CN101982070B - Water-saving method for culturing young abalones - Google Patents
Water-saving method for culturing young abalones Download PDFInfo
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Abstract
The invention discloses a water-saving method for culturing young abalones, comprising the following steps: (1) paving bricks in a culture pond and then pouring seawater into the culture pond; (2) putting the young abalones into the culture pond and starting to carry out aeration; (3) regularly feeding bait and adding a bdellovibro plastid bacterial liquid to a culture water body and/or the fed bait; changing water every 5-30 days with the volume of changed water each time as a measuring range; and (4) collecting the young abalones when the young abalones grow to a length of 1.0-1.6cm, wherein the bacterial strain is Bdellovibrio sp. BDFM05, and preserved in China Center for Preserving Type Culture Collection with the preservation number of CCTCC NO: M209172 on 7th, August 2009. The method provided by the invention has the following advantages: (1) saving energy and reducing emission and being more environment-friendly; (2) enjoying more convenient preparation and preservation of leech plastid, good tolerance and longer bactericidal action; and (3) being capable of invigorating health and enhancing immunity of the young abalones.
Description
Technical field
The present invention relates to marine shellfish cultural technique field, particularly a kind ofly improve young abalone culture survival rate, save water and energy, the cultural method of environmental protection.
Background technology
The abalone culture research of China is since the starting seventies in 20th century, and the development through nearly 30 years has obtained huge achievement and remarkable economic efficiency.In recent years, along with young Bao is propagated the expansion of scale artificially, technical defective also highlights gradually; In breeding process, particularly after young Bao peels off, cause some young Bao diseases easily; Along with the breed scale develops rapidly; It is cultured the work ubiquity and young Bao in peeling off mortality in various degree appears in the back in a couple of days phenomenon, and lethality is up to more than 80%, and survival rate of seedlings generally maintains between the 3-20%; The survival rate of seedlings of most plants is lower than 10%, and this kind situation can not solve always at present.Because Bao Miao, young Bao mortality cause production cost to increase, many families can not keep ordinary production, and a part family stops production, changes the line of production, and cause the young Bao amount of supply to fall sharply.Bring enormous economic loss to the raiser.Pathogenic infection is to cause young Bao main causes of death, and propagating artificially mainly is bacteriosis and viral disease.Common potentially pathogenic organism has vibrio parahaemolytious, vibrio fluvialis, vibrio alginolyticus etc.
The method of the conventional young Bao board falling of control mainly is regular administration of antibiotics at present, though antibiotic has the direct entry of medicine, high, fast, the eutherapeutic advantage of absorption of concentration, shortcoming is possibly produce excitant, cause new illness young Bao.If long-term a large amount of antibiotic that uses not only can make the potentially pathogenic organism antibody-resistant bacterium increase, some Bao cases are become be difficult to cure.Antibiotic residue can influence young Bao quality in the Bao body in addition, is unfavorable for selling, and then causes culturist's economic loss.The young Bao board falling of herbal control also is a kind of common way, has drug residue free, advantage that economic benefit is high, but the slow shortcoming that takes effect is also arranged, thereby seek a kind of more efficient, economic way and seem very urgent.
Bdellovibrio leech plastid is meant the growth forms before Bdellovibrio is invaded host bacteria.Can be divided into the leech plastid state that breaks away from host bacteria, has the telotroch state of flagellum, free swimming and in host bacteria, grow the history of life of Bdellovibrio.Lose flagellum in the time of periplasmic space that the telotroch Bdellovibrio is invaded the host, host cell also begins to expand, and becomes one to osmotic pressure and insensitive spherical (i.e. " leech plastid ").Compare with telotroch, the leech plastid has preparation, preservation is more convenient, and the environment tolerance is strong, and the characteristics though the bactericidal action time slightly more waits so long slowly also have potential inactivated vaccine function simultaneously.Behind telotroch Bdellovibrio invasion host cell, the respiration of host cell (respiration) is stopped very soon.Bdellovibrio also gets into leech plastid state, begins to secrete plurality of enzymes, and the big molecule of clearing up host cell is a small-molecule substance, and then is integrated into the nutriment of self, makes self growth, and is elongated.Finally, the leech plastid of strip is divided into the sections of a lot of weak points, grows flagellum, becomes telotroch again.The essential enzyme of they secretions afterwards, broken wall and going out becomes the telotroch that breaks away from host bacteria, has flagellum, free swimming once more.Between this leech plastid puberty, Bdellovibrio does not change the surface texture of host cell, makes former host still have antigenic action, but the stimulating organism body produces immune response.
