CN102017915B - Energy-saving and emission-reducing method for culturing Haliotis diversicolor supertexta - Google Patents
Energy-saving and emission-reducing method for culturing Haliotis diversicolor supertexta Download PDFInfo
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- CN102017915B CN102017915B CN2010102710736A CN201010271073A CN102017915B CN 102017915 B CN102017915 B CN 102017915B CN 2010102710736 A CN2010102710736 A CN 2010102710736A CN 201010271073 A CN201010271073 A CN 201010271073A CN 102017915 B CN102017915 B CN 102017915B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses an energy-saving and emission-reducing method for culturing Haliotis diversicolor supertexta, which comprises the following steps: selecting pollution-free deep sea water from a sea area as water for cultivation; selecting young healthy Haliotis diversicolor supertexta which are in good condition, putting into a culture pond; spraying a Bdellovibrio bacteriovorus mixed agent into the culture pond until the concentration of the Bdellovibrio bacteriovorus mixed agent in water of the pond is 10-107 pfu/mL; feeding once every day, wherein the feed amount of each time is limited to 5-15% of the body weight of the Haliotis diversicolor supertexta; prior to feeding, soaking the feed in the Bdellovibrio bacteriovorus mixed agent of which the concentration is 10-107 pfu/mL;and changing the water of the pond once every other 5-30 days, and spraying the Bdellovibrio bacteriovorus mixed agent after changing the water each time. The culture method obviously promotes the growth of Haliotis diversicolor supertexta, increases the immunity and the survival rate of Haliotis diversicolor supertexta and improves the water quality of culture environment. The Bdellovibrio bacteriovorus mixed agent has no poisonous or side effect on abalones, therefore, the invention is suitable for large-scale industrial culture of abalones.
Description
Technical field
The invention belongs to marine shellfish cultural technique field, particularly a kind of cultural method of energy-conservation, green, environmental protection of forming for haliotis diversicolor Reeve (Haliotisdiversicolor aquatilis).
Background technology
Bao is one of eight sea treasure of China, has very high economy and medical value.The abalone culture of China begins starting the seventies in 20th century, has formed large-scale production to the mid-80, and after this development is swift and violent especially, and the foster Bao Ye of coastal various places has formed bigger scale.Yet along with the aggravation of environmental pollution, the expansion of culturing scale and the raising of intensive degree, the disease of Bao also takes place in succession, and the trend that increases the weight of is year by year arranged, and the development to the foster Bao Ye of China constitutes a serious threat.Especially haliotis diversicolor Reeve, it is frequent to occur the disease problem in recent years in the abalone culture, has had a strong impact on abalone culture production, causes many raisers to abandon culturing haliotis diversicolor Reeve.Therefore, seek the new cultural method of haliotis diversicolor Reeve, improve its survival rate and growth rate, in the production technology of haliotis diversicolor Reeve and even abalone culture industry, all have very important meaning.
In the breeding process of Bao, use antibiotic to suppressing the breeding of pathogenic microorganism, reduce its metabolism toxin the aspects such as toxic action of Bao have been played positive effect, but the long-term antibiotic that uses, particularly abuse of antibiotics, its all drawback also reveals gradually.As causing the normal intestinal flora imbalance of Bao, the immunity of organisms generation harmful effect to Bao produces side effects such as drug resistance and medicament residue, has all brought potential harm for benign development and the human beings'health of aquaculture.In addition, at present in China's batch production continental rise abalone culture, main employing is extracted outer seawater and is entered the breed pond through husky filter post precipitation, or extract sand drain water and carry out the flowing water breed, waste water directly enters the sea area through pipeline, thisly directly takes out in line mode and has the energy consumption height, to the heavy-polluted shortcoming in sea area, not only increase the weight of aquaculture cost, also become the big hidden danger that disease takes place Bao Rihou.
Therefore, seek the cultural technique of novel, energy-conservation, the environmental protection of haliotis diversicolor Reeve, the nuisanceless green haliotis diversicolor Reeve product of exploitation safety, high-quality, nutrition, these have all become the common recognition of current society.
