A method for improving the survival rate of Hong Kong giant oyster under high salt conditions

Abstract

The present invention relates to the technical field of oyster aquaculture, and discloses a method for improving the survival rate of Hong Kong giant oysters under high-salinity conditions, comprising S1, preparing a bait culture solution and a mother liquor; S2, culturing Chaetoceros muelleri as bait for Hong Kong giant oysters. The present invention provides a new means for solving the problem of the survival rate of Hong Kong giant oysters in a high-salinity environment, and is helpful for coping with the drastic fluctuation of salinity during aquaculture. By feeding with bait rich in betaine, the survival rate of Hong Kong giant oysters under 35 salinity conditions is significantly improved. At the same time, in view of the fact that Hong Kong giant oysters have the characteristics of low-salinity tolerance but weak high-salinity tolerance, the present invention aims to improve their adaptability to high-salinity environments. To achieve this goal, by feeding bait that absorbs betaine, Hong Kong giant oysters indirectly absorb betaine, thereby enhancing their high-salinity tolerance, and the survival rate of Hong Kong giant oysters is increased by 50% compared with before.

Description

Technical Field

[0001] The invention relates to the technical field of oyster culture, and in particular to a method for improving the survival rate of Hong Kong giant oysters under high-salt conditions. Background Art

[0002] The Hong Kong oyster (Crassostrea hongkongensis), also known as the "Qinzhou oyster", is the main shellfish cultured variety in the Guangdong and Guangxi regions of my country.

[0003] However, as the scale of aquaculture continues to expand, problems related to Hong Kong oyster aquaculture have gradually emerged. Salinity, as an environmental factor closely related to the osmotic pressure regulation of aquatic animals, has an important impact on the growth and metabolism of aquatic animals. As an intertidal organism, the Hong Kong oyster has a suitable physiological salinity of 13-20. However, the salinity of its aquaculture environment is easily affected by rivers entering the sea. Changes in salinity force organisms to go through a series of physiological responses to adapt to the dynamic balance of osmotic pressure inside and outside the body, resulting in changes in related indicators such as respiratory metabolism and energy budget, and even death in severe cases.

[0004] Betaine, a quaternary ammonium alkaloid, has a unique molecular structure, a neutral molecular charge and three active methyl groups, which can efficiently provide methyl groups to the body. At present, betaine is mainly used as a feed additive for terrestrial farmed animals such as pigs and chickens, replacing part of choline, participating in protein and fat metabolism, and promoting the growth of farmed animals. At the same time, betaine, as an osmotic protectant, has a certain protective effect on animals under osmotic stress. However, in the field of aquaculture, especially in the osmotic stress of shellfish, its role is still unclear.

[0005] Therefore, the present invention proposes a new method for improving the survival rate of Hong Kong oysters under high-salt conditions, aiming to improve the survival ability of Hong Kong oysters in high-salt environments and provide strong support for the healthy cultivation of Hong Kong oysters. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a method for improving the survival rate of Hong Kong giant oysters under high-salt conditions, so as to improve the survival rate of Hong Kong oysters under high-salt conditions and provide strong support for the healthy cultivation of Hong Kong oysters.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for improving the survival rate of Hong Kong giant oysters under high salt conditions, comprising the following steps:

[0008] S1. Prepare bait culture solution and mother solution:

[0009] S11. Add an appropriate amount of mother solution to the sterilized seawater to provide nutrients required by algae to obtain culture solution.

[0010] In this step, the mother liquid is added to the sterilized seawater to provide the nutrients required for the algae. The mother liquid contains substances such as carbon source, nitrogen source, phosphorus source and trace elements, which are necessary for the growth and reproduction of algae. By adding the appropriate amount of mother liquid, sufficient nutrients can be provided to promote the growth and reproduction of algae, thereby providing a sufficient source of food for Hong Kong oysters.

[0011] S2. Cultivate Chaetoceros muelleri as bait for Hong Kong oysters, wherein the Hong Kong oysters are selected as the cultured individuals, which are in good growth condition, free of diseases, and have intact shells without damage. The Chaetoceros muelleri is selected from algae species that are free of pest pollution, have strong vitality, grow vigorously, and have no cracking:

[0012] S21, adding betaine to the culture solution for culturing Chaetoceros muelleri;

[0013] S22, mixing the Chaetoceros muelleri algae species and the culture solution in a volume ratio of 1:4 to culture the algae species.