" green cultivation " becomes the great demand of country in recent years, causes the extensive concern of aquatic products circle.At present, carrying out young abalone culture with Bdellovibrio leech plastid does not at home and abroad report as yet.
Existing research shows, as a kind of effective microorganism preparation, no matter is in food industry or in fields such as (ocean) aquacultures, the application of Bdellovibrio all is safe.For example: abroad; Lenz and Hespell (1978) discovers; Bdellovibrio and animal and people's cell do not had an infectivity [Lenz R.W.; HespellR.B.Attempts to grow bedellovibrios micurgically-injected into animal cells.Archives of Microbiology, 1978,119 (3): 245-248].At home, Lin Mao etc. (2006) have studied the effect of Bdellovibrio to fish cell, find that it does not have dissemination [Lin Mao, Yang Xianle, Xue Hui, Cao Haipeng, Qiu Junqiang to the fish bacterium.Bdellovibrio BDH2102 is to the effect of fish cell and pathogen.The microbiology circular, 2006,33 (1): 7-11].
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, provide a kind of raising young Bao immunity, promote young Bao growth, improve the water-saving cultural method of young Bao survival rate.
The object of the invention is realized through following proposal:
A kind of water-saving young abalone culture method is characterized in that breeding process is following:
(1) tile work in culturing the pond injects seawater then;
(2) young Bao lower storage reservoir begins aeration simultaneously;
(3) bait of regularly throwing something and feeding, and add Bdellovibrio leech plastid bacterium liquid in breeding water body and/or in the bait of throwing something and feeding; And changed water once, and changed water for full pond at every turn in every 5-30 days;
(4) when young Bao body length arrives 1.0-1.6cm, can receive young Bao;
Said Bdellovibrio is Bdellovibrio (Bdellovibrio sp.) BDFM05, and by China's typical culture collection center preservation, it abbreviates CCTCC as, and deposit number is CCTCC NO:M209172, and preservation date is on August 7th, 2009.It is unicellular that BDFM05 is, ellipse, and size is 1.43 * 0.53 μ m, end is given birth to flagellum, flagellum length at least 2 μ m; Said Bdellovibrio (Bdellovibrio sp.) BDFM05 cultivates the transparent circular plaque can form diameter 2～3mm in three days with the double-layer plate method in 28 ℃, adopts the fermentation of leech plastid fermentation process in high density, obtains certain density Bdellovibrio leech plastid.
Preferably, said tile work is earlier brick to be washed and use disinfecting solution of potassium permanganate, takes to ride folded formula pendulum brick after the sterilization again.
Preferably, said seawater process sand filtration and precipitation process before injection.
The bait of being thrown something and fed is artificial synthetic diet, and preferably, said throwing something and feeding will stop aeration earlier during bait, begins aeration behind the bait throwing in 0.5-1h again.
Preferably, behind the said young Bao lower storage reservoir, aeration rate is controlled at 10-100L/h/m
3, illumination is controlled at 50-200lux, and water temperature is controlled at 15-28 ℃.
Preferably, the said Bdellovibrio leech plastid bacterium liquid that in the bait of throwing something and feeding, adds is meant and uses concentration to be 10-10
7The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaked bait 20～40 minutes.
Preferably, the said Bdellovibrio leech plastid bacterium liquid that in breeding water body, adds makes the concentration of Bdellovibrio leech plastid in water body reach 10pfu/mL at least.
Preferably, saidly all add Bdellovibrio leech plastid bacterium liquid immediately after changing water at every turn, and make the concentration of Bdellovibrio leech plastid in water body reach 10pfu/mL at least.
Preferably, the concentration of said Bdellovibrio leech plastid in water body is 10-10
7Pfu/mL.
