A method for preparing rainbow trout tetraploid fry

Abstract

The present application relates to the field of aquatic organism breeding technology, and particularly relates to a method for preparing rainbow trout tetraploid fry. The method provided by the present application comprises the following steps: obtaining fertilized eggs by dry fertilization of naturally matured rainbow trout parents, inducing the rainbow trout fertilized eggs to obtain tetraploid rainbow trout after 6.2-6.4 hours, and the duration of the induction is 10-18 minutes; after the tetraploid rainbow trout fry is obtained by induction, the chromosome ploidy of the fish is determined by using techniques such as chromosome karyotype analysis and flow cytometry DNA content determination, and the tetraploid rainbow trout fry is identified and screened for cultivation. The present application adopts a drug induction method, and controls the starting time and duration of the induction to inhibit the first mitosis time of the fertilized eggs, thereby improving the induction rate and survival rate of the tetraploid rainbow trout, and the induction rate of the tetraploid rainbow trout can reach 25-30%, and the survival rate of the fry can reach more than 90%.

Description

Technical Field

[0001] This invention relates to the field of aquatic organism breeding technology, and in particular to a method for preparing tetraploid rainbow trout seedlings. Background Technology

[0002] Rainbow trout are an important economic fish species, named for the iridescent stripes along their lateral lines in adult individuals. Native to North America and the Pacific coast of the Russian Far East, they belong to the order Salmoniformes, family Salmonidae, and genus *Oncorhynchus*. They primarily inhabit springs and streams with temperatures below 20°C and require high water quality for aquaculture. Dissolved oxygen levels are typically maintained above 6 mg / L, and the pH value between 6.5 and 8. Rainbow trout meat is characterized by high protein, low cholesterol, and high levels of vitamins and trace elements.

[0003] Polyploid breeding of fish is an important cell engineering breeding method. Sterile triploid fish can use all the energy available for reproduction to grow their somatic cells, avoiding slow growth during gonadal development and maturation, as well as declining meat quality at sexual maturity. Mature triploid individuals have higher survival rates and disease resistance than diploids, giving them significant economic value. Rainbow trout triploids cannot reproduce normally, and each generation of triploid fry requires artificial induction. However, the technology for directly preparing triploid fry is still immature, with unstable survival rates for each batch and an unreliable triploidity rate, hindering the application and promotion of triploid fish. In contrast, tetraploid rainbow trout reproductive cells contain an even number of chromosome sets and are fertile. Fertilization of mature tetraploid parents with normal diploid parents can stably and on a large scale produce fry with a 100% triploidity rate. Therefore, it is necessary to develop efficient and stable rainbow trout tetraploid preparation technology with high hatching rates and tetraploidity rates, using tetraploid rainbow trout to breed with diploid rainbow trout to achieve stable, large-scale production of rainbow trout triploids. Summary of the Invention

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

[0005] Currently, the induction of tetraploid rainbow trout is mainly achieved using physical methods such as hydrostatic pressure and heat shock. These methods generally suffer from low hatching rates, high deformity rates, and low and unstable tetraploid rates, making large-scale production impossible. To address these issues in existing technologies, this invention provides a method for preparing tetraploid rainbow trout using drug-induced technology. This method precisely controls the development of fertilized eggs to metaphase I of mitosis by utilizing water temperature. At this stage, chromosomes have completed division and are located at the center of the equatorial plate, the site for the first division is in place, and the spindle fibers have reached their peak development, enveloping the chromosomes and about to pull them to the two poles of the egg cell. At this point, the optimal concentration of drug is used to break down the spindle fibers, preventing them from pulling the chromosomes to the two ends of the egg cell, thereby inhibiting the first mitosis of the fertilized egg and forming a tetraploid egg cell. Compared with the process of inhibiting the expulsion of the second polar body of the fertilized egg from the body during the preparation of triploid seedlings, the tetraploid seedling preparation process is more complex, requiring more precise selection of drug concentration and more precise correlation between water temperature control and the initiation time.

