Method for mass induction of triploid of green pufferfish

Abstract

The application discloses a method for batch inducing triploid of green peau fish, and belongs to the technical field of aquatic genetic breeding. The method comprises the following steps: selecting green peau fish females close to initial spawning, isolating the females from males, and extruding the eggs into a beaker with the whole inner wall evenly smeared with vaseline; artificially collecting sperm and mixing the sperm with a sperm protection solution; adding the sperm into seawater to activate, and then quickly pouring into the beaker containing the eggs; washing the fertilized eggs after 2 minutes of insemination, and then adding the washed fertilized eggs into a finishing box filled with seawater and putting into a PP material corrugated board; after 5 minutes of insemination, the corrugated board with the fertilized eggs attached is transferred to seawater with a temperature of 12 DEG C for cold shock treatment for 45 minutes; and the corrugated board is taken out and placed in seawater for continuous hatching. The method has the advantages that the batch and stable production of triploid fry is realized under the premise that the triploid induction rate is as high as 100%.

Description

Technical Field

[0001] This invention relates to a method for inducing triploidy in fish, specifically a method for mass-producing triploid greenfin pufferfish, belonging to the field of aquatic genetic breeding technology. Background Technology

[0002] The greenfin filefish (Thamnaconus modestus), belonging to the family Thamnaconidae and the genus Thamnaconus, is a warm-temperate bottom-dwelling fish that can survive in water temperatures ranging from 7°C to 32°C, with an optimal growth temperature of 18°C ​​to 25°C. This fish is highly favored by consumers due to its omnivorous nature, rapid growth rate, short culture cycle, and strong disease resistance. Furthermore, its tender flesh and lack of intramuscular bones make it a popular choice. Particularly noteworthy is its ability to nibble on netting, effectively cleaning up attached organisms in aquaculture cages, making it an ideal species for ecological polyculture in deep-sea cage culture. However, under artificial culture conditions, greenfin filefish reach sexual maturity at just one year old. The development of gonads leads to slower growth, lower survival rates, and poorer flesh quality, severely limiting the cultivation of large-sized marketable fish.

[0003] The sterility of triploid fish effectively avoids the reproductive energy expenditure caused by sexual maturation, which is beneficial for cultivating large-sized commercial fish and has broad industrial application prospects. Methods for artificially inducing triploidy include temperature shock (cold shock and heat shock), hydrostatic pressure, and chemical induction. Among these, cold shock is one of the most widely used physical induction methods. Its basic principle is to inhibit the release of the second polar body during the second meiotic division of the fertilized egg through low-temperature treatment, thereby doubling the chromosome set and obtaining triploid individuals. Due to its advantages of simple operation, low cost, and minimal damage to the fertilized egg, cold shock has been successfully applied in various fish species.

[0004] However, the greenfin filefish is a batch-spawning, asynchronous fish with small eggs that become extremely sticky upon contact with water. Sexually mature parents have a low gonadal index (GSI), with a single fish laying only 1g–30g (egg weight). This extremely low egg production severely restricts the large-scale artificial induction and seedling cultivation of triploids. Furthermore, under artificial breeding conditions, females often spawn spontaneously, making the collection of unfertilized eggs extremely difficult, further limiting the bulk acquisition of eggs. In addition, the sticky eggs easily clump together during cold shock treatment, leading to localized hypoxia in fertilized eggs, hindered development, and a sharp decline in hatching rate. This is the core technical bottleneck restricting the industrial application of triploid fish with sticky eggs.

