Cultivation method and application of triploid tracheilus fasciatus

Abstract

The invention relates to a cultivation method and application of triploid tracheilus fasciatus, and belongs to the technical field of genetic breeding of aquatic animals. The invention provides a cultivation method of triploid trachidermus fasciatus, which comprises the following steps: (1) collecting eggs and sperms of trachidermus fasciatus, and carrying out artificial insemination to obtain fertilized eggs of trachidermus fasciatus; (2) putting the fertilized eggs of the trachidermus fasciatus into seawater at a specific temperature for pretreatment; (3) carrying out heat shock treatment on the pretreated fertilized eggs to obtain trachidermus fasciatus triploid fertilized eggs; and (4) putting the trachidermus fasciatus triploid fertilized eggs into seawater, and hatching to obtain the triploid trachidermus fasciatus. Heat shock treatment is adopted to inhibit discharge of second polar bodies of fertilized eggs, so that the triploid trachidermus fasciatus is obtained, the triploid rate of the trachidermus fasciatus is 80%, the relative survival rate of the trachidermus fasciatus is 40.93%, and the survival triploid rate of the trachidermus fasciatus is 32.74%.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal genetic breeding technology, and in particular to a method for breeding and applying triploid Songjiang perch. Background Technology

[0002] The Songjiang perch (Trachidermus fasciatus), belonging to the order Perciformes, family Ctenopharynidae, and genus Trachidermus, is a migratory fish. Commonly known as the four-gilled perch, it is a marine migratory fish. Renowned for its delicious flavor, rich nutrition, and high medicinal value, it is hailed as the "Number One Fish of Jiangnan." Artificially farmed Songjiang perch has significant market potential. The Songjiang perch is a one-year-old sexually mature eurythermal fish, with an optimal breeding temperature of 4–14℃. It prefers low-temperature environments; growth slows when the water temperature exceeds 26℃, and mortality increases dramatically at 30℃. During artificial breeding, reduced or absent feeding leads to extremely high postpartum mortality rates. To reduce growth stagnation and decreased meat quality caused by gonadal development, as well as the high mortality rate due to reproduction, preparing and producing sterile Songjiang perch is the preferred path for the industrialization of artificial Songjiang perch farming.

[0003] Triploid fish, due to the presence of odd chromosomes, may experience meiotic disorders, hindering gonadal development and significantly increasing growth rate and meat quality. Currently, the artificial induction of triploidy in salmonids and some cyprinid fish has been systematically studied and commercially applied. However, triploid induction in perch has not yet been reported.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for breeding triploid Songjiang perch, in order to solve the problem of extremely high postpartum mortality rate in the artificial breeding of Songjiang perch due to reduced or no feeding during the existing technology.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for cultivating triploid Songjiang perch, comprising the following steps:

[0008] (1) Collect eggs and sperm from the Songjiang perch and artificially inseminate them to obtain fertilized eggs of the Songjiang perch;

[0009] (2) Place the fertilized eggs of the Songjiang perch in seawater at 4-6℃ and let them stand for 10-40 minutes to obtain the pretreated fertilized eggs;

[0010] (3) The pretreated fertilized eggs were subjected to heat shock treatment to obtain triploid fertilized eggs of the Songjiang perch;

[0011] (4) Place the triploid fertilized eggs of Songjiang perch in seawater and incubate for 18 to 22 days to obtain triploid Songjiang perch.

[0012] Preferably, the Songjiang perch mentioned in step (1) is an F2 generation Songjiang perch broodstock.

[0013] Preferably, the heat shock treatment in step (3) involves placing the pretreated fertilized eggs in seawater at a temperature of 26–32°C and letting them stand for 10–25 minutes.

[0014] Preferably, the incubation temperature in step (4) is 7 to 9°C.

[0015] This invention provides the application of the described cultivation method in the cultivation of triploid perch.

[0016] The present invention has the following technical effects and advantages:

[0017] This invention employs heat shock treatment to inhibit the expulsion of the second polar body from fertilized eggs, thereby obtaining triploid Songjiang perch. The invention screened parameters for the treatment temperature, start time, and duration of the heat shock treatment, ultimately determining the optimal treatment temperature for cultivating triploid Songjiang perch to be 30℃, the start time to be 40 min, and the duration to be 15 min. Under these parameters, the triploidity rate of Songjiang perch was 80%, the relative survival rate was 40.93%, and the survival triploidity rate was 32.74%. Attached Figure Description

[0018] Figure 1 Morphology and relative DNA content of diploid and triploid Songjiang perch fry;

[0019] Figure 2 The results show the relative survival rate and triploidity rate of treatment groups 1–7.

