A sea cucumber seedling cultivation method based on vibrio regulation

Abstract

The present application relates to sea cucumber aquaculture technical field, especially to a kind of sea cucumber seedling cultivation method based on vibrio regulation.The cultivation method is in the whole process of sea cucumber seedling cultivation according to the total number of bacteria and the total number of vibrio in the detected environment, the total number of bacteria / vibrio total number ratio is calculated, and the amount of probiotics is adjusted according to the total number of bacteria / vibrio total number ratio, compared with sea cucumber seedling without adding probiotics, the yield of sea cucumber seedling is increased by more than 50%, and the survival rate is increased by more than 10%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sea cucumber aquaculture, and particularly relates to a sea cucumber seedling cultivation method based on Vibrio regulation. BACKGROUND

[0002] As an important economic marine aquaculture species in China, sea cucumber (Apostichopus japonicus) seedling industry is a key link in the sea cucumber industry chain and is of great significance to the sustainable development of the entire industry. There are several modes for sea cucumber seedling production, including pond natural breeding, net cage breeding and factory breeding. Factory breeding uses modern equipment and technology to precisely control water quality, temperature and nutrition, which can effectively avoid the adverse effects of the external environment and is one of the main directions of modern breeding.

[0003] However, there are still some problems in the factory breeding of sea cucumber, such as low survival rate of seedlings and low yield due to the deterioration of water quality and the difficulty of disease control in high-density breeding. The technologies in factory breeding, such as water quality management, disease control and nutrition optimization, still need to be improved.

[0004] Vibrio is widely distributed in various marine environments. Under the condition of high-density aquaculture and poor water quality, Vibrio reproduces rapidly. Vibrio is one of the most common pathogenic bacteria in aquaculture. Vibrio genus contains multiple important pathogenic bacteria, and the species that are more harmful to aquaculture include Vibrio harveyi, Vibrio parahaemolyticus and Vibrio alginolyticus. In order to ensure the safety and sustainable development of aquaculture, it is particularly important to detect and control Vibrio. By regularly detecting the number of Vibrio in water, potential risks can be found in time and corresponding prevention and control measures can be taken.

[0005] In sea cucumber cultivation, the development and application of probiotics and microecological preparations have gradually risen in recent years. For example, CN108949615A discloses a microecological preparation for fermenting sea cucumber feed. The complex microecological preparation is a microbial mixture composed of Bacillus subtilis DB005, Bacillus thuringiensis XW008, Bacillus licheniformis XW015 and Bacillus subtilis ZF003. Or CN104367597A discloses a microecological preparation for preventing and treating sea cucumber skin rot syndrome and a preparation method. The active ingredient is the cell body of Bacillus megaterium H1 and its extracellular metabolites. Or CN111647523A discloses a probiotic composition for sea cucumber cultivation and its application. The composition includes Lactobacillus paracasei CSDN-6, Lactobacillus plantarum CLY-5 and Bacillus velezensis DY-6, and the three strains are mixed in a cell quantity ratio of 1:1:1. Or in the journal "Heilongjiang Fisheries", Vol. 43, No. 5, the author is Li Zongwen, and the title is "Effect of two microecological preparations on sea cucumber cultivation water environment and juvenile growth". The effect of bacillus and lactic acid bacteria on the growth of sea cucumber is studied. However, the current research focuses more on the development of bacterial strains and the configuration of microecological preparations. There is no scientific guidance and reference index for the use of these products in production, and the amount of use still depends on the feeling and experience of the farmers.

[0006] Based on the above situation, although new microbial preparations have been introduced for the cultivation of sea cucumber seedlings in the prior art, there is still no scientific guidance and reference index, and there is no use guidance. Most of them are added according to experience, which cannot efficiently industrialize the seedling cultivation, resulting in low survival rate of sea cucumber seedlings, unstable yield and other technical problems to be solved. SUMMARY

[0007] To solve the above technical problems, the present application provides a sea cucumber seedling cultivation method based on vibrio regulation. The cultivation method detects the total number of bacteria and the total number of vibrio in the environment according to the following formula during the whole process of sea cucumber seedling cultivation, calculates the ratio of total number of bacteria / vibrio, and adjusts the amount of probiotics added according to the ratio of total number of bacteria / vibrio:

