CN103355241A - Application of lucid ganoderma extractive in enhancement of cultured fish immunity - Google Patents
Application of lucid ganoderma extractive in enhancement of cultured fish immunity Download PDFInfo
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- CN103355241A CN103355241A CN2013103422960A CN201310342296A CN103355241A CN 103355241 A CN103355241 A CN 103355241A CN 2013103422960 A CN2013103422960 A CN 2013103422960A CN 201310342296 A CN201310342296 A CN 201310342296A CN 103355241 A CN103355241 A CN 103355241A
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses application of lucid ganoderma extractive in enhancement of cultured fish immunity. The lucid ganoderma extractive can be used as feed additives, the adding dosage of the lucid ganoderma extractive is 0.6%-3.0%, and the using effect of the lucid ganoderma extractive is related to the dosage and time. When a culture cycle is 65 days, the immune activity, the ingestion capacity and the germ resistant activity after germ infection of a fish body can be obviously improved by adding 3.0% of the lucid ganoderma extractive to mixed aquatic feed (Guangdong Haida mixed aquatic feed with a product number of 112). The immune activity and the ingestion capacity of the fish body can be improved by adding 0.6% of the lucid ganoderma extractive to the mixed aquatic feed, and the resistant activity cannot be improved yet after the fish body is infected with the germs. The environment-friendly lucid ganoderma extractive is natural extractive, is safe and free of residues and selective specificity, and can activate the activity of macrophage to induce a series of immunoreactions afterwards, and therefore the fish body is made to absorb, damage and remove causative agents in the fish body through phagocytosis, and the immunologic function of the fish body is further improved.
Description
Technical field
The invention belongs to aquatic product matched field of feed, more specifically relate to the application of a kind of Ganodenna Lucidum P.E in strengthening cultured fishes immunity.
Background technology
Along with the development of China's intensive aquaculture, disease, germplasm, the own bottleneck that develops in a healthy way through becoming the restriction culture fishery of breeding environment three large problems, wherein disease becomes current particularly distinct issues.At present, the control of aquatic animal disease has method (Turnidge, Antibiotic use in animals-prejudices, the perceptions and realities.Journal of Antimicrobial Chemotherapy such as vaccine injection and chemicals, 2004:53,26-27).Although the vaccine injection method is effective to a certain extent, it is again effort consuming time, and has the specific restriction of cause of disease.That antibiotic refers to be obtained through cultivation by microorganisms such as bacterium, actinomycetes, fungies or with the synthetic same or similar material that has killing or suppress pathogenic microorganism of the structure (Livermore of chemical synthesis, Fourteen years in resistance.International Journal of Antimicrobial Agents, 2012:39,283-294).China's feedstuff industry is started late, and the history that antibiotic is used in aquatic feeds as feed addictive is also shorter, but China is its application and increases gradually trend in recent years.Yet, because long-term use and some the unreasonable application of antibiotic in animal produces also causing many serious problems when bringing economic benefit to aquaculture.Sum up antibiosis and have following drawback:
1) use for a long time antibiotic, can make bacterium produce drug resistance, antibiotic drug effect is reduced, this just needs constantly to increase dosage, thereby causes the increase of feed and feeding cost.In addition, produce tolerance antibiotic " superbacteria ", bio-safety is produced serious harm.
2) use for a long time antibiotic, make host's body produce dependence, limited the performance of immunity function in the body, body's immunity is descended, form immunosupress, aggravated the reduction of resist the disease ability.
3) use for a long time antibiotic, antibiotic use makes some sensitive bacterias death in the aquatic livestock body, and microecological balance destroys in the body, causes ecological niche vacancy in the body, and availing oneself of the opportunity to get in for extraneous drug-fast bacteria offers an opportunity, thereby causes exogenous infection.Suprainfection also is because a large amount of administration of antibiotics when killing certain bacterium, are destroyed microecological balance, and another or multiple endogenous or exogenous germ immediately reinfect body are caused.