It is a kind of new method that developed recently gets up that microorganism formulation is applied to aquaculture, microorganism formulation just is being widely used in the aquaculture, to the purifying aquatic product cultivating environment, improve survival rate, prevent and treat disease etc. and more and more play a significant role, be world today's research and the upsurge used.Bdellovibrio is to parasitize other bacteriums, and can cause a bacterioid of host bacteria cracking; Littler than general bacterium, can pass through bacterial filter, the effect of similar phage is arranged; Gram is negative, and can cracking comprise that vibrio parahaemolytious, vibrio alginolyticus etc. cause the potentially pathogenic organism of Bao disease.With the microorganism formulation that the mix preparation of Bdellovibrio telotroch and leech plastid is formed as haliotis diversicolor Reeve, it is rapid-action to have telotroch concurrently, and energetic and leech plastid prepares, preserves easily, the more of a specified duration and stronger advantage of environment tolerance of bactericidal action time.These characteristics make the Bdellovibrio mix preparation be suitable as a kind of microorganism formulation, in the breeding process that is applied to haliotis diversicolor Reeve.In addition, what the method for newly forming among the present invention adopted is the cycle to change water, so not only reduces breeding water amount and discharge of wastewater, reduces energy consumption especially, has reduced aquaculture cost; Compare less investment, easier popularization with totally-enclosed cyclic culture method.Therefore, this method be that a kind of facility is feasible, green, the breeding way of low-carbon (LC), energy-saving and environmental protecting.
No matter existing studies show that as a kind of effective microorganism preparation, 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., Hespell R.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.The effect of the fish cell of Bdellovibrio BDH2102 and pathogen.The microbiology circular, 2006,33 (1): 7-11].
Summary of the invention
For overcoming the deficiency that exists in the above-mentioned existing cultural technique, the object of the present invention is to provide the haliotis diversicolor Reeve cultural method of a kind of novel, green, environmental protection and energy-saving and emission-reduction.
Purpose of the present invention realizes by following technical proposals: a kind of haliotis diversicolor Reeve cultural method of energy-saving and emission-reduction comprises following operating procedure:
(1) gets free of contamination deep-sea, marine site water as breeding water; Breeding water injects and cultures the pond after sand filtration, precipitation and sterilization; The lid gobo is to control intensity of illumination between 500～2000lux above culturing the pond; At the bottom of the pond of culturing the pond, ride folded formula and put four pin bricks;
(2) choose the young Bao of haliotis diversicolor Reeve healthy and in good condition and drop in the breed pond, the shell length of young Bao is 1～4cm, and throwing in density is 300～1000/m
2
(3) spray the Bdellovibrio mix preparation in the pond to culturing, make the concentration of the Bdellovibrio mix preparation of Chi Shuizhong reach 10～10
7Pfu/mL;
(4) daily ration of feeding control is in 5%～15% of haliotis diversicolor Reeve body weight; Bait is 10～10 with concentration before input
7The Bdellovibrio mix preparation of pfu/mL soaks 15～45min; Changed Chi Shui once every 5～30 days, change at every turn and spray the Bdellovibrio mix preparation behind the water, make the concentration of the Bdellovibrio mix preparation of Chi Shuizhong reach 10～10
7Pfu/mL;
(5) culture during according to aeration rate 70～110L/h/m
3Carry out aeration, every day, soil pick-up was 1～2 time, and in time cleaned out dead Bao.
The source quality of the described breeding water of step (1) 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 ".
Freshwater soaking was used in the described breed of step (1) pond before injecting breeding water, the sterilization afterflush is clean; Before described breeding water injects and cultures the pond, culturing 200 purpose mesh bags of pond water inlet pipe mouth wrapping, prevent that the fine sand in the breeding water from going into the pond.
The shading rate of the described gobo of step (1) is 80～95%.
The young Bao of the described haliotis diversicolor Reeve of step (2) is followed organic aquaculture principle and cultivates out.
The described bait of step (4) is at least a in fragrant plant mentioned in ancient texts, sea-tangle and the undaria pinnitafida of fresh chopping, or the artificial abalone manufactured feed; Bait throwing in every day once, the time of bait throwing in is between 16:00～18:00pm.