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

[0007] This invention provides a method for preparing tetraploid rainbow trout seedlings, the method comprising: placing rainbow trout eggs in water at a temperature of 6-10℃ for 6.2-6.4 hours after fertilization, and then inducing the fertilized eggs obtained by fertilization with 6-dimethylaminopurine;

[0008] In the induction treatment, the concentration of 6-dimethylaminopurine is 120-130 mg / L;

[0009] The duration of the induction treatment is 10-18 minutes.

[0010] Induction treatment with 6-dimethylaminopurine can inhibit the first mitosis of fertilized eggs, thus enabling the preparation of tetraploid rainbow trout fry. This invention reveals that, compared to physical methods such as hydrostatic pressure and heat shock for preparing tetraploid fry, using 6-dimethylaminopurine as an inducing agent, combined with optimized drug concentration, induction start time, and induction duration, can significantly improve the eye-forming rate of fertilized eggs and the hatching rate of fry, while also effectively reducing the fry deformity rate and increasing the tetraploidity rate. The selection of the inducing agent and the synergistic effect of its concentration, induction start time, and induction duration work together to promote the tetraploidity induction rate and survival rate of fertilized eggs. Changing the drug concentration and the induction start time and duration significantly reduces the eye-forming rate of fertilized eggs, the hatching rate of fry, and / or the tetraploidity rate.

[0011] Preferably, 6.25-6.30 hours after fertilization of rainbow trout eggs, the fertilized eggs are induced with 6-dimethylaminopurine.

[0012] Preferably, the duration of the induction treatment is 10-15 minutes.

[0013] Preferably, the temperature of the induction treatment is 6-10℃.

[0014] In the above method, the induction treatment is as follows: the fertilized eggs are soaked in a 6-dimethylaminopurine solution with a concentration of 120-130 mg / L.

[0015] During the above soaking process, the temperature of the 6-dimethylaminopurine solution is 7-9°C.

[0016] The above method also includes: after the induction treatment, incubating the fertilized eggs in water at 6-10℃ for 40-45 days.

[0017] Preferably, the water body is hatching water that meets national fishery water quality standards.

[0018] Preferably, after the induction treatment, the fertilized eggs are incubated in water at 7-9°C for 40-45 days. After incubation, rainbow trout larvae are obtained.

[0019] In the above method, the fertilization of the egg is in vitro fertilization.

[0020] Preferably, the in vitro fertilization is performed using the dry fertilization method.

[0021] Preferably, well-developed rainbow trout parents are selected for egg collection and fertilization.

[0022] In some embodiments of the present invention, a method for preparing tetraploid rainbow trout fry is provided, comprising the following steps: selecting well-developed rainbow trout parents for egg collection and fertilization; after fertilization, the eggs are placed in water at a temperature of 7-9℃ for 6.25-6.30 hours, and then the fertilized eggs are quickly transferred to a 120-130 mg / L 6-dimethylaminopurine solution for immersion, with an induction duration of 10-15 minutes; subsequently, the fertilized eggs are quickly transferred to water at 7-9℃ for normal incubation for 40-45 days to obtain tetraploid rainbow trout fry.

[0023] In some embodiments of the present invention, a method for preparing tetraploid rainbow trout fry is provided, comprising the following steps: selecting naturally mature and well-developed rainbow trout parents for egg collection and dry fertilization; under water temperature of 7-9℃ (preferably 8℃), after fertilization of the eggs for 6.25-6.27 h (preferably 6.25 h), the fertilized eggs are rapidly transferred into a 120-130 mg / L 6-dimethylaminopurine solution for soaking; the induction duration is 14-15 min (preferably 15 min); then the fertilized eggs are rapidly transferred into water at 7-9℃ (preferably 8℃) for normal incubation for 40-45 days to obtain tetraploid rainbow trout fry.

[0024] In the above method, after obtaining tetraploid rainbow trout seedlings through induction, the chromosome ploidy of the fish is determined by techniques such as chromosome karyotype analysis and flow cytometry DNA content determination, and tetraploid rainbow trout seedlings are identified and screened for cultivation.