Preferably, the preparation method of said Bdellovibrio leech plastid bacterium liquid is:
In the DNB liquid nutrient medium that contains vibrio parahaemolytious (Vibrio parahaemolyticus), insert Bdellovibrio BDFM05 (CCTCC M209172), constant temperature shaking table 150rpm～300rpm, 20～35 ℃ of cultivation 24～48h; Culture fluid is got deposition in 4 ℃ of centrifugal 15～20min of 6000～8000rpm, in deposition, adds the DNB liquid nutrient medium Bdellovibrio sediment that suspends again, promptly makes Bdellovibrio leech plastid bacterium liquid.
The present invention has following advantage and effect with respect to prior art:
1, Bdellovibrio leech plastid bacterium fluid power of the present invention significantly improves young Bao physique, enhancing immunity.
The effect of Bdellovibrio leech plastid bacterium liquid described in the present invention bacterium in the young abalone culture environment of control, bait, water body is remarkable, can obviously improve young Bao physique, enhancing immunity.
2, Bdellovibrio leech plastid bacterium liquid of the present invention safety in controlling and eliminate young Bao potentially pathogenic organism application is good.
Bdellovibrio leech plastid bacterium liquid is biological method through the method for cracking potentially pathogenic organism.Bdellovibrio leech plastid can infect, the characteristic of cracking host bacteria makes it to be suitable as the biological cleaning factor of restraining or removing potentially pathogenic organism in organism and the environment thereof; And it is behind the intact host bacteria of cracking; Can wither away automatically because of hungry; Thereby overcome side effect and the adverse effect of routine disinfection agent that the antibiotic abuse brings, had research simultaneously and proved that Bdellovibrio is nontoxic to people etc. young Bao.
3, described Bdellovibrio leech plastid bacterium fluid power purifies water and promotes young Bao growth.
Bdellovibrio leech plastid has catharsis to water quality, can improve the animal gastrointestinal tract environment, promotes growth.
4, contain Bdellovibrio leech plastid bacterium liquid of the present invention and be fit to be applied to the abalone culture process.
Bdellovibrio leech plastid has no side effect to people and abalone.Be suitable for the large-scale industrialized breed of abalone, can provide safeguard for the green processing production of abalone and for control with eliminate the abalone potentially pathogenic organism a kind of new method is provided.
Embodiment
Below in conjunction with specific embodiment the present invention is done further concrete detailed description the in detail, but embodiment of the present invention is not limited thereto, the technological parameter for not indicating especially can carry out with reference to routine techniques.Enforcement of the present invention status Shandong plant, young Bao of the present invention is a haliotis discus hannai Ino, is provided by this plant.
Embodiment 1
The preparation of Bdellovibrio leech plastid
Said Bdellovibrio (Bdellovibrio sp.) BDFM05 cultivates the transparent circular plaque that can form diameter 2～3mm in three days with the double-layer plate method in 28 ℃; Used Bdellovibrio leech plastid ferments through the fermentation process of high density Bdellovibrio leech plastid: 100mL nutrient broth (peptone 10g is being housed; Beef extract powder 3g; Sodium chloride 5g, pH 7.4 ± 0.2) conical flask in inoculation 0.5mL 10
7Cfu/mL vibrio parahaemolytious (Vibrio parahaemolyticus; Purchase AmericanType Culture Collection in U.S. representative microbial DSMZ; Numbering: ATCC 17802); 200rpm, 28 ℃ of shaking tables were cultivated 18 hours, and culture fluid is abandoned supernatant respectively at 4 ℃, the centrifugal 15min of 5000rpm.With 5mL physiological saline suspension deposition thalline; Join one afterwards 100mL DNB (dilute nutrient broth) liquid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolysate 0.5g, yeast extract 0.1g are housed; Sea crystal 28g; Be dissolved in 1000mL distilled water, the pH value is 7.2～7.6) conical flask in, add 1mL again and contain 10
3The Bdellovibrio BDFM05 of pfu/mL (CCTCC M209172).Constant temperature shaking table 250rpm, 28 ℃ of cultivation 36h.Culture fluid is got deposition in 4 ℃ of centrifugal 20min of 6000rpm, adds 10mL DNB liquid nutrient medium (nutrient broth 0.8g; Caseinic acid hydrolysate 0.5g, yeast extract 0.1g, sea crystal 28g; Be dissolved in the 1000mL distilled water, the pH value is 7.2～7.6) deposition that suspends thalline, obtaining concentration is 10
7-10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL.