[0020] Figure 3 The results show the relative survival rate and triploid rate of treatment groups 8–28.

[0021] Figure 4 The results show the relative survival rate and triploid rate of treatment groups 29–49. Detailed Implementation

[0022] This invention provides a method for cultivating triploid Songjiang perch, comprising the following steps:

[0023] (1) Collect eggs and sperm from the Songjiang perch and artificially inseminate them to obtain fertilized eggs of the Songjiang perch;

[0024] (2) Place the fertilized eggs of the Songjiang perch in seawater at 4-6℃ and let them stand for 10-40 minutes to obtain the pretreated fertilized eggs;

[0025] The seawater temperature is preferably 5°C; the settling time is preferably 40 min.

[0026] (3) The pretreated fertilized eggs were subjected to heat shock treatment to obtain triploid fertilized eggs of the Songjiang perch;

[0027] (4) Place the triploid fertilized eggs of Songjiang perch in seawater and incubate for 18-22 days to obtain triploid Songjiang perch;

[0028] The incubation period is preferably 20 days.

[0029] In this invention, the Songjiang perch mentioned in step (1) is an F2 generation Songjiang perch broodstock.

[0030] In this invention, the heat shock treatment in step (3) involves placing the pretreated fertilized eggs in seawater at a temperature of 26-32°C and letting them stand for 10-25 minutes, preferably 15 minutes.

[0031] The preferred temperature of the seawater is 30°C.

[0032] In this invention, the incubation temperature in step (4) is 7 to 9°C, preferably 8°C.

[0033] This invention provides the application of the described cultivation method in the cultivation of triploid perch.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Breeding method of triploid Songjiang perch

[0036] 1. Materials and Methods

[0037] The broodstock used were F2 generation Songjiang perch broodstock broodstock bred at the Beidaihe Central Experimental Station. The F2 generation Songjiang perch broodstock were cultured in flowing water at a temperature of 3.5±1℃, and were fed sandworms daily to promote gonadal development.

[0038] 2. Collection of eggs and sperm from the Songjiang perch

[0039] In mid-to-late March, healthy male Songjiang perch with golden-yellow bellies and female Songjiang perch with well-developed orange-red eggs visible through their swollen genital pores were selected. After anesthetizing with MS-222 at a concentration of 100 ppm, the area around the genital pore was wiped clean with a dry towel. The abdominal cavity was dissected with scissors to remove the testes, which were placed in a beaker containing sperm dilution. The testes were then cut into smaller pieces to collect semen. Sperm motility was examined under a microscope, and the diluted semen with longer swimming time was selected for later use. For the anesthetized females, the abdomen was gently squeezed to expel the eggs into a dissecting dish covered with plastic wrap. The clustered eggs were then scraped into a single layer and spread evenly in the dish. The dish was then stored away from light for later use.

[0040] 3. Artificial insemination

[0041] The diluted semen was poured into a dissecting dish containing the eggs laid flat. The dish was shaken horizontally to mix the sperm and eggs thoroughly for 1 minute. Then, seawater of suitable temperature was added and the dish was slowly shaken to complete the fertilization process, resulting in artificially inseminated Songjiang perch fertilized eggs.

[0042] 4. Exploration of triploid induction parameters

[0043] Experimental method: Artificially inseminated perch fertilized eggs were placed in seawater at 4-6℃ and left to stand for a certain period of time to obtain pretreated fertilized eggs. Then, the pretreated fertilized eggs were placed in seawater at a certain temperature and left to stand for a period of time to obtain triploid perch fertilized eggs.

[0044] During 2024-2025, a heat shock induction experiment was conducted on triploid perch by using single-factor experiments on three factors: the time of settling (heat shock initiation time), the temperature of the seawater (heat shock treatment temperature), and the settling time (heat shock duration). The optimal induction conditions were obtained accordingly. Based on the characteristics of perch eggs (demersal, adhesive eggs, and a small number of eggs), eggs from one female per six experimental parameters were used, and one artificially inseminated diploid per female was set up as a control group.

[0045] (1) 2024

[0046] The start times for heat shock treatment were set to 10 min, 20 min, 40 min, and 60 min; the heat shock treatment temperatures were set to 22℃, 24℃, 26℃, 28℃, 30℃, 32℃, and 34℃; and the duration of heat shock treatment was set to 15 min, i.e., treatment groups numbered 1 to 28, as shown in Table 1.