4) long-term or excessive use antibiotic is so that the original shape of antibiotic thing and metabolism thereof can accumulate or be stored in cell, tissue or the organ of animal.Produce medicine animal residual, not only reduced product quality, indirectly health is had a negative impact.The drawback that antibiotic use brings has been subject to extensive concern, forbid also surging (Landers etc. day by day of antibiotic cry, A review of antibiotic use in food animals:perspective, policy, and potential.Public Health Reports, 2012:127,4-22).The portion that the World Health Organization (WHO) delivers is about using the antibiotic medicine impact statement in edible animal, report points out that bacterium may be transferred to the people to drug tolerance in animal body.So European Union's regulation: from 2006, completely forbidding antibiotic in European Union's scope was the feed growth accelerator.Be standard and the reasonable use of instructing medicated feed additive, China has formulated a series of policies and regulations, such as " feed and feed addictive management rules ", " feed medicated premix operating specification ", " food animal forbidding veterinary drug and other compound inventories ", " forbid use in feed and drinking water for animals types of drugs catalogue " etc.
Therefore, from the reality of aquatic products production, use immunity to strengthen additive in manufactured feed, by improving the autoimmunity of cultivation object, thereby the generation of resist the disease becomes the focus of industry gradually.Immunopotentiator claims again immunopotentiating agent, refers to have promotion or brings out host defense, strengthens a class material of living organism resistance against diseases.Immunostimulant can be divided into two large classes by function: the one, strengthen the nonspecific immunity mechanism (Nyachoti etc. of animal, Weaned pig responses to Escherichia coli K88oral challenge when receiving a lysozyme supplement.Journal of Animal Science, 2012:90,252-260); Another kind of is to strengthen by vaccine-induced specific immune function.For aquatic animal research more be the former, namely strengthen the anti-disease infection ability of aquatic livestock by initiatively improving nospecific immunity.Because the classification position of aquatic livestock is lower, specific immunity is machine-processed perfect not as mammality, so the application immunopotentiator improves nonspecific immunity, thereby the general level of the health, the increase premunition that improve body have great significance and wide prospect undoubtedly.
Immunopotentiator can be divided into artificial synthesising preparation, carbohydrate, some amino acid and high unsaturated fatty acid, vitamin, nucleotide, hormone etc.Wherein, carbohydrate is the very important immunopotentiator of a class.
Lipopolysaccharides is one of gram-negative bacteria cell wall chief component composition, also is endotoxin.Even high vertebrate is also very responsive to the endotoxin of low dosage, but the low vertebrate of waiting, such as fish, the resistance of induced by endotoxin is stronger.Toll-like acceptor 4 (TLR4) is main bind receptor relevant with the lipopolysaccharides Immunestimulatory effect.Lipopolysaccharides has the expression that promotes cell factor, Acute phase protein in some fish, even also has the effect of immunopathogenesis, immune physiology, immune and endocrine system and nerve immunity.Also can promote the activation of B/T cell, macrophage and complement system.
Peptide glycan is that the procaryotic cell walls such as bacterium contain a class by polysaccharide chains, peptide subunit and a macromolecule polyalcohol that the peptide bridge forms.In the gram-positive bacteria cell wall, peptide glycan thickness is 10-90nm approximately, and content accounts for the 40%-90% of cell wall dry weight.And in the cell wall of Gram-negative bacteria, peptide glycan is positioned at the innermost layer of cell wall, and thickness only is that content is no more than the 5%-10% of cell wall dry weight about 2-3nm.Have been found that mammal existence comprises CD14, Toll-1ike acceptor 2 (TLR2) peptidoglycan recognition protein group, the peptide glycan identification receptors such as Nod1, NodZ.Have and studies show that peptide glycan can affect fish immunity power from cytophagy, lysozyme and level of complement.
Shitosan is the deacetylated product of chitin, is β-Isosorbide-5-Nitrae-2 amino-2-deoxidation-D-glucan, is cation animal fiber and the alkaline polysaccharide of up to now only discovery.Shitosan can the activating complement system, and the series of biologic of mediation complement is learned effect.The stimulating expression of macrophage activation, thereby promote its phagocytic activity, strengthen its HLA-II antigen, can also activated T cell and B cell, strengthen its synergistic effect in immune response, cellullar immunologic response and the humoral immunoresponse(HI) (Li etc. of mediation body, Effect of chitosan on intestinal bacteria of allogynogenetic crucian carp, Carassius auratus gibelio, as depicted by polymerase chain reaction-denaturing gradient gel electrophoresis.Journal of World Aquaculture Society, 2011:42,539-548).Shitosan also has antibacterial action in addition, and the product after its acidifying is poisonous to rainbow trout.
