A method for culturing oyster larvae

Abstract

The application relates to the field of aquaculture, and particularly discloses a method for cultivating oyster larvae, which comprises the following steps: S1, fertilized eggs are developed into D-shaped larvae in seawater at 22 DEG C for 20-24 hours; S2, the D-shaped larvae are cultivated under the condition that the temperature is 22-24 DEG C and the salinity is 29-31 ‰, feed and growth nutrient solution are fed daily, water is changed daily, after the D-shaped larvae appear eye spots, a larval collector is put in, and algal feed and growth promoting solution are fed daily, the larvae are cultivated into 0.8-1 mm juvenile shellfish, and are transferred to sea cultivation; the growth nutrient solution comprises the following components in weight parts: 1-2 parts of a small molecule peptide solution, 0.01-0.03 parts of neomycin sulfate powder, 0.5-1 part of corn starch, 2-4 parts of modified montmorillonite, and 10-15 parts of water. The method for cultivating oyster larvae has the advantages that the survival rate of the larvae is improved, the growth speed is accelerated, and the metamorphosis and attachment rate is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquaculture, more particularly, it relates to a method for cultivating oyster larvae. BACKGROUND

[0002] Oysters, commonly known as oysters, oysters, etc., are a kind of bivalve mollusk with group aggregation, and are also the world's first largest farmed shellfish. Oysters are diverse in species, and more than 100 species of oysters have been found so far, widely distributed in coastal areas of various countries. Oysters are a kind of aquatic food with high protein and low fat, and have unique nutritional and medicinal values. The edible soft part of oysters is rich in various nutrients such as protein, amino acids, glycogen and various trace elements. Studies have shown that the composition of amino acids is one of the important factors determining the quality of protein. Each 100g of fresh oysters contains 8100mg of protein. As a high-quality protein, the content of essential amino acids in oyster protein is significantly higher than the recommended reference standard value of the World Food Organization. In addition, oysters contain a complete range of trace elements, which have great nutritional value. Therefore, oysters have great development potential as a marine food with medicinal and edible properties.

[0003] Oyster culture plays an important role in the shellfish culture industry in China and is one of the four major cultured shellfish in China. It is cultured in various coastal areas. Although there has been a major breakthrough in artificial oyster seed production technology, there have been many reports on the research of physicochemical factors and biological feed necessary for oyster larva cultivation. However, during the process of seed production, the planktonic larval stage is extremely sensitive to environmental changes, and the cultivation period of the larval stage is long, which increases the risk of cultivation due to slow growth rate, high mortality rate and low metamorphosis and attachment rate of the larvae.

[0004] In view of the above related art, the inventors found that the current oyster larvae still have the problems of slow growth rate and long larva cultivation period. SUMMARY

[0005] In order to improve the growth rate of oyster larvae and shorten the larva cultivation period, the present application provides a method for cultivating oyster larvae.

[0006] In a first aspect, the present application provides a method for cultivating oyster larvae, which adopts the following technical solution:

[0007] A method for cultivating oyster larvae, comprising the following steps:

[0008] S1, developing the fertilized eggs into D-shaped larvae in sea water at 22-25℃ for 20-24h;

[0009] S2, the D-shaped larvae are cultured under the condition of temperature 22-24℃ and salinity 29-31‰, fed with bait and growth nutrient solution every day, and the water is changed every day. After the D-shaped larvae appear eye spots, a sampler is put in and fed with algal feed and growth promoting solution every day. The larvae are cultured to 0.8-1mm juvenile shellfish, transferred to sea cultivation, and the D-shaped larvae are cultured at a density of 5-10 per ml, and the eye spot larvae are cultured at a density of 1-3 per ml;

[0010] The growth nutrient solution comprises the following components in parts by weight: 2-4 parts of small molecule peptide solution, 0.01-0.03 parts of neomycin sulfate powder, 0.5-1 parts of corn starch, and 1-2 parts of modified montmorillonite.

[0011] By using the above technical solution, the fertilized eggs are hatched in a suitable temperature, the D-shaped larvae are obtained, and the larvae are cultured in a suitable salinity and temperature, and the bait and growth nutrient solution are used as the nutrient source. When the eye spots appear, the growth promoting solution is used, and the larvae are cultured to juvenile shellfish and then transferred to sea cultivation. The small molecule peptide solution, corn starch, and modified montmorillonite are used to prepare the growth nutrient solution, which is used for predation and absorption by the D-shaped larvae. The small molecule peptide has simple structure, small molecular weight, and can be quickly absorbed without antigenicity. In addition, it can stimulate appetite, increase food intake, promote the rapid growth of D-shaped larvae, and enhance the stress resistance of D-shaped larvae to salinity difference, temperature difference, and pH deviation, thereby improving the survival rate. The neomycin sulfate powder belongs to aminoglycoside antibiotic drugs, can produce bactericidal effect by inhibiting bacterial protein synthesis, has strong killing effect on bacteria in the stationary phase, can control the breeding of harmful organisms in the culture pond, and reduce the influence of harmful bacteria on the survival rate of D-shaped larvae. The modified montmorillonite has strong surface activity, superfine effect, and crystal structure, can play a role in adjusting and supplementing trace elements, promote larval metabolism, prevent and resist diseases, adjust water quality, improve larval immunity, promote growth, and improve survival rate.

