A method for efficiently inducing heteropolyploid fry

Abstract

This invention discloses a method for efficiently inducing allopolyploid fish fry, belonging to the field of fish breeding technology. The method of this invention includes the following technical steps: (1) Induction of spawning in distant hybridization parents: Selecting female and male parent fish from different subfamilies of Cyprinidae, and collecting mature eggs and semen respectively; (2) Obtaining hybrid fertilized eggs: Mixing the collected eggs with semen, and performing dry artificial insemination to obtain hybrid fertilized eggs; (3) Inducing allopolyploidy: Placing the fertilized eggs in a water bath for heat shock treatment to inhibit the expulsion of the second polar body or inhibit the first cleavage, and then quickly transferring the treated fertilized eggs to aquaculture water to continue hatching fry. The method of this invention combines the method of distant hybridization between subfamilies with the method of heat shock induction. After induction using this method, the multiplication rate of hatched fry can reach 100%, which significantly improves the yield of allopolyploid fish fry from distant hybridization.

Description

Technical Field

[0001] This invention belongs to the field of fish breeding technology, specifically relating to a method for efficiently inducing allopolyploid fish fry. Background Technology

[0002] Polyploids are organisms whose somatic cells contain three or more complete sets of chromosomes, and they are ubiquitous in animals and plants. Based on the origin of their chromosomes, polyploids can be classified into autopolyploids and allopolyploids. Although the formation rate of autopolyploids is higher than that of allopolyploids, allopolyploids, influenced by both polyploidization and heterozygosity, are considered to have a more significant advantage over autopolyploids in evolutionary processes. The formation of allopolyploids involves two important steps: interspecific hybridization and chromosome doubling. Due to the high plasticity of fish chromosomes, chromosome-level changes are easily observed in the offspring of distant hybridization, resulting in polyploid hybrid offspring. Breeding research utilizing distant hybridization to produce allopolyploid fish has broad application prospects. For example, in the third generation of distant hybridization between crucian carp (2n=100) and common carp (2n=100), a sexually fertile allotetraploid crucian carp (4n=200) was formed, providing direct evidence for the tetraploid speciation mechanism in nature. The allotetraploid Xiangyun crucian carp (3n=150), bred by crossing a tetraploid crucian carp with a diploid crucian carp, exhibits characteristics such as sterility, rapid growth, strong disease resistance, and good meat quality, generating significant social, economic, and ecological benefits after its widespread cultivation. Furthermore, distant hybridization between subfamilies of common carp and topmouth culter (2n=48) can yield allotetraploid common culter (3n=124) and allotetraploid common culter (4n=148). These allotetraploid fish possess faster growth rates and better survival rates, serving not only as valuable high-quality germplasm resources in aquaculture but also holding significant importance in research on biological evolution and biodiversity.

[0003] Although interfamilial distant hybridization often yields diploid, triploid, and tetraploid offspring in the F1 generation, the randomness of chromosome doubling during hybridization results in the vast majority (over 99%) of the hatched hybrid fry being diploid. Furthermore, because diploid hybrid fry have significantly lower viability than polyploid hybrid fry, they experience continuous mortality during rearing, leading to an extremely low overall survival rate (less than 1%) in the F1 generation and a very small number of polyploid hybrid fish ultimately obtained. The low polyploid yield from distant hybridization means that allogeneic polyploid fry obtained solely through distant hybridization are far from meeting breeding or research needs. Therefore, there is an urgent need to explore a more efficient method for producing allogeneic polyploid fish. Besides distant hybridization, artificial induction of polyploidy can also be achieved by intervening in the normal cell division process using extreme physicochemical conditions, including temperature shock (heat shock and cold shock), drug treatment (cytochalasin B, 6-dimethylaminopurine, caffeine, and colchicine, etc.), high-salt and high-alkali methods, electro-pulse methods, and hydrostatic pressure methods. Among these, temperature shock is relatively less harmful to fertilized eggs, simple to operate, and has a high induction rate, making it widely used in the induction of polyploid fish. However, current research on artificially inducing polyploidy in fish using temperature shock mainly focuses on the induction of autopolyploidy, with no evidence of allopolyploidy induction, and even less evidence of artificial allopolyploidy in fish induced by combining interfamilial distant hybridization with temperature shock. Furthermore, the induction effect of temperature shock is affected by multiple factors such as temperature, treatment timing, and duration. The sensitivity of fertilized eggs to temperature is also closely related to their genetic background and sperm / egg maturity. Induction conditions vary greatly among different fish species, making allopolyploidy induction based on temperature shock even more challenging. Therefore, the induction technology for allogeneic polyploid fish that combines distant hybridization and temperature shock methods urgently needs to be developed.