[0014] These steps mainly involve the cultivation and preparation of Chaetoceros muelleri as food for Hong Kong oysters. The addition of betaine helps to promote the growth of Chaetoceros muelleri because betaine is a common organic carbon source that can provide the carbon elements required by algae. Mixing Chaetoceros muelleri algae and culture medium in a volume ratio of 1:4 can provide appropriate algae concentration and nutrient concentration, providing a rich food source for oysters.

[0015] Chaetoceros muelleri has several beneficial effects as a bait for Hong Kong oysters. First, Chaetoceros muelleri is a type of algae rich in nutrients, including protein, fat, vitamins and minerals, which can provide rich nutrition for Hong Kong oysters to ingest, promote their growth and increase their survival rate. Secondly, the selection of Chaetoceros muelleri requires no contamination by predators and pests, which can avoid algae infection or reproduction of other harmful organisms, and keep the breeding environment clean and healthy. In addition, the selection of algae species with strong vitality, vigorous growth and no lysis can ensure the provision of high-quality bait and improve the feeding effect and nutrient absorption capacity of Hong Kong oysters.

[0016] Preferably, the mother liquor comprises inorganic salts and organic substances to provide nutrients required by algae, wherein the inorganic salts include sodium nitrate, potassium dihydrogen phosphate and sodium silicate, and the organic substances include ferric citrate hydrate, VB 1 and VB 12 .

[0017] Inorganic salts:

[0018] Sodium nitrate (NaNO 3 ): Provide nitrogen source to promote protein synthesis and growth of algae.

[0019] Potassium dihydrogen phosphate (KH2 PO 4 ): Provide a source of phosphorus to promote the growth and metabolic process of algae.

[0020] Sodium silicate (Na 2 SiO 3 ): Provides a source of silicon, which plays an important role in the growth and skeleton formation of some diatoms.

[0021] Organic matter:

[0022] Ferric citrate hydrate: provides a source of iron, which is one of the trace elements necessary for algae growth and is involved in photosynthesis and other metabolic processes.

[0023] VB 1 (Vitamin B 1 ): Also known as thiamine, it is a member of the B vitamins and plays an important role in the growth and metabolic processes of algae.

[0024] VB 12 (Vitamin B 12 ): Also known as cobalamin, it is a member of the B vitamin complex and plays an important role in the growth and metabolic processes of algae.

[0025] These ingredients provide the main nutrients required by algae, including nitrogen, phosphorus, silicon, iron and vitamins. Their presence in the mother solution can meet the nutritional needs of algae and promote their growth and reproduction. By adjusting the concentration and proportion of these ingredients in the mother solution, the breeding conditions can be optimized, the quality of oyster bait can be improved, and the survival rate and growth effect of Hong Kong oysters can be increased.

[0026] Preferably, the culture solution is prepared by adding 1 mL of the culture solution of Chaetoceros muelleri to each liter of sterilized seawater. This addition method can provide an appropriate amount of Chaetoceros muelleri and provide a sufficient food source for Hong Kong oysters.

[0027] In this way, oysters can obtain the nutrients they need to promote growth and survival by eating these algae. Chaetoceros muelleri is a nutrient-rich algae, including protein, fat, vitamins and minerals, which can provide rich food for oysters.

[0028] Adding an appropriate amount of Chaetoceros muelleri culture solution helps maintain the appropriate algae concentration and nutrient concentration, avoiding excessive addition leading to nutrient excess or excessive growth. At the same time, quantitative addition of Chaetoceros muelleri culture solution also helps control the growth of algae in the culture environment to avoid excessive algal blooms or other adverse effects.

[0029] Preferably, the mother solution of the culture solution of Chaetoceros muelleri is 60 mg of sodium nitrate, 4.5 mg of potassium dihydrogen phosphate, 5 mg of sodium silicate, 0.5 mg of ferric citrate hydrate, and VB 1 :150μg、VB 12 : 200ng and then made into 1000× mother solution, the preparation steps are as follows:

[0030] Prepare sterile distilled water: Sterilize the distilled water to ensure the sterility of the culture medium.