Embodiment 2
Bdellovibrio leech plastid bacterium liquid is used in breed
The present invention implements the breed pond on ground and build in the breed booth, and each pond specification is 2 * 2 * 0.75m, and the depth of water is 0.45m.8 of aeration tubes are placed in every pond.It is 60% gobo that pond top covers obscurity, avoids illumination strong excessively.Intensity of illumination generally is controlled at (50-200) lux.
The concrete condition of water quality of implementing the marine site, ground is: water temperature 15-28 ℃, salinity 27-33 ‰, and pH is 6.8-7.2, and seawater is through sand filtration, precipitation process, and get into culture the pond before, one 200 purpose mesh bag of water inlet pipe mouth wrapping in the pond prevents that fine sand from going into the pond.Source quality meets State Standard of the People's Republic of China's water quality standard for fishery (GB11607-89) requirement, meets (NY5052-2001) requirement of Ministry of Agriculture's " pollution-free food mariculture water water quality ".
Before the tile work, four jiaos of cement square bricks are washed and use disinfecting solution of potassium permanganate, put into again after the sterilization at the bottom of the pond, put 60 four pin cement bricks at the bottom of the pond altogether, bilateral symmetry, ride folded formula and place 6 of every row, every side 5 row.Disinfecting solution of potassium permanganate is all used in each pond after putting brick, rinses well repeatedly, injects the seawater through sand filtration and precipitation process then.Young Bao lower storage reservoir, selected spatfall corrugated plating attach young Bao amount at 30-2000 grain/plate, and young Bao body length is at 2.0-4.5mm, and young Bao is healthy, vigor is good, and the young Bao overburden amount in every pond is 10000, promptly begins aeration behind the throwing Bao, and aeration rate is 50L/h/m
3
The present invention is provided with one and produces control group-normal flowing water, and day quantity of exchanged water is 2 times of the pond water yield, and every morning, 6:00 used the high pressure water washing pond; The normal bait throwing in of 18:00 in evening; Do not add Bdellovibrio leech plastid bacterium liquid during this time, factory's breeding way is compiled the group for A with it completely.Other concrete layout schemes are following: Bdellovibrio leech plastid bacterium liquid is added in (1) in breeding water body, but does not add in the bait.
In breeding water body, add Bdellovibrio leech plastid bacterium liquid immediately after changing water at every turn; Required 48 ponds are divided into B-1, B-2, B-3, B-4, C-1, C-2, C-3, C-4, D-1, D-2, D-3, D-4; E-1, E-2, E-3, E-4; F-1, F-2, F-3, F-4, G-1, G-2, G-3, G-4, every group of 2 repetitions.The exchange water cycle of each group and to add bacterial concentration following:
B organizes (B-1, B-2, B-3, B-4): water once to change (full pond) in 5 days;
C organizes (C-1, C-2, C-3, C-4): water once to change (full pond) in 10 days;
D organizes (D-1, D-2, D-3, D-4): water once to change (full pond) in 15 days;
E organizes (E-1, E-2, E-3, E-4): water once to change (full pond) in 20 days;
F organizes (F-1, F-2, F-3, F-4): water once to change (full pond) in 25 days;
G organizes (G-1, G-2, G-3, G-4): water once to change (full pond) in 30 days;
B-1, C-1, D-1, E-1, F-1, G-1 group are changed behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, make that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
1Pfu/mL;
B-2, C-2, D-2, E-2, F-2, G-2 group are changed behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, make that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
3Pfu/mL;
B-3, C-3, D-3, E-3, F-3, G-3 group be for change behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
5Pfu/mL;
B-4, C-4, D-4, E-4, F-4, G-4 group be for change behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
7Pfu/mL.
(2) in bait, add Bdellovibrio leech plastid bacterium liquid, but in breeding water body, do not add.