[0047] Table 1. Triploid Induction Experiment Grouping in 2024

[0048]

[0049]

[0050] (2) 2025

[0051] The parameters for inducing triploid growth of Songjiang perch were refined by setting 21 sets of parameters. Specifically, the start time of heat shock treatment was set to 40 min; the heat shock treatment temperatures were 22℃, 24℃, 26℃, 28℃, 32℃ and 34℃; and the duration of heat shock treatment was 10 min, 20 min and 25 min, respectively, which are groups numbered 29 to 49, as shown in Table 2.

[0052] Table 2 Grouping of Triploid Induction Experiments in 2025

[0053]

[0054]

[0055] During 2024–2025, after the heat shock treatment of groups 1–49, the relative survival rate, ploidy, and triploidy rate of each group were determined.

[0056] The relative survival rate was calculated as follows: After the heat shock period, the fertilized eggs in each group were weighed and then returned to filtered seawater at a temperature of 8±1℃ for further cultivation. Flowing water stimulated development, and embryonic development was observed regularly. Emergence occurred around 20 days later. Three days after hatching, the number of surviving individuals in each group was counted, and the number was divided by the egg weight of each group to obtain the survival rate. The relative survival rate of each group was then calculated based on the survival rate of the control group.

[0057] The ploidy was determined by measuring the relative DNA content of cells using flow cytometry. The procedure for determining DNA content in the control group using flow cytometry was as follows: 0.1 mL of blood was collected from the tail vein of a common Songjiang perch using a disposable syringe moistened with heparin sodium. A small amount of blood was dissolved at the needle tip in 1 mL of DNA staining solution DAPI-B containing nuclear extraction solution DAPI-A (provided by Partec GmbH, Germany). The solution was then protected from light for 10 min. The sample was filtered through a 20 μm nylon filter before being analyzed.

[0058] The procedure for determining the DNA content of triploid fish fry by flow cytometry is as follows: Take 30 fish fry that have been fed for 7 days and immerse them in 0.6 mL of DNA staining solution DAPI-B containing nuclear extract DAPI-A. Grind them manually for 1 min until no tissue blocks are visible to the naked eye. Add 1.4 mL of DNA staining solution DAPI-B containing nuclear extract DAPI-A and treat in the dark for 10 min. Filter the samples through a 20 μm nylon filter before testing.

[0059] The triploid rate was calculated as follows: After determining ploidy by flow cytometry, the relative DNA content of the tested fish fry was compared with the relative DNA content of standard diploid blood cells. The ratio was expressed as the DNA index (DI). If DI = 1.5 ± 0.05, the fish was triploid; if DI = 1.25 ± 0.05 or DI = 1.75 ± 0.05, the fish was chimera; and if DI = 1.0 ± 0.05, the fish was diploid. The ratio of the number of triploid fry detected to the total number of samples tested was used as the triploid rate for each treatment group.

[0060] (3) Test results

[0061] 1. The morphology of diploid and triploid Songjiang perch fry was observed and the relative DNA content of the two was determined. The results are as follows: Figure 1 As shown in the figure, A represents the morphology and relative DNA content of diploid Songjiang perch fry, and B represents the morphology and relative DNA content of triploid Songjiang perch fry.

[0062] according to Figure 1 It can be seen that the triploid larvae of the Songjiang perch are no different in appearance from ordinary diploid larvae, the only difference being the relative amount of DNA.

[0063] 2. The relative survival rate and triploidy rate of groups 1–49 were determined during 2024–2025, and the results are as follows: Figures 2-4 As shown, Figure 2 The results show the relative survival rate and triploid rate of treatment groups 1–7. Figure 3 The results show the relative survival rate and triploid rate of treatment groups 8–28. Figure 4 The results show the relative survival rate and triploid rate of treatment groups 29–49.

[0064] according to Figures 1-3 It was found that the triploid induction rate was 0 at treatment temperatures of 22℃ and 24℃, indicating that temperatures below 26℃ were insufficient to inhibit the release of the second polar body from the eggs of the *Perequa maculata*. At a treatment temperature of 34℃, the survival rate was 0, suggesting that 34℃ may be the extreme mortality temperature for *Perequa maculata* fertilized eggs. Therefore, the treatment temperatures for triploid induction in *Perequa maculata* were set at 26℃, 28℃, 30℃, and 32℃.

