A method for efficient production of triploid oysters

Abstract

This application discloses a method for efficiently producing triploid oysters, belonging to the field of aquaculture breeding technology. The method includes: S1, obtaining sperm and eggs from diploid oysters and performing sperm-egg fertilization; S2, inducing the fertilized eggs to inhibit the release of the second polar body; S3, collecting the induced fertilized eggs, washing them, and hatching them to the larval stage; S4, 24-72 hours after fertilization, screening based on differences in larval size and collecting the target larval population; S5, culturing the larvae obtained in S4. Using the method provided in this application, even with an initial induction rate of only around 70%, a stable triploid larval population with a triploid rate exceeding 90% can be obtained, with high hatching and survival rates. The method is simple to operate, has low requirements for induction conditions, and is suitable for triploid preparation of precious oyster varieties or oyster strains with significant economic traits where it is difficult to obtain a large number of fertilized eggs through strong induction conditions.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture breeding technology, specifically relating to a method for efficiently producing triploid oysters. Background Technology

[0002] As an important aquaculture species globally, oysters are increasingly valued for their industry and the demand for genetic improvement is growing. my country is the world's largest producer of oysters, with a production of 7.25 million tons in 2024. Among them, triploid oysters have advantages such as low fertility, fast growth rate, and strong resistance to adverse conditions, and have become a major aquaculture species in my country in recent years.

[0003] Currently, existing technologies for obtaining triploid oysters mainly fall into two categories: The first category involves hybridizing tetraploid oysters with diploid oysters to obtain triploids. However, this method usually requires obtaining surviving tetraploid oysters, which depends on the pre-cultivation process of "fertile triploid → tetraploid." This method is not feasible for some new strains and varieties that have not yet been developed for triploid production. The second category involves directly inducing triploid oysters to obtain triploid oysters. The principle is to increase the number of chromosomes by inhibiting the release of the first or second polar body of the fertilized egg. This is the mainstream technology used for the first development of triploids in new varieties and strains. The induction is usually divided into physical induction (high and low temperature treatment, hyperosmolar treatment, hydrostatic pressure treatment, etc.) and chemical induction (treatment with drugs such as cytochalasin B, 6-diaminomethylpurine, and caffeine).

[0004] However, existing induction methods have the following drawbacks: First, the induced triploid rate is unstable; second, the induction conditions that can achieve a high triploid induction rate are often quite stringent and technically difficult to implement. Moreover, while stringent induction conditions bring a high induction rate, they can also lead to a low hatching rate and a high mortality rate. For example, in chemical induction, it is usually necessary to increase the concentration of the chemical inducer or extend the induction time to improve the induction rate. However, this can lead to a low hatching rate and a high mortality rate during the later stages of fertilized egg hatching due to the excessive toxicity of the chemical inducer, ultimately making it difficult to obtain a large number of surviving triploid oysters. Therefore, there is an urgent need for a triploid oyster production method that can achieve a stable high triploid rate, is highly operable, and has low requirements for induction conditions. Summary of the Invention

[0005] The purpose of this application is to provide a method for producing triploid oysters with high efficiency, in order to solve the problems of unstable triploidity rate, high operation difficulty, strict induction conditions, and difficulty in achieving high induction rate, high hatching rate and high survival rate in the above-mentioned prior art.

[0006] The embodiments of this application can be implemented through the following technical solutions:

[0007] A method for efficiently producing triploid oysters includes the following steps:

[0008] S1, obtain sperm and eggs from diploid oysters and perform sperm-egg fertilization;

[0009] S2, induced treatment of fertilized eggs to inhibit the release of the second polar body;

[0010] S3: Collect the fertilized eggs after induction treatment, wash them, and hatch them into larvae.

[0011] S4. 24h~72h after fertilization, the target larval population is collected based on the differences in individual larval size.

[0012] S5, the larvae selected in S4 are cultured.

[0013] Furthermore, in S4, the larvae are screened using a graded sieve, which includes an upper sieve and a lower sieve. The upper sieve has a larger aperture than the lower sieve, and the collected target larval population passes through the upper sieve and is retained by the lower sieve.

