Method for physically inducing tetraploid of micropterus salmoides and application thereof

Abstract

This invention provides a physical induction method for tetraploid largemouth bass, belonging to the field of aquaculture genetic breeding technology. A method for inducing tetraploid largemouth bass through physical cold shock involves placing the fertilized eggs in water at 2-8°C for 3-30 minutes during the stage when the blastodisc bulges and the first cleavage is about to begin. This method uses purely physical cold shock treatment throughout, without the addition of oxytocin, meeting the requirements of green aquaculture. The entire operation is compact, with good repeatability. Under optimized parameters, the hatching rate can reach over 80%, the early tetraploid larval rate over 70%, and the seedling survival rate over 75%. This method can be applied to the large-scale breeding of triploid largemouth bass.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture genetic breeding technology, specifically involving a physical induction method for tetraploid largemouth bass. Background Technology

[0002] Triploid largemouth bass exhibit gonadal sterility, avoiding the depletion of nutrients during gonadal development. They also possess excellent traits such as rapid growth, superior meat quality, strong resistance to adverse conditions, and a short breeding cycle, making them an excellent material for largemouth bass farming. Compared to directly induced triploidy, hybridization-bred triploids offer significant advantages, including uniform spawning, high juvenile survival rates, near 100% triploid induction rate, and stable and controllable traits. Stable and breedable tetraploid largemouth bass parent stock is essential for this breeding model. Currently, the artificial induction of tetraploidy in fish in the aquaculture field primarily involves intervening in the division process of fertilized egg cells through external stress, inhibiting cytoplasmic division during the first cleavage, and promoting chromosome doubling to form tetraploids. However, chemical induction is prone to reagent residues posing food safety risks; hydrostatic induction equipment is expensive; and temperature induction results in significant embryonic hatching losses, limiting its application.

[0003] There are few existing studies on the induction of tetraploid embryos in largemouth bass using low temperatures. This is because largemouth bass fertilized eggs have thin membranes and a unique yolk structure, resulting in significant differences in low-temperature tolerance at different developmental stages. Any deviation from the optimal range in induction timing, treatment temperature, or treatment duration will lead to either extremely low tetraploid induction rates or large-scale necrosis and death of fertilized eggs. In existing published studies, most cold shock experiments only yield a small number of tetraploid embryos, with a large number dying during the larval stage, and very few reaching sexual maturity, making them unsuitable for use as breeding stock. Summary of the Invention

[0004] The purpose of this invention is to provide a method for inducing tetraploid largemouth bass through physical cold shock. By precisely implementing cold shock in the early stage of the first cleavage, a large number of stable tetraploid individuals can be obtained and cultured to sexual maturity.

[0005] This invention provides a method for inducing tetraploid largemouth bass through physical cold shock, comprising the following steps: During the stage when the fertilized eggs of largemouth bass develop to the point where the blastodisc bulges and the first cleavage is about to begin, the fertilized eggs of largemouth bass are placed in water at 2-8℃ for cold shock treatment for 3-30 minutes.

[0006] Preferably, the temperature of the cold shock treatment is 3~7°C.

[0007] Preferably, the temperature of the cold shock treatment is 4~6℃.

[0008] Preferably, the temperature of the cold shock treatment is 5~5.5℃.

[0009] Preferably, the cold shock treatment time is 8 to 20 minutes.

[0010] Preferably, the cold shock treatment time is 10~16.5 min.

[0011] Preferably, the cold shock treatment time is 13~13.5 min.

[0012] Preferably, a room-temperature control group of largemouth bass fertilized eggs is also included; In the normal temperature control group, the fertilized eggs of largemouth bass were cultured normally in a culture water temperature environment. When 60% to 80% of the fertilized eggs developed to the stage of blastodisc bulging and about to start the first cleavage, cold shock treatment was immediately carried out.

[0013] This invention provides the application of the method in improving at least one of the yield, hatching rate and seedling survival rate of largemouth bass tetraploids, in storing largemouth bass triploid breeding materials or in breeding largemouth bass triploids.

[0014] This invention provides a method for large-scale breeding of triploid largemouth bass, comprising the following steps: The tetraploid and diploid largemouth bass obtained by the aforementioned method were naturally fertilized to obtain fertilized eggs, which were then incubated and raised to produce triploid largemouth bass.