[0031] Weigh ingredients: According to the above formula, sodium nitrate, potassium dihydrogen phosphate, sodium silicate, ferric citrate hydrate, VB 1 and VB 12 Weigh out an appropriate amount.

[0032] Mix into mother liquor: Dissolve the weighed ingredients separately with a small amount of sterile distilled water, then transfer all into a 1000mL volumetric flask, make up to volume with sterile distilled water, and mix evenly by inverting.

[0033] Filtration and sterilization: Filter the mixed mother liquor with a filter to remove suspended solid particles. Then, use a suitable sterilization method (such as high temperature and high pressure sterilization) to sterilize the mother liquor to ensure the sterility of the culture solution.

[0034] Storage: Store the sterilized Chaetoceros muelleri culture stock solution in a suitable container and seal it properly. It is best to store it in a cool, dry and dark place.

[0035] When in use, the mother solution of the culture solution of Chaetoceros muelleri is added into the sterilized seawater in an appropriate proportion as required to achieve the required algae concentration and nutrient concentration.

[0036] Preferably, the added amount of betaine is 100 mg / L.

[0037] Preferably, the culture time of Chaetoceros muehii is 5-7 days.

[0038] Preferably, the pH value of the culture solution of Chaetoceros muehii is adjusted in the range of 7.5 to 8.5.

[0039] Preferably, the culture solution of Chaetoceros muehii is sterilized to ensure the purity and safety of the bait, and the sterilization treatment includes high temperature and high pressure sterilization and filtration sterilization.

[0040] Preferably, the Chaetoceros muelleri in the culture solution is used as bait for Hong Kong oysters, and the number of baits fed to each oyster per liter of water is 3×10 4 cells.

[0041] The present invention provides a method for improving the survival rate of Hong Kong giant oysters under high-salinity conditions. It has the following beneficial effects:

[0042] 1. The present invention provides a new means to solve the problem of survival rate of Hong Kong oysters in high-salinity environment, which helps to cope with the drastic fluctuation of salinity during aquaculture. By feeding with bait rich in 100 mg / L betaine, the survival rate of Hong Kong oysters under 35 salinity conditions is significantly improved.

[0043] 2. Considering that Hong Kong oysters have low salt tolerance but weak high salt tolerance, the present invention aims to improve their adaptability to high salt environment. To achieve this goal, by feeding baits that absorb betaine, Hong Kong oysters can indirectly absorb betaine, thereby enhancing their high salt tolerance, and the survival rate of Hong Kong oysters has increased by 50% compared with before.

[0044] 3. The present invention selects Chaetoceros muelleri as bait and adds betaine for bait cultivation. The experimental results show that the reproduction rate of Chaetoceros muelleri is increased by 5.96 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Embodiment 1:

[0048] Selection of oysters: Select healthy Hong Kong oysters with a shell height of 10.0 cm, a shell age of 2 years, complete bivalve, smooth shell surface with few attachments, and vigorous growth of scales on the outer edge of the shell without disease as the breeding individuals;

[0049] Selection of algae species: select algae species with clear algae liquid, no obvious flocs, and no lysis under the microscope for cultivation;

[0050] Selection of aquaculture equipment: The volume of the oyster culture barrel is 15L, and the microalgae culture uses a 2L transparent conical bottle. Before starting the culture, the culture barrel and air stone required for oyster culture need to be soaked in potassium permanganate for more than 3 hours, and the transparent conical bottle and seawater used for microalgae culture need to be sterilized by high temperature and high pressure.

[0051] Preparation of mother solution: The bait algae used is Chaetoceros muelleri, which is a diatom. The culture solution formula is as follows: sodium nitrate: 60 mg, potassium dihydrogen phosphate: 4.5 mg, sodium silicate: 5 mg, ferric citrate hydrate: 0.5 mg, VB 1 :150μg、VB 12 : 200ng, seawater: 1000mL, according to this ratio, prepare 1000× mother solution. When culturing microalgae, add 1mL of mother solution for every liter of seawater.

[0052] Betaine addition: Add 1L of sterilized seawater with a salinity of 30 to a conical flask, add 1mL of the mother solution, shake well, and then add 100mg of betaine.