[0004] This invention, by combining temperature shock and distant hybridization between fish subfamilies, has pioneered a new and efficient method for inducing allopolyploid fish fry. Using this method to induce allopolyploid fish fry, the ploidization rate of hatched fry can reach 100%, and the polyploid yield is much higher than that of the single distant hybridization method. It has important application value in fish distant hybridization, polyploid breeding, and evolutionary biology research. Summary of the Invention

[0005] To address the above problems, this invention provides a method for efficiently inducing allopolyploid fish fry, which can induce a large number of allotriploid and allotetraploid fish fry, significantly improving the polyploidization rate compared with traditional methods, and the induction results are stable.

[0006] This invention is achieved through the following technical solution:

[0007] A method for efficiently inducing allopolyploid fish fry includes the following steps:

[0008] (1) Inducing spawning of distant hybrid parents: Select female fish with obvious sexual maturity characteristics and good physical condition from different subfamilies of Cyprinidae fish as maternal parents, and male fish that can squeeze out milky white sperm as paternal parents, and collect mature eggs and sperm respectively.

[0009] (2) Obtaining hybrid fertilized eggs: The collected eggs are mixed with semen and artificially inseminated using the dry method. After being gently stirred and mixed evenly, the mixture is spread evenly in a container with aquaculture water and shaken slowly to fertilize the eggs and obtain hybrid fertilized eggs.

[0010] (3) Inducing allopolyploids: Fertilized eggs are placed in a water bath for heat shock treatment to inhibit the expulsion of the second polar body or inhibit the first cleavage. Then, the treated fertilized eggs are quickly transferred to aquaculture water to continue hatching into fry.

[0011] Furthermore, in step (3), after the second polar body is suppressed, the fertilized egg continues to hatch and then an allogeneic triploid fry is obtained; after the first cleavage is suppressed, the fertilized egg continues to hatch and then an allogeneic tetraploid fry is obtained.

[0012] Further, in step (3), the specific operation of inhibiting the expulsion of the second polar body is as follows: placing the fertilized egg in a 40-41°C water bath for heat shock treatment for 1-2 minutes after fertilization (0-1 minutes); or placing the fertilized egg in a 40-41°C water bath for heat shock treatment for 2-3 minutes after fertilization (5 minutes); or placing the fertilized egg in a 40-41°C water bath for heat shock treatment for 1-2 minutes after fertilization (8 minutes).

[0013] Further, in step (3), the specific operation of inhibiting the expulsion of the second polar body is as follows: the fertilized egg is placed in a water bath at 40-41°C for 2 minutes after fertilization, 0-1 minutes after fertilization, for heat shock treatment.

[0014] Further, in step (3), the specific operation of inhibiting the first cleavage is as follows: 30 min after fertilization, the fertilized egg is placed in a water bath at 40-41°C for heat shock treatment for 2-3 min; or 50 min after fertilization, the fertilized egg is placed in a water bath at 40-41°C for heat shock treatment for 1 min.

[0015] Further, in step (3), the specific operation of inhibiting the first cleavage is as follows: 30 min after fertilization, the fertilized egg is placed in a water bath at 40-41°C for 2 min of heat shock treatment; or 50 min after fertilization, the fertilized egg is placed in a water bath at 40-41°C for 1 min of heat shock treatment.