Beta glucan is a kind of natural polysaccharide, and molecule is greatly about more than 6500, and great majority are the particle of water-insoluble or colloid, mainly are present in the cell wall of bacterium, saccharomycete, fungi (glossy ganoderma), also is present in higher plant seed coated.Beta glucan is different from general common carbohydrates (such as starch, glycogen, dextrin etc.), topmost difference is that bond closes the mode difference, general carbohydrate is with 1,4 glycosidic bond keys combine and become the linear molecule structure, and beta glucan is with 1,3 keys are main body, and contain the side chain of some 1,6 keys.Scleroglucan, schizophan, lentinan and laminarin all are the derivative of glucan different sugar glycosidic bond.The existence that beta glucan closes mode and intramolecular hydrogen bond because of its special bond causes spiral molecular structure, and the configuration of this uniqueness is easy to be identified by immune system.Experiment can both prove that glucan has the immunity of raising and disease-resistant ability in existing considerable experiment in vitro and the body.The specific receptor of the beta glucan that exists on the surface of fish macrophage, beta glucan is after macrophage is combined, activity that can activating macrophage, then bring out a series of immune response, thereby make body by the pathogene in phagocytosis absorption, destruction and the removing body, and then improved the immunologic function of fish.
Glossy ganoderma is a kind of fungi that originates in East Asia, because the effect of its anticancer and immune enhancing, the Asian usually uses it as a kind of promotion health and long-lived prescription (Chan etc., The effects of beta-glucan on human immune and cancer cells.Journal of Hematology and Oncology, 2009:2:25).From glossy ganoderma, many active ingredients be can extract, GL-B, β-1,3/1 comprised, 6 glucans and ganodenic acid.Many β-1,3/1 that studies show that, 6 glucans and ganodenic acid are the main active in the glossy ganoderma.β-1,3/1,6 glucans are that 1,3-glycosidic bond is main chain, and 1,6-glycosidic bond is the glucose polymer of side chain.The immunity of this polysaccharide strengthens characteristic broad research (Gupta etc., Microbial levan in the diet of Labeo rohita Hamilton juveniles:effect on non-specific immunity and histopathological changes after challenge with Aeromonas hydrophila.Journal of Fish Diseases, 2008:31,649-657), verified its has the animal white of promotion cytophagy vigor, the respiratory burst vigor, SC is active, strengthens body and resists the characteristics such as germ ability.The main Ganodenna Lucidum P.E that is comprised of glucan and these two kinds of active substances of ganodenic acid is a kind of novel developing human health product now.Yet whether it also can be applicable to fish, thereby the report that strengthens its immune response is also rare.
Summary of the invention
The objective of the invention is to be to provide the application of a kind of Ganodenna Lucidum P.E in strengthening cultured fishes immunity, Ganodenna Lucidum P.E is added in the aquatic feeds, easy to implement the method, easy and simple to handle, the immunity of fish body and the infection ability of opposing germ can be effectively strengthened, ingesting of fish can be significantly improved.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
The purposes of a kind of Ganodenna Lucidum P.E in strengthening cultured fishes immunity the steps include:
A, Ganodenna Lucidum P.E is added in the aquatic product matched feed (the large aquatic products manufactured feed in Guangdong sea, production number 112), the dosage that Ganodenna Lucidum P.E adds is 0.6%-3.0%, obtains a kind of Ganodenna Lucidum P.E feed; The addition 0.6%-3.0% addition of described aquatic product matched feed usage amount 97.0%-99.4% and Ganodenna Lucidum P.E equals 100%, and experiment effect is very good, asks for an interview the effect described in the embodiment 1 to 4.
Result of use and the additive capacity (0.6%-3.0%) of Ganodenna Lucidum P.E and the service time relevant (bimestrial service time be advisable, generally be no less than one month) of this feed of the Ganodenna Lucidum P.E feed that obtains in B, the steps A in strengthening cultured fishes immunity;
C, according to the aquatic product matched feedstuff feeding cultured fishes of interpolation 3.0% Ganodenna Lucidum P.E that obtains in the steps A after 65 days, can improve significantly ingestion ability and the immunity of fish body, mainly comprise the leucocyte respiratory burst vigor 51.69% that can improve the fish body, improve leukocytes phagocytic vigor 20.81%, improve serum myeloperoxidase enzyme activity 46.87%; Infect the fish body after 7 days at tarda, can on the basis of control group 79.36% survival rate, improve 23.79% survival rate, reach 98.25% survival rate;
D, according to the aquatic product matched feedstuff feeding cultured fishes of interpolation 0.6% Ganodenna Lucidum P.E that obtains in the steps A after 65 days, can improve ingestion ability and the immunity of fish body, mainly comprise the leucocyte respiratory burst vigor 15.58% that can improve the fish body, improve leukocytes phagocytic vigor 5.28%, improve serum myeloperoxidase enzyme activity 29.19%; But can not improve the resistivity behind the courses of infection;
E, according to steps A, B, C and D, when the culture-cycle was 65 days, the recommendation additive capacity of aquatic product matched feed Ganodenna Lucidum P.E was 3.0%.