[0008] Formula 1: y1=-3.2196x1+41.237;

[0009] wherein R1 2 = 0.9986;

[0010] y1 is the Ct value detected by double-fluorescence quantitative PCR with double primers when the total number of bacteria is detected;

[0011] x1 is the total number of bacteria, unit: GC / mL;

[0012] Formula 2: y2=-3.2196x2+35.489;

[0013] wherein R22 = 0.9986;

[0014] y2 is the Ct value of the double primer duplex fluorescent quantitative PCR detection of total Vibrio count;

[0015] x2 is the total Vibrio count, in GC / mL;

[0016] wherein, GC is the copy number (Gene Copy Number);

[0017] The primer sequence used in the PCR detection of the total number of bacteria is shown as SEQ ID NO: 1 and SEQ ID NO: 2;

[0018] SEQ ID NO: 1: CCTACGGGAGGCAGCAG;

[0019] SEQ ID NO: 2: GTATTACCGCGGCTGCTG;

[0020] The primer sequence used in the determination of the total number of Vibrio is shown as SEQ ID NO: 3 and SEQ ID NO: 4;

[0021] SEQ ID NO: 3: CCAAGAACTTAACCGTATC;

[0022] SEQ ID NO: 4: TACCCATAGAAAGCATCA;

[0023] Compared with sea cucumber seedlings without the addition of probiotics, the cultivation method increases the yield of sea cucumber seedlings by more than 50%, and the survival rate is increased by more than 10%.

[0024] Further, the probiotics are Bacillus, photosynthetic bacteria, lactic acid bacteria and Clostridium butyricum.

[0025] Further, the primers are all labeled with fluorescein.

[0026] Further, the fluorescein includes but is not limited to carboxyfluorescein (FAM, 6-Carboxyfluorescein) or hexachlorofluorescein (HEX, 6-isomer);

[0027] The carboxyfluorescein presents green light when detected by double primer duplex fluorescent quantitative PCR;

[0028] The hexachlorofluorescein presents yellow-green light when detected by double primer duplex fluorescent quantitative PCR.

[0029] Further, the primers used in the PCR assay of the total number of bacteria and the primers used in the assay of the total number of Vibrio are different in the use of fluorescein, so as to distinguish between the two and count the total number.

[0030] Further, when x1 / x2> 50:1, the administration amount of the probiotic is:

[0031] Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added in the aquaculture water every 7 days, and the administration concentration is independently 0.1-0.2 g / m 3 ;

[0032] Based on the total weight of the daily feeding bait, 0.5 g / kg of lactic acid bacteria and 0.5 g / kg of Clostridium butyricum are added in the feeding bait every day.

[0033] Further, when 10:1≤x1 / x2≤50:1, the administration amount of the probiotic is:

[0034] Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added in the aquaculture water every 3 days, and the administration concentration is independently 0.1-0.2 g / m 3 ;

[0035] Based on the total weight of the daily feeding bait, 2 g / kg of lactic acid bacteria and 2 g / kg of Clostridium butyricum are added in the feeding bait every day.

[0036] Further, when x1 / x2< 10:1, the administration amount of the probiotic is:

[0037] Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added in the aquaculture water every 1 day, and the administration concentration is independently 0.2-0.4 g / m 3 ;

[0038] Based on the total weight of the daily feeding bait, 2 g / kg of lactic acid bacteria and 2 g / kg of Clostridium butyricum are added in the feeding bait every day.

[0039] Further, the probiotic is a powder.

[0040] Further, the number of viable bacteria in each of the probiotics is independently >10 9 GC / g.

[0041] The beneficial effects of the present application are:

[0042] 1. The application provides a sea cucumber seedling cultivation method based on Vibrio regulation. The cultivation method is to calculate the ratio of total bacteria count to Vibrio count according to the total bacteria count and Vibrio count in the environment during the whole process of sea cucumber seedling cultivation, and to adjust the amount of probiotics added according to the ratio of total bacteria count to Vibrio count. Compared with sea cucumber seedlings without the addition of probiotics, the yield of sea cucumber seedlings is increased by more than 50%, and the survival rate is increased by more than 10%.