[0012] Optionally, the modified montmorillonite is prepared by the following method:

[0013] (1) 2-5 parts of montmorillonite are added to 5-8 parts of water to prepare a suspension, 0.2-0.3 parts of bis-quaternary ammonium salt is added, and the mixture is water-bathed at 60-65℃ for 2-2.5h, and then cooled to room temperature. After drying and grinding to 200-300 mesh, the modified montmorillonite is obtained.

[0014] (2) 0.01-0.03 parts of yeast cell wall, 0.03-0.05 parts of botryosphaerio polysaccharide, 0.01-0.05 parts of disintegrating agent, 2-3 parts of polyvinyl alcohol, and 10-15 parts of water are mixed to prepare a coating solution. The coating solution is uniformly sprayed on the montmorillonite obtained in step (1), and then dried to obtain the modified montmorillonite.

[0015] By adopting the technical scheme, first, the double quaternary ammonium salt is used to intercalate modify the montmorillonite, so that the montmorillonite has super strong antibacterial performance, then the coating liquid formed by the yeast cell wall, the polystyrene and the disintegrating agent, polyvinyl alcohol and the like is sprayed on the modified montmorillonite, under the adhesion of the polyvinyl alcohol, the coating liquid is wrapped on the double quaternary ammonium salt modified montmorillonite, when the modified montmorillonite is put into the water body, under the action of the disintegrating agent, the modified montmorillonite is rapidly disintegrated and diffused, the particle size of the modified montmorillonite is reduced, the D-shaped larvae are easily preyed, with the disintegration and diffusion of the modified montmorillonite, the yeast cell wall and the polystyrene are diffused in the water body, the yeast cell wall can enhance the immunity, prevent diseases, promote growth and supplement nutrition, the polystyrene can enhance the immunity and promote growth, so that the immunity of the D-shaped larvae to the water environment is improved, and the survival rate is improved.

[0016] Optionally, the small molecule peptide solution is prepared by mixing Cistanche, wheat and yeast, and then centrifuging and concentrating the mixture after fermentation at 25-30 DEG C for 48-72 h, the mass ratio of Cistanche, wheat and yeast being 1:0.3-0.5:0.001-0.003.

[0017] By adopting the technical scheme, the Cistanche and the wheat are enzymatically hydrolyzed to obtain Cistanche small molecule peptides and wheat oligopeptides containing a large amount of nutrients, which provide the required nutrients for the growth and development of the larvae, promote the synthesis of enzymes and proteins in the cells of the larvae, and accelerate the growth of the D-shaped larvae.

[0018] Optionally, the growth promoting liquid comprises the following components in parts by weight: 0.3-0.5 parts of Rhipilia, 0.3-0.5 parts of fish oil, 0.4-0.8 parts of Clostridium ethanolium protein, 0.25-0.5 parts of threonine, 0.5-1 parts of fish meal, 1-1.5 parts of denatured starch, 2-2.5 parts of sodium caseinate, 0.08-0.1 parts of Bacillus subtilis and 10-15 parts of water.

[0019] By adopting the technical scheme, the oyster feed prepared by using sodium caseinate and modified starch as wall materials to coat chaetoceros, fish oil, clostridium ethanolium protein, threonine and fish meal can be used for feeding of the eye spot worm, the bacillus subtilis produces inhibitory substances to hinder pathogenic bacteria, compete for adhesion sites, inhibit colonization of pathogenic bacteria, compete for nutrients, cause pathogenic bacteria to be unable to obtain nutrients, enhance immunity, improve immune level, can prevent diseases of the eye spot larvae, can also improve water quality, prevent diseases, enhance immune response, and the nutrient substances can meet the growth needs of the larvae, and the growth and metamorphosis of the larvae can meet the production requirements. Since the larvae are small and thin, the feeding capacity is low, the ability to cope with changes in external environmental conditions is poor, and at the same time, the larvae cultivation makes the larvae have the highest growth rate in the highest peak of the life cycle, and the level of metabolism is high, and the larvae have higher requirements for the nutrients and morphology of the bait. After the multiple components are coated by sodium caseinate and modified starch, the particles can be prevented from swelling when soaked in seawater, causing some particles to be difficult to be ingested and adhered to the oyster shell or sink to the seabed, causing a layer of sticky substances to be attached to the seabed, leading to deterioration of water quality. The surface coating of the particles can prevent the particles from swelling easily, protect the particles, and meet the growth needs of the larvae.

[0020] Optionally, the growth promoting liquid is prepared by the following method:

[0021] The chaetoceros, fish oil, clostridium ethanolium protein, threonine, fish meal, modified starch and sodium caseinate are uniformly mixed, water with a weight of 7-8 times the total weight of the raw materials is added, the temperature is raised to 70-80℃, constant temperature stirring is performed for 20-30min, spray drying is performed, sieving is performed, grinding is performed, and mixed particles are prepared.

[0022] The bacillus subtilis is mixed with water, the mixed particles are added, and ultrasonic treatment is performed for 20-30min to prepare the growth promoting liquid.

[0023] By adopting the technical scheme, spray drying is adopted to form a film of sodium caseinate and modified starch, coat the effective components, and perform superfine pulverization after filtration and grinding, so that the nutritional ingredients and particle size meet the needs of the larvae.

[0024] Optionally, the method for preparing the seedling collector is as follows: the adhesion promoting liquid is uniformly brushed on the surface of the concrete base column, the oyster shell particles are uniformly adhered to the adhesion promoting liquid, and drying is performed. The adhesion promoting liquid is prepared by the following method: 1-2 parts of chitosan are dissolved in 5-10 parts of an acetic acid solution with a concentration of 3-5wt%, 0.4-0.6 parts of bovine bone powder and 0.1-0.3 parts of gypsum powder are added, and the adhesion promoting liquid is prepared after uniform mixing.