Step (3) and (4) described Bdellovibrio are Bdellovibrio (Bdellovibrio sp.) BDFM05, described Bdellovibrio is preserved in Chinese typical culture collection center in the Wuhan University of Luojia Mountain, Wuhan, Hubei Province on August 7th, 2009, and deposit number is CCTCC NO:M 209172.
The described Bdellovibrio mix preparation in step (3) and (4) is that Bdellovibrio telotroch bacterium liquid and Bdellovibrio leech plastid bacterium liquid mix by the bacterium number at 1: 1.
Described Bdellovibrio leech plastid bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, insert Bdellovibrio BDFM05, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid behind centrifugal 15～20min, is removed the supernatant that contains the Bdellovibrio telotroch with 6000～8000rpm under 4 ℃ of temperature, the precipitation that obtains is Bdellovibrio leech plastid; Be that 7.2～7.6 phosphate buffer suspends with Bdellovibrio leech plastid with DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, obtaining concentration is 10
6～10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL;
Described Bdellovibrio telotroch bacterium liquid prepares in accordance with the following methods: with the supernatant that contains the Bdellovibrio telotroch among the Bdellovibrio leech plastid preparation method behind the centrifugal 15～20min of 16000～18000rpm, precipitation is the suspension of 7.2～7.6 phosphate buffer with DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
Described host is Aeromonas hydrophila (Aeromonas hydrophila).
Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeast extract 0.1g are dissolved in the 1000mL distilled water, regulates pH value to 7.2～7.6.
Adopt separately to initial culturing pool and change in the breed pond after the water and spray the Bdellovibrio mix preparation, perhaps employing is 10～10 with concentration with bait before input separately
7The mode that the Bdellovibrio mix preparation of pfu/mL soaks perhaps adopts the mode of both combinations, reaches the purpose of adding Bdellovibrio, all can reach the technology of the present invention effect.
Compared with prior art, the present invention has following beneficial effect:
(1) Bdellovibrio mix preparation of the present invention has stronger cracking strength to most aquatic products pathogenic bacteria, and Bdellovibrio mix preparation itself can improve the aquaculture organism intestinal environment, promotion growth, raising abalone immunity.Therefore, compare with other microorganism formulation, the Bdellovibrio mix preparation among the present invention has the immunity that improves Bao Zishen, accelerates the characteristics of the growth of Bao.
What (2) existing abalone culture technology adopted all is water-flowing type aquaculture, has not only expended great amount of manpower and material resources, financial resources, also can pollute environment.And the Bao Yangcheng method of utilizing the Bdellovibrio mix preparation can reduce the demand of output, has saved production cost to a great extent, and has not polluted the environment, and the sustainable development of Bao Ye and other aquacultures is had great importance.
(3) the Bdellovibrio mix preparation comprises Bdellovibrio leech plastid, and Bdellovibrio leech plastid also has potential inactivated vaccine function.Between the leech plastid puberty, Bdellovibrio does not change the surface texture of host cell, makes former host still have immunogenicity, but the stimulating organism body produces immune response.Therefore, can effectively prevent the generation of the bacteriosis of haliotis diversicolor Reeve, improve survival rate.
(4) in addition, the author experiment showed, the growth rate that adopts this Bdellovibrio mix preparation can improve haliotis diversicolor Reeve significantly.Therefore, the method also can be generalized to the breed of other abalone or aquatic livestock, increases economic benefit, is that the robust techniques of new period fishery synergy increasing peasant income supports.
Embodiment
Do further detailed description below in conjunction with the present invention of embodiment, but embodiments of the present invention are not limited thereto.Technological parameter for not indicating especially can carry out with reference to routine techniques.