[0015] This invention provides a method for inducing tetraploidity in largemouth bass using physical cold shock. During the development of largemouth bass fertilized eggs to the stage where the blastodisc bulges and the first cleavage is about to begin, the fertilized eggs are placed in water at 2–8°C for cold shock treatment for 3–30 minutes. This invention takes advantage of the fact that largemouth bass cannot be artificially induced to spawn and that fertilization occurs naturally. The entire process uses purely physical cold shock treatment, without the addition of oxytocin or chemical mutagen, resulting in seedlings with no drug residues and high product safety for consumption, meeting the requirements of green aquaculture. The entire operation is compact, with rapid integration from egg collection to group induction, good process repeatability, and under optimized parameters, a hatching rate of over 80%, an early tetraploidity rate of over 70%, and a seedling survival rate of over 75%. Furthermore, it only requires conventional constant temperature refrigeration equipment, resulting in low purchase and maintenance costs and a low operational threshold, suitable for both laboratory breeding experiments and large-scale aquaculture seedling production. The tetraploid largemouth bass bred can develop normally to sexual maturity. The tetraploid parent and diploid parent can be crossed to stably breed fully triploid offspring, solving the industry pain point of uneven emergence and low survival rate of directly induced triploids from the source.

[0016] This invention further limits the temperature and time of cold shock treatment. Cold shock treatment at 4~6℃ for 10~16.5min can greatly improve the yield of tetraploids, while ensuring the hatching rate and fry survival rate. It has the characteristics of simple method, easy operation and good repeatability. Attached Figure Description

[0017] Figure 1 and Figure 2 To obtain partial results of tetraploid ploidy in largemouth bass using flow cytometry; Figures 3-5 The results of optimizing cold shock process parameters using the response surface method; Figure 6 For flow cytometry analysis of triploid ploidy in hybrid offspring; Figure 7 A bar chart comparing the growth performance of triploid and diploid largemouth bass at 180 days. Figure 8 A bar chart comparing sex hormone levels in diploid and triploid largemouth bass; Figure 9 A comparison of the gonads of triploid and diploid largemouth bass. Detailed Implementation

[0018] This invention provides a method for inducing tetraploid largemouth bass through physical cold shock, comprising the following steps: During the stage when the fertilized eggs of largemouth bass develop to the point where the blastodisc bulges and the first cleavage is about to begin, the fertilized eggs of largemouth bass are placed in water at 2-8℃ for cold shock treatment for 3-30 minutes.

[0019] In this invention, the method for preparing fertilized eggs of largemouth bass preferably involves culturing broodstock to obtain naturally matured broodstock with developed gonads. These broodstock are then placed in a rearing tank, with regular inspections and collection of naturally fertilized eggs. The preferred selection criteria for the broodstock are diploid largemouth bass aged 2-3 years, weighing over 550g, with intact bodies and excellent vitality. The preferred conditions for culturing broodstock are: water temperature 22-26℃, dissolved oxygen ≥5.0mg / L, feeding with a high-protein formulated feed containing ≥42% crude protein, regular daily water changes and waste removal, and culturing for 30-40 days. Palm flakes are preferably placed in the rearing tank to facilitate attachment of the eggs or fertilized eggs. Regular inspections are preferably conducted hourly.

[0020] In this invention, to ensure the continuity of the processing, the method also includes a room-temperature control group for largemouth bass fertilized eggs. The fertilized eggs in the room-temperature control group are used to observe the embryonic development status, so that the cold shock treatment of the experimental group's fertilized eggs can be carried out based on the embryonic development status of the fertilized eggs in the room-temperature control group, ensuring the continuity of the experimental group's operation. The largemouth bass fertilized eggs in the room-temperature control group are cultured normally in a culture water environment. When 60%–80% of the fertilized eggs have developed to the stage of blastodisc bulging and are about to initiate the first cleavage, cold shock treatment is immediately performed. The culture water temperature is preferably 24–26°C, and can be 25°C.

[0021] In this invention, the preferred temperature for the cold shock treatment is 3-7°C, which can be 4-6°C or 5-5.5°C. The preferred duration of the cold shock treatment is 8-20 min, which can be 10-16.5 min or 13-13.5 min. In an embodiment of this invention, a two-factor optimization experiment was conducted using a central composite experimental design (CCD) to induce tetraploidy in largemouth bass. The study found that the main effects, interaction effects, and nonlinear effects of the cold shock treatment temperature of 5.211°C and the treatment time of 13.168 min on embryonic development and tetraploidy induction efficiency were investigated. The optimal cold shock process parameters were selected to balance high tetraploidy rate, high hatching rate, and high seedling survival rate.