[0025] The present invention also provides the application of the above-described method for preparing tetraploid rainbow trout seedlings in the preparation of tetraploid rainbow trout or in the preparation of triploid rainbow trout from tetraploid rainbow trout prepared by the method.

[0026] The beneficial effects of this invention include at least the following: This invention utilizes 6-dimethylaminopurine to induce tetraploid rainbow trout fertilized eggs to prepare tetraploid rainbow trout. Compared with other methods, it has advantages such as simple equipment, short processing time, stable effect, high eye development rate of fertilized eggs (up to about 65%), high hatching rate (i.e., survival rate) of seedlings (up to over 90%), high tetraploidity rate (up to 25%-30%), and low deformity rate. The eye development rate of fertilized eggs and the hatching rate of seedlings are not much different from the normal rainbow trout reproductive indicators. The tetraploidity rate of seedlings meets the design requirements, thereby establishing a mature and stable high-quality tetraploid rainbow trout seedling production system, which is conducive to the large-scale production of tetraploid rainbow trout populations and can obtain tetraploid rainbow trout seedlings in batches. The tetraploid seedlings obtained using the method of this invention are screened and then further cultivated until sexual maturity to form a tetraploid reserve parent population. This population is then bred with normal diploid rainbow trout parents, laying the foundation for the subsequent stable and large-scale production of seedlings with a 100% triploid rate. This also lays the foundation for solving the problem of the source of triploid salmon and trout seedlings, and has high economic benefits and market value. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art.

[0029] The rainbow trout parent fish used in this invention were provided by the Fisheries Research Institute of Beijing Academy of Agricultural and Forestry Sciences.

[0030] In the following embodiments, the formula for calculating the fertilized egg eye development rate is: fertilized egg eye development rate (%) = number of eye-developing eggs / total number of fertilized eggs × 100%; the formula for calculating the seedling hatching rate is: seedling hatching rate (%) = number of hatched seedlings / number of eye-developing eggs × 100%; the formula for calculating the seedling deformity rate is: seedling deformity rate (%) = number of deformed fish / number of hatched seedlings × 100%.

[0031] Example 1: Preparation method of tetraploid rainbow trout seedlings (1)

[0032] This embodiment provides a method for preparing tetraploid rainbow trout seedlings, which includes the following steps:

[0033] Select well-developed rainbow trout broodstock for egg collection, and use dry fertilization. First, squeeze the mature rainbow trout females into a clean porcelain basin using the squeezing method, with 4 females per group; then squeeze the semen of 2 males into a water basin, stir immediately, add incubation water at a volume ratio of 1:3000 (semen to incubation water), stir gently, pour out the water after 1.5 minutes, repeat 4 times to clean the semen, and place at a water temperature of 8℃ for induction treatment.

[0034] At a water temperature of 8℃, after 6.25 hours of fertilization, the fertilized eggs were quickly transferred to a prepared 120 mg / L solution of 6-dimethylaminopurine for immersion; the induction period was 15 minutes; after that, the fertilized eggs were quickly transferred to water at 8℃ for normal incubation.

[0035] After about 42 days of hatching, the fry were observed to have an eye development rate of 65%, a hatching rate of 91%, and a deformity rate of 0.5%. After hatching, the tetraploid rate of the fry was detected by flow cytometry to be 25%.

[0036] Example 2: Preparation method of tetraploid rainbow trout seedlings (2)

[0037] This embodiment provides a method for preparing tetraploid rainbow trout seedlings, which includes the following steps:

[0038] Select well-developed rainbow trout broodstock for egg collection, and use dry fertilization. First, squeeze the mature rainbow trout females into a clean porcelain basin using the squeezing method, with 4 females per group; then squeeze the semen of 2 males into a water basin, stir immediately, add incubation water at a volume ratio of 1:3000 (semen to incubation water), stir gently, pour out the water after 1.5 minutes, repeat 4 times to clean the semen, and place at a water temperature of 8℃ for induction treatment.