[0025] By using the above technical solution, the concrete is used as the base column, and the promoting adhesion liquid made of chitosan and the like is brushed. The viscosity of the chitosan makes the promoting adhesion liquid adhere to the base column. The organic calcium material, i.e. the bovine bone powder and the gypsum powder calcium sulfate, improves the effective calcium ion concentration of the base column. The calcium ion can promote the adhesion and metamorphosis of the larvae. The chitosan component contained therein can provide convenient conditions for the generation of the microbial membrane and the bacterial colony of the base column. In turn, the polysaccharides and glycoproteins and the like on the bacterial cell membrane are combined with the exogenous agglutinin on the body surface of the larvae, thereby inducing the metamorphosis of the D. plicata larvae.

[0026] The D. plicata shell particles are adhered to the base column by using the dry promoting adhesion liquid, thereby increasing the surface roughness of the base column. The rough base column provides better tactile stimulation for the crawling and adhesion of the D. plicata larvae, thereby assisting the larvae to stay on the base column. On the other hand, the rough surface has a larger area and a potentially more abundant and diverse microbial environment than the smooth surface, which can promote the adhesion of the D. plicata larvae.

[0027] Optionally, the brushing amount of the promoting adhesion liquid on the base column is 20-30 g / m 2 , and the adhering amount of the D. plicata shell particles on the base column is 10-20 pieces / m 2 .

[0028] By using the above technical solution, the promoting adhesion liquid is brushed on the base column, which can provide a high calcium ion concentration, thereby inducing the adhesion and metamorphosis of the D. plicata larvae. The D. plicata shell particles can increase the roughness of the base column, thereby facilitating the adhesion of the D. plicata larvae.

[0029] Optionally, the bait is selected from one or more of Isochrysis galbana, Platymonas subcordiformis, Chlorella vulgaris and Nannochloropsis oculata.

[0030] The algal feed is selected from one or more of I. galbana, C. muelleri and P. lutzii.

[0031] Optionally, the daily feeding amount of the bait is (5-6)×10 4 cells / ml, which is fed in 4-6 times.

[0032] The daily feeding amount of the growth nutrient liquid is 0.1-0.3 g / ml, which is fed in 4-6 times, and is fed before the feeding of the bait.

[0033] The daily feeding amount of the algal feed is (7-8)×10 4 cells / ml, which is fed in 3-5 times.

[0034] The daily feeding amount of the promoting growth liquid is 0.2-0.4 g / ml, which is fed in 3-5 times, and is fed before the feeding of the algal feed.

[0035] By adopting the technical scheme, the feeding density of the algal feed is not prone to be too large, and overfeeding is not conducive to the growth of the larvae, and if the excess feed cannot be removed in time, the water quality will be affected, and the living environment of the larvae will be affected, the feeding amount is low, the larvae cannot meet the requirements of nutrition and energy due to insufficient feeding amount, and the larvae can grow rapidly at a suitable feeding density; and the growth nutrient solution is fed first, and then the feed is fed, so that the utilization rate of the growth nutrient solution is high.

[0036] Optionally, the fertilized eggs are prepared by mixing and stirring the eggs produced by the diploid female oysters and the sperm produced by the tetraploid male oysters at a number ratio of 1:10-20.

[0037] By adopting the technical scheme, the fertilized eggs produced by the diploid female oysters and the tetraploid male oysters are hatched to obtain triploid larvae, and the triploid oysters have the advantages of strong environmental adaptability, fast growth speed and rich nutrition.

[0038] In summary, the present application has the following beneficial effects:

[0039] 1. In the present application, the growth nutrient solution is matched with the feed when the D-shaped larvae of oysters are cultivated, the growth nutrient solution is prepared from modified montmorillonite, small molecule peptide solution and other components, which can promote the growth of the D-shaped larvae, improve the immunity of the larvae, improve the water quality, reduce the breeding of bacteria and reduce the influence of harmful bacteria on the survival rate of the D-shaped larvae.

[0040] 2. In the present application, the montmorillonite is modified by using the double quaternary ammonium salt, and then a solution prepared from the yeast cell wall, the botryospongia polysaccharide, the disintegrating agent and the polyvinyl alcohol is sprayed, under the action of the disintegrating agent, the modified montmorillonite rapidly diffuses in the water body, the particle size is reduced, the larvae can easily eat, and with the disintegration of the modified montmorillonite, the coated yeast cell wall and botryospongia polysaccharide diffuse in the water body, thereby providing nutrient substances for the growth of the D-shaped larvae, improving the immunity and improving the growth speed.

[0041] 3. In the present application, the small molecule peptide solution is prepared by mixing and enzymolysis of the cistanche, wheat and yeast, which provides nutrient components for the growth of the D-shaped larvae, promotes the synthesis of enzymes and proteins, and accelerates the growth of the D-shaped larvae.