With Bdellovibrio leech plastid bacterium liquid immersion bait half an hour, and breeding water body does not add Bdellovibrio leech plastid bacterium liquid after changing water at every turn before bait throwing in every day.Required 48 ponds are divided into b-1, b-2, b-3, b-4; C-1, c-2, c-3, c-4; D-1, d-2, d-3, d-4; E-1, e-2, e-3, e-4; F-1, f-2, f-3, f-4; G-1, g-2, g-3, every group of 2 repetitions of g-4.The exchange water cycle of each group and to add bacterial concentration following:
B organizes (b-1, b-2, b-3, b-4): water once to change (full pond) in 5 days;
C organizes (c-1, c-2, c-3, c-4): water once to change (full pond) in 10 days;
D organizes (d-1, d-2, d-3, d-4): water once to change (full pond) in 15 days;
E organizes (e-1, e-2, e-3, e-4): water once to change (full pond) in 20 days;
F organizes (f-1, f-2, f-3, f-4): water once to change (full pond) in 25 days;
G organizes (g-1, g-2, g-3, g-4): water once to change (full pond) in 30 days;
The bait that b-1, c-1, d-1, e-1, f-1, g-1 group are thrown is 10 through over-richness
1The bait that the Bdellovibrio leech plastid bacterium liquid bacterium liquid of pfu/mL soaked; Change water at every turn and do not add Bdellovibrio leech plastid bacterium liquid at water body.
The bait that b-2, c-2, d-2, e-2, f-2, g-2 group are thrown is 10 through over-richness
3The bait that the Bdellovibrio leech plastid bacterium liquid bacterium liquid of pfu/mL soaked; Change water at every turn and do not add Bdellovibrio leech plastid bacterium liquid at water body.
The bait that b-3, c-3, d-3, e-3, f-3, g-3 group are thrown is 10 through over-richness
5The bait that the Bdellovibrio leech plastid bacterium liquid bacterium liquid of pfu/mL soaked; Change water at every turn and do not add Bdellovibrio leech plastid bacterium liquid at water body.
The bait that b-4, c-4, d-4, e-4, f-4, g-4 group are thrown is 10 through over-richness
7The bait that the Bdellovibrio leech plastid bacterium liquid bacterium liquid of pfu/mL soaked; Change water at every turn and do not add Bdellovibrio leech plastid bacterium liquid at water body.
(3) in breeding water body and bait, all add Bdellovibrio leech plastid bacterium liquid.
Before bait throwing in every day with Bdellovibrio leech plastid bacterium liquid immersion bait half an hour, change water at every turn after breeding water body also add Bdellovibrio leech plastid bacterium liquid.Required 48 ponds are divided into Bb-1, Bb-2, Bb-3, Bb-4; Cc-1, Cc-2, Cc-3, Cc-4; Dd-1, Dd-2, Dd-3, Dd-4; Ee-1, Ee-2, Ee-3, Ee-4;
Ff-1, Ff-2, Ff-3, Ff-4; Gg-1, Gg-2, Gg-3, every group of 2 repetitions of Gg-4.The exchange water cycle of each group and to add bacterial concentration following:
Bb organizes (Bb-1, Bb-2, Bb-3, Bb-4): water once to change (full pond) in 5 days;
Cc organizes (Cc-1, Cc-2, Cc-3, Cc-4): water once to change (full pond) in 10 days;
Dd organizes (Dd-1, Dd-2, Dd-3, Dd-4): water once to change (full pond) in 15 days;
Ee organizes (Ee-1, Ee-2, Ee-3, Ee-4): water once to change (full pond) in 20 days;
Ff organizes (Ff-1, Ff-2, Ff-3, Ff-4): water once to change (full pond) in 25 days;
Gg organizes (Gg-1, Gg-2, Gg-3, Gg-4): water once to change (full pond) in 30 days;
Bb-1, Cc-1, Dd-1, Ee-1, Ff-1, Gg-1 group be for change behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
1Pfu/mL; It is 10 that the bait of each bait throwing in uses concentration
1The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks half an hour;
Bb-2, Cc-2, Dd-2, Ee-2, Ff-2, Gg-2 group be for change behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
3Pfu/mL; It is 10 that the bait of each bait throwing in uses concentration
3The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks half an hour.