[0039] At a water temperature of 8℃, 6.25 hours after fertilization, the fertilized eggs were quickly transferred into a prepared 130 mg / L solution of 6-dimethylaminopurine for immersion; the induction period was 15 minutes; after that, the fertilized eggs were quickly transferred into water at 8℃ for normal incubation.

[0040] After about 43 days of hatching, the fry were observed to have an eye development rate of 63% during this period, a hatching rate of 90%, a malformation rate of 1%, and a tetraploid rate of 27% after the fry opened, as determined by flow cytometry.

[0041] Example 3: Preparation method of tetraploid rainbow trout seedlings (3)

[0042] This embodiment provides a method for preparing tetraploid rainbow trout seedlings, which includes the following steps:

[0043] Select well-developed rainbow trout broodstock for egg collection, and use dry fertilization. First, squeeze the mature rainbow trout females into a clean porcelain basin using the squeezing method, with 4 females per group; then squeeze the semen of 2 males into a water basin, stir immediately, add incubation water at a volume ratio of 1:3000 (semen to incubation water), stir gently, pour out the water after 1.5 minutes, repeat 4 times to clean the semen, and place at a water temperature of 8℃ for induction treatment.

[0044] At a water temperature of 8℃, after 6.40 hours of fertilization, the fertilized eggs were quickly transferred to a prepared 130 mg / L solution of 6-dimethylaminopurine for immersion; the induction period was 15 minutes; after that, the fertilized eggs were quickly transferred to water at 8℃ for normal incubation.

[0045] After about 43 days of hatching, the fry were observed to have an eye development rate of 41%, a hatching rate of 90%, and a deformity rate of 1%. After hatching, the tetraploid rate of the fry was 15% as determined by flow cytometry.

[0046] Example 4: Preparation method of tetraploid rainbow trout seedlings (4)

[0047] This embodiment provides a method for preparing tetraploid rainbow trout seedlings, which includes the following steps:

[0048] Select well-developed rainbow trout broodstock for egg collection, and use dry fertilization. First, squeeze the mature rainbow trout females into a clean porcelain basin using the squeezing method, with 4 females per group; then squeeze the semen of 2 males into a water basin, stir immediately, add incubation water at a volume ratio of 1:3000 (semen to incubation water), stir gently, pour out the water after 1.5 minutes, repeat 4 times to clean the semen, and place at a water temperature of 8℃ for induction treatment.

[0049] At a water temperature of 8℃, 6.25 hours after fertilization, the fertilized eggs were quickly transferred into a prepared 130 mg / L solution of 6-dimethylaminopurine for immersion; the induction period was 18 minutes; after that, the fertilized eggs were quickly transferred into water at 8℃ for normal incubation.

[0050] After about 43 days of hatching, the fry were observed to have an eye development rate of 41% during this period, a hatching rate of 90%, a malformation rate of 1.5%, and a tetraploid rate of 15% after hatching by flow cytometry.

[0051] Example 5: Method for preparing tetraploid rainbow trout seedlings (5)

[0052] This embodiment provides a method for preparing tetraploid rainbow trout seedlings, which includes the following steps:

[0053] Select well-developed rainbow trout broodstock for egg collection, and use dry fertilization. First, squeeze the mature rainbow trout females into a clean porcelain basin using the squeezing method, with 4 females per group; then squeeze the semen of 2 males into a water basin, stir immediately, add incubation water at a volume ratio of 1:3000 (semen to incubation water), stir gently, pour out the water after 1.5 minutes, repeat 4 times to clean the semen, and place at a water temperature of 8℃ for induction treatment.

[0054] At a water temperature of 8℃, after 6.25 hours of fertilization, the fertilized eggs were quickly transferred to a prepared 120 mg / L solution of 6-dimethylaminopurine for immersion; the induction period was 10 minutes; after that, the fertilized eggs were quickly transferred to water at 8℃ for normal incubation.

