Vibrio regulation-based stichopus japonicus seedling cultivation method

Abstract

The invention relates to the technical field of stichopus japonicus aquaculture, in particular to a stichopus japonicus fry breeding method based on vibrio regulation and control. According to the cultivation method, in the whole cultivation process of the stichopus japonicus fry, the total bacterial count / total vibrio count ratio is calculated according to the total bacterial count and total vibrio count in a detection environment, the amount of added probiotics is adjusted according to the total bacterial count / total vibrio count ratio, and compared with stichopus japonicus fry without probiotics, the yield of the stichopus japonicus fry is increased by 50% or above through the cultivation method, and the yield of the stichopus japonicus fry is increased by 50% or above. The survival rate is increased by 10% or above.

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 appropriate prevention and control measures can be taken.

[0005] In recent years, the development and application of probiotics and microecological preparations in sea cucumber farming have gradually emerged. For example, CN108949615A discloses a microecological preparation for fermenting sea cucumber feed, wherein the compound microecological preparation is a microbial mixture composed of Bacillus subtilis DB005, Bacillus thuringiensis XW008, Bacillus licheniformis XW015, and Bacillus subtilis ZF003. Alternatively, CN104367597A discloses a microecological preparation and its preparation method for preventing and treating sea cucumber skin rot syndrome, wherein the active ingredient is the cells of Bacillus megaterium H1 and its extracellular metabolites. Or, CN111647523A discloses a probiotic composition for sea cucumber farming and its application, wherein the composition includes Lactobacillus paracasei CSDN-6, Lactobacillus plantarum CLY-5, and Bacillus belyssus DY-6, the three strains mixed in a cell ratio of 1:1:1. Alternatively, the study on the effects of Bacillus and Lactobacillus on sea cucumber growth was conducted in the journal *Heilongjiang Fisheries*, Volume 43, Issue 5, authored by Li Zongwen. The study focused on the development of microbial strains and the formulation of microbial preparations. However, current research primarily concentrates on strain development and the preparation of microbial preparations, while the application of these products in production lacks scientific guidance and reference indicators. Farmers still rely on intuition and experience to determine the dosage.

[0006] Based on the above situation, although new microbial agents have been introduced into the cultivation of sea cucumber seedlings in the existing technology, there are still technical problems that urgently need to be solved, such as the lack of scientific guidance and reference indicators, the lack of usage guidelines, the fact that most of them are added based on experience, the inability to efficiently cultivate seedlings in factories, and the low survival rate and unstable yield of sea cucumber seedlings. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for cultivating sea cucumber seedlings based on Vibrio regulation. The method involves detecting the total bacterial count and total Vibrio count in the environment throughout the entire sea cucumber seedling cultivation process using the following formula, calculating the ratio of total bacterial count to total Vibrio count, and adjusting the amount of probiotics added based on this ratio. 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 is the total number of Vibrio, in units of GC / mL; wherein GC is the gene copy number; The primer sequences used in the PCR detection of the total number of bacteria 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 are shown in SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 3: CCAAGAACTTAACCGTATC; SEQ ID NO: 4: TACCCATAGAAAGCATCA; 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 by more than 10%.

[0008] Further, the probiotics are Bacillus, photosynthetic bacteria, lactic acid bacteria, and Clostridium butyricum.

[0009] Further, the primers are all labeled with fluorescein.

[0010] Further, the fluorescein includes but is not limited to carboxyfluorescein (FAM, 6-Carboxyfluorescein) or hexachlorofluorescein (HEX, 6-isomer); The carboxyfluorescein presents green light in double primer duplex fluorescent quantitative PCR detection; The hexachlorofluorescein presents yellow-green light in double primer duplex fluorescent quantitative PCR detection.

[0011] Further, the fluorescein used in the primers in the PCR determination of the total number of bacteria and the primers in the determination of the total number of Vibrio is different, so as to distinguish between the two and count the total number.

[0012] Further, when x1 / x2>50:1, the application amount of the probiotics is: Based on the total volume of the aquaculture water, Bacillus and photosynthetic bacteria are added in the aquaculture water every 7 days, and the application concentration is independently 0.1-0.2 g / m 3 ; 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.