[0005] Currently, there are reports on triploid induction in fish. For example, Chinese invention patent CN102106292B discloses a method for mass production of triploid flounder, and Chinese invention patent application CN102246708A discloses a method for inducing triploid flounder. However, none of these methods offer a systematic solution to the unique biological characteristics of greenfin pufferfish, such as low batch spawning rates and high egg viscosity. There is currently no complete technical solution specifically for the mass induction of triploid pufferfish. Therefore, to achieve large-scale production of greenfin pufferfish triploids, it is urgent to establish a complete technical system that includes methods for promoting the maturation and collection of mature unfertilized eggs, low-temperature cryopreservation of sperm, and cold shock treatment processes suitable for adhesive eggs. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for mass-producing triploid greenfin pufferfish.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for mass inducing triploid greenfin pufferfish includes the following steps: (1) Obtaining mature eggs: Select female greenfin pufferfish with well-developed gonads and close to their first spawning, isolate them from males, collect eggs when the time is right, and squeeze the eggs into a beaker whose inner wall is evenly coated with Vaseline. (2) Obtaining mature sperm: Select male greenfin pufferfish with mature gonads, collect semen artificially, mix the collected raw semen with sperm preservation solution pre-cooled at 4℃, and store it in a refrigerator at 4℃ away from light for later use. (3) Wet insemination: Semen stored at 4℃ is added to seawater to activate it, and then quickly poured into a beaker containing eggs. Stir gently with a dry glass rod. The moment the stirring begins is the start of insemination. After 2 minutes of insemination, the fertilized eggs are washed quickly with excess seawater for 15 seconds. After washing, the fertilized eggs are immediately added to a container filled with seawater and stirred for 10 seconds. Then, PP corrugated plates are immediately placed into the container to make the fertilized eggs evenly adhere to the corrugated plates. (4) Cold shock induction of fertilized eggs: 5 minutes after fertilization, the corrugated plate with fertilized eggs attached was immediately transferred to seawater at 12°C for cold shock treatment for 45 minutes. (5) Hatching: Remove the corrugated board and place it in seawater to continue hatching.

[0008] Preferably, the method for inducing triploid greenfin pufferfish in batches also includes a step of promoting the maturation of broodstock. Specifically, three months before the breeding season, select male and female greenfin pufferfish broodstock with intact body shape and good vitality, stabilize the culture water temperature at 13℃–14℃, and promote the development and maturation of the gonads of the broodstock by artificially simulating the seasonal changes of natural light and combining nutritional enhancement measures.

[0009] Preferably, in step (1), the culture water is fresh seawater that has been disinfected by ultraviolet light and filtered by sand, the light-dark cycle is 16h:8h, and the culture water temperature is 18.0±0.5℃.

[0010] Preferably, in step (2), the sperm protection solution has the following formula: sodium chloride 8.5 g / L, potassium chloride 0.14 g / L, calcium chloride 0.12 g / L, sodium dihydrogen phosphate 0.28 g / L, sodium bicarbonate 0.20 g / L, and glucose 2.00 g / L.

[0011] Preferably, in step (3), the ratio of raw semen to egg is 1 mL: 20 g.

[0012] Preferably, in steps (3) and (5), the seawater is fresh seawater at 18±0.5℃ that has been disinfected by ultraviolet light and filtered by sand.

[0013] The advantages of this invention are: (1) In view of the problem of low natural spawning and difficulty in obtaining mature eggs of greenfin pufferfish, this invention establishes a batch mature egg acquisition method that integrates parent selection, environmental control, male and female separation and artificial egg extraction, which significantly improves the quantity and quality of usable eggs; (2) In view of the problem that sperm has a short survival time in vitro and a limited fertilization window, this invention has developed a low-temperature cryopreservation technology for sperm and a special sperm preservation solution for greenfin pufferfish, which effectively extends the survival time and fertilization window of sperm in vitro, and provides a stable and controllable source of sperm for batch insemination; (3) In view of the technical bottleneck that sticky eggs are prone to sticking together and clumping during cold shock treatment, which leads to a sharp drop in hatching rate, the present invention attaches fertilized eggs evenly to a corrugated plate and places the corrugated plates carrying fertilized eggs in a cold shock water bath in an overlapping manner, which effectively avoids the problem of eggs sticking together when directly subjected to cold shock treatment and greatly improves hatching rate. (4) This invention precisely determined the key process parameters for cold shock-induced triploid production of greenfin pufferfish: the start time was 5 minutes after fertilization, the treatment water temperature was 12℃, and the treatment duration was 45 minutes. Under the premise of ensuring a triploid induction rate of up to 100%, the mass and stable production of triploid seedlings was realized. Attached Figure Description

