for 6.25 - 6.30 h, the fertilized eggs are quickly transferred into a 6-dimethylaminopurine solution with a concentration of 120 - 130 mg / L for soaking, and the induction duration is 10 - 15 min; then the fertilized eggs are quickly transferred into water at 7 - 9 °C for normal hatching for 40 - 45 days to obtain tetraploid fry of rainbow trout.

[0021] In some embodiments of the present invention, a method for preparing tetraploid fry of rainbow trout is provided, which includes the following steps: selecting naturally mature and well-developed rainbow trout parents for egg collection and performing dry fertilization; under the condition of a water temperature of 7 - 9 °C (preferably 8 °C), after the eggs are fertilized for 6.25 - 6.27 h (preferably 6.25 h), the fertilized eggs are quickly transferred into a 6-dimethylaminopurine solution with a concentration of 120 - 130 mg / L for soaking; the induction duration is 14 - 15 min (preferably 15 min); then the fertilized eggs are quickly transferred into water at 7 - 9 °C (preferably 8 °C) for normal hatching for 40 - 45 days to obtain tetraploid fry of rainbow trout.

[0022] In the above method, after inducing the rainbow trout tetraploid fry, techniques such as chromosome karyotype analysis and flow cytometry DNA content determination are used to measure the chromosome ploidy of the above fish bodies, and the rainbow trout tetraploid fry are identified and screened for cultivation.

[0023] The present invention also provides the application of the above-mentioned method for preparing rainbow trout tetraploid fry in the preparation of tetraploid rainbow trout or in the preparation of triploid rainbow trout from the tetraploid rainbow trout prepared by using the method.

[0024] The beneficial effects of the present invention at least include: The present invention uses 6-dimethylaminopurine to induce rainbow trout fertilized eggs to prepare tetraploid rainbow trout. Compared with other methods, it has the advantages of simple equipment, short treatment time, stable effect, high fertilized egg eyed rate (up to about 65%), high fry hatching rate (i.e., survival rate) (up to more than 90%), high tetraploid rate (up to 25%-30%) and low malformation rate. The fertilized egg eyed rate and fry hatching rate are not much different from the normal rainbow trout breeding indicators. The tetraploid rate of the fry reaches the design requirements, and then a mature and stable high-quality fry production system for tetraploid rainbow trout is established, which is beneficial to the large-scale production of tetraploid rainbow trout populations and can obtain tetraploid rainbow trout fry in batches. The tetraploid fry obtained by using the method of the present invention are screened and then intensively cultivated. After being cultivated to sexual maturity, they form a tetraploid reserve parent population, which is bred with normal diploid rainbow trout parents, laying a foundation for the subsequent stable and large-scale production of fry with a triploid rate of 100%, and laying a foundation for solving the problem of the source of triploid fry of salmonid fish, and having high economic benefits and market value. Specific Embodiments

[0025] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0026] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art.

[0027] The rainbow trout parents used in the present invention are provided by the Fisheries Research Institute of Beijing Academy of Agriculture and Forestry Sciences.

[0028] In the following examples, the calculation formula for the hatching rate of fertilized eggs with visible eyes is: Hatching rate of fertilized eggs with visible eyes (%) = Number of fertilized eggs with visible eyes / Total number of fertilized eggs × 100%; the calculation formula for the hatching rate of fry is: Hatching rate of fry (%) = Number of hatched fry / Number of fertilized eggs with visible eyes × 100%; the calculation formula for the malformation rate of fry is: Malformation rate of fry (%) = Number of malformed fish / Number of hatched fry × 100%.

[0029] Example 1 Method for preparing tetraploid fry of rainbow trout (1) This example provides a method for preparing tetraploid fry of rainbow trout, which includes the following steps: Select well-developed rainbow trout parents for egg collection, and dry fertilization is used for the fish eggs. First, squeeze the eggs of mature rainbow trout females into a clean porcelain basin by the extrusion method, with every 4 female fish as a group; then squeeze the semen of 2 male fish into a water basin, stir evenly immediately, add hatching water with a volume ratio of semen to hatching water of 1:3000, stir gently, pour out the water after 1.5 minutes, repeat 4 times, wash the semen clean, and place it at a water temperature of 8°C to wait for induction treatment.

[0030] At a water temperature of 8°C, 6.25 hours after egg fertilization, quickly transfer the fertilized eggs into a prepared 6-dimethylaminopurine solution with a concentration of 120 mg / L for soaking; the induction duration is 15 minutes; then quickly transfer the fertilized eggs into an 8°C water body for normal hatching.

[0031] After about 42 days, fry hatch out. During this period, the hatching rate of fertilized eggs with visible eyes is 65%, the hatching rate of fry is 91%, the malformation rate of fry is 0.5%, and the tetraploid rate is 25% after the fry start feeding, detected by a flow cytometer.