[0016] Further, in step (1), the mother parent fish is a female carp and the father parent fish is a male mandarin fish.

[0017] Further, in step (1), the method for collecting mature eggs and semen is as follows:

[0018] a. Artificial spawning induction: The pectoral fin base injection method is used. The female broodstock is injected with 14-16 μg / kg luteinizing hormone-releasing hormone A2, 700-800 IU / kg human chorionic gonadotropin and 2.5-3 mg / kg domperidone. The male broodstock is injected with 7-8 μg / kg luteinizing hormone-releasing hormone A2 and 350-400 IU / kg human chorionic gonadotropin. The injection dosage is based on the weight of the broodstock. The broodstock injected with spawning induction hormones are placed in the culture water. The effect time is 10-16 hours.

[0019] b. Sperm and egg collection: After the effective time is reached, that is, when the abdomen is gently pressed and eggs flow smoothly out of the female parent fish’s genital opening, and the male parent fish can squeeze out milky white semen, sperm and egg collection is carried out. When collecting, gently press the abdomen of the female parent fish to squeeze the eggs into a clean container. Collect the male parent fish’s semen in the same way. Both eggs and semen should be protected from direct sunlight.

[0020] Furthermore, the female parent fish were injected with 15 μg / kg luteinizing hormone-releasing hormone A2, 800 IU / kg human chorionic gonadotropin and 3 mg / kg domperidone, and the male parent fish were injected with 7.5 μg / kg luteinizing hormone-releasing hormone A2 and 400 IU / kg human chorionic gonadotropin.

[0021] Furthermore, in step (2), the volume ratio of the egg to the semen is 90 to 100:1.

[0022] The temperature of the aquaculture water used in the method of this invention is 22-25℃.

[0023] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0024] This invention establishes a highly efficient method for inducing allopolyploid fish fry through a combination of interfamilial distant hybridization and heat shock. The specific methods and conditions for inhibiting the expulsion of the second polar body and the first cleavage in fertilized eggs induced by heat shock were determined, successfully inducing allotriploid and allotetraploid fry with a triploid and tetraploid rate of 100%, significantly improving the yield of allopolyploid fish fry. The method for inducing allopolyploid fish fry provided by this invention has a clear process, is simple to operate, and is highly efficient, possessing significant application value in fish distant hybridization, polyploid breeding, and evolutionary biology research. Attached Figure Description

[0025] Figure 1This is a graph showing the relative DNA content of allodiploid carp fry in Example 3.

[0026] Figure 2 This is a graph showing the relative DNA content of allogeneic triploid carp fry in Example 3.

[0027] Figure 3 This is a graph showing the relative DNA content of allotetraploid carp fry in Example 3.

[0028] Figure 4 This is a phase diagram of metaphase division of the embryonic chromosomes of the allodiploid carp in Example 3.

[0029] Figure 5 This is a phase diagram of the metaphase division of the embryonic chromosomes of the allotriploid carp in Example 3.

[0030] Figure 6 This is a phase diagram of metaphase division of the embryonic chromosomes of the allotetraploid carp in Example 3. Detailed Implementation

[0031] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0032] Example 1: Induction of allogeneic triploid carp fry

[0033] (1) Two-year-old or older female carp with obvious sexual maturity and good physical condition were selected as the female parent fish, and male culter fish that could squeeze out milky white semen were selected as the male parent fish. The female parent fish were injected with 15 μg / kg luteinizing hormone-releasing hormone A2, 800 IU / kg human chorionic gonadotropin and 3 mg / kg domperidone, and the male parent fish were injected with 7.5 μg / kg luteinizing hormone-releasing hormone A2 and 400 IU / kg human chorionic gonadotropin to induce artificial spawning. The injected parent fish were placed in 24℃ culture water. After the effect time of 12 hours, mature eggs and semen were collected separately. The eggs and semen were mixed at a volume ratio of 100:1 and artificially inseminated by dry method. The obtained hybrid fertilized eggs were evenly spread in a culture dish and placed in 24℃ culture water for incubation.