[0014] Figure 1 This is a statistical chart showing the relative hatching rate and triploid rate of greenfin pufferfish fertilized eggs under different cold shock treatment water temperatures; Figure 2 This is a statistical chart showing the relative hatching rate and triploid rate of greenfin pufferfish fertilized eggs at different cold shock initiation times. Figure 3 This is a statistical chart showing the relative hatching rate and triploid rate of greenfin pufferfish fertilized eggs under different durations of cold shock treatment; Figure 4 This is a flow cytometry diagram of a diploid greenfin pufferfish. Figure 5 This is a flow cytometry diagram of a triploid greenfin pufferfish. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0016] I. Determination of Technical Parameters 1. Determining the ovulation method Select female greenfin triggerfish with well-developed gonads, entering the breeding season and nearing their first spawning, and weighing 0.5kg–1.0kg.

[0017] The female fish were divided into three groups: (1) Group 1: 20 female fish were randomly selected and placed into the parent fish rearing pond at 6:00 am. No male fish were present. (2) Second group: 20 female fish were randomly selected and injected with human chorionic gonadotropin (HCG) 500U / kg and luteinizing hormone-releasing hormone A2 (LHRH-A2) 10μg / kg respectively at 6:00 am according to their body weight. They were raised separately without male fish under the same conditions as the first group and marked with T-shaped tags. (3) The third group: 20 female fish and 20 male fish were randomly selected and kept together in another parent fish breeding pond at 6:00 am without injecting hormones.

[0018] The aquaculture water is fresh seawater that has been disinfected with ultraviolet light and filtered through sand, and the water temperature is 18.0±0.5℃.

[0019] During the breeding period, the light-dark cycle is 16h:8h.

[0020] Check three times a day (6:00, 14:00, 20:00), record the time of the first spawning of each group of parent fish, the number of female fish that spawned on the first spawning day, and the amount of artificial spawning, and determine the best method for obtaining mature eggs.

[0021] Table 1 shows the results of obtaining mature eggs from female greenfin pufferfish in each group.

[0022] Table 1. Acquisition of mature eggs from female greenfin filefish (Pteranodon spp.)

[0023] Note: The number of eggs laid refers to the cumulative number of eggs collected from female fish that were found to have laid eggs during a single inspection on the day of their first spawning.

[0024] As shown in Table 1, the first spawning time of the second group was significantly earlier, but the number of eggs laid was not significantly increased; the first spawning time of the first group was later than that of the second group, but the number of spawning females and the number of eggs laid were not significantly different from those of the second group; when the third group was checked at 6:00 on the 5th day (96 hours after rearing), fertilized eggs could be seen at the bottom of the pond, the number of spawning females was significantly reduced, and the artificial spawning was significantly reduced.

[0025] Therefore, the method of raising well-developed female fish separately without injecting hormones (to avoid male fish chasing each other and causing natural ovulation) was chosen for subsequent triploid induction experiments.

[0026] 2. Determining the method of sperm preservation Male greenfin filefish with mature gonads were selected, and semen was artificially collected. The collected semen was divided into two groups: Control group: The collected semen was not processed in any way and was directly refrigerated at 4°C; Experimental group: The collected semen was diluted with the sperm preservation solution provided by this invention (the volume ratio of semen to sperm preservation solution was 1:9), and then refrigerated at 4°C.

[0027] Sperm viability was measured under a microscope at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h and 4 h after the start of refrigeration.