Embodiment 1
(1) is used for the preparation of the Bdellovibrio mix preparation that haliotis diversicolor Reeve (Haliotis diversicolor aquatilis) forms
The preparation of Bdellovibrio leech plastid: inoculation 0.5mL 10 in a conical flask that 100mL nutrient broth liquid nutrient medium (sodium chloride 5g, pH 7.4 ± 0.2 for peptone 10g, beef extract powder 3g) are housed
7Cfu/mL Aeromonas hydrophila (Aeromonas hydrophila, bacterium numbering: GIM 1.172 derive from microorganism fungus kind preservation center, Guangdong Province), 200rpm, 35 ℃ of shaking tables were cultivated 12 hours, and culture fluid is abandoned supernatant in 4 ℃, the centrifugal 15min of 5000rpm.With 5mL physiological saline suspension precipitation thalline, join one afterwards 100mL DNB liquid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolysate 0.5g are housed, yeast extract 0.1g, be dissolved in the 1000mL distilled water, the pH value is 7.2～7.6) conical flask in, insert 1mL again and contain 10
3Pfu/mL Bdellovibrio BDFM05 (CCTCC M209172).Constant temperature shaking table 250rpm, 28 ℃ of cultivation 36h.Culture fluid is in 4 ℃ of centrifugal 20min of 6000rpm, removes the supernatant that contains the Bdellovibrio telotroch, keeps precipitation (being Bdellovibrio leech plastid), adds the 10mL DNB liquid nutrient medium Bdellovibrio leech plastid that suspends again in the precipitation, and obtaining concentration is 10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL;
The preparation of Bdellovibrio telotroch: with the supernatant that contains the Bdellovibrio telotroch among the above-mentioned Bdellovibrio leech plastid preparation method behind the centrifugal 20min of 16000rpm, the precipitation that obtains namely is the Bdellovibrio telotroch, add 10mLDNB liquid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolysate 0.5g, yeast extract 0.1g, be dissolved in the 1000mL distilled water, the pH value is 7.2～7.6) suspension Bdellovibrio telotroch, obtaining concentration is 10
9Pfu/mL Bdellovibrio telotroch bacterium liquid.。
The Bdellovibrio mix preparation is mixed by bacterial population by Bdellovibrio BDFM05 telotroch bacterium liquid and Bdellovibrio BDFM05 leech plastid bacterium liquid and forms at 1: 1;
(2) use the Bdellovibrio mix preparation and carry out forming of haliotis diversicolor Reeve
Be abalone culture field, Fujian with implementing.The source quality of implementing the marine site, ground 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 ".Seawater is through sand filtration, precipitation process, and enter culture the pond before, at 200 purpose mesh bags of pond water inlet pipe mouth wrapping, prevent that fine sand from going into the pond.
The abalone culture pond is built in and is cultured in the booth, and each tank floor is long-pending to be 20m
2, high 1.0m is put 200 four pin bricks altogether at the bottom of the pond, divide both sides to place.Four pin bricks are laterally ridden folded formula and are placed, every row 10 (each 5 of both sides), totally 20 row.Before young Bao was placed, the freshwater soaking two weeks was all used in each pond, rinses well stand-by repeatedly.It is 80% gobo that pond top covers obscurity, avoids illumination strong excessively.Each pond depth of water 45cm, the haliotis diversicolor Reeve that shell is about 3.2cm (counterpoise 4.59g), 8000 in every pond are thrown in each pond.The present invention arranges one and produces control group, and it is compiled the group for A, and two parallel, adopt normal flowing water, day quantity of exchanged water is 3 times of the pond water yield, and every morning, 7:00 used the high pressure water washing pond, normal bait throwing in, during do not add the Bdellovibrio mix preparation, factory forms mode completely.It is as follows that other organize concrete layout scheme:
1) in breeding water body, adds the Bdellovibrio mix preparation, but do not add in the bait
Need 48 Kou Shui ponds altogether, be respectively 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-4,2 every group parallel.
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): 30 change (full pond), and water once;
B-1, C-1, D-1, E-1, F-1, G-1 group be for change behind the water immediately the Bdellovibrio mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
1Pfu/mL;
B-2, C-2, D-2, E-2, F-2, G-2 group be for change behind the water immediately the Bdellovibrio mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation 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 mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
5Pfu/mL;
B-4, C-4, D-4, E-4, F-4, G-4 group is for change behind the water immediately the Bdellovibrio mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
7Pfu/mL;
2) in bait, add the Bdellovibrio mix preparation, but in breeding water body, do not add
Soak bait half an hour with the Bdellovibrio mix preparation before each bait throwing in, and breeding water body does not add the Bdellovibrio mix preparation after changing water at every turn.Required 48 Kou Shui 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-4,2 every group parallel.