[0034] (2) Divide the fertilized eggs into 5 groups:

[0035] Group 1: Control group, no treatment was given, and the fertilized eggs were directly placed in aquaculture water at 24℃ for incubation;

[0036] Group 2: 0-1 min after fertilization, the fertilized eggs were placed in a 41℃ water bath for 1 min, 2 min and 3 min respectively;

[0037] Group 3: 5 minutes after fertilization, the fertilized eggs were placed in a 41℃ water bath for 1 minute, 2 minutes and 3 minutes respectively.

[0038] Group 4: 8 minutes after fertilization, the fertilized eggs were placed in a 41℃ water bath for 1 minute, 2 minutes and 3 minutes respectively;

[0039] Group 5: 10 minutes after fertilization, the fertilized eggs were placed in a 41℃ water bath for 1 minute, 2 minutes and 3 minutes respectively;

[0040] After the above treatments are completed, the cells are quickly transferred to aquaculture water at 24°C to continue incubation.

[0041] (3) When the hybrid embryos develop to the gastrulation stage, the fertilization rate is calculated as follows: Fertilization rate = (Number of surviving eggs in the gastrulation stage / Total number of eggs) × 100%.

[0042] (4) After the fry hatch, the hatching rate is calculated as follows: Hatching rate = (Number of hatched fry / Number of fertilized eggs) × 100%.

[0043] (5) The hatched fry from each group were transferred to nursery trays and fed with Artemia. After 7 days, the DNA content of the fry was measured by flow cytometry, and the triploidity rate and triploid yield of each group were calculated as follows: Triploidity rate = (number of triploid individuals / total number of fry tested) × 100%; Triploid yield = (hatching rate × triploidity rate) × 100%.

[0044] The results are shown in Table 1.

[0045] Table 1. Fertilization rate, hatching rate, and triploid rate of distant hybridization of carp and bream under different induction conditions.

[0046]

[0047]

[0048] The results showed that when fertilized eggs from distant hybridization of *Culter alburnus* were placed in a 41℃ water bath for 1 min and 2 min respectively 0–1 min after fertilization, 2 min and 3 min respectively 5 min after fertilization, and 1 min and 2 min respectively 8 min after fertilization, the hatched fry all exhibited triploidy. Among the above induction conditions, especially the 2 min treatment in a 41℃ water bath 0–1 min after fertilization, the triploidy rate of the fry reached the highest, reaching 100%, while the triploid yield reached 3.12%. Under these conditions, the triploidy induction effect was the best.

[0049] In Table 1, although the fertilization rate and fry hatching rate were high in the control group and other induction conditions, some of the hatched fry did not exhibit triploidity. For example, the fertilization rate in the control group was 98.93% and the fry hatching rate was 72.78%. In the case of fertilization followed by treatment in a 41°C water bath for 1 minute after 5 minutes, the fertilization rate was 96.36% and the fry hatching rate was 37.58%. Although the fertilization rate and fry hatching rate were high under both conditions, the triploidity rate of the fry was 0, and no triploid fry were observed, thus failing to achieve the goal of successfully inducing allopolyploid fry.

[0050] Example 2: Induction of allotetraploid carp fry

[0051] (1) Two-year-old or older female carp with obvious sexual maturity and good physical condition were selected as the female parent fish, and male culter fish that could squeeze out milky white semen were selected as the male parent fish. The female parent fish were injected with 15 μg / kg luteinizing hormone-releasing hormone A2, 800 IU / kg human chorionic gonadotropin and 3 mg / kg domperidone, and the male parent fish were injected with 7.5 μg / kg luteinizing hormone-releasing hormone A2 and 400 IU / kg human chorionic gonadotropin to induce artificial spawning. The injected parent fish were placed in 24℃ culture water. After the effect time of 12 hours, mature eggs and semen were collected separately. The eggs and semen were mixed at a volume ratio of 100:1 and artificially inseminated by dry method. The obtained hybrid fertilized eggs were evenly spread in a culture dish and placed in 24℃ culture water for incubation.