[0013] Further, when 10:1≤x1 / x2≤50:1, the application amount of the probiotic is: On the basis of the total volume of the aquaculture water, Bacillus, photosynthetic bacteria are added in the aquaculture water every 3 days, and the application concentration is independently 0.1-0.2 g / m 3 ; On the basis of 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.

[0014] Further, when x1 / x2<10:1, the application amount of the probiotic is: On the basis of the total volume of the aquaculture water, Bacillus, photosynthetic bacteria are added in the aquaculture water every 1 day, and the application concentration is independently 0.2-0.4 g / m 3 ; On the basis of 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.

[0015] Further, the probiotic is a powder.

[0016] Further, the viable count in each of the probiotics is independently >10 9 GC / g.

[0017] The beneficial effects of the present application are: 1. The present application provides a sea cucumber seedling cultivation method based on Vibrio regulation. The cultivation method is to calculate the ratio of total bacteria to total Vibrio according to the total number of bacteria and the total number of Vibrio in the environment during the whole process of sea cucumber seedling cultivation, and to adjust the amount of added probiotics according to the ratio of total bacteria to total Vibrio. Compared with sea cucumber seedlings without added probiotics, the yield of sea cucumber seedlings is increased by more than 50%, and the survival rate is increased by more than 10%. 2. The present application selects specific types of probiotics, such as Bacillus which can secrete hydrolytic enzymes (such as protease, 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; lactic acid bacteria and Clostridium butyricum probiotics can colonize in the intestinal tract, regulate intestinal microbial community and intestinal health, stimulate the intestinal mucosa of cultured animals to produce immune factors, and enhance non-specific immune ability; at the same time, the four probiotics produce digestive enzymes (such as amylase, protease) to promote the decomposition of nutrients in the feed; 3. The present application adds probiotics according to formula 1 and formula 2, which can make the probiotics compete with the pathogenic bacteria in the environment for survival resources, inhibit the growth of pathogenic bacteria, and at the same time secrete organic acids, hydrogen peroxide, antibacterial peptides and other antibacterial substances to directly inhibit the reproduction of pathogenic bacteria, and the metabolites (such as vitamins, short-chain fatty acids) produced can also directly promote the growth of sea cucumbers. 4. The present invention applies Bacillus, photosynthetic bacteria, lactic acid bacteria and Clostridium butyricum throughout the entire process of sea cucumber seedling cultivation. Through the comprehensive effect, it regulates the number of Vibrio in the aquatic environment and can also effectively prevent the occurrence of diseases. 5. The application of probiotics in this invention is guided by the ratio of total bacterial count to total Vibrio count in the aquaculture environment, reducing the blind addition of probiotics in the existing technology. That is, the seedling yield is lower than the yield based on Vibrio regulation using this invention when using conventional experience and fixed methods; the survival rate of sea cucumber seedlings is lower than the survival rate based on Vibrio regulation using this invention. 6. The targeted addition of probiotics in this invention can improve the non-specific immunity of sea cucumbers, enhance their resistance, effectively prevent disease occurrence, and reduce mortality, thereby increasing the survival rate and yield of sea cucumbers, and ultimately promoting the weight gain rate of farmed sea cucumbers and improving farming efficiency. Attached Figure Description

[0018] Figure 1 This is the standard curve for the determination of total bacterial count in this invention; Figure 2 This is the standard curve for the determination of total Vibrio count in this invention; Figure 3 This is the fluorescence quantitative PCR amplification curve for the double primer dual fluorescence quantitative PCR detection of the present invention. Detailed Implementation