[0022] This invention provides the application of the method in improving at least one of the yield, hatching rate and seedling survival rate of largemouth bass tetraploids, in storing largemouth bass triploid breeding materials or in breeding largemouth bass triploids.

[0023] This invention provides a method for large-scale breeding of triploid largemouth bass, comprising the following steps: The tetraploid and diploid largemouth bass obtained by the aforementioned method were naturally fertilized to obtain fertilized eggs, which were then incubated and raised to produce triploid largemouth bass.

[0024] This invention does not impose any particular restrictions on the method of natural fertilization; any method known in the art can be used. The fertilization rate of the fertilized eggs reaches 85.2%, the hatching rate reaches 78.6%, and the survival rate of offspring fry reaches 72.3%. Triploid largemouth bass exhibits a significant advantage in size and weight compared to diploid individuals. However, it lacks gonadal tissue, testes, vas deferens, and other reproductive-related structures; its gonadal net weight is 0, and its gonadal index is 0, completely lacking the ability to produce gametes and reproduce, thus exhibiting complete infertility. Gonadal development in female triploids is also significantly inhibited, with an average gonadal index of only 1.20%, less than one-quarter of that of the diploid female control group. No individual reaches the gonadal index threshold (≥15%) for female largemouth bass sexual maturity, resulting in no effective reproductive capacity. Furthermore, hormone testing results show that female fish have lower levels of sex hormone (E2), and male fish also have lower serum testosterone levels, avoiding nutritional depletion during the breeding season and achieving rapid growth throughout the body, thus adapting to the needs of intensive aquaculture.

[0025] The following detailed description, in conjunction with embodiments, illustrates a method for physically inducing tetraploid largemouth bass provided by the present invention and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 Optimization experiment of cold shock temperature and treatment time 1. Broodstock selection and pre-spawning maturation: At the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences, diploid largemouth bass aged 2-3 years, weighing over 550g, with intact bodies free from injury or disease, and exhibiting excellent swimming vitality were selected. Male and female broodstock were mixed and cultured in a 1:1 ratio in a rearing pond with palm fronds. The rearing water temperature was 22-26℃, the dissolved oxygen in the water was ≥5.0mg / L, and they were fed a high-protein formulated feed with a crude protein content of ≥42%. The water was changed and waste was discharged daily. The maturation was promoted for 30-40 days, and the gonads were naturally developed and matured by relying on environmental regulation. No exogenous hormones were used to artificially induce spawning throughout the entire process.

[0027] 2. Regularly inspect and collect naturally fertilized fish eggs: After the gonads of the parent fish mature, inspect the spawning pond once an hour. Collect any fertilized eggs found attached to palm leaves immediately. All the collected fish eggs have been naturally fertilized in the aquaculture water.

[0028] 3. Rapid grouping of fertilized eggs: After filtering out impurities, the fertilized eggs were rapidly and randomly divided into a room temperature control group and a cold shock treatment group. The control group was placed in a culture water environment for normal culture and served as a reference for embryo development. The time spent in the static state after egg removal was shortened throughout the process, ensuring the continuity of embryo development.

[0029] 4. Pre-cleavage site-specific cold shock induction: Continuous microscopic sampling was used to observe the embryonic development of the control group. When 60%–80% of the fertilized eggs in the control group reached the critical period of blastodisc protrusion and were about to enter the first cleavage, the fertilized eggs in the treatment group were rapidly transferred to pre-cooled water at a constant temperature for cold shock treatment. Water temperature fluctuations during the treatment were controlled within ±0.5℃. The temperature gradient for cold shock treatment was set at 2℃–8℃; the time gradient for cold shock treatment was set at 3 min–30 min. The control group was cultured at room temperature (25℃) without cold shock treatment.