The Ganodenna Lucidum P.E feed that F, steps A obtain can strengthen the immunity of the cultured fishes such as crucian, grass carp, carp, black carp, bream and to the resistivity behind the courses of infection.
Described Ganodenna Lucidum P.E main component is 30% GL-B, is provided by Wuhan Yuancheng Technology Development Co., Ltd..
The amount that described Ganodenna Lucidum P.E adds in the aquatic product matched feed is 0.6%-3.0%, and wherein the main component of the Ganodenna Lucidum P.E of each addition is 30% GL-B.
Described cultured fishes are specially wherein any of crucian, grass carp, carp, black carp, bream.
A kind of Ganodenna Lucidum P.E that strengthens cultured fishes immunity can use as green, safe feed addictive, and the dosage that adds in the aquatic product matched feed is 0.6%-3.0%.The result of use of this Ganodenna Lucidum P.E and dosage, time are relevant, and when the culture-cycle was 65 days, to recommend additive capacity be 3.0% to Ganodenna Lucidum P.E in the feed.Described aquatic product matched feed is the large aquatic products manufactured feed (production number 112) in sea, Guangdong.
The Ganodenna Lucidum P.E of interpolation 3.0% can improve immune vigor and the ingestion ability of fish body significantly in the aquatic product matched feed; After tarda infected 7 days, cumulative survival rate improved 23.79% than control group (79.36%).
Add immune vigor and the ingestion ability that 0.6% Ganodenna Lucidum P.E can improve the fish body in the aquatic product matched feed, but can not improve the resistivity behind the courses of infection.
At present, the control of aquatic animal disease mainly relies on the methods such as chemicals and vaccine injection.But because not only there is pesticide resistance in chemotherapy, and the accumulation meeting of medicine in water pollutes destruction breeding ecological environment, thereby formation vicious circle to breeding environment.Although the vaccine injection method is effective to a certain extent, it is again effort consuming time, and has the specific restriction of cause of disease.
The present invention compared with prior art has following advantage:
The Ganodenna Lucidum P.E of this green, environmental protection is a kind of natural extract, and safety, noresidue, nothing are selected specificity; Activity that can activating macrophage is brought out a series of immune response then, thereby makes the fish body absorb, destroy by phagocytosis and remove pathogene in the body, and then has improved the immunologic function of fish.Its advantage is:
1. the present invention is the application of the natural glossy ganoderma extract of environmental protection, healthy and helpful to body.The advantages such as that this additive has is pollution-free, non-specific, noresidue easy to use, safe.
2. the Ganodenna Lucidum P.E that the present invention relates to can activating macrophage activity, then bring out a series of immune response, thereby make the fish body absorb, destroy by phagocytosis and remove pathogene in the body, and then improved the immunologic function of fish.
3. the Ganodenna Lucidum P.E that the present invention relates to is tested by aquatic livestock, show can improve the fish body immunocompetence, can improve the infection ability of opposing germ, can promote the ingestion ability of fish body.
Embodiment
Below the specific embodiment of the present invention is specifically described.
Embodiment 1:
The purposes of a kind of Ganodenna Lucidum P.E in strengthening cultured fishes immunity the steps include:
Test feed: control feed is not added Ganodenna Lucidum P.E, and control feed is the large aquatic products manufactured feed (production number 112) in sea, Guangdong.Test feed is to reduce on the bentonitic basis of 3.0% filler (total usage amount of control feed is 97.0%) in control feed, adds 3.0% Ganodenna Lucidum P.E again, keeps toatl proportion 100% constant.Test feed uses minitype fodder-pelleting machine (SLR-150, fishery machinery research institute of Chinese aquatic science institute) to make the heavy property particle of diameter 2mm, and 60 ℃ of oven dry also are stored in-20 ℃.
Described Ganodenna Lucidum P.E main component is 30% GL-B, is provided by Wuhan Yuancheng Technology Development Co., Ltd..