[0019] A quantitative PCR (qPCR) standard curve is a common tool used in quantitative analysis to determine the copy number or concentration of a target sample. Its principle is to establish a linear relationship between the Ct value and the logarithm of the initial template amount by serially diluting a standard of known concentration. In this embodiment of the invention, the standard curve is prepared according to a conventional procedure, the steps of which are as follows: S1, prepare bacterial and Vibrio standards of known concentrations; S2, dilute the bacterial and Vibrio standards of known concentrations into samples of different concentrations according to a 10-fold gradient, and obtain multiple sets of bacterial concentration data and multiple sets of Vibrio concentration data; S3, detect the samples in step S2 using the dual-primer double fluorescence quantitative PCR detection method of this invention, and measure each sample three times on average and take the average value to obtain the Ct values ​​corresponding to bacteria and Vibrio in each sample, and screen them; the screening criteria are: the Ct value of the sample is less than 30, and the range of Ct values ​​(maximum value - minimum value) of the three replicates should be ≤0.5, and the coefficient of variation (CV) should be ≤1%; S4, after removing abnormal results in S2, based on the concentration in step S2 and the Ct value in step S3, generate a fitting curve with log10 (initial copy number) as the x-axis and the average Ct value as the y-axis, that is, draw the standard curve for the determination of total bacterial count (e.g. Figure 1 (as shown) and the standard curve for the determination of total Vibrio count (as shown)Figure 2 (as shown); and in the standard curve R 2 ≥0.99; The selection of the number of standard curve sample points shall comply with the requirement in GB / T 22554-2010 "Linear Calibration Based on Standard Samples" that the number of standard samples N shall be at least 3.

[0020] Example 1 This embodiment provides a method for cultivating sea cucumber seedlings based on Vibrio regulation, which was implemented in a sea cucumber breeding workshop in Xinhu Town, Hekou District, Dongying City, Shandong Province.

[0021] The sea cucumber seedling cultivation period was from May 4th to June 15th, 2024. Six cultivation ponds were used: three control ponds and three experimental ponds. Each pond measured 3 meters × 10 meters with a water depth of 1.5 meters. The water temperature was controlled at 21-22℃, and the salinity at 27-28‰. Continuous aeration was maintained throughout the cultivation process to ensure dissolved oxygen levels remained above 5 mg / ml. The initial sea cucumber seedling size for the experiment was 10,000 individuals / kg, with 5 kg of seedlings stocked in each pond. The water was changed once daily at a fixed time each morning, replacing 1 / 3 to 1 / 2 of the total water volume each time. After water changes, the feed amount was 5% of the total weight of the sea cucumbers. The feed consisted of any commercially available juvenile sea cucumber feed and sea mud, with a mass ratio of 1:10.

[0022] In this embodiment, the juvenile sea cucumber feed comprises, by weight, 30wt% Sargassum powder, 30wt% Sargassum powder, 20wt% Ulva powder, 5wt% defatted fish meal, 5wt% scallop edge powder, 3wt% marine red yeast powder, 2wt% Dunaliella powder, 3wt% soybean peptide, and 2wt% dry yeast.

[0023] None of the three control tanks received probiotic treatment. The three experimental tanks were fed probiotics according to the following culture method: During the cultivation period, 1 ml of water sample was taken from the culture tank before each daily water change and centrifuged. The solid precipitate after centrifugation was used to extract DNA from the sample using a bacterial genome extraction kit (Vazyme DNA Extraction Kit). The ratio of total bacterial count to total Vibrio count was determined using a double primer doubly fluorescent quantitative PCR method.

[0024] In this embodiment, the primer sequences used in the double primer doubly real-time quantitative PCR detection 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; Wherein, 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).

[0025] The primer sequences used in the determination of the total number of Vibrio are shown in SEQ ID NO: 3 and SEQ ID NO: 4: SEQ ID NO: 3: CCAAGAACTTAACCGTATC; SEQ ID NO: 4: TACCCATAGAAAGCATCA; 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).

[0026] 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.

[0027] The results of the double-primer duplex fluorescent quantitative PCR detection are calculated according to the following formula for the total number of bacteria / the total number of Vibrio ratio: Formula 1: y1=-3.2196x1+41.237; Wherein R1 2 = 0.9986; and the standard curve is shown in Figure 1 ; y1 is the Ct value of the double-primer duplex fluorescent quantitative PCR detection when determining the total number of bacteria; x1 is the total number of bacteria; Formula 2: y2=-3.2196x2+35.489; Wherein R2 2 = 0.9986; and the standard curve is shown in Figure 2 ; y2 is the Ct value of the double-primer duplex fluorescent quantitative PCR detection when determining the total number of Vibrio; x2 is the total number of Vibrio; According to the total number of bacteria / the total number of Vibrio ratio, probiotics are added to the experimental group.