[0053] Cultivation of bait: Add algae species and culture solution in a ratio of 1:4 into a conical flask for cultivation. Count the algae regularly every day and observe the growth rate of the algae. Start feeding after 5 to 7 days of algae cultivation.

[0054] Feeding: Before each feeding, the algae in the conical bottle need to be shaken and counted. The number of feeds per oyster per liter of water is 3×10 4 cells, calculate the required number of baits based on the actual aquaculture water, calculate the volume of algae liquid required for feeding, and use a measuring cup to measure the required amount of algae liquid.

[0055] Breeding: The seawater used for breeding comes from Qinzhou Bay. The salinity of the breeding water is adjusted to the required salinity. It needs to be aerated for 24 hours before it can be used. Then, 30 oysters are placed in each breeding barrel. Oxygen is continuously supplied during the breeding period and the oysters are fed three times a day.

[0056] Embodiment 2:

[0057] The oysters were treated according to the method described in Example 1 above, and the results were as follows:

[0058] 1. Experimental location: Aquaculture Room, Beibu Gulf University

[0059] 2. Experimental time: May 2022

[0060] 3. Experimental design: The species of Chaetoceros muelleri were purchased from Fenggeng Algae Liquid Co., Ltd. for expansion. The experiment set up two groups, control group 1 and experimental group 2;

[0061] Control group 1: Three replicate groups were set up, the culture water volume of each group was 1L, and no betaine was added.

[0062] Experimental group 2: Three replicate groups were set up, the culture water volume of each group was 1L, and the added concentration of betaine was 100mg / L.

[0063] The initial algae density in each group of the experimental group and the control group was 1.85×10 4 Pieces / mL.

[0064] During the culture period, the algae were shaken three times a day, counted every day, the number of Chaetoceros muelleri was counted, the growth rate of the algae was calculated, and the changes in the betaine content in the algae on the 1st day and the 8th day were detected.

[0065] in,

[0066] Growth rate = (number on the current day - number on the previous day) / number on the previous day;

[0067] Betaine was detected using the Elisa kit from Shanghai ELISA.

[0068] Experimental results and analysis: The experimental results were statistically analyzed using GraphPad Prism, and the independent sample T test was used to analyze the experimental results. When P < 0.05, the difference was significant, and when P < 0.01, the difference was extremely significant. All data are expressed as mean ± standard deviation (Mean ± SD).

[0069] As shown in Table 1, after adding betaine, the cell concentration of Chaetoceros muelleri in the experimental group was effectively increased. From the 5th day, the cell concentration of Chaetoceros muelleri in the experimental group (×10 4 On the 8th day, the cell concentration of Chaetoceros muelleri in the experimental group reached 2.5 times that of the control group.

[0070] As shown in Table 2, the changes in betaine content in Chaetoceros muelleri of the control group and the experimental group on the 1st day and the 8th day. On the 8th day, the betaine content in Chaetoceros muelleri of the experimental group was significantly higher than that of the control group.

[0071] Table 1 Effect of adding betaine on the growth of Chaetoceros muelleri (unit: ×10 4 pcs / ml)

[0072]

[0073]

[0074] Note: The superscript "*" in the same column indicates significant difference (P<0.05), and the superscript "**" indicates extremely significant difference (P<0.01), the same below.

[0075] Table 2 Changes in betaine content in Chaetoceros muelleri after adding betaine (unit: ug / L)

[0076]

[0077] Embodiment 3:

[0078] The oysters were treated according to the method described in Example 1 above, and the results were as follows:

[0079] 1. Experimental location: Aquaculture Room of the Ocean College;

[0080] 2. Trial time: May 2022;

[0081] 3. Experimental design: 180 two-year-old Hong Kong oysters were taken from the Hong Kong oyster farming group in Maowei Sea, Guangxi for a breeding experiment. The experiment set up two groups, namely control group one and experimental group two.

[0082] In the control group 1, three replicate groups were set up, and 30 oysters were put into each group. The Hong Kong oysters in this group were fed with Chaetoceros muelleri without adding betaine as bait.

[0083] Experimental group 2: 3 replicate groups were set up, and 30 oysters were placed in each group. The Hong Kong oysters in this group were fed with Chaetoceros muelleri with a betaine addition of 100 mg / L as bait.