[0052] (2) Divide the fertilized eggs into 4 groups:

[0053] Group 1: Control group, no treatment was given, and the fertilized eggs were directly placed in aquaculture water at 24℃ for incubation;

[0054] Group 2: 30 minutes after fertilization, the fertilized eggs were placed in a 41℃ water bath for 1 minute, 2 minutes and 3 minutes respectively.

[0055] Group 3: 40 minutes after fertilization, the fertilized eggs were placed in a 41℃ water bath for 1 minute, 2 minutes and 3 minutes respectively.

[0056] Group 4: 50 minutes after fertilization, the fertilized eggs were placed in a 41℃ water bath for 1 minute, 2 minutes and 3 minutes respectively.

[0057] After the above treatments are completed, the cells are quickly transferred to aquaculture water at 24°C to continue incubation.

[0058] (3) When the hybrid embryos develop to the gastrulation stage, the fertilization rate is calculated as follows: Fertilization rate = (Number of surviving eggs in the gastrulation stage / Total number of eggs) × 100%.

[0059] (4) After the fry hatch, the hatching rate is calculated as follows: Hatching rate = (Number of hatched fry / Number of fertilized eggs) × 100%.

[0060] (5) The hatched fry from each group were transferred to brooder trays and fed with Artemia. After 7 days, the DNA content of the fry was determined by flow cytometry. At least 75 samples were tested from each group (if less than 75, all samples were tested). The tetraploidization rate and tetraploid yield of each group were calculated as follows: Tetraploidization rate = (number of tetraploid individuals / total number of fry tested) × 100%; Tetraploid yield = (hatching rate × tetraploidization rate) × 100%.

[0061] The results are shown in Table 2.

[0062] Table 2. Fertilization rate, hatching rate, and tetraploid rate of distant hybridization of carp and bream under different induction conditions.

[0063]

[0064] The results showed that when the fertilized eggs of distant hybrids of *Culter alburnus* were placed in a 41℃ water bath for 2 min and 3 min after fertilization, and also placed in a 41℃ water bath for 1 min after 50 min after fertilization, the hatched fry all exhibited tetraploidy. Among the above induction conditions, the tetraploidization rate reached 100% and the tetraploid yield reached 4.51% when placed in a 41℃ water bath for 2 min after 30 min after fertilization, and the tetraploidization rate also reached 100% and the tetraploid yield reached 13.75% when placed in a 41℃ water bath for 1 min after 50 min after fertilization. These two conditions showed the best tetraploid induction effect.

[0065] In Table 2, although the fertilization rate and fry hatching rate were high in the control group and other induction conditions, some hatched fry did not exhibit tetraploidy. For example, the fertilization rate in the control group was 98.93% and the fry hatching rate was 72.78%. In the case of fertilization followed by treatment in a 41°C water bath for 1 minute after 30 minutes, the fertilization rate was 96.21% and the fry hatching rate was 6.53%. Although the fertilization rate and fry hatching rate were high under both conditions, the tetraploidy rate of the fry was 0, and no tetraploidy was observed in the fry. Therefore, the goal of successfully inducing allopolyploid fry was not achieved.

[0066] Example 3: Identification of the induction results of distant hybridization in carp treated with heat shock.