[0042] 4. In the present application, the base column is formed by pouring the concrete, the surface of the base column is brushed with the promoting adhesion liquid containing chitosan, bovine bone powder and plaster powder, and then the oyster shell particles are adhered, so as to improve the concentration of calcium ions on the base column and improve the roughness of the base column, so that the larvae can metamorphose and adhere rapidly, the growth speed of the oysters is improved, and the cultivation period of the larvae is shortened. DETAILED DESCRIPTION

[0043] Preparation examples 1-5 of modified montmorillonite

[0044] Preparation Example 1: (1) 5 kg of montmorillonite was added to 8 kg of water to form a suspension, 0.3 kg of a diquaternary ammonium salt was added, and the mixture was water-bathed at 65°C for 2 h, cooled to room temperature, and dried at 60°C, and then ground to 300 mesh. The diquaternary ammonium salt was dioctadecyldimethylammonium chloride.

[0045] (2) 0.03 kg of yeast cell walls, 0.05 kg of a Sphaerotilus polysaccharide, 0.05 kg of a disintegrant, 3 kg of polyvinyl alcohol, and 15 kg of water were mixed to form a coating liquid, which was uniformly sprayed onto the montmorillonite obtained in step (1), and dried at 60°C to obtain modified montmorillonite. The disintegrant was microcrystalline cellulose.

[0046] Preparation Example 2: (1) 2 kg of montmorillonite was added to 5 kg of water to form a suspension, 0.2 kg of a diquaternary ammonium salt was added, and the mixture was water-bathed at 60°C for 2.5 h, cooled to room temperature, and dried at 60°C, and then ground to 200 mesh. The diquaternary ammonium salt was dioctadecyldimethylammonium chloride.

[0047] (2) 0.01 kg of yeast cell walls, 0.03 kg of a Sphaerotilus polysaccharide, 0.01 kg of a disintegrant, 2 kg of polyvinyl alcohol, and 10 kg of water were mixed to form a coating liquid, which was uniformly sprayed onto the montmorillonite obtained in step (1), and dried at 60°C to obtain modified montmorillonite. The disintegrant was microcrystalline cellulose.

[0048] Preparation Example 3: The difference from Preparation Example 1 is that the montmorillonite was not modified with a diquaternary ammonium salt, i.e., step (1) was not performed.

[0049] Preparation Example 4: The difference from Preparation Example 1 is that yeast cell walls were not added in step (2).

[0050] Preparation Example 5: The difference from Preparation Example 1 is that a Sphaerotilus polysaccharide was not added in step (2).

[0051] Preparation Examples 6-11 of the growth-promoting liquid

[0052] Preparation Example 6: (1) 0.5 kg of Chaetomorpha, 0.5 kg of fish oil, 0.8 kg of Clostridium aceticum protein, 0.5 kg of threonine, 1 kg of fish meal, 1.5 kg of denatured starch, and 2.5 kg of sodium caseinate were uniformly mixed, 8 times the total weight of the raw materials of water was added, heated to 80°C, and stirred at constant temperature for 20 min, then spray-dried, passed through a 325 mesh sieve, and ground to form a mixed granule. The inlet air temperature for spray-drying was 130°C, and the outlet air temperature was 85°C.

[0053] (2) 0.1 kg of Bacillus subtilis and 10 kg of water were mixed, and the mixed granule prepared in step (1) was added, and ultrasonicated for 20 min to obtain a growth-promoting liquid.

[0054] Preparation Example 7: (1) 0.3 kg of Chaetomorpha, 0.3 kg of fish oil, 0.4 kg of Clostridium aceticum protein, 0.25 kg of threonine, 0.5 kg of fish meal, 1 kg of denatured starch, and 2 kg of sodium caseinate were uniformly mixed, 7 times the weight of water of the total weight of the raw materials was added, heated to 70℃, and stirred at constant temperature for 30 min. The mixture was spray dried, sieved through a 325 mesh sieve, ground, and mixed into granules. The inlet air temperature of the spray dryer was 130℃, and the outlet air temperature was 85℃.

[0055] (2) 0.08 kg of Bacillus subtilis and 15 kg of water were mixed, and the mixed granules prepared in step (1) were added. The mixture was ultrasonicated for 20 min to prepare a growth promoting solution.

[0056] Preparation Example 8: The difference from Preparation Example 6 is that Bacillus subtilis is not added.

[0057] Preparation Example 9: The difference from Preparation Example 6 is that threonine is not added.

[0058] Preparation Example 10: The difference from Preparation Example 6 is that Clostridium aceticum protein is not added.

[0059] Preparation Example 11: The difference from Preparation Example 6 is that Chaetomorpha is not added.

[0060] Example

[0061] Example 1: A method for cultivating oyster larvae, comprising the following steps:

[0062] S1, fertilized eggs are developed into D-shaped larvae in seawater at 22℃, the hatching density is 20 per ml, and the fertilized eggs are prepared by mixing and stirring the eggs produced by diploid female oysters and the sperm produced by tetraploid male oysters at a mass ratio of 1:10;

[0063] S2, the D-shaped larvae are cultured under conditions of 22℃ and a salinity of 29, fed with feed and growth nutrient solution daily, and the water is changed daily. After the D-shaped larvae develop eyespots, oyster shell strings are used as a larval collector, and algal feed and growth promoting solution are fed daily. The larvae are cultured to 0.8 mm juvenile oysters, and then transferred to sea cultivation. The D-shaped larvae have a cultivation density of 5 per ml, the eyed larvae have a cultivation density of 1 per ml, and the daily feed amount is 6 x 10 4cell / ml, the feed is Isochrysis galbana, the daily feeding amount of the growth nutrient solution is 0.3g / ml, and the growth nutrient solution is fed in four times, the feed is fed after the growth nutrient solution is fed for 20min, the growth nutrient solution is prepared by mixing the following raw materials in a certain weight: 4kg of small molecule peptide solution, 0.03kg of neomycin sulfate powder, 1kg of corn starch, 2kg of modified montmorillonite and 15kg of water, the small molecule peptide solution is prepared by mixing Cistanche deserticola, wheat and yeast, and then centrifuging and concentrating after fermentation at 30℃ for 48h, the mass ratio of Cistanche deserticola, wheat and yeast is 1:0.5:0.003, and the modified montmorillonite is prepared according to Preparation Example 1;