[0028] When x1 / x2>50:1, the application amount of the probiotics is: 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 / m3 ; 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.

[0029] 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 feed fed daily, 2g / kg of lactic acid bacteria and 2g / kg of butyric acid clostridium were added to the feed daily.

[0030] 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 ; 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.

[0031] 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.

[0032] Table 1. Bacterial / Vibrio Detection and Corresponding Probiotic Treatment Measures in Experimental Pool No. 1 (5.4-5.22)

[0033] Table 2. Bacterial / Vibrio Detection and Corresponding Probiotic Treatment Measures in Experimental Pool No. 1 (May 23 - June 15)

[0034] Table 3 Bacteria / vibrio detection and corresponding probiotic treatment measures for No. 2 experimental pool (5.4-5.22)

[0035] Table 4 Bacteria / vibrio detection and corresponding probiotic treatment measures for No. 2 experimental pool (5.23-6.15)

[0036] Table 5 Bacteria / vibrio detection and corresponding probiotic treatment measures for No. 3 experimental pool (5.4-5.22)

[0037] Table 6 Bacteria / vibrio detection and corresponding probiotic treatment measures for No. 3 experimental pool (5.23-6.15)

[0038] 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, with an average of 10.28 kg per cultivation pool. The total harvest of sea cucumber seedlings in the experimental group was 56.83 kg, with an average of 18.94 kg per cultivation pool. The yield of sea cucumber seedlings cultivated using 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 using the sea cucumber seedling cultivation method based on vibrio regulation was increased by 15.03%.

[0039] Example 2 The sea cucumber seedling cultivation method based on vibrio regulation is implemented in a sea cucumber cultivation workshop on Kongdong Island in Yantai, Shandong.

[0040] The sea cucumber seedling cultivation time is from May 28, 2024 to July 8, 2024. There are 6 cultivation pools, divided into 3 control pools and 3 experimental pools. The cultivation pool is 2.8 meters x 12 meters in size, with a water depth of 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 to maintain the dissolved oxygen content of the cultivation water above 5 mg / ml. The sea cucumber seedlings at the start of the cultivation experiment are 6000 heads / kg, and 10 kg of seedlings are put into each cultivation pool. Water is replaced once a day in the morning at a fixed time, with a replacement of 1 / 3-1 / 2 of the total water volume each time. 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 commercially available juvenile sea cucumber feed and sea mud, with a mass ratio of 1:10.

[0041] In the embodiment, the juvenile sea cucumber feed comprises 25 wt% of F. serra powder, 30 wt% of S. thunbergii powder, 25 wt% of U. prolifera powder, 4 wt% of defatted fish meal, 6 wt% of scallop edge powder, 3 wt% of R. marina powder, 2 wt% of D. tertiolecta powder, 4 wt% of soybean peptide, and 1 wt% of dry yeast.

[0042] The 3 control pools are not subjected to probiotic administration treatment, and the 3 experimental pools are fed with probiotics according to the following cultivation method. During the cultivation, 1 ml of water sample is taken from the cultivation pool before water change every day for centrifugation. The solid precipitate after centrifugation is used for sample DNA extraction by using a bacterial genome extraction kit (Vazyme DNA Extraction Kit). The ratio of total bacteria count to total Vibrio count is determined by using a double-primer duplex fluorescent quantitative PCR detection method.

[0043] In the embodiment, when the double-primer duplex fluorescent quantitative PCR detection is performed, the primer sequences used in the PCR determination of the total bacteria count are as shown in SEQ ID NO: 1 and SEQ ID NO: 2: SEQ ID NO: 1: CCTACGGGAGGCAGCAG; SEQ ID NO: 2: GTATTACCGCGGCTGCTG; 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).

[0044] The primer sequences used in the determination of the total Vibrio count are as shown in SEQ ID NO: 3 and SEQ ID NO: 4: SEQ ID NO: 3: CCAAGAACTTAACCGTATC; SEQ ID NO: 4: TACCCATAGAAAGCATCA; Among them, 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).