[0032] Example 2 Method for preparing tetraploid fry of rainbow trout (2) This example provides a method for preparing tetraploid fry of rainbow trout, which includes the following steps: Select well-developed rainbow trout parents for egg collection, and dry fertilization is used for the fish eggs. First, squeeze the eggs of mature rainbow trout females into a clean porcelain basin by the extrusion method, with every 4 female fish as a group; then squeeze the semen of 2 male fish into a water basin, stir evenly immediately, add hatching water with a volume ratio of semen to hatching water of 1:3000, stir gently, pour out the water after 1.5 minutes, repeat 4 times, wash the semen clean, and place it at a water temperature of 8°C to wait for induction treatment.

[0033] At a water temperature of 8°C, 6.25 hours after egg fertilization, quickly transfer the fertilized eggs into a prepared 6-dimethylaminopurine solution with a concentration of 130 mg / L for soaking; the induction duration is 15 minutes; then quickly transfer the fertilized eggs into an 8°C water body for normal hatching.

[0034] The fry hatch after about 43 days. During this period, the eye emergence rate of the fertilized eggs is 63%, the fry hatching rate is 90%, the malformation rate of the fry is 1%, and the tetraploid rate detected by flow cytometry after the fry start feeding is 27%.

[0035] Example 3 Method for preparing tetraploid fry of rainbow trout (3) This example provides a method for preparing tetraploid fry of rainbow trout, which includes the following steps: Select well-developed rainbow trout parents for egg collection, and dry fertilization is used for the fish eggs. First, squeeze the eggs of the mature rainbow trout female fish into a clean porcelain basin by the extrusion method, with every 4 female fish as a group; then squeeze the semen of 2 male fish into the water basin, stir evenly immediately, add hatching water with a volume ratio of semen to hatching water of 1:3000, stir gently, pour out the water after 1.5 minutes, repeat 4 times, wash the semen clean, and place it at a water temperature of 8°C to wait for induction treatment.

[0036] At a water temperature of 8°C, 6.40 hours after egg fertilization, quickly transfer the fertilized eggs into a prepared 6-dimethylaminopurine solution with a concentration of 130 mg / L for soaking; the induction duration is 15 minutes; then quickly transfer the fertilized eggs into an 8°C water body for normal hatching.

[0037] The fry hatch after about 43 days. During this period, the eye emergence rate of the fertilized eggs is 41%, the fry hatching rate is 90%, the malformation rate of the fry is 1%, and the tetraploid rate detected by flow cytometry after the fry start feeding is 15%.

[0038] Example 4 Method for preparing tetraploid fry of rainbow trout (4) This example provides a method for preparing tetraploid fry of rainbow trout, which includes the following steps: Select well-developed rainbow trout parents for egg collection, and dry fertilization is used for the fish eggs. First, squeeze the eggs of the mature rainbow trout female fish into a clean porcelain basin by the extrusion method, with every 4 female fish as a group; then squeeze the semen of 2 male fish into the water basin, stir evenly immediately, add hatching water with a volume ratio of semen to hatching water of 1:3000, stir gently, pour out the water after 1.5 minutes, repeat 4 times, wash the semen clean, and place it at a water temperature of 8°C to wait for induction treatment.

[0039] At a water temperature of 8°C, 6.25 hours after egg fertilization, quickly transfer the fertilized eggs into a prepared 6-dimethylaminopurine solution with a concentration of 130 mg / L for soaking; the induction duration is 18 minutes; then quickly transfer the fertilized eggs into an 8°C water body for normal hatching.

[0040] After about 43 days, fry hatched. During this period, the eye emergence rate of fertilized eggs was 41%, the hatching rate of fry was 90%, the malformation rate of fry was 1.5%, and the tetraploid rate detected by flow cytometry after the fry started feeding was 15%.

[0041] Example 5 Method for preparing tetraploid fry of rainbow trout (5) This example provides a method for preparing tetraploid fry of rainbow trout, which includes the following steps: Select well-developed rainbow trout parents for egg collection, and fertilize the eggs by dry method. First, use the extrusion method to squeeze the eggs of mature rainbow trout female fish into a clean porcelain basin, with every 4 female fish as a group; then squeeze the semen of 2 male fish into the basin, stir evenly immediately, add hatching water with a volume ratio of semen to hatching water of 1:3000, stir gently, pour out the water after 1.5 minutes, repeat 4 times, wash the semen clean, and place it at a water temperature of 8°C to wait for induction treatment.

[0042] At a water temperature of 8°C, 6.25 hours after egg fertilization, quickly transfer the fertilized eggs into a prepared 6-dimethylaminopurine solution with a concentration of 120 mg / L for soaking; the induction duration is 10 minutes; then quickly transfer the fertilized eggs into an 8°C water body for normal hatching.