[0030] 5. Methods for detecting ploidy in largemouth bass. Immediately after the cold shock period for each group of largemouth bass, the fertilized eggs were transferred to aeration tanks at 23-26℃ for incubation. During incubation, whitish and necrotic eggs were routinely removed. After hatching, the larvae were raised according to the standardized largemouth bass fry rearing process. Samples were taken 2-4 days after hatching, and cellular DNA content was determined using PI staining combined with flow cytometry. The specific steps are as follows: Sample preparation: Take 20 intact larvae from each group, 2-4 days after the membrane is removed, and place them in pre-cooled PBS buffer (pH 7.4) for storage in an ice bath; Cell suspension preparation: Cut the tissue sample into small pieces, add 1 mL of PBS buffer, mix repeatedly by pipetting, filter through a 300-mesh sieve to remove tissue residue, collect the filtrate in a 1.5 mL centrifuge tube, centrifuge at 1000 r / min for 5 min, discard the supernatant, and retain the cell pellet; Cell staining: Add 1 mL of PI staining solution (containing 50 μg / mL PI, 100 μg / mL RNase A, and 0.1% Triton X-100) to the cell pellet, mix gently, and stain at 4°C in the dark for 10 min. Flow cytometry analysis: The stained cell suspension was filtered again through a 300-mesh sieve and loaded onto a flow cytometer. The fluorescence intensity of the FL2 channel was detected using an excitation wavelength of 488 nm. At least 10,000 cells were analyzed for each sample. Ploidy determination: Using the peak fluorescence intensity of diploid largemouth bass samples as a reference, the peak fluorescence intensity of tetraploid samples is twice that of diploid samples. This is used to determine the ploidy of individuals, distinguishing between diploid and tetraploid. Hatching rate, tetraploid rate, and seedling survival rate are statistically analyzed using the following formulas: Hatching rate (%) = (Total number of normal fry hatched / Total number of valid fertilized eggs participating in hatching) × 100% Seedling survival rate (%) = Total number of normal seedlings surviving at the end of the rearing period / Total number of normal larvae initially hatched × 100% Tetraploidy rate (%) = Total number of larvae identified as tetraploid / Total number of larvae tested in the same batch × 100%.

[0031] The experimental results are shown in Table 1 and Figures 1-2 As shown. Compared with the 25℃ group, cold shock treatment reduced hatching rate and seedling survival rate, but at the same treatment temperature, shorter treatment time resulted in relatively higher hatching rate and seedling survival rate. Different cold shock treatment temperatures and times had a significant impact on the tetraploid rate results: The tetraploid rates were 0.1, 0.2, and 0.1 when treated at 8℃ for 3 min, 16.5 min, and 30 min, respectively. Simultaneously, the tetraploid rate was 0.4 when treated at 7℃ for 16.5 min. The tetraploid rates were 0.6 and 0.5 when treated at 6℃ for 16.5 min and 30 min, respectively. The tetraploid rate was 0.4 when treated at 4℃ and 3℃ for 30 min. The tetraploid rate was 0.7 when treated at 5℃ for 30 min, 0.8 when treated at 16.5 min, and 0.2 when treated at 5℃ for 3 min. Simultaneously, the tetraploid rates were 0.2, 0.9, and 0.6 when treated at 2℃ for 3 min, 16.5 min, and 30 min, respectively. This indicates that the cold shock time is a crucial factor affecting the tetraploid yield; too short a treatment time is detrimental to obtaining tetraploids, while too long a treatment time also reduces the tetraploid rate.

[0032] Table 1. Results of hatching rate and seedling survival rate in the cold shock test.

[0033] Example 2 This embodiment employs a central composite experimental design (CCD) to conduct a two-factor optimization experiment on cold shock induction of tetraploid largemouth bass. It clarifies the main effects, interaction effects, and nonlinear influences of the two core factors, cold shock temperature and treatment time, on embryonic development and tetraploid induction efficiency. The optimal cold shock process parameters that balance high tetraploid rate, high hatching rate, and high seedling survival rate are selected, providing a standardized technical solution for subsequent industrial applications.

[0034] The test results are as follows Figures 3-5 As shown. With the goal of maximizing the combined weight of hatching rate, tetraploid rate, and seedling survival rate, multi-response value optimization was performed, and the optimal cold shock process parameters were obtained as follows: cold shock temperature 5.211℃, treatment time 13.168min. Under these conditions, the predicted hatching rate was 0.810, tetraploid rate was 0.701, seedling survival rate was 0.795, and the overall satisfaction (Desirability) was 0.808, which is the best overall performance among all experimental combinations.

[0035] Example 3 This embodiment verifies the reproductive performance of the tetraploid largemouth bass bred according to the present invention. By crossing tetraploid male largemouth bass with diploid female largemouth bass, large-scale breeding of fully triploid largemouth bass was achieved. The growth performance and sex hormone levels of the triploid and diploid strains were compared. The specific experiment is as follows: 1. Experimental materials Six-month-old sexually mature tetraploid male largemouth bass bred using the optimized process of Example 2 of this invention, along with healthy diploid female largemouth bass bred in the same batch, were both two years old, weighing over 600g, with intact body surfaces, excellent swimming vitality, and fully developed gonads. Their ploidy was confirmed in advance by flow cytometry. The breeding environment was consistent with the basic process of this invention: a breeding water temperature of 22–26℃, dissolved oxygen ≥5.0mg / L, feeding with a high-protein formulated feed with a crude protein content ≥42%, daily water changes and wastewater removal, and no exogenous hormones used throughout the breeding process.