[0028] The specific method for detecting sperm viability is as follows: Semen is mixed with 0.5% eosin Y solution at a volume ratio of 1:1, then dropped onto a clean glass slide, covered with a coverslip, and observed under a microscope. 200 sperm are randomly observed and counted under a high-power microscope. Those that are not stained are live sperm, and those that are stained red are dead sperm. The proportion of live sperm to the total number of sperm is calculated, which is the sperm viability rate.

[0029] The formula for sperm protection solution is as follows: sodium chloride 8.5 g / L, potassium chloride 0.14 g / L, calcium chloride 0.12 g / L, sodium dihydrogen phosphate 0.28 g / L, sodium bicarbonate 0.20 g / L, and glucose 2.00 g / L.

[0030] Table 2 shows the sperm survival rate of each group of greenfin pufferfish under low-temperature refrigeration. Table 2. Results of sperm viability testing of greenfin pufferfish under low-temperature refrigeration.

[0031] As shown in Table 2, the sperm survival rate of the control group was only 10% after being refrigerated in vitro for 1 hour, which was insufficient for normal fertilization; while the sperm survival rate of the experimental group was still above 80% after being refrigerated in vitro for 3 hours, which met the requirements for fertilization.

[0032] It is evident that the sperm preservation solution provided by this invention can significantly prolong the in vitro survival time of greenfin pufferfish sperm under low-temperature refrigeration conditions, and prevent the sperm from being activated and inactivated due to contamination by urine, body fluids, etc., thus providing a sufficient operating time window for the synchronous fertilization of oocytes in batch triploid induction.

[0033] 3. Determination of triploid induction conditions This invention uses cold shock treatment to inhibit the release of the second polar body of the fertilized egg and induce triploidy by doubling the chromosome set.

[0034] (1) Obtaining eggs, sperm and artificial insemination Female greenfin filefish with well-developed gonads, in their reproductive period, and not yet having experienced their first ovulation were selected and separated from the males for individual rearing in fresh seawater that had been disinfected with ultraviolet light and filtered through sand. Light (16h:8h light-dark cycle) and temperature (18.0±0.5℃) were controlled. When the females were ready to be spawned (their abdomens softened, and eggs could be easily expelled by gentle abdominal pressure), the eggs were collected by manually squeezing their abdomens and pressing them into a beaker whose inner wall was evenly coated with petroleum jelly to prevent adhesion. The collected eggs were examined under a microscope. Eggs with uniform diameter, transparent yolks, and a moderate and evenly distributed number of oil globules were selected and stored in a 18.0℃ water bath in the dark for later use.

[0035] Sexually mature male greenfin filefish were selected, and semen was artificially collected. An experimental group and a control group were set up, wherein: (i) Experimental group: The collected raw semen was placed in a sperm preservation solution pre-cooled at 4°C. The volume ratio of raw semen (1 mL) to sperm preservation solution (9 mL) was 1:9. The solution was stored in a refrigerator at 4°C in the dark for later use.

[0036] (ii) Control group: The raw semen collected was not processed in any way and was directly refrigerated at 4°C.

[0037] Before insemination, sperm motility is tested under a microscope, and sperm with high motility and long motility time are selected for insemination.

[0038] Semen stored at 4℃ (10mL for the experimental group and 1mL for the control group) was added to fresh seawater that had been disinfected by UV and filtered by sand at 18±0.5℃ (190mL for the experimental group and 199mL for the control group) to activate sperm. The volume ratio of semen to seawater in the experimental group was 1:19, and the volume ratio of semen to seawater in the control group was 1:199. Then, the activated semen was quickly poured into a container containing eggs (20g). The mixing ratio of the original semen to eggs was 1mL:20g. The mixture was continuously and gently stirred with a dry glass rod to prevent the eggs from sticking together. The moment the stirring started was the moment of insemination.

[0039] (2) Methods for determining the ploidy of fish fry Five days after hatching, fish fry were used for ploidy identification. After briefly rinsing the fry with distilled water, they were placed in a 1.5 mL centrifuge tube and 1 drop of distilled water was added.