[0043] After about 42 days, fry hatched. During this period, the eye emergence rate of fertilized eggs was 66%, the hatching rate of fry was 90%, the malformation rate of fry was 0.4%, and the tetraploid rate detected by flow cytometry after the fry started feeding was 15%.

[0044] Example 6 Method for preparing tetraploid fry of rainbow trout (6) This example provides a method for preparing tetraploid fry of rainbow trout, which includes the following steps: Select well-developed rainbow trout parents for egg collection, and fertilize the eggs by dry method. First, use the extrusion method to squeeze the eggs of mature rainbow trout female fish into a clean porcelain basin, with every 4 female fish as a group; then squeeze the semen of 2 male fish into the basin, stir evenly immediately, add hatching water with a volume ratio of semen to hatching water of 1:3000, stir gently, pour out the water after 1.5 minutes, repeat 4 times, wash the semen clean, and place it at a water temperature of 8°C to wait for induction treatment.

[0045] At a water temperature of 8°C, 6.0 hours after egg fertilization, quickly transfer the fertilized eggs into a prepared 6-dimethylaminopurine solution with a concentration of 120 mg / L for soaking; the induction duration is 15 minutes; then quickly transfer the fertilized eggs into an 8°C water body for normal hatching.

[0046] After about 42 days, fry are hatched. During this period, the eye emergence rate of fertilized eggs is 58%, the hatching rate of fry is 92%, the malformation rate of fry is 0.5%, and the tetraploid rate detected by flow cytometry after the fry start feeding is 5%.

[0047] Example 7 Method for preparing tetraploid fry of rainbow trout (7) This example provides a method for preparing tetraploid fry of rainbow trout, which includes the following steps: Select well-developed rainbow trout parents for egg collection, and dry fertilization is used for the fish eggs. First, squeeze the eggs of mature rainbow trout female fish into a clean porcelain basin by extrusion method, with every 4 female fish as a group; then squeeze the semen of 2 male fish into the water basin, immediately stir evenly, add hatching water with a volume ratio of semen to hatching water of 1:3000, stir gently, pour out the water after 1.5 minutes, repeat 4 times, wash the semen clean, and place it under the condition of water temperature of 8°C waiting for induction treatment.

[0048] Under the condition of water temperature of 8°C, 6.25 hours after the eggs are fertilized, quickly transfer the fertilized eggs into a prepared 6-dimethylaminopurine solution with a concentration of 150 mg / L for soaking; the induction duration is 15 minutes; then quickly transfer the fertilized eggs into the water body at 8°C for normal hatching.

[0049] After about 42 days, fry are hatched. During this period, the eye emergence rate of fertilized eggs is 50%, the hatching rate of fry is 78%, the malformation rate of fry is 3%, and the tetraploid rate detected by flow cytometry after the fry start feeding is 30%.

[0050] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various examples of the present invention.

Claims

1. A method for preparing tetraploid rainbow trout seedlings, characterized in that: The method comprises: placing rainbow trout eggs in water at 6-10°C for 6.2-6.4 hours after fertilization, and then inducing the fertilized eggs obtained by fertilizing the eggs with 6-dimethylaminopurine; In the induction treatment, the concentration of 6-dimethylaminopurine is 120-130 mg / L; The duration of the induction treatment is 10-18 minutes.

2. The method for preparing tetraploid rainbow trout seedlings according to claim 1, characterized in that: 6.25-6.30 hours after the rainbow trout eggs are fertilized, the fertilized eggs obtained by fertilizing the eggs are induced with 6-dimethylaminopurine.

3. The method for preparing tetraploid rainbow trout seedlings according to claim 1 or 2, characterized in that: The duration of the induction treatment is 10-15 minutes.

4. The method for preparing tetraploid rainbow trout seedlings according to any one of claims 1 to 3, characterized in that: The temperature of the induction treatment is 6-10°C.

5. The method for preparing tetraploid rainbow trout seedlings according to any one of claims 1 to 4, characterized in that: The induction treatment is: immersing the fertilized eggs in a 6-dimethylaminopurine solution with a concentration of 120-130 mg / L.

6. The method for preparing tetraploid rainbow trout seedlings according to any one of claims 1 to 5, characterized in that: The method further comprises: after the induction treatment, incubating the fertilized eggs in water at 6-10° C. for 40-45 days.

7. The method for preparing tetraploid rainbow trout seedlings according to any one of claims 1 to 6, characterized in that: The egg fertilization is in vitro fertilization.

8. The method for preparing tetraploid rainbow trout seedlings according to claim 7, characterized in that: The in vitro fertilization adopts dry fertilization.

9. Use of the method for preparing tetraploid rainbow trout seedlings according to any one of claims 1 to 8 in the preparation of tetraploid rainbow trout or in the preparation of triploid rainbow trout using the tetraploid rainbow trout prepared by the method.