[0045] In the PCR system of the embodiment: water body genomic DNA 1 μL, 2.5 mmol / L dNTP mixture 2 μL, 10×PCR buffer (concentrated 10 times of PCR buffer) 2.5 μL, forward primer (10 μmol / mL) 1 μL, reverse primer (10 μmol / mL) 1 μL, DNA polymerase 1 U (0.5 μL), and autoclaved ddH2O is added to 25 μL.

[0046] The total bacteria count / total Vibrio count ratio is calculated according to the following formula: Formula 1: y1=-3.2196x1+41.237; wherein R1 2 = 0.9986; y1 is the Ct value of the double-primer duplex fluorescent quantitative PCR detection when determining the total bacteria count; x1 is the total bacteria count; Formula 2: y2=-3.2196x2+35.489; wherein R2 2 = 0.9986; y2 is the Ct value of the double-primer duplex fluorescent quantitative PCR detection when determining the total Vibrio count; x2 is the total Vibrio count; According to the total bacteria count / total Vibrio count ratio, probiotics are added to the experimental group.

[0047] When x1 / x2> 50:1, the application amount of the probiotics is: Based on the total volume of the aquaculture water, Bacillus, photosynthetic bacteria are added to the aquaculture water every 7 days, and the application concentration is independently 0.1-0.2 g / m 3 . 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 to the feeding bait every day.

[0048] When 10:1≤x1 / x2≤50:1, the application amount of the probiotics is: Based on the total volume of the aquaculture water, Bacillus, photosynthetic bacteria are added to the aquaculture water every 3 days, and the application concentration is independently 0.1-0.2 g / m 3 . 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 to the feeding bait every day.

[0049] When x1 / x2< 10:1, the application amount of the probiotics is: Based on the total volume of the aquaculture water, Bacillus, photosynthetic bacteria are added to the aquaculture water every 1 day, and the application concentration is independently 0.2-0.4 g / m 3 . 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 to the feeding bait every day.

[0050] After the aquaculture experiment, the growth of sea cucumber seedlings in the experimental and control ponds was compared. The control ponds yielded a total of 56.58 kg of seedlings, averaging 18.86 kg per pond. The experimental ponds yielded a total of 93.24 kg of seedlings, averaging 31.08 kg per pond. The yield of sea cucumber seedlings cultivated using the Vibrio-regulated method of this invention increased by 64.79%. The survival rate of sea cucumber seedlings in the control ponds was 73.26%, while the survival rate in the experimental ponds was 84.75%. The survival rate of sea cucumber seedlings cultivated using the Vibrio-regulated method of this invention increased by 15.68%.

[0051] Comparative Example 1 This comparative example is based on the existing technology of adding probiotics based on experience, and was implemented in a sea cucumber breeding workshop in Xinhu Town, Hekou District, Dongying City, Shandong Province, and was carried out at the same time as Example 2.

[0052] The sea cucumber seedling cultivation period was from May 4th to June 15th, 2024, with three control ponds. Each control pond measured 3 meters × 10 meters with a water depth of 1.5 meters. Water temperature was controlled at 21-22℃, and salinity was controlled at 27-28‰. Continuous aeration was maintained throughout the cultivation process to keep the dissolved oxygen content in the water above 5 mg / ml. The initial sea cucumber seedling size for the cultivation experiment was 10,000 heads / kg, with 5 kg of seedlings stocked in each cultivation pond. Water was changed once every morning at a fixed time, replacing 1 / 3 to 1 / 2 of the total water volume each time. After water changes, the feed amount was 5% of the total weight of the sea cucumbers, consisting of any commercially available juvenile sea cucumber feed and sea mud at a mass ratio of 1:10.

[0053] Unlike the previous example where daily double-primer quantitative real-time PCR detection was performed and probiotic application was guided by the Vibrio index based on the detection results, this aquaculture experiment used a conventional, empirically-based, and fixed probiotic application procedure. Bacillus and photosynthetic bacteria were added to the aquaculture water every 7 days at independent concentrations of 0.1-0.2 g / m³. 3 Add 0.5g / kg of lactic acid bacteria and 0.5g / kg of butyric acid clostridium to the feed daily.