[0043] 2. The application selects specific types of probiotics, such as Bacillus, which can secrete hydrolytic enzymes (such as protease and lipase) to decompose organic matter in residual feed and excrement; photosynthetic bacteria use toxic substances such as sulfides as nutrient sources to purify and improve water quality; Lactobacillus and butyrate Clostridium probiotics can colonize in the intestinal tract to regulate intestinal microbial communities and intestinal health, stimulate the intestinal mucosa of farmed animals to produce immune factors, and enhance non-specific immune ability; at the same time, the four types of probiotics produce digestive enzymes (such as amylase and protease) to promote the decomposition of nutrients in the feed;

[0044] 3. According to formula 1 and formula 2, the application adds probiotics, which can make probiotics compete with pathogenic bacteria in the environment for survival resources, inhibit the growth of pathogenic bacteria, and at the same time secrete organic acids, hydrogen peroxide, and antibacterial peptides to directly inhibit the reproduction of pathogenic bacteria. The metabolic products (such as vitamins and short-chain fatty acids) produced can also directly promote the growth of sea cucumbers;

[0045] 4. The application applies Bacillus, photosynthetic bacteria, Lactobacillus, and butyrate Clostridium throughout the whole process of sea cucumber seedling cultivation to regulate the number of Vibrio in the water environment through comprehensive effects, and can also effectively prevent diseases from occurring;

[0046] 5. The application uses the ratio of total bacteria count to Vibrio count in the breeding environment as a guide to reduce the blind addition of probiotics in the prior art, i.e., using conventional experience to apply probiotics, the yield of seedlings is lower than that using the application based on Vibrio regulation; the survival rate of sea cucumber seedlings is lower than that using the application based on Vibrio regulation;

[0047] 6. The directional addition of probiotics in the application can improve the non-specific immunity of sea cucumbers, enhance the resistance of sea cucumbers, effectively prevent diseases from occurring, reduce mortality, improve the survival rate and yield of sea cucumbers, and ultimately achieve the purpose of promoting the weight gain rate of farmed sea cucumbers and improving the breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 It is a standard curve for measuring total bacteria count in the application;

[0049] Figure 2 It is a standard curve for measuring total Vibrio count in the application;

[0050] Figure 3 The fluorescence quantitative PCR amplification curve for the double-priming duplex fluorescent quantitative PCR detection of the application. DETAILED DESCRIPTION

[0051] The fluorescence quantitative PCR (qPCR) standard curve is a common tool for determining the copy number or concentration of a target detection sample in quantitative analysis. The principle is to establish a linear relationship between the Ct value and the logarithm of the initial template amount by gradient dilution of standard samples with known concentrations. In the embodiment of the application, the standard curve is prepared according to the conventional process, and the steps are as follows:

[0052] S1, configuring bacterial standard samples and vibrio standard samples with known concentrations;

[0053] S2, diluting the bacterial standard samples and the vibrio standard samples with known concentrations into different concentration samples according to a 10-fold gradient, respectively, to obtain multiple sets of bacterial concentration data and multiple sets of vibrio concentration data;

[0054] S3, detecting the samples in step S2 using the double-priming duplex fluorescent quantitative PCR detection method in the application, determining each sample an average of three times to take an average value, respectively obtaining the Ct values of the bacteria and the vibrio in each sample, and screening the Ct values;

[0055] The screening criteria are that the Ct value of the sample is less than 30, the range of the Ct values of the three repetitions (the maximum value minus the minimum value) should be ≤0.5, and the coefficient of variation (CV) should be ≤1%;

[0056] S4, after removing the abnormal results in S2, generating a fitting curve according to the concentrations in step S2 and the Ct values in step S3, taking log10 (initial copy number) as the abscissa x and the average Ct value as the ordinate y, to draw the standard curve for determining the total number of bacteria (as shown in Figure 1 ) and the standard curve for determining the total number of vibrio (as shown in Figure 2 ), and the R 2 of the standard curve is ≥0.99;

[0057] The number of standard curve sample points is selected in accordance with the requirement in GB / T 22554-2010 “Linear Calibration Based on Standard Samples” that the number of standard samples N is at least 3.

