Method for salinity induction of haliotis discus hannai triploid

Abstract

The invention discloses a method for inducing triploid of haliotis discus hannai by salinity, which comprises the following steps: (1) synchronously fertilizing sperms and ova of haliotis discus hannai to obtain fertilized ova; (2) when the appearance proportion of the first polar bodies of the fertilized eggs obtained in the step (1) is 60-70%, performing high-salt treatment or low-salt treatment on the fertilized eggs; (3) the fertilized eggs treated in the step (2) are subjected to salinity gradient sand filtration seawater re-infiltration treatment, the osmotic pressure is gradually adjusted through salinity gradient sand filtration seawater, damage to the fertilized eggs is relieved, then the fertilized eggs are resuspended in fresh sand filtration seawater with normal salinity, and conventional incubation and cultivation are conducted. According to the method, efficient triploid induction can be achieved through salinity optimization and re-infiltration steps, the problems of drug residues and equipment requirements of traditional chemical and physical methods are solved, and the method has the advantages of being safe, simple, convenient and efficient.

Description

Technical Field

[0001] The invention belongs to the technical field of shellfish breeding, and in particular relates to a method for inducing triploid abalone of Haliotis discus hannai with salinity. Background Art

[0002] Abalone is an important economic shellfish for marine aquaculture. Its gastropod muscle is the main edible part and is highly favored in domestic and foreign consumer markets. my country is the world's largest producer and consumer of abalone. In 2023, the total output of abalone reached 244,000 tons, of which the wrinkled disc abalone (Haliotis discus hannai) was the main farmed species. However, with the rapid development of the abalone farming industry, problems such as slow individual growth rate, germplasm degradation and increased production costs have become increasingly prominent. Usually, abalone needs to be farmed for 2 to 3 years to reach market specifications, and the continued rise in labor and infrastructure costs has further aggravated the constraints of its growth characteristics on industrial development.

[0003] Triploids bred based on chromosome engineering technology have attracted widespread attention because they can significantly improve the growth rate and individual quality of shellfish. Due to their sterility, triploid shellfish can transfer the energy used for reproduction to individual growth, thereby improving the economic benefits during the breeding cycle. Since Stanley first used cytochalasin B to successfully induce triploid American oysters in 1956, scholars at home and abroad have conducted triploid breeding research in a variety of shellfish such as oysters, scallops, mussels, clams and abalone. At present, except for some oyster species that achieve full triploid production through interploid hybridization, the acquisition of triploids in other shellfish mainly relies on physical and chemical induction methods.

[0004] In the prior art, commonly used methods for inducing triploid shellfish include:

[0005] 1. Chemical induction: using chemical drugs such as cytochalasin B, 6-dimethylaminopurine (6-DMAP), caffeine, colchicine and nocodazole to induce triploidy by inhibiting the release of fertilized egg polar bodies. However, this method has the problem of drug residues, which may affect embryonic development and shellfish consumption safety, and cause potential pollution to the environment.

[0006] 2. Physical induction: Inducing triploidy by interfering with the normal development process of fertilized eggs through physical means such as temperature shock, hydrostatic pressure or electric pulses. This method relies on expensive special equipment and has high requirements for operating conditions. The induction efficiency is unstable, which limits its application in large-scale production.

[0007] 3. Exploration of salinity induction: In recent years, researchers have tried to use high salt and low salt to change the osmotic pressure to inhibit the release of fertilized egg polar bodies to induce triploidy, and have made some progress in shellfish such as oysters and scallops. The wrinkled abalone is a narrow salt shellfish, and the fertilized eggs are more sensitive to changes in environmental salinity. The existing salinity induction technology has not yet formed a mature application for this species. For example, Guo Deqiang (2014) showed that the highest induction rates of low-salt and high-salt induced wrinkled abalone triploids were only 45.34% and 33.46%, respectively, which is far from the ideal level of production application, and the induction efficiency and stability still need to be improved. Summary of the invention

[0008] The invention aims to overcome the defects of the prior art and provide a method for inducing triploidy of Haliotis discus hannai with salinity.

[0009] The technical solution of the present invention is as follows:

[0010] A method for inducing triploid abalone in salinity, comprising the following steps:

[0011] (1) synchronously fertilizing the sperm and ovum of Haliotis discus hannai to obtain a fertilized egg;

[0012] (2) when the proportion of the first polar body of the fertilized eggs obtained in step (1) is 60-70%, the fertilized eggs are subjected to high salt treatment or low salt treatment;

[0013] (3) The fertilized eggs treated in step (2) are subjected to a salinity gradient sand-filtered seawater re-infiltration treatment to gradually adjust the osmotic pressure through the salinity gradient sand-filtered seawater to reduce damage to the fertilized eggs, and then resuspended in fresh sand-filtered seawater of normal salinity for conventional incubation and cultivation.