[0064] The daily feeding amount of the algal feed is 8x10 4 cell / ml, the feed is Isochrysis galbana, the daily feeding amount of the growth nutrient solution is 0.3g / ml, and the growth nutrient solution is fed in four times, the feed is fed after the growth nutrient solution is fed for 20min, the growth nutrient solution is prepared by mixing the following raw materials in a certain weight: 4kg of small molecule peptide solution, 0.03kg of neomycin sulfate powder, 1kg of corn starch, 2kg of modified montmorillonite and 15kg of water, the small molecule peptide solution is prepared by mixing Cistanche deserticola, wheat and yeast, and then centrifuging and concentrating after fermentation at 30℃ for 48h, the mass ratio of Cistanche deserticola, wheat and yeast is 1:0.5:0.003, and the modified montmorillonite is prepared according to Preparation Example 1;

[0065] Example 2: A method for cultivating oyster larvae, comprising the following steps:

[0066] S1, the fertilized eggs are developed to D-shaped larvae in seawater at 25℃, the hatching density is 30 / ml, and the fertilized eggs are prepared by mixing and stirring the eggs produced by a diploid female oyster and the sperm produced by a tetraploid male oyster according to a mass ratio of 1:20;

[0067] S2, the D-shaped larvae are cultivated under conditions of 25℃ and a salinity of 31, the feed and the growth nutrient solution are fed daily, the water is changed daily, after the D-shaped larvae appear eye spots, oyster shell strings are put as a larval collector, and the algal feed and the growth promoting solution are fed daily, and the cultivation is carried out to 1mm juvenile shellfish, and then the cultivation is transferred to the sea, the cultivation density of the D-shaped larvae is 10 / ml, the cultivation density of the eyed larvae is 3 / ml, and the daily feeding amount of the feed is 5x10 4 cell / ml, the feed is Isochrysis galbana, the daily feeding amount of the growth nutrient solution is 0.3g / ml, and the growth nutrient solution is fed in four times, the feed is fed after the growth nutrient solution is fed for 20min, the growth nutrient solution is prepared by mixing the following raw materials in a certain weight: 4kg of small molecule peptide solution, 0.03kg of neomycin sulfate powder, 1kg of corn starch, 2kg of modified montmorillonite and 15kg of water, the small molecule peptide solution is prepared by mixing Cistanche deserticola, wheat and yeast, and then centrifuging and concentrating after fermentation at 30℃ for 48h, the mass ratio of Cistanche deserticola, wheat and yeast is 1:0.5:0.003, and the modified montmorillonite is prepared according to Preparation Example 1;

[0068] The daily feeding amount of the algal feed is 8x10 4Example 1: A method for cultivating oyster larvae, wherein the modified montmorillonite in the growth nutrient solution is prepared by the method of Preparation Example 1, the algae feed is Chaetoceros muelleri, the daily feeding amount of the growth promoting solution is 0.2 g / ml, the algae feed is fed 20 minutes after the growth promoting solution is fed, and the growth promoting solution is prepared by Preparation Example 7.

[0069] Example 3: A method for cultivating oyster larvae, which is different from Example 1 in that the modified montmorillonite in the growth nutrient solution is prepared by the method of Preparation Example 3.

[0070] Example 4: A method for cultivating oyster larvae, which is different from Example 1 in that the modified montmorillonite in the growth nutrient solution is prepared by the method of Preparation Example 4.

[0071] Example 5: A method for cultivating oyster larvae, which is different from Example 1 in that the modified montmorillonite in the growth nutrient solution is prepared by the method of Preparation Example 5.

[0072] Example 6: A method for cultivating oyster larvae, which is different from Example 1 in that Cistanche deserticola is not added during the fermentation of the small-molecule peptide solution in the growth nutrient solution.

[0073] Example 7: A method for cultivating oyster larvae, which is different from Example 1 in that the growth promoting solution is prepared by the method of Preparation Example 8.

[0074] Example 8: A method for cultivating oyster larvae, which is different from Example 1 in that the growth promoting solution is prepared by the method of Preparation Example 9.

[0075] Example 9: A method for cultivating oyster larvae, which is different from Example 1 in that the growth promoting solution is prepared by the method of Preparation Example 10.

[0076] Example 10: A method for cultivating oyster larvae, which is different from Example 1 in that the growth promoting solution is prepared by the method of Preparation Example 11.