[0058] Example 1

[0059] The embodiment provides a sea cucumber seedling cultivation method based on vibrio regulation, which is implemented in a sea cucumber breeding workshop in Xinhu Town, Dongying Estuary, Shandong.

[0060] The sea cucumber seed cultivation time is from May 4, 2024 to June 15, 2024, and the cultivation pool is 6, which is divided into 3 control pools and 3 experimental pools. The size of the cultivation pool is 3m x 10m, and the water depth is 1.5m. The water temperature is controlled at 21-22℃, and the salinity range is controlled at 27-28‰. The cultivation process is continuously aerated, and the dissolved oxygen content of the water body is maintained above 5mg / ml. The starting size of the sea cucumber seed is 10000 heads / kg, and 5kg of seed is put into each cultivation pool. Water is replaced once a day in the morning at a fixed time, and each time the whole water volume is replaced by 1 / 3-1 / 2. The feeding amount of the feeding feed after water replacement is 5% of the total weight of the sea cucumber, and the feeding feed is any kind of juvenile sea cucumber feed and sea mud on the market, and the mass ratio of the two is 1:10.

[0061] In this embodiment, the juvenile sea cucumber feed comprises 30wt% of Gracilaria dura powder, 30wt% of Sargassum powder, 20wt% of Enteromorpha powder, 5wt% of defatted fish meal, 5wt% of scallop edge powder, 3wt% of marine red yeast powder, 2wt% of Dunaliella powder, 3wt% of soybean peptide, and 2wt% of dry yeast.

[0062] The 3 control pools do not perform probiotic administration treatment, and the 3 experimental pools are fed with probiotics according to the following cultivation method, which is as follows:

[0063] During the cultivation period, 1ml of water sample is taken from the cultivation pool before water replacement every day, and the solid precipitate after centrifugation is used to extract sample DNA using a bacterial genome extraction kit (Vazyme DNA Extraction Kit). The ratio of total bacteria and total Vibrio is determined by the method of double-primer duplex fluorescent quantitative PCR detection.

[0064] In this embodiment, when the double-primer duplex fluorescent quantitative PCR detection is performed, the primer sequences used in the PCR determination of the total bacteria are as shown in SEQ ID NO:1 and SEQ ID NO:2:

[0065] SEQ ID NO:1: CCTACGGGAGGCAGCAG;

[0066] SEQ ID NO:2: GTATTACCGCGGCTGCTG;

[0067] Among them, SEQ ID NO:1 is a forward primer and SEQ ID NO:2 is a reverse primer, both of which are labeled with carboxyfluorescein (FAM).

[0068] The primer sequences used in the determination of the total number of Vibrio are as shown in SEQ ID NO:3 and SEQ ID NO:4:

[0069] SEQ ID NO:3: CCAAGAACTTAACCGTATC;

[0070] SEQ ID NO: 4: TACCCATAGAAAGCATCA;

[0071] wherein SEQ ID NO: 3 is a forward primer and SEQ ID NO: 4 is a reverse primer, both of which are labeled with hexachlorofluorescein (HEX).

[0072] In the PCR system of the present embodiment: 1 μL of water body genomic DNA, 2 μL of 2.5 mmol / L dNTP mixture, 2.5 μL of 10×PCR buffer (10-fold concentrated PCR buffer), 1 μL of forward primer (10 μmol / mL), 1 μL of reverse primer (10 μmol / mL), 1 U of DNA polymerase (0.5 μL), and high-pressure sterilized ddH2O are added to 25 μL.

[0073] The total number of bacteria / total number of Vibrio ratio is calculated according to the following formula based on the double-primer duplex fluorescent quantitative PCR detection result:

[0074] Formula 1: y1=-3.2196x1+41.237;

[0075] wherein R1 2 = 0.9986; and the standard curve thereof is shown in Figure 1

[0076] y1 is the Ct value of the double-primer duplex fluorescent quantitative PCR detection when the total number of bacteria is determined;

[0077] x1 is the total number of bacteria;

[0078] Formula 2: y2=-3.2196x2+35.489;

[0079] wherein R2 2 = 0.9986; and the standard curve thereof is shown in Figure 2

[0080] y2 is the Ct value of the double-primer duplex fluorescent quantitative PCR detection when the total number of Vibrio is determined;

[0081] x2 is the total number of Vibrio;

[0082] According to the total number of bacteria / total number of Vibrio ratio, probiotics are added to the experimental group.