[0014] In a preferred embodiment of the present invention, the temperature of the synchronous fertilization is 18-23.5°C.

[0015] In a preferred embodiment of the present invention, the high-salt treatment comprises: placing the fertilized eggs in sand-filtered seawater with a salinity of 41-52 ppm for 10-25 minutes.

[0016] Further preferably, the salinity gradient sand-filtered seawater re-infiltration treatment includes: first placing in 37-39 ppm sand-filtered seawater for 2-3 minutes, and then placing in 32-34 ppm sand-filtered seawater for 2-5 minutes.

[0017] In a preferred embodiment of the present invention, the low-salinity treatment comprises: placing the fertilized eggs in sand-filtered seawater with a salinity of 12-18 ppm for 10-25 minutes.

[0018] Further preferably, the salinity gradient sand-filtered seawater re-infiltration treatment comprises: first placing in 24-26 ppm sand-filtered seawater for 2-3 minutes, and then placing in 30-32 ppm sand-filtered seawater for 2-5 minutes.

[0019] In a preferred embodiment of the present invention, the salinity of the normal salinity fresh sand-filtered seawater is 30-35 ppm.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present invention completely avoids the use of chemical drugs, eliminates the potential impact of drug residues on embryonic development and food safety, and does not cause pollution to the environment, meeting the development requirements of green breeding.

[0022] 2. The present invention does not require expensive special equipment and can achieve induction only by adjusting the salinity of seawater. It is simple to operate and easy to promote and apply, which significantly reduces production costs.

[0023] 3. By optimizing the salinity (high salt 41-52ppm or low salt 12-18ppm) and treatment time (10-25min), and combining with re-infiltration treatment, the present invention can achieve a triploid induction rate of up to 100%, far exceeding the induction efficiency of traditional physical methods.

[0024] 4. The present invention introduces a salinity gradient re-osmosis step, which effectively slows down the damage to the fertilized eggs caused by the rapid change of osmotic pressure, and can improve the normal development ratio of the fertilized eggs as well as the induction efficiency and stability.

[0025] 5. The present invention not only meets the technical requirements of the production of triploid Haliotis discus hannai, but also establishes a safe, simple, stable and efficient triploid induction method for Haliotis discus hannai, which is conducive to promoting the sustainable development of the abalone farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram of DNA content analysis of the juvenile disc of Abalone discus hannai obtained in Example 1 of the present invention (2N: diploid, 3N: triploid).

[0027] Figure 2 This is a diagram of DNA content analysis of the juvenile surface disc of the Haliotis discus hannai obtained in Example 4 of the present invention (3N: triploid). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0029] The preparation methods of sand-filtered seawater of different salinities in the following examples are based on “Sub-low salinity impact on survival, growth and meat quality of the pacific abalone (Haliotisdiscus hannai) and hybrids” (Boamah GA, Wang T, Chowdhury IA, Luo X, Huang MQ, Xu CA, Ke CH, You WW. Aquaculture Research, 2020, 51: 5184–5193).

[0030] Example 1

[0031] (1) The fertilization water temperature is 21.7° C., and the fertilized eggs of Haliotis discus hannai are taken. When the proportion of the first polar body of the fertilized eggs is 60%, the fertilized eggs are placed in low-salinity sand-filtered seawater with a salinity of 12 ppm for 15 min;

[0032] (2) The fertilized eggs treated in step (1) are placed in sand-filtered seawater with salinity of 24-25 ppm and 30-31 ppm for re-infiltration for 2-3 min, and then resuspended in fresh sand-filtered seawater with normal salinity (salinity of 33.5 ppm), and then conventionally incubated and cultured to obtain the larvae of Haliotis discus henna.

[0033] The larvae of Haliotis discus hannai obtained in step (2) were cultured to the veliger stage, and the ploidy was determined by flow cytometry. The triploid induction rate was 85.47±1.49%. The DNA content analysis chart is as follows: Figure 1 shown.