[0077] Example 11: A method for cultivating oyster larvae, which is different from Example 1 in that the larva sampler is prepared by the following method: 144 kg / m 3 water, 220 kg / m 3 PO42.5 Portland cement, 76 kg / m 3 Class I fly ash, 92 kg / m 3 slag, 1108 kg / m 3 stone, 750 kg / m 3 river sand, 3.4 kg / m 3The BL-J type polycarboxylic acid superplasticizer is mixed to prepare a concrete slurry, and a concrete base column is prepared after pouring and curing. A promoting adhesion liquid is uniformly brushed on the surface of the concrete base column, and oyster shell particles are uniformly adhered on the promoting adhesion liquid. The promoting adhesion liquid is dried at 80 ℃. The promoting adhesion liquid is prepared by dissolving 1 kg of chitosan in 5 kg of an acetic acid solution with a concentration of 3 wt%, and then uniformly mixing 0.4 kg of bovine bone powder and 0.1 kg of gypsum powder. The brushing amount of the promoting adhesion liquid on the base column is 30 g / m 2 The oyster shell particles are prepared by crushing oyster shells and hot-melt extruding the crushed oyster shells and black polyethylene masterbatch at a mass ratio of 1:0.1. The particle size of the oyster shell particles is 3 cm, and the adhering amount of the oyster shell particles on the base column is 20 g / m 2 .

[0078] Example 12: A method for cultivating oyster larvae, which is different from example 11 in that the promoting adhesion liquid is prepared by dissolving 2 kg of chitosan in 10 kg of an acetic acid solution with a concentration of 5 wt%, and then uniformly mixing 0.6 kg of bovine bone powder and 0.3 kg of gypsum powder. The brushing amount of the promoting adhesion liquid on the base column is 20 g / m 2 The oyster shell particles are prepared by crushing oyster shells and hot-melt extruding the crushed oyster shells and black polyethylene masterbatch at a mass ratio of 1:0.1. The particle size of the oyster shell particles is 3 cm, and the adhering amount of the oyster shell particles on the base column is 20 g / m 2 .

[0079] Example 13: A method for cultivating oyster larvae, which is different from example 11 in that no oyster shell particles are adhered on the base column.

[0080] Example 14: A method for cultivating oyster larvae, which is different from example 11 in that the oyster shell particles are prepared by crushing oyster shells.

[0081] Example 15: A method for cultivating oyster larvae, which is different from example 11 in that an equal amount of calcium carbonate powder is used to replace the bovine bone powder.

[0082] Example 16: A method for cultivating oyster larvae, which is different from example 11 in that the bovine bone powder, the gypsum powder and the oyster shell particles are directly mixed into the concrete slurry to prepare the base column through pouring and curing.

[0083] Comparative example

[0084] Comparative example 1: A method for cultivating oyster larvae, which is different from example 1 in that no modified montmorillonite is added to the growth nutrient solution.

[0085] Comparative example 2: A method for cultivating oyster larvae, which is different from example 1 in that no small molecule peptide solution is added to the growth nutrient solution.

[0086] Comparative Example 3: A method for culturing D-shaped oyster larvae, which is different from Example 1 in that the growth nutrient solution is not added with neomycin sulfate powder.

[0087] Comparative Example 4: A method for culturing D-shaped oyster larvae, which is different from Example 1 in that the growth nutrient solution is not fed.

[0088] Comparative Example 5: A method for culturing D-shaped oyster larvae, which is different from Example 1 in that the growth promoting solution is not fed.

[0089] Performance test

[0090] I. According to the method in Examples 1-6 and Comparative Examples 1-4, each example is set up in 4 parallel, for measuring growth rate mortality, shell mortality every 5 days, for 15 days, the survival rate is calculated as follows: R = d1 / d0 x 100%, d1 represents the number of individuals of larvae at the end of the test, d0 represents the number of individuals of larvae at the beginning of the test, when the shell is taken, 30 larvae are taken as the detection object, the test results are taken as the average value, and the shell growth rate at 15 days is calculated: L = (L0-L1) / L0 x 100%, L1 represents the final shell of larvae at 15 days, L0 represents the initial shell of larvae, and each detection result is recorded in Table 1.

[0091] Table 1 Growth rate and survival rate of D-shaped larvae

[0092]

[0093] In Examples 1 and 2, D-shaped larvae are cultured using growth nutrient solution, and the growth rate of D-shaped larvae is fast and the survival rate is high when D-shaped larvae are cultured into eye-spotted larvae, which can shorten the culture period.

[0094] In Example 3, modified montmorillonite prepared in Preparation Example 3 is used, and Table 1 shows that the growth rate of D-shaped larvae is slowed down and the growth rate is reduced, indicating that modification of montmorillonite with bis-quaternary ammonium salt can increase the growth rate of D-shaped larvae, but does not affect the survival rate of D-shaped larvae.

[0095] In Example 4, modified montmorillonite prepared in Preparation Example 4 is used, and compared with Preparation Example 1, yeast cell wall is not added, and the data in Table 1 shows that the growth rate of D-shaped larvae cultured in Example 4 is slowed down, the daily growth shell length is reduced, and the survival rate is also decreased, indicating that yeast cell wall can increase the growth rate of D-shaped larvae and improve the survival rate.

[0096] In Example 5, modified montmorillonite prepared in Example 5 is used, wherein no nostoc polysaccharide is added, and compared with Example 1, the survival rate of D-shaped larvae changes little, but the growth rate of D-shaped larvae is reduced.

[0097] Example 6 compared with Example 1, no Cistanche miltiorrhiza was added in the small molecule peptide solution, and the growth rate of the D-shaped larvae was much slower than that of Example 1, and the daily growth was also decreased, but the survival rate changed little, as shown in Table 1.

[0098] Comparative Example 1 compared with Example 1, no modified montmorillonite was added in the growth nutrient solution, and the growth rate of the D-shaped larvae in Comparative Example 1 was slower than that in Example 1, Example 3, Example 4, Example 5, and the survival rate was smaller than that in Example 1, indicating that the addition of modified montmorillonite significantly improved the growth rate and survival rate of the D-shaped larvae.