[0083] When x1 / x2>50:1, the application amount of the probiotics is:

[0084] Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added to the aquaculture water every 7 days, and the application concentration is independently 0.1-0.2 g / m 3 .​​

[0085] Based on the total weight of the daily feed, 0.5 g / kg of lactic acid bacteria and 0.5 g / kg of butyric acid clostridium were added to the feed daily.

[0086] When 10:1 ≤ x1 / x2 ≤ 50:1, the dosage of the probiotics is:

[0087] Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria were added to the aquaculture water every 3 days at an independent concentration of 0.1-0.2 g / m³. 3 ;

[0088] Based on the total weight of the feed fed daily, 2g / kg of lactic acid bacteria and 2g / kg of butyric acid clostridium were added to the feed daily.

[0089] When x1 / x2 < 10:1, the dosage of the probiotics is:

[0090] Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added to the aquaculture water daily at an independent concentration of 0.2-0.4 g / m³. 3 ;

[0091] like Figure 3 As shown, the curves of quantitative real-time PCR amplification during the double primer doubly real-time PCR detection in this embodiment are as follows: Curve 1 is the curve of Vibrio in the sample to be tested in this embodiment; Curve 2 is the curve of bacteria in the sample to be tested in this embodiment; Curve 3 is the positive test curve of Vibrio in the control kit in this embodiment; Curve 4 is the positive test curve of bacteria in the control kit in this embodiment; Curve 5 is the negative test curve of Vibrio in the control kit in this embodiment; and Curve 6 is the negative test curve of bacteria in the control kit in this embodiment.

[0092] Based on the total daily feed weight, 2 g / kg of lactic acid bacteria and 2 g / kg of butyric acid clostridium were added to the feed daily. The following tables show the probiotic treatment measures for the three experimental ponds according to the ratio of total bacterial count to total Vibrio count: Table 1 (May 4-22) and Table 2 (May 23-15) for Experimental Pond 1, Table 3 (May 4-22) and Table 4 (May 23-15) for Experimental Pond 2, and Table 5 (May 4-22) and Table 6 (May 23-15) for Experimental Pond 3.

[0093] Table 1. Bacterial / Vibrio Detection and Corresponding Probiotic Treatment Measures in Experimental Pool No. 1 (5.4-5.22)

[0094]

[0095] Table 2 Bacteria / vibrio detection and corresponding probiotic treatment measures of No. 1 experimental pool (5.23-6.15)

[0096]

[0097] Table 3 Bacteria / vibrio detection and corresponding probiotic treatment measures of No. 2 experimental pool (5.4-5.22)

[0098]

[0099] Table 4 Bacteria / vibrio detection and corresponding probiotic treatment measures of No. 2 experimental pool (5.23-6.15)

[0100]

[0101] Table 5 Bacteria / vibrio detection and corresponding probiotic treatment measures of No. 3 experimental pool (5.4-5.22)

[0102]

[0103] Table 6 Bacteria / vibrio detection and corresponding probiotic treatment measures of No. 3 experimental pool (5.23-6.15)

[0104]

[0105] After the end of the cultivation experiment, the growth of sea cucumber seedlings in the experimental pool and the control pool was compared. The total harvest of sea cucumber seedlings in the control pool was 30.84 kg, and the average output of sea cucumber seedlings in each cultivation pool was 10.28 kg. The total harvest of sea cucumber seedlings in the experimental group was 56.83 kg, and the average output of sea cucumber seedlings in each cultivation pool was 18.94 kg. The yield of sea cucumber seedlings cultivated by the sea cucumber seedling cultivation method based on vibrio regulation was increased by 84.24%. The survival rate of sea cucumber seedlings in the experimental pool was 82.26%, and the survival rate of sea cucumber seedlings in the control group was 71.51%. The survival rate of sea cucumber seedlings cultivated by the sea cucumber seedling cultivation method based on vibrio regulation was increased by 15.03%.