[0014] Furthermore, the upper layer of sieve silk has a mesh size of 300 mesh, and the lower layer of sieve silk has a mesh size of 500 mesh.

[0015] Furthermore, the screening operation in S4 is performed 24 hours after fertilization.

[0016] Furthermore, the induction treatment in S2 is performed using cytochalasin B at a concentration of 0.5~1.0 mg / L for 15~20 min.

[0017] Furthermore, the induction treatment in S2 begins 15 minutes after fertilization.

[0018] Furthermore, S1 specifically includes the following steps:

[0019] S11, Parental selection: Selective diploid oyster females and males with well-developed glands were used as parents;

[0020] S12, Egg preparation: The parent plants were dissected and examined under a microscope. Female individuals were selected to obtain eggs, which were then placed in natural seawater at 24°C for 30 minutes to mature.

[0021] S13, Sperm preparation: Sperm is obtained by dissecting a male individual that has been identified under a microscope 10 minutes before sperm and egg mixing;

[0022] S14, Oocyte pretreatment: After maturation, the oocytes are examined under a microscope to confirm that there is no sperm contamination. The oocytes are then collected using an 800-mesh sieve and the concentrated oocytes are placed in a container and natural seawater is added.

[0023] S15, Sperm-Egg Mixing: Mix the sperm from S13 with the egg from S14 and start timing.

[0024] Furthermore, in step S3, fertilized eggs are collected using an 800-mesh sieve.

[0025] Furthermore, the incubation in S3 is carried out at a seawater temperature of 24°C.

[0026] Furthermore, the density of cells cultured in S5 is 1-2 cells / mL.

[0027] The embodiments of this application provide a method for efficiently producing triploid oysters, which has at least the following beneficial effects:

[0028] This method is based on the discovery of the anomaly that triploid larvae are smaller than diploid larvae in a specific time stage of early post-fertilization development. It uses physical screening to efficiently enrich the mixed larval population. Even if the initial triploid induction rate is relatively low (around 70%), the triploid rate can be stably increased to over 90% after screening, effectively solving the problem of unstable induction rate in existing technologies.

[0029] This method reduces the dependence on chemical induction conditions and eliminates the need for harsh high-concentration, long-term induction protocols, thereby significantly mitigating the impact of inducer toxicity on embryos. It achieves an extremely high triploidy rate while ensuring high hatching and high survival rates.

[0030] This method is simple and easy to operate, requires no complex and expensive equipment, and has good repeatability and stable results. It is easy to promote and apply, and is especially suitable for triploid preparation of precious oyster varieties or oyster strains with important economic traits that are difficult to obtain a large number of fertilized eggs through strong induction conditions. Attached Figure Description

[0031] Figure 1 A schematic diagram illustrating the steps of a method for efficiently producing triploid oysters according to embodiments of this application;

[0032] Figure 2 This is a schematic diagram illustrating the size changes of diploid and triploid larvae with fertilization time in the embodiments of this application. Detailed Implementation

[0033] The present application will now be further described based on preferred embodiments and with reference to the accompanying drawings.

[0034] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] The triploidy rate detection method in each embodiment of this application is to use the commonly used method of 4',6-diamidinyl-2-phenylindole (DAPI) staining followed by flow cytometry. It will not be described again in the following embodiments.

[0036] As described in the background section, the existing methods for producing triploid oysters using induction, especially chemical induction, typically involve steps such as parent selection, sperm and egg collection, fertilization, induction treatment, hatching, and cultivation. Technological improvements often focus on optimizing the type, concentration, and timing of inducing agents to directly increase the initial induction rate. However, due to the toxicity of chemical inducing agents, high induction rates are often accompanied by low hatching rates and high mortality rates, making it difficult to achieve a balance between high induction rate, high hatching rate, and high survival rate.