[0036] 2. Test Methods Tetraploid males and diploid females were mixed at a sex ratio of 1:2 in a spawning pond with palm-strip nests. The breeding environment was kept stable, and the spawning pond was inspected once every hour. Fertilized eggs attached to the palm strips were collected immediately. All the collected fish eggs had been naturally fertilized in the breeding water. Hatching and seedling cultivation were completed according to the unified basic process of this invention to obtain hybrid offspring seedlings.

[0037] Samples were taken from hybrid offspring fry within 2-4 days after hatching. Twenty fry were randomly selected from each group. The DNA content of the cells was detected by PI staining and flow cytometry as described in Example 1 of this invention. The ploidy of the individuals was determined and the triploid rate of the offspring was calculated.

[0038] Hybrid triploid offspring and diploid largemouth bass hatched in the same batch were raised in separate ponds under completely identical culture conditions, with identical stocking density, feeding strategies, and water environment management. Samples were taken at 180 days of rearing, with 30 fish randomly selected from each group to measure body length and weight, and the differences in growth performance between triploid and diploid were compared.

[0039] At sexual maturity (180 days), diploid females, diploid males, triploid females, and triploid males were randomly selected (10 fish per group). Blood was collected from the tail vein, incubated at 4°C for 2 hours, and then centrifuged at 3000 rpm for 10 minutes to separate serum. A largemouth bass-specific ELISA kit was used to detect serum estradiol (E2) levels in females and serum testosterone (T) levels in males, comparing the differences in sex hormone levels between diploid and triploid individuals. Gonadal tissue was also collected to compare the differences in gonads and gonadal indices between diploid and triploid individuals. The gonadal index was calculated using the following formula: Gonadal Index (GSI, %) = (Net weight of gonads / Net weight of fish after visceration) × 100% Formula I 3. Test Results The tetraploid male and diploid female were crossbred, and the fertilization rate of the fertilized eggs reached 85.2%, the hatching rate reached 78.6%, and the survival rate of the offspring fry reached 72.3%. Flow cytometry ploidy identification showed that... Figure 6As shown, the triploid rate of the hybrid offspring reached 98.7%, with only a very small number of diploid individuals, achieving stable and large-scale breeding of all triploid largemouth bass.

[0040] Under the same breeding conditions, after 180 days of cultivation, if Figure 7 As shown, the triploid largemouth bass had an average weight of 452.3g, while the diploid largemouth bass of the same batch had an average weight of 301.5g, meaning the triploid weight was 1.50 times that of the diploid. The triploid had an average body length of 28.6cm, while the diploid had an average body length of 19.1cm, meaning the triploid body length was 1.50 times that of the diploid. Both the body length and weight of the triploid were significantly higher than those of the diploid. P <0.05).

[0041] like Figure 8 As shown, in female individuals, the average serum estradiol (E2) content in diploid females was 128.6 pg / mL, while the average serum estradiol content in triploid females was 42.3 pg / mL. The sex hormone levels in diploid females were significantly higher than those in triploid females. P <0.05); In male individuals, the average serum testosterone (T) content in diploid males was 215.4 ng / mL, while the average serum testosterone content in triploid males was 68.7 ng / mL. The sex hormone levels in diploid males were significantly higher than those in triploid males. P <0.05).

[0042] As shown in Table 2 and Figure 9 As shown, all male triploid individuals sampled showed no visible gonadal tissue after dissection, and lacked reproductive-related structures such as testes and vas deferens. The net weight of the gonads was 0, and the gonadal index was 0, indicating a complete lack of gamete production and reproductive capacity, resulting in complete infertility. Gonadal development in female triploids was also significantly inhibited, with an average gonadal index of only 1.20%, less than 1 / 4 of that in the diploid female control group. No individual reached the gonadal index threshold (≥15%) for sexual maturity in female largemouth bass, indicating no effective reproductive capacity.