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 for the bait thrown for being 10 through over-richness
1The bait that the Bdellovibrio mix preparation of pfu/mL soaked;
B-2, c-2, d-2, e-2, f-2, g-2 group for the bait thrown for being 10 through over-richness
3The bait that the Bdellovibrio mix preparation of pfu/mL soaked;
B-3, c-3, d-3, e-3, f-3, g-3 group for the bait thrown for being 10 through over-richness
5The bait that the Bdellovibrio mix preparation of pfu/mL soaked;
B-4, c-4, d-4, e-4, f-4, g-4 group for the bait thrown for being 10 through over-richness
7The bait that the Bdellovibrio mix preparation of pfu/mL soaked;
3) in breeding water body and bait, all add the Bdellovibrio mix preparation
Bait soaks half an hour with the Bdellovibrio mix preparation of variable concentrations before each bait throwing in, change water at every turn after breeding water body also add the Bdellovibrio mix preparation.Need 48 Kou Shui ponds altogether, be 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-2,2 every group of Gg-3, Gg-4 are parallel.
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 mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
1Pfu/mL, simultaneously, the bait concentration of each bait throwing in is 10
1The Bdellovibrio mix preparation 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 mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
3Pfu/mL, simultaneously, the bait concentration of each bait throwing in is 10
3The Bdellovibrio mix preparation 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 mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
5Pfu/mL, simultaneously, the bait concentration of each bait throwing in is 10
5The Bdellovibrio mix preparation 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 mix preparation of splashing in the pond at every turn, makes that Bdellovibrio mix preparation cell concentration reaches 10 in the water body
7Pfu/mL, simultaneously, the bait concentration of each bait throwing in is 10
7The Bdellovibrio mix preparation of pfu/mL soaks half an hour.
Each group is in breeding process, and every day, 18:00pm carried out bait throwing in, and bait is the artificial abalone manufactured feed, and injected volume is controlled at 7% of Bao Tichong.The control of the aeration rate in pond is at 100L/h/m in the breeding process
3, adopt the soil pick-up at the bottom of siphonage carries out the pond every day, and clean out dead Bao, record data.
(3) growing state formed of haliotis diversicolor Reeve
After going through 6 months, add up the abalone number of surviving in final every mouthful of pond, randomly draw 100 haliotis diversicolor Reeves respectively from every mouthful of pond, with its shell of ruler measurement long (cm), electronic balance is surveyed its weight (g), calculates following parameter:
1) rate of body weight gain (%)=(W
t-W
o)/W
o* 100%;
2) the long growth rate (%) of shell=(L
t-L
o)/L
o* 100%;
3) the long per day increment (μ m) of shell=(final shell long-3.2)/180 * 10000
4) weight average daily growth amount (mg)=(final weight-4.59)/180 * 1000
5) survival rate=survival number/8000 * 100%
(4) the abalone immune indexes is measured
After the off-test, get 5 haliotis diversicolor Reeves, detect its immune indexes respectively for every group.Use the 1mL syringe, No. 5 syringe needles insert the heart extracting blood of Bao, because blood volume very little, is cut from the centre with the abdominal foot of blade with Bao again, draw the blue blood that oozes out.Place little centrifuge tube to spend the night in 4 ℃ the blood of drawing, the blue serum that will separate out then directly inclines to, and carries out every mensuration.
1) mensuration of superoxide dismutase (SOD) vigor
The mensuration of 1,2,3,-thrihydroxy-benzene autoxidation speed, under 25 ℃, in 4.5mL 50mmol/L, the K of Ph=8.30
2HPO
4-KH
2PO
4Add 10 μ L 50mmol/L 1,2,3,-thrihydroxy-benzenes in the buffer solution, shake up rapidly, pour in the cuvette of optical path 1cm, under the 325nm wavelength, survey the A value once every 30s, require autoxidation speed about 0.070OD/min.Enzyme assay before adding 1,2,3,-thrihydroxy-benzene, adds SOD sample to be measured, records data, is calculated as follows enzymic activity:
Enzymic activity=(0.070-A
325nm)/min * 100% * reactant liquor cumulative volume * sample liquid extension rate/(0.070 * 50% * sample liquid is long-pending)
The enzyme unit definition of living: in every milliliter of reactant liquor, per minute suppresses 1,2,3,-thrihydroxy-benzene autoxidation speed and reaches 50% enzyme amount and be defined as the enzyme unit (U/mL) that lives.