[0055] After about 42 days of hatching, the fry were observed to have an eye development rate of 66% during this period, a hatching rate of 90%, a deformity rate of 0.4%, and a tetraploid rate of 15% after hatching by flow cytometry.

[0056] Example 6: Method for preparing tetraploid rainbow trout seedlings (6)

[0057] This embodiment provides a method for preparing tetraploid rainbow trout seedlings, which includes the following steps:

[0058] Select well-developed rainbow trout broodstock for egg collection, and use dry fertilization. First, squeeze the mature rainbow trout females into a clean porcelain basin using the squeezing method, with 4 females per group; then squeeze the semen of 2 males into a water basin, stir immediately, add incubation water at a volume ratio of 1:3000 (semen to incubation water), stir gently, pour out the water after 1.5 minutes, repeat 4 times to clean the semen, and place at a water temperature of 8℃ for induction treatment.

[0059] At a water temperature of 8℃, after 6.0 hours of fertilization, the fertilized eggs were quickly transferred to a prepared 120 mg / L solution of 6-dimethylaminopurine for immersion; the induction period was 15 minutes; after that, the fertilized eggs were quickly transferred to water at 8℃ for normal incubation.

[0060] After about 42 days of hatching, the fry were observed to have an eye development rate of 58%, a hatching rate of 92%, and a deformity rate of 0.5%. After hatching, the tetraploid rate of the fry was detected by flow cytometry to be 5%.

[0061] Example 7 Method for preparing tetraploid rainbow trout seedlings (7)

[0062] This embodiment provides a method for preparing tetraploid rainbow trout seedlings, which includes the following steps:

[0063] Select well-developed rainbow trout broodstock for egg collection, and use dry fertilization. First, squeeze the mature rainbow trout females into a clean porcelain basin using the squeezing method, with 4 females per group; then squeeze the semen of 2 males into a water basin, stir immediately, add incubation water at a volume ratio of 1:3000 (semen to incubation water), stir gently, pour out the water after 1.5 minutes, repeat 4 times to clean the semen, and place at a water temperature of 8℃ for induction treatment.

[0064] At a water temperature of 8℃, 6.25 hours after fertilization, the fertilized eggs were quickly transferred to a prepared 150 mg / L solution of 6-dimethylaminopurine for immersion; the induction period was 15 minutes; after that, the fertilized eggs were quickly transferred to water at 8℃ for normal incubation.

[0065] After about 42 days of hatching, the fry were observed to have an eye development rate of 50%, a hatching rate of 78%, and a deformity rate of 3%. After hatching, the tetraploid rate of the fry was detected by flow cytometry and found to be 30%.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing tetraploid rainbow trout fry, characterized in that, The method includes: placing rainbow trout eggs at a water temperature of 6-10℃ for 6.25-6.30 hours after fertilization, and then inducing the fertilized eggs with 6-dimethylaminopurine. In the induction treatment, the concentration of 6-dimethylaminopurine is 120-130 mg / L; The induction treatment temperature is 6-10℃; The duration of the induction treatment is 10-15 minutes.

2. The method for preparing tetraploid rainbow trout seedlings according to claim 1, characterized in that, The induction treatment involves immersing the fertilized eggs in a 6-dimethylaminopurine solution with a concentration of 120-130 mg / L.

3. The method for preparing tetraploid rainbow trout seedlings according to claim 1, characterized in that, The method further includes: after the induction treatment, incubating the fertilized eggs in water at 6-10℃ for 40-45 days.

4. The method for preparing tetraploid rainbow trout seedlings according to any one of claims 1 to 3, characterized in that, The fertilization of the egg was performed through in vitro fertilization.

5. The method for preparing tetraploid rainbow trout seedlings according to claim 4, characterized in that, The in vitro fertilization method used was dry fertilization.

6. The application of the method for preparing tetraploid rainbow trout seedlings according to any one of claims 1 to 5 in the preparation of tetraploid rainbow trout or in the preparation of triploid rainbow trout from tetraploid rainbow trout prepared by the method.