Bb-3, Cc-3, Dd-3, Ee-3, Ff-3, Gg-3 group be for change behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
5Pfu/mL; It is 10 that the bait of each bait throwing in uses concentration
5The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks half an hour;
Bb-4, Cc-4, Dd-4, Ee-4, Ff-4, Gg-4 group be for change behind the water immediately the Bdellovibrio leech plastid bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio leech plastid cell concentration reaches 10 in the water body
7Pfu/mL; It is 10 that the bait of each bait throwing in uses concentration
7The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks half an hour.
Of the present invention respectively the group in the pond, the bait of being thrown something and fed is artificial synthetic diet, the abundance of food changes with young Bao size, in the time will throwing something and feeding bait, stop aeration earlier, begins aeration behind the bait throwing in 1h again.In order to reduce the destruction of residual bait to water quality, need every day to except that A group is respectively organize at the bottom of the pond and four jiaos of square bricks on residual bait carry out siphon, also can the interval residual bait be carried out siphon in 5 days.In addition, need observe young Bao active situation every night, food situation etc.
Add up young Bao to the end from peeling off lower plate, the time is 60 days, in order to understand the influence promotion growing ability of Bdellovibrio leech plastid, need count the young Bao that lives at last to the survival rate of young Bao, and by the young Bao of certain indicator-specific statistics.
(4) culture index and measurement
1. young Bao primary quantity, every pond 10,000 young Baos.
2. it is long to receive the average shell of young Bao, randomly draws 50 young Baos, and it is long to measure shell, averages.
3. young Bao average weight is randomly drawed 50 young Baos, claims to make even all after its gross weight
4. receive the Bao total amount, take by weighing the young Bao of 10g, calculate its number, claim overall weight estimate grain number young Bao.
5. survival rate, certain stage when finishing young Bao amount account for the percentage that this stage throws young Bao amount.
(5) young Bao immune indexes is measured
18 of every group of off-test samplings are shelled, and 3/pipe is put in 6 2ml-Eppendorf pipes that fill 1mL Hank ' s buffer solution (pH value 7-8) respectively, stores in-80 ℃ of mensuration of preparing against anti-immune indexes.Take out sample during mensuration, it is being melted on ice.Carry out homogenate, 6000r/min then ,-4 ℃ of centrifugal 5min. get the mensuration that supernatant is used for immune indexes.
1. the mensuration of phenol oxide enzyme
The determination of activity of phenol oxide enzyme is to calculate through the light absorption value of measuring with the dopamine of L-DOPA (levodopa) reaction generation.Each sample 100 μ L in the Eppendorf of 1.5mL pipe, each sample triplicate.The 0.5mg/mL sodium alginate that adds again with the preparation of PBS liquid reacts 30min down at 26-27 ℃.Add the L-DOPA (levodopa) of 50 μ L, behind the 10min, read light absorption value at the 490nm place with the 3mg/mL of PBS liquid preparation.Contrast liquid replaces sodium alginate with corresponding PBS liquid.
2. antalzyme activity is measured
With the micrococcus lysodeikticus freeze-dried powder is substrate.Use 0.1mol/L, the phosphate buffer of pH=6.4 is made into substrate suspension (OD570nm ≈ 0.3), gets this suspension of 3.0mL in the built-in ice bath of test tube, adds 50 μ L test serums again, mixes, and surveys the A0 value.Then test solution is moved into 37 ℃ of temperature and bathe mid-30min, place ice bath 10min after the taking-up again,, survey its A value with cessation reaction.Bacteriolyze vigor UL calculates by (A0-A)/A formula.