Test processed group: throw something and feed control feed 65 days of two processed group of growth test design, control group; And the test feed 65 days that experimental group is thrown something and fed and added 3.0% Ganodenna Lucidum P.E; 3 tests of each test processed group are parallel.
Growth test: growth test carries out in the flowing water system that contains 6 glass fibre cylinders (diameter 80cm, water volume is 300L), and this flowing water system is furnished with an aeration basin of filling zeolite and activated carbon, and replenish fresh tap water and account for 1/6 of system's Total Water every day.The water inlet flow velocity of each cylinder is 2.5L/min.Duration of test by aeration, keeps the dissolved oxygen 6mg/L in the water, and water temperature maintains 25 ℃ to 29 ℃ (27 ± 2 ℃), and periodicity of illumination is illumination (08:00-20:00) in 12 hours, and water surface light intensity is between 120lx to 160lx.Ammonia nitrogen and chlorine residue are monitored weekly once, ammonia nitrogen＜0.1mg/L, chlorine residue＜0.05mg/L.Water pH value is about 6.9.
Hybridized prussian carp then postlarva obtains from Guan Qiao plant of Inst. of Hydrobiology, Chinese Academy of Sciences, and before formal test, the test fish is supported temporarily in cultivating system, and each cylinder is put 100 tail fishes.Supporting the phase temporarily is one month, and during this period, every day, twice (09:00 and 15:00) used control feed feeding experiment fish.When on-test, neat specification is chosen in all fish fasting one day at random, and 45 tail fishes of apparent health (approximately 6.7g/ tail) are put into each cylinder, totally 6 test cylinder (jar)s.Duration of test is satiated with food and throws something and feeds twice (09:00 and 15:00) every day, and whole culture experiment continues 65 days.
Challenge test: tarda (Edwardsiella tarda) strain is applied to dull and stereotyped 37 ℃ of TSB and cultivates 12h.Use the PBS washing plate 3 times, collect bacterium liquid and use the centrifugal 10min collection of centrifuge 500g bacterium.Bacterial concentration is adjusted to 2.76 * 10
6Cfu/mL.
That carries out in test carried out challenge test on the 66th day.After sampling in 66 days, remaining 20 tail fishes are put back to continuation raising in the former cylinder after adopting lumbar injection tarda liquid 0.3mL in every cylinder.The lethality of adding up 7 days continuously.
The result shows: compare with control group, feed 3.0% Ganodenna Lucidum P.E feed 65 days can improve the leucocyte respiratory burst vigor 51.69% of fish body, improves leukocytes phagocytic vigor 20.81%, improve serum myeloperoxidase enzyme activity 46.87%, increase the grazing rate 6.86% of fish body; Tarda infected the fish body after 7 days, and the control group survival rate is 79.36%, and the survival rate of processed group is 98.25%, and survival rate has improved 23.79%.Therefore, this Ganodenna Lucidum P.E can improve the immune vigor of fish body, improves the infection ability of opposing tarda, can promote the ingestion ability of fish body.
Described cultured fishes are specially wherein any of crucian, grass carp, carp, black carp, bream.
Embodiment 2:
The purposes of a kind of Ganodenna Lucidum P.E in strengthening cultured fishes immunity the steps include:
Test feed: control feed is not added Ganodenna Lucidum P.E, and control feed is the large aquatic products manufactured feed (production number 112) in sea, Guangdong.Test feed is to reduce on the bentonitic basis of 3.0% filler (total usage amount of control feed is 97.0%) in control feed, adds 3.0% Ganodenna Lucidum P.E again, keeps toatl proportion 100% constant.Test feed uses minitype fodder-pelleting machine (SLR-150, fishery machinery research institute of Chinese aquatic science institute) to make the heavy property particle of diameter 2mm, and 60 ℃ of oven dry also are stored in-20 ℃.
Described Ganodenna Lucidum P.E main component is 30% GL-B, is provided by Wuhan Yuancheng Technology Development Co., Ltd..
Test processed group: throw something and feed control feed 65 days of two processed group of growth test design, control group; And the test feed after 21 days that experimental group is thrown something and fed and added 3.0% Ganodenna Lucidum P.E, the control feed of throwing something and feeding again 44 days; 3 tests of each test processed group are parallel.