[0065] The triploid induction rate was 0 when the starting time was 60 min. However, a certain proportion of triploids were present when the treatment temperature was set at 26℃ with a starting time of 40 min, the treatment temperatures were set at 28℃ and 30℃ with a starting time of 10 min, and the treatment temperature was set at 32℃ with a starting time of 20 min. This indicates that the release time of the second polar body of the Songjiang perch egg is within 10 to 40 min. This may be related to the breeding water temperature (low temperature) and ovulation mode of the Songjiang perch (single ovulation, asynchronous egg development). Therefore, the starting time for triploid induction of Songjiang perch was set to the longest 40 min.

[0066] Triploids appeared at treatment durations of 10 min, 15 min, 20 min, and 25 min. The highest triploid induction rate (80.00%) was observed at a treatment temperature of 30℃, an induction time of 40 min, and a treatment duration of 15 min.

[0067] (4) Data Analysis

[0068] Treatment temperatures were set at 26℃, 28℃, 30℃, and 32℃, with start times of 10 min, 20 min, and 40 min, and durations of 10 min, 15 min, 20 min, and 25 min. Orthogonal experiments were conducted to induce triploid Songjiang perch, taking treatment temperature, start time, and duration as factors. The relative survival rate (%), triploidity rate (%), and survival triploidity rate (%) of different treatments were measured. The detection results of triploid Songjiang perch induced by orthogonal experiments are shown in Table 3.

[0069] The triploid survival rate is calculated by multiplying the relative survival rate of each group by its triploid survival rate.

[0070] Table 3. Results of orthogonal experiment induced triploid Songjiang perch.

[0071]

[0072] Table 3 shows that the treatment group with a treatment temperature of 30℃, a start time of 40 min, and a duration of 15 min had the highest triploid rate of 80% for Songjiang perch, a relative survival rate of 40.93%, and a triploid survival rate of 32.74%. The treatment temperature, start time, and duration of this treatment group are the optimal parameters for triploid cultivation of Songjiang perch. On the other hand, the treatment group with a treatment temperature of 26℃, a start time of 40 min, and a duration of 15 min had the highest relative survival rate of 87.75% for Songjiang perch, a triploid rate of 28.57%, and a triploid survival rate of 25.07%. The treatment temperature, start time, and duration of this treatment group are the second-best parameters for triploid cultivation of Songjiang perch.

[0073] In summary, the breeding method for triploid Songjiang perch is as follows:

[0074] Eggs and sperm from *Perequa maculata* were collected, and artificial insemination was performed to obtain fertilized eggs. The fertilized eggs were then placed in seawater at 5°C for 40 minutes to obtain pretreated fertilized eggs. The pretreated fertilized eggs were then placed in seawater at 30°C for 20 minutes to obtain triploid fertilized eggs. The triploid fertilized eggs were then placed in seawater at 8°C and incubated for 20 days to obtain triploid *Perequa maculata*.

[0075] As shown in the above embodiments, this invention provides a method for cultivating triploid Songjiang perch. This invention employs heat shock treatment to inhibit the expulsion of the second polar body of the fertilized egg, thereby obtaining triploid Songjiang perch. This invention screened parameters for the treatment temperature, start time, and duration of the heat shock treatment, ultimately determining the optimal treatment temperature for cultivating triploid Songjiang perch to be 30℃, the start time to be 40 min, and the duration to be 15 min. Under these parameters, the triploidity rate of Songjiang perch was 80%, the relative survival rate was 40.93%, and the survival triploidity rate was 32.74%.

[0076] 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 cultivating triploid Songjiang perch, characterized in that, Includes the following steps: (1) Collect eggs and sperm from the Songjiang perch and artificially inseminate them to obtain fertilized eggs of the Songjiang perch; (2) Place the fertilized eggs of the Songjiang perch in seawater at 4-6℃ and let them stand for 10-40 minutes to obtain the pretreated fertilized eggs; (3) The pretreated fertilized eggs were subjected to heat shock treatment to obtain triploid fertilized eggs of the Songjiang perch; (4) Place the triploid fertilized eggs of Songjiang perch in seawater and incubate for 18 to 22 days to obtain triploid Songjiang perch.

2. The cultivation method according to claim 1, characterized in that, The Songjiang perch mentioned in step (1) is the F2 generation Songjiang perch parent fish.

3. The cultivation method according to claim 1, characterized in that, The heat shock treatment in step (3) involves placing the pretreated fertilized eggs in seawater at a temperature of 26–32°C and letting them stand for 10–25 minutes.

4. The cultivation method according to claim 1, characterized in that, The incubation temperature in step (4) is 7 to 9°C.

5. The application of the cultivation method according to any one of claims 1 to 4 in the cultivation of triploid perch.

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