[0099] In Comparative Example 2, no small molecule peptide solution was added, and compared with Example 1, the growth rate of the D-shaped larvae in Comparative Example 2 was slower, but the survival rate changed little.

[0100] Comparative Example 3 compared with Example 1, no neomycin sulfate powder was added, and as shown in Table 1, the survival rate of the D-shaped larvae in Comparative Example 3 was significantly reduced, and the growth rate was also slowed down.

[0101] Comparative Example 4 compared with Example 1, only feed was fed during the cultivation of D-shaped larvae, and no growth nutrient solution was fed, and the growth rate of the D-shaped larvae in Comparative Example 4 was slow, and the survival rate was low.

[0102] II. Growth rate and survival rate detection of eye spot larvae

[0103] According to the method in Examples 7-11 and Comparative Example 5, each example was set with 4 parallel, for measuring growth rate and mortality, and the mortality was measured every 5 days for 15 days, and the survival rate was calculated according to the formula: R = d1 / d0 x 100%, d1 represented the number of larvae at the end of the test, and d0 represented the number of larvae at the beginning of the test, and 30 larvae were taken as the detection object when measuring, the test results were averaged, and the growth rate at 15 days was calculated: L = (L0-L1) / L0 x 100%, L1 represented the final at 15 days, and L0 represented the initial of the larvae, and the detection results were recorded in Table 2.

[0104] Table 2 Growth rate and survival rate detection of eye spot larvae

[0105]

[0106] Example 1 and Example 2 used the growth promoting solution prepared by Preparation Example 6 and Preparation Example 7, and as shown in Table 2, the eye spot larvae cultivated in Example 1 and Example 2 grew faster, and could reach more than 1100 microns in about 15 days, and the daily growth reached more than 50 microns, and the water quality was not easily polluted after feeding the growth promoting solution.

[0107] Example 7: The growth promoting liquid prepared in Preparation Example 8 was used, without adding Bacillus subtilis, and the water quality was similar to that of Example 1, but the survival rate of the D-larvae was slightly lower after 15 days of cultivation.

[0108] Example 8: The growth promoting liquid prepared in Preparation Example 9 was used, without adding threonine, and the growth rate of the D-larvae was slower, the survival rate was slightly lower, and the water quality was similar to that of Example 1.

[0109] Example 9 and Example 10: The growth promoting liquids prepared in Preparation Example 10 and Preparation Example 11 were used, respectively, without adding Clostridium protein in Preparation Example 10 and without adding Prorocentrum in Preparation Example 11, and the growth rate of the D-larvae was slower and the survival rate was lower in Example 9 and Example 10.

[0110] Comparative Example 5: No growth promoting liquid was used, and the growth rate of the D-larvae was significantly slower than that of Example 1, the water quality was poor, and the survival rate of the larvae was lower.

[0111] III. Attachment rate detection of D-larvae:

[0112] The D-larvae were cultured according to the methods in Examples 1-16, and after 20 days of cultivation, the sampling device was removed, the concrete surface was gently wiped with a dry towel, and a photograph was taken for record. The number of oysters attached to the six surfaces of the base column was counted using the Photoshop software grid method. After counting, the sampling device was returned to its original position, and the number of surviving oysters was counted after 100 days. The results were recorded in Table 3.

[0113] Table 3: Attachment performance detection of D-larvae

[0114]

[0115]

[0116] As can be seen from the data in Table 3, the number of oysters attached to the oyster string as the sampling device was high in Examples 1-2, and the survival rate was more than 85% after secondary counting. In Examples 3 and 4, the number of oyster larvae attached was reduced due to the absence of bis-quaternary ammonium salt and yeast cell wall, respectively, and the survival rate of the D-larvae may have decreased during cultivation, but after induction of attachment, the number of oysters attached to the sampling device was reduced in Examples 5-6 and Examples 9-10. In Example 7, the number of oysters attached was reduced due to the absence of Bacillus subtilis, and in Example 8, the number of oysters attached was reduced due to the absence of threonine, which made the growth of oyster larvae susceptible to infection or slower.

[0117] The base pillar formed by the concrete casting maintenance in Example 11 and Example 12 is used as the base body of the seed collector, then the adhesion promoting liquid formed by the chitosan, the bovine bone powder and the gypsum powder is sprayed on the base pillar, and the oyster shell particles are uniformly adhered on the adhesion promoting liquid. As can be seen from the data in Table 3, the number of the oysters adhered on the seed collector is significantly increased by the induction of the calcium ions and the adhesion promotion of the eye spot larvae in Example 11 and Example 13.

[0118] The difference between Example 13 and Example 11 is that the oyster shell particles are not adhered, and the adhesion of the oysters on the seed collector is significantly reduced. In Example 14, the black polyethylene master batch is not added when the oyster shell particles are prepared. As shown in Table 3, the adhesion amount of the oysters in Example 14 is less than that in Example 11, but higher than that in Example 13, which indicates that the black polyethylene master batch can increase the adhesion of the master batch.

[0119] In Example 15, the calcium carbonate is used to replace the organic calcium bovine bone powder. The adhesion amount of the oyster larvae in Example 15 is reduced, which indicates that the calcium carbonate has a lower adhesion promotion effect on the larvae than the bovine bone powder.