[0034] Example 2

[0035] (1) The fertilization water temperature is 22.4° C., and the fertilized eggs of Haliotis discus hannai are taken. When the proportion of the first polar body of the fertilized eggs is 60%, the fertilized eggs are placed in high-salinity sand-filtered seawater with a salinity of 52 ppm for 15 min;

[0036] (2) The fertilized eggs treated in step (1) are placed in sand-filtered seawater with salinity of 38-39 ppm and 32-33 ppm for re-infiltration for 2-3 min and 3-4 min, respectively, and then resuspended in fresh sand-filtered seawater with normal salinity (salinity of 32.4 ppm), and the larvae of Haliotis discus discus are obtained after conventional incubation and cultivation.

[0037] The larvae of the Haliotis discus hannai obtained in step (2) were cultured to the veliger stage, and the ploidy was determined by flow cytometry. The triploid induction rate was 96.94±2.74%.

[0038] Example 3

[0039] (1) The fertilization water temperature is 19.5° C., and the fertilized eggs of Haliotis discus hannai are taken. When the proportion of the first polar body of the fertilized eggs is 70%, the fertilized eggs are placed in high-salinity sand-filtered seawater with a salinity of 41 ppm for 25 minutes;

[0040] (2) The fertilized eggs treated in step (1) are placed in sand-filtered seawater with salinity of 38-39 ppm and 33-34 ppm for re-infiltration for 2-3 min and 3-4 min, respectively, and then resuspended in fresh sand-filtered seawater with normal salinity (salinity of 33.1 ppm), and the larvae of Haliotis discus discus are obtained after conventional incubation and cultivation.

[0041] The larvae of the Haliotis discus hannai obtained in step (2) were cultured to the veliger stage, and the ploidy was determined by flow cytometry. The triploid induction rate was 93.42±4.22%.

[0042] Example 4

[0043] (1) The fertilization water temperature is 23.0° C., and the fertilized eggs of Haliotis discus hannai are taken. When the proportion of the first polar body of the fertilized eggs is 60%, the fertilized eggs are placed in high-salinity sand-filtered seawater with a salinity of 52 ppm for 10 minutes;

[0044] (2) The fertilized eggs treated in step (1) are placed in sand-filtered seawater with salinity of 38-39 ppm and 32-33 ppm for re-infiltration for 2-3 min and 4-5 min, respectively, and then resuspended in fresh sand-filtered seawater with normal salinity (salinity of 33.5 ppm), and the larvae of Haliotis discus discus are obtained after conventional incubation and cultivation.

[0045] The larvae of Haliotis discus hannai obtained in step (2) were cultured to the veliger stage, and the ploidy was determined by flow cytometry. The triploid induction rate was 100.00±0.00%. The DNA content analysis chart is as follows: Figure 2 shown.

[0046] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for inducing triploid Haliotis discus hannai with salinity, characterized in that: The steps include: (1) synchronously fertilizing the sperm and ovum of Haliotis discus hannai to obtain a fertilized egg; (2) when the proportion of the first polar body of the fertilized eggs obtained in step (1) is 60-70%, the fertilized eggs are subjected to high salt treatment or low salt treatment; (3) The fertilized eggs treated in step (2) are subjected to a salinity gradient sand-filtered seawater re-infiltration treatment to gradually adjust the osmotic pressure through the salinity gradient sand-filtered seawater to reduce damage to the fertilized eggs, and then resuspended in fresh sand-filtered seawater of normal salinity for conventional incubation and cultivation.

2. The method according to claim 1, characterized in that: The temperature of the synchronous fertilization is 18-23.5°C.

3. The method according to claim 1, characterized in that: The high-salinity treatment comprises placing the fertilized eggs in sand-filtered seawater with a salinity of 41-52 ppm for 10-25 minutes.

4. The method according to claim 3, characterized in that: The salinity gradient sand-filtered seawater re-infiltration treatment comprises: first placing in 37-39 ppm sand-filtered seawater for 2-3 minutes, and then placing in 32-34 ppm sand-filtered seawater for 2-5 minutes.

5. The method according to claim 1, characterized in that: The low-salinity treatment comprises placing the fertilized eggs in sand-filtered seawater with a salinity of 12-18 ppm for 10-25 minutes.

6. The method according to claim 5, characterized in that: The salinity gradient sand-filtered seawater re-infiltration treatment comprises: first placing in 24-26 ppm sand-filtered seawater for 2-3 minutes, and then placing in 30-32 ppm sand-filtered seawater for 2-5 minutes.

7. The method according to claim 1, characterized in that: The salinity of the normal salinity fresh sand-filtered seawater is 30-35 ppm.

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