[0067] Pluripotency was determined using chromosome counting for the distant hybridization embryos of *Culter alburnus* in Examples 1 and 2. The specific method is as follows: Untreated distant hybridization embryos of *Culter alburnus* served as the control group; embryos subjected to heat shock treatment in a 41°C water bath for 2 minutes after fertilization (0-1 min post-fertilization) served as induction group 1; and embryos subjected to heat shock treatment in a 41°C water bath for 1 minute after fertilization (50 min post-fertilization) served as induction group 2. From each group, distant hybridization embryos of *Culter alburnus* that had developed to the organogenesis stage were collected. Metaphase chromosomes were prepared using embryo chromosome preparation techniques, stained with Giemsa, and observed under a microscope. The results are as follows: Figures 4-6 As shown.

[0068] The female carp has a chromosome number of 2n = 100, the male culter cichlid has a chromosome number of 2n = 48, and the control group has a chromosome number of 74, indicating that this group is an allodiploid carp (2n = 74). The induced group 1 has a chromosome number of 124, indicating that this group is an allotriploid carp (3n = 124), which is consistent with the induction result of inhibiting the expulsion of the second polar body. The induced group 2 has a chromosome number of 148, indicating that this group is an allotetraploid carp (4n = 148), which is consistent with the induction result of inhibiting the first cleavage.

[0069] Ploidy identification of fish fry hatched from two induction groups and a control group was performed using DNA content determination. The specific method was as follows: The fish fry were wrapped in 200-mesh 10×10cm nylon gauze, crushed with tweezers, and rinsed with 500μL ACD solution into EP tubes. After staining with DAPI solution at 4℃ for 5 minutes in the dark, a blood sample of red crucian carp with known DNA content was used as a control. DNA content and ploidy identification were performed using flow cytometry. The ploidy identification criteria were as follows: using the Mean value in the flow cytometry peak diagram as a reference, the Mean value for the female carp was approximately 100, and for the male Culter alburnus, it was approximately 70. If the Mean value of the tested sample was in the range of 80–90, it was an allodiploid carp; in the range of 120–140, it was an allotriploid carp; and in the range of 160–190, it was an allotetraploid carp. Results are as follows: Figures 1-3 As shown.

[0070] Flow cytometry analysis showed that the mean value of the control group sample was 87.05, indicating it was an allodiploid Culter alburnus; the mean value of the induced group 1 sample was 135.79, indicating it was an allotriploid Culter alburnus, consistent with the induction result of inhibiting the expulsion of the second polar body; and the mean value of the induced group 2 sample was 181.39, indicating it was an allotetraploid Culter alburnus, consistent with the induction result of inhibiting the first cleavage.

[0071] Example 4: Evaluation of the culture effect of highly efficient induction of polyploid fry from distant hybridization of carp and bream

[0072] The rearing effect of polyploid fry obtained by the efficient induction methods of Examples 1 and 2 was evaluated using survival rate and body length parameters. The specific methods are as follows: Since the number of fry obtained in each induction group was too small to be grouped separately, the fry obtained under all induction conditions in Examples 1 and 2 were combined as the induction group (10,000 fry). Untreated polyploid fry were used as the control group (10,000 fry). Both groups were reared under the same conditions, with Artemia feeding four times daily during the rearing period. Fry survival rate was calculated every month, and 30 fry samples were randomly selected to measure body length and detect ploidy. After two months of rearing, some fry did not show ploidy, while after three months, ploidy became more stable. Fiber ploidy rate = (number of polyploid individuals / total number of fry tested) × 100%; Polyploid yield = (survival rate × ploidy rate) × 100%.

[0073] The results are shown in Table 3.

[0074] Table 3. Body length, survival rate, multiplication rate, and polyploid yield of fry in the distant hybridization induction group and control group of *Culter alburnus*.