[0106] Example 2

[0107] The sea cucumber seedling cultivation method based on vibrio regulation is implemented in a sea cucumber cultivation workshop on Kongdong Island in Yantai, Shandong.

[0108] The sea cucumber seed cultivation time is from May 28, 2024 to July 8, 2024, and the cultivation pool is 6, which is divided into 3 control pools and 3 experimental pools. The specification of the cultivation pool is 2.8 meters x 12 meters, and the water depth is 1.5 meters. The water temperature is controlled at 19-22℃, and the salinity range is controlled at 27-28‰. The cultivation process is continuously aerated, and the dissolved oxygen content of the water body is maintained above 5mg / ml. The starting size of the sea cucumber seed is 6000 heads / kg, and 10kg of seed is put into each cultivation pool. Water is replaced once a day in the morning at a fixed time, and each time the whole water volume is replaced by 1 / 3-1 / 2. The feeding amount of the feeding feed after water replacement is 5% of the total weight of the sea cucumber, and the feeding feed is any kind of juvenile sea cucumber feed and sea mud on the market, and the mass ratio of the two is 1:10.

[0109] In this embodiment, the juvenile sea cucumber feed comprises 25wt% of Gracilaria dura powder, 30wt% of Sargassum thunbergii powder, 25wt% of Enteromorpha prolifera powder, 4wt% of defatted fish meal, 6wt% of scallop edge powder, 3wt% of marine red yeast powder, 2wt% of Dunaliella salina powder, 4wt% of soybean peptide, and 1wt% of dry yeast.

[0110] The 3 control pools do not perform probiotic administration treatment, and the 3 experimental pools are fed with probiotics according to the following cultivation method, which is as follows:

[0111] During the cultivation period, 1ml of water sample is taken from the cultivation pool before water replacement every day, and the solid precipitate after centrifugation is used to extract sample DNA using a bacterial genome extraction kit (Vazyme DNA Extraction Kit). The total number of bacteria and the total number of Vibrio are determined by the method of double-primer duplex fluorescent quantitative PCR detection.

[0112] In this embodiment, when the double-primer duplex fluorescent quantitative PCR detection is performed, the primer sequences used in the PCR determination of the total number of bacteria are as shown in SEQ ID NO:1 and SEQ ID NO:2:

[0113] SEQ ID NO:1: CCTACGGGAGGCAGCAG;

[0114] SEQ ID NO:2: GTATTACCGCGGCTGCTG;

[0115] Among them, SEQ ID NO:1 is a forward primer and SEQ ID NO:2 is a reverse primer, both of which are labeled with carboxyfluorescein (FAM).

[0116] The primer sequences used in the determination of the total number of Vibrio are as shown in SEQ ID NO:3 and SEQ ID NO:4:

[0117] SEQ ID NO:3: CCAAGAACTTAACCGTATC;

[0118] SEQ ID NO: 4: TACCCATAGAAAGCATCA;

[0119] wherein SEQ ID NO: 3 is a forward primer and SEQ ID NO: 4 is a reverse primer, both of which are labeled with hexachlorofluorescein (HEX).

[0120] In the PCR system of the present embodiment: 1 μL of water body genomic DNA, 2 μL of 2.5 mmol / L dNTP mixture, 2.5 μL of 10x PCR buffer (10-fold concentrated PCR buffer), 1 μL of forward primer (10 μmol / mL), 1 μL of reverse primer (10 μmol / mL), 1 U of DNA polymerase (0.5 μL), and high-pressure sterilized ddH2O are added to 25 μL.

[0121] The total number of bacteria / total number of Vibrio ratio is calculated according to the following formula based on the results of the double-fluorescent quantitative PCR detection by double primers:

[0122] Formula 1: y1=-3.2196x1+41.237;

[0123] wherein R1 2 = 0.9986;

[0124] y1 is the Ct value of the double-fluorescent quantitative PCR detection by double primers when the total number of bacteria is determined;

[0125] x1 is the total number of bacteria;

[0126] Formula 2: y2=-3.2196x2+35.489;

[0127] wherein R2 2 = 0.9986;

[0128] y2 is the Ct value of the double-fluorescent quantitative PCR detection by double primers when the total number of Vibrio is determined;

[0129] x2 is the total number of Vibrio;

[0130] According to the total number of bacteria / total number of Vibrio ratio, probiotics are added to the experimental group.