3. infection experiment
Infection experiment is carried out in the transparent aquarium that fills 15L sand filtration seawater (salinity 29～30 ‰) of sterilization in advance, and from A, B-1, each group of B-2.....Gg-4 are got 200 of young Baos at random, establish two repetitions.In each group water body, add vibrio parahaemolytious liquid, make that the vibrio parahaemolytious cell concentration reaches 10 in the water body
8Cfu/mL.Experimental session not flowing water is cultured, and normal bait throwing in is with 5L/h/m
3Continuous aeration, water temperature is 22 ℃, pH is controlled at 7.2.Behind the artificial infection, observed continuously 5 days, finally be calculated to be motility rate, the employing relative survival rate (Relative Percentage Survival, RPS) calculating formula:
RPS (%)=(1-immune group lethality/control group lethality) * 100%
This kind method specifically sees the following form to each item index influence of young abalone culture:
Table 1 changed the influence of water to young Bao growing state and immunity in 5 days
Table 2 changed the influence of water to young Bao growing state and immunity in 10 days
Table 3 changed the influence of water to young Bao growing state and immunity in 15 days
Table 4 changed the influence of water to young Bao growing state and immunity in 20 days
Table 5 changed the influence of water to young Bao growing state and immunity in 25 days
Table 6 changed the influence of water to young Bao growing state and immunity in 30 days
1-6 can find out from table, under identical exchange water cycle, along with the concentration rising of Bdellovibrio leech plastid; Survival rate all has certain rising, and all apparently higher than the A group, the method that adopts novel interpolation Bdellovibrio leech plastid is described; Obviously practicing thrift aquaculture cost, is example with the pond of 2 * 2 * 0.75m, by the pipeline mode of two exchange capacities every day; Depth of water 0.45m calculates, and need 3.6 tons of seawater every day, with 0.264 yuan/ton of the cost (data that plant provides) of seawater; The water consumption expense of a bite pond every day reaches 0.95 yuan like this, and a day water consumption expense of using Bdellovibrio leech plastid has only 0.047 yuan, and both differ 0.9 yuan.Therefore this new method has not only been practiced thrift water consumption greatly, has also saved great amount of manpower, and material resources has been practiced thrift aquaculture cost, has improved survival rate.
From different exchange water cycles, respectively organize young Bao survival rate under the same concentrations, the long grade of body all not have too big variation, and the novel cultural method of employing is described, do not change the growth that leech vibrios leech plastid still can promote young Bao in 5-30 days.In general, under 10pfu/mL concentration, 30 days do not change water is both economical mode, adopts such method, and effect is preferable.
The immunity aspect is added the immune indexes of respectively organizing of Bdellovibrio leech plastid and is organized apparently higher than A; Infection experiment is the result show, the survival rate of A group is 0%, other all added Bdellovibrio leech plastid the relative survival rate of respectively organizing be 84.5-100%.Explain that Bdellovibrio leech plastid can significantly improve the immunity of young Bao.
The water quality aspect, ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91); Nitrite nitrogen is with diazonium-azo spectrphotometric method for measuring (GB12763.4-91).When application test finishes, the water quality of each group is measured.The result shows that each item water-quality determination value in the A group pond all is higher than experiment group B, C, D...G group.Wherein, the testing result of A group ammoniacal nitrogen is 0.0356mg/L, between B-1 and the Gg-4 minimum for 0.0258mg/L, be up to 0.0354mg/L; It is 0.051mg/L that A group detects the nitrite nitrogen result, minimum between B-1 and the Gg-4 be 0.043mg/L, be up to 0.051mg/L.
Can know that by above interpretation of result application concentration is 10
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid can effectively improve the breeding water body environment, regulates the content of ammoniacal nitrogen, nitrite nitrogen in the water, thereby provides the water body environment that is beneficial to young Bao existence.Vibrios is counted the aspect, adopts coating TCBS flat band method to detect, and control group A water vibrios number is 2.67 * 10
2About cfu/mL, however add Bdellovibrio leech plastid bacterium liquid respectively organize the vibrios sum generally in 2.35-3.82 * 10
1Cfu/mL explains that Bdellovibrio leech plastid can effectively control the vibrios number of water body, and reaches the purpose bacterial population aspect of improving water quality; In general, no matter be, still at bait at water body; Or all add Bdellovibrio leech plastid bacterium liquid among both, can both significantly improve the physique of young Bao, strengthen the immunity of young Bao; And can improve water quality, reach the good result of saving water and energy, improve survival rate.
From Bdellovibrio leech plastid addition manner, no matter at water body, bait, or both all use Bdellovibrio leech plastid, three kinds of methods can both improve greatly young Bao survival rate, promote the growth of young Bao.But in breeding water body and bait, all add the method best results of Bdellovibrio leech plastid.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not limited by the foregoing description; Other are any not to deviate from change, the modification that spirit of the present invention and principle do, the displacement that substitutes, all should be equivalence, all within protection scope of the present invention.