[0040] Before identification, 1 mL of one-step staining reagent (produced by Partec GmbH, Germany, product model DAPI) was added to each centrifuge tube. The fish fry were crushed with a grinder to make a single-cell suspension. After filtration through a filter membrane, the filtrate was transferred to a 5 mL test tube that comes with the CyFlow ploidy analyzer (CyFlow PA, a simplified flow cytometry system produced by Partec GmbH, Germany). 1.5 mL of one-step staining reagent was added, and the mixture was gently pipetted to mix. Then, the ploidy was measured using the CyFlow ploidy analyzer.

[0041] Using common diploid embryos of the greenfin pufferfish as a control, the fluorescence intensity corresponding to the absorbance peak of the diploid DNA was set to 100 (see flow cytometry diagram for diploid identification). Figure 4 When the fluorescence intensity corresponding to the peak absorbance of the DNA sample is 150 (see flow cytometry diagram for triploid identification), Figure 5 This means that the sample being tested is triploid.

[0042] (3) Determination of cold shock water temperature Five minutes after fertilization, the fertilized eggs were immediately placed in cold shock water baths at 3℃, 6℃, 9℃, 12℃, and 15℃ for 45 minutes each. After treatment, the fertilized eggs were removed and placed in fresh seawater that had been disinfected with ultraviolet light and filtered through sand at 18±0.5℃ for further incubation. After incubation, the relative hatching rate (experimental group hatching rate ÷ control group hatching rate × 100%) and triploid rate were calculated.

[0043] The statistical results of the relative hatching rate and triploid rate of greenfin triggerfish fertilized eggs under different cold shock treatment water temperatures are shown in the figure. Figure 1 .Depend on Figure 1It can be seen that when the water temperature of the cold shock treatment is 12℃, the relative hatching rate of the greenfin pufferfish fertilized eggs is the highest (88.73±10.84%) and the triploid rate reaches 100%.

[0044] Based on the relative hatching rate and triploidity rate, the optimal cold shock treatment water temperature for greenfin pufferfish fertilized eggs was determined to be 12℃.

[0045] (4) Determining the starting time of cold shock treatment Immediately after fertilization (1 min, 3 min, 5 min, 7 min, and 9 min), the fertilized eggs were placed in fresh seawater at 12℃ (UV-sterilized and sand-filtered) for 45 min of cold shock treatment. After treatment, the fertilized eggs were removed and placed in fresh seawater at 18±0.5℃ (UV-sterilized and sand-filtered) for further incubation. After incubation, the relative hatching rate and triploid rate were calculated.

[0046] The statistical results of the relative hatching rate and triploid rate of greenfin triggerfish fertilized eggs at different cold shock initiation times are shown in the figure. Figure 2 .Depend on Figure 2 It can be seen that when the cold shock treatment is initiated 5 minutes after fertilization, the relative hatching rate of the greenfin pufferfish eggs is the highest (81.50±6.28%) and the triploidity rate reaches 100%.

[0047] Based on the relative hatching rate and triploid rate, the optimal time to begin cold shock treatment on greenfin pufferfish eggs was determined to be 5 minutes after fertilization.

[0048] (5) Determination of the duration of cold shock treatment Five minutes after fertilization, the fertilized eggs were immediately placed in fresh seawater at 12℃ that had been disinfected with UV light and filtered through sand for cold shock treatment for 15, 30, 45, 60, and 75 minutes, respectively. After treatment, the fertilized eggs were removed and placed in fresh seawater at 18±0.5℃ that had been disinfected with UV light and filtered through sand for further incubation. After incubation, the relative hatching rate and triploid rate were calculated.

[0049] The statistical results of the relative hatching rate and triploid rate of greenfin triggerfish fertilized eggs under different durations of cold shock treatment are shown in the figure. Figure 3 .Depend on Figure 3 It can be seen that when the cold shock treatment lasts for 45 minutes, the relative hatching rate of the greenfin pufferfish fertilized eggs is the highest (91.58±7.28%) and the triploidity rate reaches 100%.