[0131] When x1 / x2>50:1, the application amount of the probiotics is:

[0132] Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added to the aquaculture water every 7 days, and the application concentration is independently 0.1-0.2 g / m 3 ;

[0133] 0.5 g / kg of the lactobacillus and 0.5 g / kg of the clostridium butyricum is added in the feeding feed every day, on the basis of the total weight of the feeding feed.

[0134] When 10:1≤x1 / x2≤50:1, the application amount of the probiotic is:

[0135] On the basis of the total volume of the aquaculture water, bacillus, photosynthetic bacteria is added in the aquaculture water every 3 days, and the application concentration is independently 0.1-0.2 g / m 3 ;

[0136] 2 g / kg of the lactobacillus and 2 g / kg of the clostridium butyricum is added in the feeding feed every day, on the basis of the total weight of the feeding feed.

[0137] When x1 / x2<10:1, the application amount of the probiotic is:

[0138] On the basis of the total volume of the aquaculture water, bacillus, photosynthetic bacteria is added in the aquaculture water every 1 day, and the application concentration is independently 0.2-0.4 g / m 3 ;

[0139] 2 g / kg of the lactobacillus and 2 g / kg of the clostridium butyricum is added in the feeding feed every day, on the basis of the total weight of the feeding feed.

[0140] After the end of the aquaculture experiment, the growth of the sea cucumber seedlings in the comparative experiment pool and the control pool is compared, the total harvest of the sea cucumber seedlings in the three cultivation pools in the control pool is 56.58 kg, and the average output of the sea cucumber seedlings in each cultivation pool is 18.86 kg; the total harvest of the sea cucumber seedlings in the three cultivation pools in the experimental pool is 93.24 kg, and the average output of the sea cucumber seedlings in each cultivation pool is 31.08 kg, the yield of the sea cucumber seedlings cultivated by the sea cucumber seedling cultivation method based on the vibrio regulation of the application is increased by 64.79%. The survival rate of the sea cucumber seedlings in the control pool is 73.26%, and the survival rate of the sea cucumber seedlings in the experimental pool is 84.75%, the survival rate of the sea cucumber seedlings cultivated by the sea cucumber seedling cultivation method based on the vibrio regulation of the application is increased by 15.68%.

[0141] Comparative Example 1

[0142] This comparative example is an existing technology, and the probiotic is added by experience, which is implemented in a sea cucumber aquaculture workshop in Xinhu Town, Dongying Estuary, Shandong, and is carried out at the same period as Example 2.

[0143] The sea cucumber seed cultivation time is from May 4, 2024 to June 15, and there are three comparison pools. The comparison pool is 3 meters x 10 meters in size, and the water depth is 1.5 meters. The water temperature is controlled at 21-22℃, and the salinity range is controlled at 27-28‰. The cultivation process is continuously aerated, and the dissolved oxygen content of the water body is maintained above 5 mg / ml. The starting size of the sea cucumber seed is 10,000 heads / kg, and 5 kg of seed is put into each cultivation pool. Water is replaced once a day in the morning at a fixed time, and each time the whole water volume is replaced by 1 / 3-1 / 2. The feeding amount of the feeding after water replacement is 5% of the total weight of the sea cucumber, and the feeding is any commercially available juvenile sea cucumber feed and sea mud, and the mass ratio of the two is 1:10.

[0144] Unlike the double primer duplex fluorescent quantitative PCR detection performed daily in the example, the probiotic application treatment guided by the vibrio index according to the detection results, the probiotic application is applied in the conventional experience fixed method during the cultivation experiment. Bacillus and photosynthetic bacteria are added in the water body every 7 days, and the application concentration is independently 0.1-0.2 g / m 3 ; 0.5 g / kg of lactic acid bacteria and 0.5 g / kg of butyrate clostridium are added in the feeding every day.