Claims (10)
1. a water-saving young abalone culture method is characterized in that, comprises the steps:
(1) tile work in culturing the pond injects seawater then;
(2) young Bao lower storage reservoir begins aeration simultaneously;
(3) bait of regularly throwing something and feeding, and add Bdellovibrio leech plastid bacterium liquid in breeding water body and/or in the bait of throwing something and feeding; And changed water once, and changed water for full pond at every turn in every 5-30 days;
(4) when young Bao body length arrives 1.0-1.6cm, the young Bao of can gathering;
Said Bdellovibrio is Bdellovibrio (Bdellovibrio sp.) BDFM05, and by China's typical culture collection center preservation, it abbreviates CCTCC as, and deposit number is CCTCC NO:M209172, and preservation date is on August 7th, 2009.
2. cultural method according to claim 1 is characterized in that, said tile work is earlier brick to be washed and use disinfecting solution of potassium permanganate, takes to ride folded formula pendulum brick after the sterilization again.
3. cultural method according to claim 1 is characterized in that, said seawater is process sand filtration and precipitation process before injection.
4. cultural method according to claim 1 is characterized in that, said throwing something and feeding will stop aeration earlier during bait, begins aeration behind the bait throwing in 0.5-1h again.
5. cultural method according to claim 1 is characterized in that, behind the said young Bao lower storage reservoir, aeration rate is controlled at 10-100L/h/m
3, illumination is controlled at 50-200lux, and water temperature is controlled at 15-28 ℃.
6. cultural method according to claim 1 is characterized in that, the said Bdellovibrio leech plastid bacterium liquid that in the bait of throwing something and feeding, adds is meant and uses concentration to be 10-10
7The Bdellovibrio leech plastid bacterium liquid of pfu/mL soaked bait 20～40 minutes.
7. cultural method according to claim 1 is characterized in that, the said Bdellovibrio leech plastid bacterium liquid that in breeding water body, adds makes the concentration of Bdellovibrio leech plastid in water body reach 10pfu/mL at least.
8. cultural method according to claim 7 is characterized in that, saidly all adds Bdellovibrio leech plastid bacterium liquid immediately after changing water at every turn, and makes the concentration of Bdellovibrio leech plastid in water body reach 10pfu/mL at least.
9. according to claim 7 or 8 described cultural methods, it is characterized in that the concentration of said Bdellovibrio leech plastid in water body is 10-10
7Pfu/mL.
10. according to any described cultural method of claim 1～8, it is characterized in that the preparation method of said Bdellovibrio leech plastid bacterium liquid is:
In containing the DNB liquid nutrient medium of vibrio parahaemolytious, insert Bdellovibrio BDFM05, constant temperature shaking table 150rpm～300rpm, 20～35 ℃ of cultivation 24～48h; Culture fluid is got deposition in 4 ℃ of centrifugal 15～20min of 6000～8000rpm, in deposition, adds the DNB liquid nutrient medium Bdellovibrio sediment that suspends again, promptly makes Bdellovibrio leech plastid bacterium liquid.
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| CN101356927A (en) * | 2008-03-31 | 2009-02-04 | 华南理工大学 | Use of Bdellovibrio in eliminating pathogenicity vibrio in marine products and breeding water body thereof |
| CN101638629A (en) * | 2009-08-28 | 2010-02-03 | 华南理工大学 | Bdellovibrio bacteriovorus for preventing and curing rice bacterial diseases and application thereof |
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| CN101356927A (en) * | 2008-03-31 | 2009-02-04 | 华南理工大学 | Use of Bdellovibrio in eliminating pathogenicity vibrio in marine products and breeding water body thereof |
| CN101638629A (en) * | 2009-08-28 | 2010-02-03 | 华南理工大学 | Bdellovibrio bacteriovorus for preventing and curing rice bacterial diseases and application thereof |
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| 宋志萍等.一种消除九孔鲍苗细菌性病原的无公害绿色生物方法的研究.《海洋科学》.2006,第30卷(第1期),第44-48页. * |
| 秦生巨.噬菌28系列之三——海特灵在海水养殖上的应用.《科学种养》.2008,(第6期),第39-40页. * |
| 钟幼平等.国内南方鲍鱼工厂化育苗和养殖技术.《集美大学学报（自然科学版）》.1999,第4卷(第1期),第52-58页. * |
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