[0037] However, during in-depth research into the aforementioned methods, the applicant discovered that in the early stages of larval development after fertilization, the size difference between diploid and triploid larvae exhibits a pattern contrary to common understanding. Generally, those skilled in the art generally believe that triploid oysters should be larger than diploid oysters; even with the same cell number, the cell volume of triploid oysters is larger than that of diploid oysters (e.g., triploid oyster eggs are approximately 60 μm in diameter, while diploid oyster eggs are approximately 50 μm in diameter). However, through extensive experiments, the applicant found that in the early stages of larval development, from 24 to 72 hours after fertilization, the individual size of triploid larvae is significantly smaller than that of diploid larvae at the same stage, and their size mostly remains within a specific range, such as... Figure 2 The diagram is shown below.

[0038] Based on the aforementioned unexpected findings, this application provides a method for efficiently producing triploid oysters. The core of this method lies in utilizing the reverse size difference between triploid and diploid larvae in the early developmental stage. Efficient enrichment is achieved through physical sieving within a specific time window, thus resolving the contradiction between the toxicity and high induction rate of chemically induced oysters in existing technologies. Even with a relatively low initial induction rate, subsequent sieving can stably yield a high-quality larval population with a triploid rate exceeding 90%, achieving both high hatching and high survival rates. Figure 1 As shown, the method specifically includes the following steps:

[0039] S1, obtain sperm and eggs from diploid oysters and perform sperm-egg fertilization;

[0040] Specifically, its operation includes the following steps:

[0041] S11, Parental selection: Selective diploid oyster females and males with well-developed glands were used as parents;

[0042] S12, Egg preparation: The parent plants were dissected and examined under a microscope. Female individuals were selected to obtain eggs, which were then placed in natural seawater at 24°C for 30 minutes to mature.

[0043] S13, Sperm preparation: Sperm was obtained from male individuals identified by a dissecting microscope 10 minutes before sperm and egg mixing;

[0044] S14, Oocyte pretreatment: After maturation, the oocytes are examined under a microscope to confirm that there is no sperm contamination. The oocytes are then collected using an 800-mesh sieve and the concentrated oocytes are placed in a container and natural seawater is added.

[0045] S15, Sperm-Egg Mixing: Mix the sperm from S13 with the egg from S14 and start timing.

[0046] S2, induced treatment of fertilized eggs to inhibit the release of the second polar body;

[0047] Specifically, the induction treatment is chemical induction. Further, the inducing agent can be chemical reagents commonly used by those skilled in the art, such as cytochalasin B, 6-diaminomethylpurine, and caffeine. In some preferred embodiments, the induction treatment uses 0.5~1.0 mg / L of cytochalasin B to inhibit the release of the second polar body. This treatment is performed approximately 15 minutes after fertilization and lasts for 15~20 minutes. More preferably, the concentration of cytochalasin B is 0.5 mg / L, which is much lower than the concentration used by conventional inducing agents, thus limiting the toxic effects of cytochalasin B.

[0048] S3: Collect the fertilized eggs after induction treatment, wash them, and hatch them into larvae.

[0049] Specifically, after induction treatment, fertilized eggs are collected using an 800-mesh sieve and rinsed with running water for 3-5 minutes. The cleaned fertilized eggs are then transferred to an incubation container for incubation. Furthermore, the incubation conditions are carried out in seawater at 24°C.

[0050] S4. 24h~72h after fertilization, the target larval population is collected based on the differences in individual larval size.

[0051] Specifically, the screening is performed using graded sieves, which include an upper sieve and a lower sieve. The upper sieve has a larger pore size than the lower sieve, and the collected target larval population passes through the upper sieve but is trapped by the lower sieve. Further, the upper sieve has a pore size of 300 mesh, and the lower sieve has a pore size of 500 mesh. In some preferred embodiments, the screening operation is preferably performed 24 hours after fertilization. Experiments have shown that screening at this time point is significantly more efficient than later screening. Specifically, the number of live triploid larvae obtained by screening 24 hours after fertilization is approximately five times the number obtained by screening 72 hours after fertilization. This is mainly due to two reasons: firstly, as the development time after fertilization increases, the size difference between diploid and triploid larvae gradually decreases; secondly, triploid larvae exhibit a higher mortality rate in the early developmental stages, thus reducing the overall yield available in later screening. Therefore, screening 24 hours after fertilization can maximize the capture and enrichment of vigorous triploid larvae populations during this developmental window, thus laying the foundation for efficient production.