[0145] At the end of the cultivation experiment, the growth of the sea cucumber seed is measured, and the total harvested sea cucumber seed of the three comparison pools is 42.36 kg, and the average yield of each comparison pool is 14.12 kg. During the cultivation experiment, the probiotic is applied in the conventional experience fixed method, and the yield of the seed is 25.45% lower than that in the experimental pool of example 1 using the sea cucumber seed cultivation method based on the vibrio regulation of the application, and 37.35% higher than that in the control pool. The survival rate of the sea cucumber seed is 78.20%, which is 4.4% lower than that in the experimental pool of example 1, and 9.36% higher than that in the control pool. The overall comparative example uses the conventional experience fixed method to apply the probiotic, and the sea cucumber seed cultivation effect is better than that without the application of the probiotic, but the cultivation yield and survival rate are lower than those using the sea cucumber seed cultivation method based on the vibrio regulation of the application.

[0146] It should be understood that the present application is not limited to what has been described above and that various modifications and changes can be made without departing from its scope. The scope of the application is defined only by the appended claims.

Claims

1. A method for cultivating sea cucumber seedlings based on Vibrio regulation, characterized in that, The cultivation method involves detecting the total bacterial count and total Vibrio count in the environment throughout the entire process of sea cucumber seedling cultivation using the following formula, and adjusting the amount of probiotics added based on the ratio of total bacterial count to total Vibrio count: Formula 1: y1=-3.2196x1+41.237; Where R1 2 = 0.9986; y1 represents the Ct value detected by double primer doubly real-time PCR when determining the total bacterial count; x1 represents the total bacterial count, in GC / mL; Formula 2: y2=-3.2196x2+35.489; R2 2 = 0.9986; y2 represents the Ct value detected by double primer doubly real-time quantitative PCR when determining the total number of Vibrio bacteria; x2 represents the total number of Vibrio cells, in GC / mL. The probiotics are Bacillus, photosynthetic bacteria, lactic acid bacteria, and Clostridium butyricum; When x1 / x2 > 50:1, the dosage of the probiotics is: Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria were added to the aquaculture water every 7 days at an independent concentration of 0.1-0.2 g / m³. 3 ; Based on the total weight of the daily feed, 0.5 g / kg of lactic acid bacteria and 0.5 g / kg of butyric acid clostridium were added to the feed daily. When 10:1 ≤ x1 / x2 ≤ 50:1, the dosage of the probiotics is: Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria were added to the aquaculture water every 3 days at an independent concentration of 0.1-0.2 g / m³. 3 ; Based on the total weight of the daily feed, add 2g / kg of lactic acid bacteria and 2g / kg of butyric acid clostridium to the feed daily. When x1 / x2 < 10:1, the dosage of the probiotics is: Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added to the aquaculture water daily at an independent concentration of 0.2-0.4 g / m³. 3 ; Based on the total weight of the daily feed, add 2g / kg of lactic acid bacteria and 2g / kg of butyric acid clostridium to the feed daily. The primer sequences used in the PCR assay for the total bacterial count are shown in SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO: 1: CCTACGGGAGGCAGCAG; SEQ ID NO:2: GTATTACCGCGGCTGCTG; The primer sequences used in the determination of the total number of Vibrio bacteria are shown in SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:3: CCAAGAACTTAACCGTATC; SEQ ID NO:4: TACCCATGAAAGCATCA.

2. The method for cultivating sea cucumber seedlings based on Vibrio regulation according to claim 1, characterized in that, All primers were labeled with fluorescein.

3. The method for cultivating sea cucumber seedlings based on Vibrio regulation according to claim 2, characterized in that, The fluorescein is either carboxyfluorescein or hexachlorofluorescein.

4. The method for cultivating sea cucumber seedlings based on Vibrio regulation according to claim 2, characterized in that, The primers used in the PCR assay for total bacterial count are different from those used in the assay for total vibrio count.

5. The method for cultivating sea cucumber seedlings based on Vibrio regulation according to claim 1, characterized in that, The probiotics are in powder form.

6. The method for cultivating sea cucumber seedlings based on Vibrio regulation according to any one of claims 1-5, characterized in that, The number of live bacteria in each of the probiotics is independently >10. 9 GC / g.

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