2) antalzyme activity is measured
Be substrate with the micrococcus lysodeikticus freeze-dried powder.Use 0.1mol/L, the phosphate buffer of pH=6.4 is made into substrate suspension (OD
570nm≈ 0.3), get this suspension of 3.0mL in the built-in ice bath of test tube, add 50 μ L test serums again, mix, survey 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, with cessation reaction, survey its A value.Bacteriolyze vigor U
LBy (A
0-A)/calculating of A formula.
(5) attack the poison experiment
After experiment finishes, randomly draw 100 abalones, the long 6.2～7.0cm of shell respectively from control group (A group) and each experimental group (B-G group, b-g group, Bb-Gg group).It is 1 * 10 that every Bao is injected 50 μ L concentration
8Aeromonas hydrophila bacterium liquid.After Bao abdomen foot bacterial injection, by the normal breed respectively of different groups.The experimental session water-flowing type aquaculture, day quantity of exchanged water is understood 4 times of water body, and normal bait throwing in is with 50L/h/m
3Continuous aeration, water temperature are 20 ℃, and pH controls 7.2.Mortality respectively organized in record behind the 3d, finally is calculated to be motility rate, the employing relative survival rate (Relative Percent Survival, RPS) calculating formula:
RPS (%)=(1-immune group lethality/control group lethality) * 100%
Experimental result is by shown in the table 1-12.Wherein, table 1-6 refers to (5 days, 10 days, 15 days, 20 days, 25 days, 30 days) under the different exchange water cycles respectively, adds the Bdellovibrio mix preparation by three kinds of modes, the influence of Bao growing state during it is formed haliotis diversicolor Reeve.As seen, under identical exchange water cycle, along with the rising (10 of concentration
1, 10
3, 10
5, 10
7Pfu/mL), the shell of haliotis diversicolor Reeve length, weight and survival rate have to a certain degree increase or raising; And, no matter adopting which kind of exchange water cycle, the experimental group that is added with the Bdellovibrio mix preparation all can promote the growth of haliotis diversicolor Reeve better than control group (A group).As can be seen from the table, in three kinds of different Bdellovibrio mix preparation addition manners, directly the effect of water body interpolation is better than the bait immersion, and the effect optimum that the both adds.
Table 7-12 refers to respectively that under different exchange water cycles add the Bdellovibrio mix preparation by three kinds of modes, it is to the influence of haliotis diversicolor Reeve immunity.As can be seen, no matter adopt which kind of exchange water cycle in the table, added the experimental group of Bdellovibrio mix preparation, the superoxide dismutase of its abalone (SOD) is active, the lysozyme activity value all is higher than control group.As seen, no matter be at water body, still at bait, or all add the Bdellovibrio mix preparation among both, can both strengthen the immunity of abalone significantly.
Table 13 refers to attack the relative survival rate of each group in the poison experiment.The result shows that the relative survival rate of A group is 0, and the abalone of other experimental group all has higher relative survival rate, all reaches more than 85%.Under identical exchange water cycle, along with the rising of Bdellovibrio mix preparation concentration, relative survival rate also raises gradually; From different exchange water cycles, the prawn relative survival rate of respectively organizing under the same concentrations does not change significantly.In three kinds of different Bdellovibrio mix preparation addition manners, directly the effect of water body interpolation is better than the bait immersion, and the effect optimum that the both adds.Bdellovibrio leech plastid in this side light Bdellovibrio mix preparation can be brought into play the effect of a potential vaccine, strengthens the immunity of haliotis diversicolor Reeve.