[0052] S5, the larvae selected in S4 are cultured.

[0053] Specifically, the selected larvae are transferred to a rearing tank for culture at a density of 1-2 larvae / mL.

[0054] Examples 1-3

[0055] Examples 1-3 all used a single parent pair as the experimental subjects, namely one male oyster and one female oyster with selectively mature glands as parents. Three parallel replicate experiments were conducted using the same operating procedure to verify the repeatability and stability of the method. The specific operating steps are as follows:

[0056] Step 1: Dissect the parent plants and examine them under a microscope. Select one female individual and obtain the eggs. Then, place the eggs in natural seawater at 24 ℃ for 30 min to mature.

[0057] Step 2: 10 minutes before sperm and egg mixing, collect sperm from one male after microscopic identification;

[0058] Step 3: Examine the matured eggs under a microscope to ensure that there is no sperm contamination. Collect the eggs using an 800-mesh sieve and place the concentrated eggs in a small bucket with an appropriate amount of natural seawater added.

[0059] Step 4: Mix the sperm and egg and start timing, stirring gently;

[0060] Step 5: 15 min after sperm and egg mixing, induce the fertilized egg with 0.5 mg / L cytochalasin B for 20 min to inhibit the release of the second polar body.

[0061] Step 6: After the induction treatment is completed, collect the fertilized eggs using an 800-mesh sieve and rinse them with running water for 5 minutes. Transfer the cleaned fertilized eggs to an incubation container for incubation. The seawater temperature for incubation is 24°C.

[0062] Step 7: 24 hours after fertilization, take an appropriate amount of larvae for the first triploidity test, and then filter them using 300-mesh and 500-mesh sieves, with the 300-mesh sieve on top and the 500-mesh sieve on the bottom. Collect the larvae that can pass through the upper 300-mesh sieve and are trapped by the lower 500-mesh sieve.

[0063] Step 8: Take an appropriate amount of the larvae collected in Step 7 for a second triploidity test, and transfer the collected larvae to a culture tank for culture at a density of 1-2 larvae / mL.

[0064] Test results:

[0065] In Examples 1-3, the triploid rates detected for the first time 24 hours after fertilization were 72.34%, 75.80%, and 73.86%, respectively; after screening, the triploid rates detected for the second time were 95.26%, 98.15%, and 98.34%, respectively.

[0066] Example 4

[0067] The only difference between Example 4 and Example 1 is that the parent oysters used in Example 4 are four male oysters and four female oysters with mature gonads. Also, the concentration of cytochalasin B used in step 5 of Example 4 during the induction treatment is 1.0 mg / L. All other experimental steps and conditions are the same as in Example 1 and can be carried out with reference to Example 1. They will not be repeated here.

[0068] Test results:

[0069] In Example 4, the first triploidy rate test at 24 hours after fertilization showed a result of 85.72%; after screening, the second triploidy rate test showed a result of 98.65%.

[0070] Example 5

[0071] The only difference between Example 5 and Example 1 is that step 7 is a screening operation performed 72 hours after fertilization. The remaining experimental steps and conditions are the same as in Example 1, and will not be repeated here.

[0072] Test results:

[0073] In Example 5, the first triploidity test at 72 hours after fertilization showed a triploidity rate of 76.46%; after screening, the second test showed a triploidity rate of 92.64%, but the number of larvae screened at 72 hours was reduced by about 80% compared to the screening at 24 hours.

[0074] Example 6

[0075] The only difference between Example 6 and Example 1 is that the parent oysters used are three male oysters and three female oysters with mature gonads. The other experimental steps and conditions are the same as in Example 1 and can be carried out with reference to Example 1. They will not be repeated here.

[0076] For Example 6, the first triploidity rate was detected 24 hours after fertilization, the second triploidity rate was detected after screening, and the triploidity rate was detected 360 days after culture. The results were 72.35%, 98.64%, and 76.67%, respectively.