[0054] After the end of the cultivation experiment, the growth of sea cucumber seedlings is measured. The total harvest of sea cucumber seedlings in the three comparison pools is 42.36 kg, and the average output of seedlings in each comparison pool is 14.12 kg. In the process of this cultivation experiment, the probiotics are applied by using the conventional experience fixed method, and the yield of seedlings is 25.45% lower than that in the experimental pool of Example 1 using the sea cucumber seedling cultivation method based on Vibrio regulation, and 37.35% higher than that in the control pool. The survival rate of sea cucumber seedlings 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. Overall, the cultivation effect of sea cucumber seedlings using the conventional experience fixed method to apply probiotics is better than that without applying probiotics, but the yield and survival rate of cultivation are lower than those using the sea cucumber seedling cultivation method based on Vibrio regulation.

[0055] 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 limited only by the appended claims.

Claims

1. A sea cucumber seedling cultivation method based on Vibrio regulation, characterized in that, The cultivation method is to detect 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 seed cultivation, and to adjust the amount of probiotics added according to the ratio of the total number of bacteria / the total number of Vibrio: Formula 1: y1=-3.2196x1+41.237; wherein R1 2 = 0.9986; y1 is the Ct value detected by double-primer duplex fluorescent quantitative PCR when detecting the total number of bacteria; x1 is the total number of bacteria, and the unit is GC / mL; Formula 2: y2=-3.2196x2+35.489; wherein R2 2 = 0.9986; y2 is the Ct value detected by double-primer duplex fluorescent quantitative PCR when detecting the total number of Vibrio; x2 is the total number of Vibrio, and the unit is GC / mL; 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; SEQ ID NO: 1: CCTACGGGAGGCAGCAG; SEQ ID NO: 2: GTATTACCGCGGCTGCTG; The primer sequence used in the detection of the total number of Vibrio is shown as SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 3: CCAAGAACTTAACCGTATC; SEQ ID NO: 4: TACCCATAGAAAGCATCA.

2. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 1, characterized in that, The probiotics are Bacillus, photosynthetic bacteria, lactic acid bacteria and Clostridium butyricum.

3. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 1, characterized in that, All the primers are labeled with fluorescein.

4. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 3, characterized in that, The fluorescein is carboxyfluorescein or hexachlorofluorescein.

5. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 3, characterized in that, The fluorescein used in the primers in the PCR detection of the total number of bacteria is different from that used in the primers in the detection of the total number of Vibrio.

6. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 1, characterized in that, When x1 / x2>50:1, the application amount of the probiotics is: On the basis of the total volume of the aquaculture water, spores of Bacillus, photosynthetic bacteria are added every 7 days in the aquaculture water, and the application concentration is independently 0.1-0.2g / m 3 ; Based on the total weight of the daily feeding feed, 0.5 g / kg of lactic acid bacteria and 0.5 g / kg of Clostridium butyricum are added in the daily feeding feed.

7. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 1, characterized in that, When 10:1≤x1 / x2≤50:1, the application amount of the probiotics is: On the basis of the total volume of the aquaculture water, spore bacillus and photosynthetic bacteria are added every 3 days in the aquaculture water, and the concentration is independently 0.1-0.2g / m 3 ; Based on the total weight of the daily feeding feed, 2 g / kg of lactic acid bacteria and 2 g / kg of Clostridium butyricum are added in the daily feeding feed.

8. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 1, characterized in that, When x1 / x2<10:1, the application amount of the probiotics is: On the basis of the total volume of the aquaculture water, the bacillus and the photosynthetic bacteria are added in the aquaculture water at a concentration of 0.2-0.4 g / m 3 ; Based on the total weight of the daily feeding feed, 2 g / kg of lactic acid bacteria and 2 g / kg of Clostridium butyricum are added in the daily feeding feed.

9. The sea cucumber seedling cultivation method based on Vibrio regulation according to claim 1, characterized in that, The probiotics are powders.

10. The sea cucumber seed culture method based on Vibrio regulation according to any one of claims 1-9, characterized in that, The number of viable bacteria in each of the probiotics is independently > 10 9 GC / g.

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