[0120] In Example 16, the bovine bone powder, the gypsum powder and the like are directly blended with the concrete slurry to form the base pillar. Compared with Example 11, the induction adhesion amount of the oyster larvae in Example 16 is reduced, which indicates that the seed collector in the present application can induce the metamorphosis of the oyster larvae, and promote the adhesion and growth of the oyster larvae.

[0121] The adhesion amount of the oyster larvae cultivated in Comparative Examples 1-5 on the seed collector is small, and the survival rate is still different from that in Example 1.

[0122] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, and the modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for culturing oyster larvae, characterized by, The method comprises the following steps: S1, developing the fertilized egg into D-shaped larvae in seawater at 22-25℃ for 20-24h; S2, cultivating the D-shaped larvae under the condition of temperature 22-24℃ and salinity 29-31‰, feeding the D-shaped larvae with feed and growth nutrient solution every day, changing water every day, and after the D-shaped larvae appear eye spots, putting the larvae into a sampler and feeding the larvae with algal feed and growth promoting solution every day, cultivating the larvae into 0.8-1mm juvenile shellfish, and transferring the larvae to sea for cultivation, the cultivation density of the D-shaped larvae being 5-10 pieces / ml, and the cultivation density of the eye spot larvae being 1-3 pieces / ml; The growth nutrient solution comprises the following components in parts by weight: 2-4 parts of small molecule peptide solution, 0.01-0.03 parts of neomycin sulfate powder, 0.5-1 parts of corn starch, and 1-2 parts of modified montmorillonite; The small molecule peptide solution is prepared by mixing Cistanche, wheat and yeast, and then fermenting at 25-30℃ for 48-72h, and then centrifuging and concentrating; the mass ratio of Cistanche, wheat and yeast is 1:0.3-0.5:0.001-0.003; The modified montmorillonite is prepared by the following method: (1) adding 2-5 parts of montmorillonite to 5-8 parts of water to prepare a suspension, adding 0.2-0.3 parts of bis-quaternary ammonium salt, and then water-bathing at 60-65℃ for 2-2.5h, and then cooling to room temperature, and then drying and grinding to 200-300 meshes; (2) mixing 0.01-0.03 parts of yeast cell wall, 0.03-0.05 parts of nostoc polysaccharide, 0.01-0.05 parts of disintegrating agent, 2-3 parts of polyvinyl alcohol and 10-15 parts of water to prepare a coating liquid, uniformly spraying the coating liquid on the montmorillonite obtained in step (1), and then drying to obtain the modified montmorillonite; The growth promoting solution comprises the following components in parts by weight: 0.3-0.5 parts of biddulphia, 0.3-0.5 parts of fish oil, 0.4-0.8 parts of clostridium ethanolium protein, 0.25-0.5 parts of threonine, 0.5-1 parts of fish meal, 1-1.5 parts of denatured starch, 2-2.5 parts of sodium caseinate, 0.08-0.1 parts of bacillus subtilis, and 10-15 parts of water.

2. The method of claim 1, wherein: The growth promoting solution is prepared by the following method: uniformly mixing biddulphia, fish oil, clostridium ethanolium protein, threonine, fish meal, denatured starch and sodium caseinate, adding water with a weight of 7-8 times the total weight of the raw materials, heating to 70-80℃, constant temperature stirring for 20-30min, spray drying, sieving and grinding to obtain mixed particles; Mixing bacillus subtilis and water, adding the mixed particles, and ultrasonicating for 20-30min to obtain the growth promoting solution.

3. The method of claim 1, wherein the oyster larvae are cultured in a water temperature of 20 to 30°C. The sampler is prepared by uniformly brushing an adhesion promoting liquid on the surface of a concrete base column, and then uniformly adhering oyster shell particles to the adhesion promoting liquid, and then drying; the adhesion promoting liquid is prepared by the following method: dissolving 1-2 parts of chitosan in 5-10 parts of acetic acid solution with a concentration of 3-5wt%, adding 0.4-0.6 parts of bovine bone powder and 0.1-0.3 parts of gypsum powder, and then uniformly mixing to obtain the adhesion promoting liquid.

4. The method for cultivating oyster larvae according to claim 3, characterized in that, The brushing amount of the adhesion promoting liquid on the base column is 20-30 g / m 2 The adhesion amount of the oyster shell particles on the base column is 10-20 pieces / m 2 .

5. The method of claim 1, wherein the oyster larvae are cultured in a water temperature of 20 to 30°C. The bait is selected from one or more of Isochrysis galbana, Platymonas subcordiformis, Chlorella vulgaris and Tetraselmis subcordiformis; the algal feed is selected from one or more of Sphalerococcus globosus, Chaetoceros muelleri and Phaeodactylum tricornutum.

6. The method of claim 1, wherein the oyster larvae are cultured in a water temperature of 20 to 30°C. The daily feeding amount of the bait is (5-6) x 10 4 cells / ml, fed in 4-6 times; The daily feeding amount of the growth nutrient solution is 0.1-0.3 g / ml, and the feeding is performed 4-6 times, and the feeding is performed before the bait feeding; The daily feeding amount of the algal feed is (7-8) x 10 4 cells / ml, fed in 3-5 times; The daily feeding amount of the growth nutrient solution is 0.1-0.3 g / ml, and the feeding is performed 4-6 times, and the feeding is performed before the bait feeding; 7. The method of claim 1, wherein the oyster larvae are cultured in a water temperature of 20 to 30°C. The fertilized eggs are prepared by mixing and stirring the eggs produced by the diploid female oysters and the sperms produced by the tetraploid male oysters according to a number ratio of 1:10-20.

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