[0077] Comparative Example 1

[0078] The only difference between Comparative Example 1 and Example 6 is that Comparative Example 1 does not include the screening operation in step 7. That is, after the first triploidity test was performed 24 hours after fertilization, all larvae in Comparative Example 1 were directly transferred to the culture tank for cultivation. The remaining experimental steps and conditions in Comparative Example 1 are the same as those in Example 6, and will not be repeated here.

[0079] The triploidity rate of Comparative Example 1 was first detected 24 hours after fertilization and 360 days after culture. The results were 75.62% and 33.33%, respectively.

[0080] Comparative Example 2

[0081] The only difference between Comparative Example 2 and Comparative Example 1 is that the concentration of cytochalasin B used in the induction treatment in step 5 is 1.0 mg / L. The rest of the experimental steps and conditions are the same as those in Comparative Example 1 and can be carried out with reference to Comparative Example 1. They will not be repeated here.

[0082] The triploidity rate of Comparative Example 2 was first tested 24 hours after fertilization and again after 360 days of culture. The results showed that the triploidity rate was 92.38% at 24 hours after fertilization, and almost all of the triploids died by 18 days after fertilization.

[0083] Comparative Example 3

[0084] The only difference between Comparative Example 3 and Comparative Example 1 is that the treatment time of cytochalasin B used in the induction process in step 5 is 40 min. The other experimental steps and conditions are the same as those in Comparative Example 1 and can be referred to Comparative Example 1. They will not be repeated here.

[0085] The triploidity rate of Comparative Example 3 was first detected 24 hours after fertilization and again after 360 days of culture. The results were 90.62% and 14.29%, respectively.

[0086] The specific embodiments of this application have been described in detail above. For those skilled in the art, several improvements and modifications can be made to this application without departing from the principle of this application, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A method for efficiently producing triploid oysters, characterized in that, Includes the following steps: S1, obtain sperm and eggs from diploid oysters and perform sperm-egg fertilization; S2, The fertilized egg is subjected to an induction treatment to inhibit the release of the second polar body, the induction treatment starting 15 minutes after fertilization; S3: Collect the fertilized eggs after induction treatment, wash them, and hatch them into larvae. S4. 24h to 72h after fertilization, the triploid larvae are smaller than the diploid larvae. Screening is carried out based on the difference in larval size to collect the target larval population. Specifically, the screening is carried out through a graded sieve, which includes an upper sieve and a lower sieve. The pore size of the upper sieve is larger than that of the lower sieve. The collected target larval population passes through the upper sieve and is trapped by the lower sieve. S5, the larvae selected in S4 are cultured.

2. The method according to claim 1, characterized in that, The upper sieve silk has a mesh size of 300 mesh, and the lower sieve silk has a mesh size of 500 mesh.

3. The method according to claim 1, characterized in that, The screening operation in S4 is performed 24 hours after fertilization.

4. The method according to claim 1, characterized in that, The induction treatment in S2 is performed using cytochalasin B at a concentration of 0.5-1.0 mg / L for 15-20 minutes.

5. The method according to claim 1, characterized in that, S1 specifically includes the following steps: S11, Parental selection: Selective diploid oyster females and males with well-developed glands were used as parents; S12, Egg preparation: The parent plants were dissected and examined under a microscope. Female individuals were selected to obtain eggs, which were then placed in natural seawater at 24°C for 30 minutes to mature. S13, Sperm preparation: Sperm is obtained by dissecting a male individual that has been identified under a microscope 10 minutes before sperm and egg mixing; S14, Oocyte pretreatment: After maturation, the oocytes are examined under a microscope to confirm that there is no sperm contamination. The oocytes are then collected using an 800-mesh sieve and the concentrated oocytes are placed in a container and natural seawater is added. S15, Sperm-Egg Mixing: Mix the sperm from S13 with the egg from S14 and start timing.

6. The method according to claim 1, characterized in that, In step S3, fertilized eggs are collected using an 800-mesh sieve.

7. The method according to claim 1, characterized in that, The incubation in S3 is carried out at a seawater temperature of 24°C.

8. The method according to claim 1, characterized in that, The density of cells cultured in S5 is 1-2 cells / mL.

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