[0050] Based on the relative hatching rate and triploidity rate, the optimal duration of cold shock treatment for greenfin pufferfish fertilized eggs was determined to be 45 minutes.

[0051] II. Methods for Mass Induction of Triploidy in Greenfin Pufferfish 1. Promoting maturation in parent fish Three months before the breeding season, select healthy, vigorous male and female greenfin filefish broodstock and maintain the water temperature at a stable 13℃-14℃, avoiding drastic fluctuations. Simultaneously, artificially simulate seasonal changes in natural light (increasing the daylight hours daily until reaching 16h:8h) and combine this with nutritional fortification measures (changing the feeding from fresh mixed fish to squid and sandworms with added vitamins) to promote gonadal maturation in the broodstock.

[0052] 2. Retrieve mature eggs After entering the breeding season, the gonadal development and reproductive behavior of the female fish were observed. Fifty female greenfin filefish with well-developed gonads and close to their first spawning were selected and isolated from the males for separate rearing. The rearing water was fresh seawater that had been disinfected with ultraviolet light and filtered with sand. Light (light-dark cycle of 16h:8h) and temperature (rearing water temperature of 18.0±0.5℃) were controlled.

[0053] When the time is right for egg collection, the female greenfin filefish is squeezed by hand to collect the eggs into a beaker whose inner wall is evenly coated with petroleum jelly. The collected eggs are examined under a microscope, and high-quality eggs with uniform diameter, transparent yolk, and a moderate number and even distribution of oil globules are selected for subsequent artificial insemination.

[0054] In this operation, 120 mL of high-quality eggs were obtained from 50 female greenfin filefish. These high-quality eggs were stored in a water bath at 18.0℃ in the dark for later use.

[0055] 3. Obtain mature sperm After entering the breeding season, the gonadal development and reproductive behavior of the male fish were observed. Four male greenfin filefish with mature gonads were selected, and their semen was collected artificially.

[0056] In this operation, a total of 5.5 mL of raw semen was collected from 4 male greenfin pufferfish, which is designated as semen A.

[0057] The collected semen A was mixed with pre-cooled sperm preservation solution at 4℃ at a volume ratio of 1:9 to obtain diluted semen, which was designated as semen B and stored in a 4℃ refrigerator away from light for later use.

[0058] The nutrient composition of the sperm preservation solution is as follows: sodium chloride 8.5 g / L, potassium chloride 0.14 g / L, calcium chloride 0.12 g / L, sodium dihydrogen phosphate 0.28 g / L, sodium bicarbonate 0.20 g / L, and glucose 2.00 g / L.

[0059] 4. Wet insemination Semen B, stored at 4℃, was added to fresh seawater at 18±0.5℃ that had been disinfected by ultraviolet light and filtered by sand to activate it. The volume ratio of semen B to seawater was 1:19, resulting in activated semen, which was denoted as semen C.

[0060] Quickly pour semen C into the beaker containing the eggs. The ratio of semen C to eggs is 200mL:20g (the ratio of raw semen (sperm A) to eggs is 1mL:20g). Stir gently and continuously with a dry glass rod to prevent the eggs from sticking together.

[0061] The moment stirring begins is the start of fertilization. Two minutes after fertilization, immediately wash the fertilized eggs with an excess of seawater for 15 seconds to remove excess semen and ovarian mucus.

[0062] After washing, the fertilized eggs were immediately added to a 50L container filled with fresh seawater at a temperature of 18±0.5℃ that had been disinfected by UV and sand filtered. The mixture was stirred for 10 seconds, and then a PP corrugated plate was immediately added to the container. After 1 minute, the fertilized eggs were evenly attached to the corrugated plate.

[0063] The entire process of wet insemination is quite time-sensitive, requiring each step to be completed quickly and efficiently.