[0043] Table 2 Comparison of gonadal indices between triploid and diploid largemouth bass

[0044] It is evident that the tetraploid male largemouth bass bred in this invention can be stably hybridized with diploid female largemouth bass to produce nearly 100% triploid offspring, solving the industry problems of uneven emergence and unstable triploidity rates in directly induced triploids. The growth performance of the hybridized triploid largemouth bass is 1.5 times that of the diploid, showing a significant growth advantage. Simultaneously, the sex hormone levels of both male and female individuals are significantly lower than those of the diploid, resulting in gonadal infertility. Figure 9 This avoids the nutritional depletion during the breeding season, allows for rapid growth throughout the entire process, and gives it excellent commercial aquaculture traits, making it perfectly suited to the industry needs of intensive largemouth bass farming.

[0045] Comparative Example 1 A gradient experiment was conducted to induce tetraploids in largemouth bass by hydrostatic pressure, clarifying the effects of two core parameters, hydrostatic pressure and treatment time, on embryonic development and tetraploid induction efficiency in largemouth bass. The comprehensive performance differences between hydrostatic pressure induction and the cold shock induction technology of this invention were compared, verifying the industrial adaptability advantages of the cold shock technology of this invention.

[0046] The experiment used largemouth bass broodstock, collected fertilized eggs, and established a basic rearing environment. Broodstock selection, fertilized egg collection, and grouping all followed the unified basic process of this invention. Hydrostatic pressure treatment was performed using a laboratory-grade hydrostatic pressure machine specifically designed for aquaculture breeding, with pressure control accuracy of ±0.5 MPa and constant temperature control of the treatment chamber water. Continuous sampling and microscopic observation of embryonic development in the control group were conducted. Once 60%–80% of the fertilized eggs in the control group reached the critical period of blastodisc protrusion and imminent first cleavage, the fertilized eggs in the treatment group were rapidly transferred to the hydrostatic pressure machine treatment chamber. Hydrostatic pressure induction was performed using different pressure and treatment time gradients, with the chamber water temperature maintained at a constant 25°C, consistent with the rearing water temperature. After hydrostatic pressure treatment, the fertilized eggs were immediately transferred to a 23–26°C micro-flow incubation tank for aeration and incubation. Incubation management, seedling rearing, ploidy identification methods, and statistical indicators fully followed the unified basic process of this invention and the testing standards of Example 1. Hatching rate, seedling survival rate, and tetraploid rate were statistically analyzed for each group.

[0047] Table 3 shows the effect of hydrostatic pressure on the induction of largemouth bass. The tetraploid rate of largemouth bass induced by hydrostatic pressure was 0, indicating that largemouth bass is not suitable for preparation using the hydrostatic pressure induction method.

[0048] Table 3 Results of core indicators of hydrostatic pressure gradient test

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for inducing tetraploid largemouth bass through physical cold shock, characterized in that, Includes the following steps: During the stage when the fertilized eggs of largemouth bass develop to the point where the blastodisc bulges and the first cleavage is about to begin, the fertilized eggs of largemouth bass are placed in water at 2-8℃ for cold shock treatment for 3-30 minutes.

2. The method according to claim 1, characterized in that, The temperature for the cold shock treatment is 3~7℃.

3. The method according to claim 1, characterized in that, The temperature for the cold shock treatment is 4~6℃.

4. The method according to claim 1, characterized in that, The temperature for the cold shock treatment is 5~5.5℃.

5. The method according to claim 1, characterized in that, The duration of the cold shock treatment is 8 to 20 minutes.

6. The method according to claim 1, characterized in that, The duration of the cold shock treatment is 10 to 16.5 minutes.

7. The method according to claim 1, characterized in that, The duration of the cold shock treatment is 13 to 13.5 minutes.

8. The method according to any one of claims 1 to 7, characterized in that, It also includes a room-temperature control group of fertilized largemouth bass eggs; In the normal temperature control group, the fertilized eggs of largemouth bass were cultured normally in a culture water temperature environment. When 60% to 80% of the fertilized eggs developed to the stage of blastodisc bulging and about to start the first cleavage, cold shock treatment was immediately carried out.

9. The application of the method according to any one of claims 1 to 8 in improving at least one of the yield, hatching rate and seedling survival rate of largemouth bass tetraploids, in storing largemouth bass triploid breeding materials or in breeding largemouth bass triploids.

10. A method for large-scale breeding of triploid largemouth bass, characterized in that, Includes the following steps: Largemouth bass tetraploids and largemouth bass diploids obtained by the method described in any one of claims 1 to 8 are naturally fertilized to obtain fertilized eggs, which are then incubated and seedlings are cultivated to obtain largemouth bass triploids.

Images