[0084] The salinity of the culture water is 35, which puts the oysters in a state of high salt stress. During the culture period, the feeding amount is 3×10 4 The oysters were fed with 1 / 3 of the actual water volume per day, and the dead individuals were removed in time, and the number of deaths was recorded and the survival rate was calculated. The betaine content in the adductor muscle of Hong Kong oysters under high salt stress was detected and recorded.

[0085] Survival rate = number of survivors during breeding period / initial breeding number.

[0086] Judging the survival of individuals: slight disturbance of the water and rapid closing of the double hulls.

[0087] Experimental results and analysis: The experimental results were statistically analyzed using GraphPad Prism, and the independent sample T test was used to analyze the experimental results. When P < 0.05, the difference was significant, and when P < 0.01, the difference was extremely significant. All data are expressed as mean ± standard deviation.

[0088] As shown in Table 3, under high-salinity conditions, the survival rate of the Hong Kong oyster in the experimental group fed with Chaetoceros muelleri at an added amount of 100 mg / L betaine on the 9th day was 50% higher than that of the control group.

[0089] As shown in Table 4, under high-salt conditions, the betaine content in the adductor muscle tissue of the Hong Kong oyster in the experimental group fed with 100 mg / L of Chaetoceros muelleri was significantly different from that in the control group at 120 h.

[0090] Therefore, adding exogenous betaine can effectively improve the survival rate of Hong Kong oysters under high salt conditions.

[0091] Table 3 Effects of feeding betaine-added bait on the survival rate of Hong Kong oysters under 35 salinity conditions

[0092]

[0093] Table 435 Betaine content in adductor muscle of Hong Kong oyster after feeding betaine under salinity conditions (ug / g)

[0094]

[0095] The present invention applies betaine to the cultivation of Hong Kong oysters for the first time. By first providing betaine to bait algae for absorption and then feeding the bait algae to oysters for ingestion, the ability of Hong Kong oysters to cope with high salt stress can be effectively improved, providing a new solution for improving the salt tolerance of Hong Kong oysters.

[0096] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the survival rate of Hong Kong giant oysters under high-salinity conditions, characterized in that: The following steps are involved: S1. Prepare bait culture solution and mother solution: S11, adding an appropriate amount of mother solution to the sterilized seawater to provide nutrients required by the algae to obtain a culture solution; S2. Cultivation of Chaetoceros muelleri as bait for Crassostrea gigas: S21, adding betaine to the culture solution for culturing Chaetoceros muelleri; S22, mixing the Chaetoceros muelleri algae species and the culture solution in a volume ratio of 1:4 to culture the algae species; The mother liquor comprises inorganic salts and organic substances to provide nutrients required by algae. The inorganic salts comprise sodium nitrate, potassium dihydrogen phosphate and sodium silicate. The organic substances comprise ferric citrate hydrate, VB1 and VB 12； The culture solution is prepared by adding 1 mL of the culture solution of Chaetoceros muelleri to each liter of sterilized seawater; The added amount of betaine is 100 mg / L; The Chaetoceros muelleri in the culture solution is used as bait for the giant oyster Crassostrea gigas, and the number of baits fed to each oyster per liter of water is 3×10 4 cells.

2. The method for improving the survival rate of Crassostrea gigas under high-salinity conditions according to claim 1, characterized in that: The mother solution of the culture solution of Chaetoceros muelleri is 60 mg of sodium nitrate, 4.5 mg of potassium dihydrogen phosphate, 5 mg of sodium silicate, 0.5 mg of ferric citrate hydrate, 150 μg of VB1, and VB 12 : 200ng and then make it into 1000× mother solution.

3. The method for improving the survival rate of Crassostrea gigas under high-salinity conditions according to claim 1, characterized in that: The culture time of the Chaetoceros muehii is 5-7 days.

4. The method for improving the survival rate of Crassostrea gigas under high-salinity conditions according to claim 1, characterized in that: The pH value of the culture solution of Chaetoceros muelleri is adjusted in the range of 7.5 to 8.

5.

5. The method for improving the survival rate of Crassostrea gigas under high-salinity conditions according to claim 1, characterized in that: The culture solution of the Chaetoceros muelleri is sterilized to ensure the purity and safety of the bait, and the sterilization treatment includes high-temperature and high-pressure sterilization and filtration sterilization.

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