[0064] 5. Cold shock induction of fertilized eggs Five minutes after insemination, the corrugated plate with the fertilized eggs attached was immediately transferred to fresh seawater at 12°C that had been disinfected by ultraviolet light and sand-filtered for 45 minutes for cold shock treatment.

[0065] 6. Incubation After the cold shock treatment, the corrugated plate was removed and placed in fresh seawater that had been disinfected with ultraviolet light and filtered with sand at a temperature of 18±0.5℃ to continue incubation.

[0066] In this operation, the fry began to hatch one after another after 2 days, and all the fry were hatched after 4 days.

[0067] According to statistics, a total of 117,500 newly hatched fry were obtained, with a hatching rate of 65.28%.

[0068] The ploidy test results of 5-day-old fry showed that the triploid rate was as high as 100%.

[0069] The above results indicate that the method provided by this invention is stable and reproducible, and is suitable for the mass induction and production of triploid seedlings of greenfin pufferfish.

[0070] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for mass-producing triploid greenfin pufferfish, characterized in that, Includes the following steps: (1) Obtaining mature eggs: Select female greenfin pufferfish with well-developed gonads and close to their first spawning, isolate them from males, collect eggs when the time is right, and squeeze the eggs into a beaker whose inner wall is evenly coated with Vaseline. (2) Obtaining mature sperm: Select male greenfin pufferfish with mature gonads, collect semen artificially, mix the collected raw semen with sperm preservation solution pre-cooled at 4℃, and store it in a refrigerator at 4℃ away from light for later use. (3) Wet insemination: Semen stored at 4℃ is added to seawater to activate it, and then quickly poured into a beaker containing eggs. Stir gently with a dry glass rod. The moment the stirring begins is the start of insemination. After 2 minutes of insemination, the fertilized eggs are washed quickly with excess seawater for 15 seconds. After washing, the fertilized eggs are immediately added to a container filled with seawater and stirred for 10 seconds. Then, PP corrugated plates are immediately placed into the container to make the fertilized eggs evenly adhere to the corrugated plates. (4) Cold shock induction of fertilized eggs: 5 minutes after fertilization, the corrugated plate with fertilized eggs attached was immediately transferred to seawater at 12°C for cold shock treatment for 45 minutes. (5) Hatching: Remove the corrugated board and place it in seawater to continue hatching.

2. The method for mass-producing triploid greenfin pufferfish according to claim 1, characterized in that, It also includes steps to promote maturation of the parent fish, specifically: Three months before the breeding season, select male and female greenfin pufferfish broodstock that are in good condition and have good vitality. Keep the water temperature stable at 13℃–14℃, artificially simulate the seasonal changes of natural light, and combine with nutritional enhancement measures to promote the development and maturation of the broodstock gonads.

3. The method for mass-producing triploid greenfin pufferfish according to claim 2, characterized in that, Nutritional fortification measures are as follows: Feed them squid and sandworms and add vitamins and nutrients.

4. The method for mass-producing triploid greenfin pufferfish according to claim 1, characterized in that, In step (1), the culture water is fresh seawater that has been disinfected by ultraviolet light and filtered by sand, the light-dark cycle is 16h:8h, and the culture water temperature is 18.0±0.5℃.

5. The method for mass-producing triploid greenfin pufferfish according to claim 1, characterized in that, In step (2), the formula for the sperm preservation solution is as follows: Sodium chloride 8.5 g / L, potassium chloride 0.14 g / L, calcium chloride 0.12 g / L, sodium dihydrogen phosphate 0.28 g / L, sodium bicarbonate 0.20 g / L, glucose 2.00 g / L.

6. The method for mass-producing triploid greenfin pufferfish according to claim 1, characterized in that, In step (3), the ratio of raw semen to egg is 1 mL: 20 g.

7. The method for mass-producing triploid greenfin pufferfish according to claim 1, characterized in that, In steps (3) and (5), the seawater is fresh seawater at 18±0.5℃, which has been disinfected by ultraviolet light and filtered by sand.

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