[0075]

[0076] The results showed significant differences in the rearing effects between the carp-breeding distant hybridization induction group and the control group. The body length of the induced group fry was significantly longer than that of the control group (P < 0.05), and the difference became more significant with longer rearing time. The survival rate of the control group fry was significantly lower than that of the induction group (P < 0.05). The doubling rate of the 1-month-old and 2-month-old induction group was significantly higher than that of the control group (P < 0.05), but the doubling rate of the 3-month-old control group reached 100%, with a fry survival rate of only 0.24% at this stage, indicating that all surviving fry were all allopolyploids, while allodiploid hybrid fry died. Regarding polyploid yield, the induction group consistently showed a significantly higher yield than the control group (P < 0.05). Therefore, under the same rearing conditions, the carp-breeding hybrid offspring treated with induction showed significantly better rearing effects than the control group, exhibiting faster growth, higher fry survival rates, and greater yields of allopolyploid carp-breeding.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for efficiently inducing a heteropolyploid fry, characterized by, Includes the following steps: (1) Inducing spawning of distant hybrid parents: Select female fish with obvious sexual maturity characteristics and good physical condition from different subfamilies of Cyprinidae fish as maternal parents, and male fish that can squeeze out milky white sperm as paternal parents, and collect mature eggs and sperm respectively. The female parent fish is a female carp, and the male parent fish is a male topmouth culter. (2) Obtaining hybrid fertilized eggs: The collected eggs are mixed with semen and artificially inseminated using the dry method. After being gently stirred and mixed evenly, the mixture is spread evenly in a container with aquaculture water and shaken slowly to fertilize the eggs and obtain hybrid fertilized eggs. (3) Inducing allopolyploids: The fertilized eggs were placed in a water bath for heat shock treatment to inhibit the expulsion of the second polar body or inhibit the first cleavage. Then the treated fertilized eggs were quickly transferred to the aquaculture water to continue hatching into fry. The specific operation to inhibit the expulsion of the second polar body is as follows: fertilized eggs are placed in a 40-41°C water bath for 2 minutes after fertilization (0-1 minute after fertilization) for heat shock treatment; after inhibiting the expulsion of the second polar body, the fertilized eggs continue to hatch to obtain allogeneic triploid fry with a triploidity rate of 100%; The specific operation to inhibit the first cleavage is as follows: fertilized eggs are placed in a water bath at 40-41°C for 2 minutes after fertilization, or fertilized eggs are placed in a water bath at 40-41°C for 1 minute after fertilization, 30 minutes after fertilization. After inhibiting the first cleavage, the fertilized eggs continue to hatch to obtain allotetraploid fry with a tetraploidization rate of 100%.

2. The method of efficiently inducing allopolyploid fish fry according to claim 1, wherein, Step (1), the method for collecting mature eggs and semen is as follows: a. Artificial spawning induction: The pectoral fin base injection method is used. The female broodstock is injected with 14-16 μg / kg luteinizing hormone-releasing hormone A2, 700-800 IU / kg human chorionic gonadotropin and 2.5-3 mg / kg domperidone. The male broodstock is injected with 7-8 μg / kg luteinizing hormone-releasing hormone A2 and 350-400 IU / kg human chorionic gonadotropin. The injection dosage is based on the weight of the broodstock. The broodstock injected with spawning induction hormones are placed in the culture water. The effect time is 10-16 hours. b. Sperm and egg collection: After the effective time is reached, that is, when the abdomen is gently pressed and eggs flow smoothly out of the female parent fish’s genital opening, and the male parent fish can squeeze out milky white semen, sperm and egg collection is carried out. When collecting, gently press the abdomen of the female parent fish to squeeze the eggs into a clean container. Collect the male parent fish’s semen in the same way. Both eggs and semen should be protected from direct sunlight.

3. The method of efficiently inducing allopolyploid fish fry according to claim 2, wherein, The female parent fish were injected with 15 μg / kg luteinizing hormone-releasing hormone A2, 800 IU / kg human chorionic gonadotropin and 3 mg / kg domperidone, and the male parent fish were injected with 7.5 μg / kg luteinizing hormone-releasing hormone A2 and 400 IU / kg human chorionic gonadotropin.

4. The method of efficiently inducing allopolyploid fish fry according to claim 1, wherein, In step (2), the volume ratio of the egg to the semen is 90 to 100:1.

Images