The water quality aspect, ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91); Nitrite nitrogen diazonium-azo spectrphotometric method for measuring (GB12763.4-91).Through measuring, the every water-quality determination value in the A group pond all is higher than experimental group.Wherein, the testing result of A group ammoniacal nitrogen is 0.083mg/L, the 0.072-0.075mg/L that is higher than 5 days exchange water cycle groups (B group, b group, Bb group), the 0.072-0.076mg/L of 10 days exchange water cycle groups (C group, c group, Cc group), the 0.073-0.078mg/L of 15 days exchange water cycle groups (D group, d group, Dd group), the 0.075-0.079mg/L of 20 days exchange water cycle groups (E group, e group, Ee group), the 0.075-0.080mg/L of 25 days exchange water cycle groups (F group, f group, Ff group), 30 days the exchange water cycle group (G group, g group, Gg group) 0.076-0.080mg/L; The testing result of A group nitrite nitrogen is 0.012mg/L, the 0.005-0.007mg/L that is higher than 5 days exchange water cycle groups (B group, b group, Bb group), the 0.005-0.007mg/L of 10 days exchange water cycle groups (C group, c group, Cc group), the 0.006-0.008mg/L of 15 days exchange water cycle groups (D group, d group, Dd group), the 0.007-0.008mg/L of 20 days exchange water cycle groups (E group, e group, Ee group), the 0.007-0.009mg/L of 25 days exchange water cycle groups (F group, f group, Ff group), 30 days the exchange water cycle group (G group, g group, Gg group) 0.007-0.010mg/L.The result shows, no matter is at water body, still at bait, or all adds the Bdellovibrio mix preparation among both, can both significantly improve water quality, reaches the good result of saving water and energy.
In addition, total number of bacteria and vibrios sum context of detection adopt coating nutrient broth flat band method and TCBS flat band method to detect respectively.The result shows that total number of bacteria and vibrios sum in the A group water body reach 10 respectively
5With 10
3The order of magnitude (cfu/mL), the total number of bacteria and the vibrios sum that add the experimental group of Bdellovibrio mix preparation are respectively 10
2～10
3With 10
1～10
2The order of magnitude (cfu/mL), lower than control group, illustrate that the Bdellovibrio mix preparation can effectively control the number of bacteria of water body.
Comprehensive each side result adds the Bdellovibrio mix preparation, can significantly improve the growth rate of abalone, improves survival rate, strengthens immunity, and can improve water quality, reaches the good result of saving water and energy.
5 days exchange water cycles of table 1 are to the influence of haliotis diversicolor Reeve growing state
Data are two groups of parallel mean values in the table
10 days exchange water cycles of table 2 are to the influence of haliotis diversicolor Reeve growing state
Data are two groups of parallel mean values in the table
15 days exchange water cycles of table 3 are to the influence of haliotis diversicolor Reeve growing state
Data are two groups of parallel mean values in the table
20 days exchange water cycles of table 4 are to the influence of haliotis diversicolor Reeve growing state
Data are two groups of parallel mean values in the table
25 days exchange water cycles of table 5 are to the influence of haliotis diversicolor Reeve growing state
Data are two groups of parallel mean values in the table
30 days exchange water cycles of table 6 are to the influence of haliotis diversicolor Reeve growing state
Data are two groups of parallel mean values in the table
5 days exchange water cycles of table 7 are to the influence of haliotis diversicolor Reeve immunity
10 days exchange water cycles of table 8 are to the influence of haliotis diversicolor Reeve immunity
15 days exchange water cycles of table 9 are to the influence of haliotis diversicolor Reeve immunity
20 days exchange water cycles of table 10 are to the influence of haliotis diversicolor Reeve immunity
25 days exchange water cycles of table 11 are to the influence of haliotis diversicolor Reeve immunity
30 days exchange water cycles of table 12 are to the influence of haliotis diversicolor Reeve immunity
Each group of table 13 attack malicious experimental result
Above-described embodiment is preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spiritual essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (2)
1. the haliotis diversicolor Reeve cultural method of energy-saving and emission-reduction is characterized in that comprising following operating procedure:
(1) gets free of contamination deep-sea, marine site water as breeding water; Breeding water injects and cultures the pond after sand filtration, precipitation and sterilization; The lid gobo is to control intensity of illumination between 500～2000 lux above culturing the pond; At the bottom of the pond of culturing the pond, ride folded formula and put four pin bricks;
(2) choose the young Bao of haliotis diversicolor Reeve healthy and in good condition and drop in the breed pond, the shell length of young Bao is 1～4 cm, and throwing in density is 300～1000/m
2
(3) spray the Bdellovibrio mix preparation in the pond to culturing, make the concentration of the Bdellovibrio mix preparation of Chi Shuizhong reach 10～10
7Pfu/mL;
(4) daily ration of feeding control is in 5%～15% of haliotis diversicolor Reeve body weight; Bait is 10～10 with concentration before input
7The Bdellovibrio mix preparation of pfu/mL soaks 15～45 min; Changed Chi Shui once every 5～30 days, change at every turn and spray the Bdellovibrio mix preparation behind the water, make the concentration of the Bdellovibrio mix preparation of Chi Shuizhong reach 10～10
7Pfu/mL;
(5) culture during according to aeration rate 70～110 L/h/m
3Carry out aeration, every day, soil pick-up was 1～2 time, and in time cleaned out dead Bao;
The described Bdellovibrio mix preparation in step (3) and (4) is that Bdellovibrio telotroch bacterium liquid and Bdellovibrio leech plastid bacterium liquid mix by bacterium number 1:1;
Described Bdellovibrio leech plastid bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, insert Bdellovibrio BDFM05, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300 rpm shaking tables, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid behind centrifugal 15～20min, is removed the supernatant that contains the Bdellovibrio telotroch with 6000～8000rpm under 4 ℃ of temperature, the precipitation that obtains is Bdellovibrio leech plastid; Be that 7.2～7.6 phosphate buffer suspends with Bdellovibrio leech plastid with DNB liquid nutrient medium, water, physiological saline or 0.2mol/L and pH value, obtaining concentration is 10
6～10
9The Bdellovibrio leech plastid bacterium liquid of pfu/mL;
Described Bdellovibrio telotroch bacterium liquid prepares in accordance with the following methods: with the supernatant that contains the Bdellovibrio telotroch among the Bdellovibrio leech plastid preparation method behind the centrifugal 15～20min of 16000～18000rpm, precipitation is the suspension of 7.2～7.6 phosphate buffer with DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL;
Described host is Aeromonas hydrophila; Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeast extract 0.1g are dissolved in the 1000mL distilled water, regulates pH value to 7.2～7.6;
Described Bdellovibrio BDFM05 is preserved in Chinese typical culture collection center on August 7th, 2009, and deposit number is CCTCC NO:M 209172.
2. the haliotis diversicolor Reeve cultural method of a kind of energy-saving and emission-reduction according to claim 1 is characterized in that: freshwater soaking use in the described breed of step (1) pond before injecting breeding water, and the afterflush of sterilizing is clean; Before described breeding water injects and cultures the pond, culturing 200 purpose mesh bags of pond water inlet pipe mouth wrapping, prevent that the fine sand in the breeding water from going into the pond.
3, the haliotis diversicolor Reeve cultural method of a kind of energy-saving and emission-reduction according to claim 1 is characterized in that: the shading rate of the described gobo of step (1) is 80～95%.
4, the haliotis diversicolor Reeve cultural method of a kind of energy-saving and emission-reduction according to claim 1 is characterized in that: the described bait of step (4) is at least a in fragrant plant mentioned in ancient texts, sea-tangle and the undaria pinnitafida of fresh chopping, or the artificial abalone manufactured feed; Bait throwing in every day once, the time of bait throwing in is between 16:00～18:00 pm.
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| CN101275119A (en) * | 2008-01-24 | 2008-10-01 | 华南理工大学 | Application of bdellovibrio to eliminating pathogen in freshwater product and culture water thereof |
| 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 |
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| CN101275119A (en) * | 2008-01-24 | 2008-10-01 | 华南理工大学 | Application of bdellovibrio to eliminating pathogen in freshwater product and culture water thereof |
| 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 |
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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. * |
| 钟幼平 等.国内南方鲍苗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51-58页. * |
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