Growth test: growth test carries out in the flowing water system that contains 6 glass fibre cylinders (diameter 80cm, water volume is 300L), and this flowing water system is furnished with an aeration basin of filling zeolite and activated carbon, and replenish fresh tap water and account for 1/6 of system's Total Water every day.The water inlet flow velocity of each cylinder is 2.5L/min.Duration of test by aeration, keeps the dissolved oxygen 6mg/L in the water, and water temperature maintains 25 ℃ to 29 ℃ (27 ± 2 ℃), and periodicity of illumination is illumination (08:00-20:00) in 12 hours, and water surface light intensity is between 120lx to 160lx.Ammonia nitrogen and chlorine residue are monitored weekly once, ammonia nitrogen＜0.1mg/L, chlorine residue＜0.05mg/L.Water pH value is about 6.9.
Hybridized prussian carp then postlarva obtains from Guan Qiao plant of Inst. of Hydrobiology, Chinese Academy of Sciences, and before formal test, the test fish is supported temporarily in cultivating system, and each cylinder is put 100 tail fishes.Supporting the phase temporarily is one month, and during this period, every day, twice (09:00 and 15:00) used control feed feeding experiment fish.When on-test, neat specification is chosen in all fish fasting one day at random, and 45 tail fishes of apparent health (approximately 6.7g/ tail) are put into each cylinder, totally 6 test cylinder (jar)s.Duration of test is satiated with food and throws something and feeds twice (09:00 and 15:00) every day, and whole culture experiment continues 65 days.
Challenge test: tarda (Edwardsiella tarda) strain is applied to dull and stereotyped 37 ℃ of TSB and cultivates 12h.Use the PBS washing plate 3 times, collect bacterium liquid and use the centrifugal 10min collection of centrifuge 500g bacterium.Bacterial concentration is adjusted to 2.76 * 10
6Cfu/mL.
That carries out in test carried out challenge test on the 66th day.After sampling in 66 days, remaining 20 tail fishes are put back to continuation raising in the former cylinder after adopting lumbar injection tarda liquid 0.3mL in every cylinder.The lethality of adding up 7 days continuously.
The result shows: compare with control group, feed 3.0% Ganodenna Lucidum P.E feed after 21 days, the control feed of throwing something and feeding again 44 days can improve the leucocyte respiratory burst vigor 24.92% of fish body, improves leukocytes phagocytic vigor 14.31%, increases the grazing rate 9.80% of fish body; Tarda infected the fish body after 7 days, and the control group survival rate is 79.36%, and the survival rate of processed group is 89.87%, and survival rate has improved 13.23.0%.Therefore, this Ganodenna Lucidum P.E can improve the immune vigor of fish body, improves the infection ability of opposing tarda, can promote the ingestion ability of fish body.
Other step is identical with embodiment 1.
Embodiment 3:
The purposes of a kind of Ganodenna Lucidum P.E in strengthening cultured fishes immunity the steps include:
Test feed: control feed is not added Ganodenna Lucidum P.E, and control feed is the large aquatic products manufactured feed (production number 112) in sea, Guangdong.Test feed is to reduce on the bentonitic basis of 0.6% filler (total usage amount of control feed is 99.4%) in control feed, adds 0.6% Ganodenna Lucidum P.E again, keeps toatl proportion 100% constant.Test feed uses minitype fodder-pelleting machine (SLR-150, fishery machinery research institute of Chinese aquatic science institute) to make the heavy property particle of diameter 2mm, and 60 ℃ of oven dry also are stored in-20 ℃.
Described Ganodenna Lucidum P.E main component is 30% GL-B, is provided by Wuhan Yuancheng Technology Development Co., Ltd..
Test processed group: throw something and feed control feed 65 days of two processed group of growth test design, control group; And the test feed 65 days that experimental group is thrown something and fed and added 0.6% Ganodenna Lucidum P.E; 3 tests of each test processed group are parallel.
Growth test: growth test carries out in the flowing water system that contains 6 glass fibre cylinders (diameter 80cm, water volume is 300L), and this flowing water system is furnished with an aeration basin of filling zeolite and activated carbon, and replenish fresh tap water and account for 1/6 of system's Total Water every day.The water inlet flow velocity of each cylinder is 2.5L/min.Duration of test by aeration, keeps the dissolved oxygen 6mg/L in the water, and water temperature maintains 25 ℃ to 29 ℃ (27 ± 2 ℃), and periodicity of illumination is illumination (08:00-20:00) in 12 hours, and water surface light intensity is between 120lx to 160lx.Ammonia nitrogen and chlorine residue are monitored weekly once, ammonia nitrogen＜0.1mg/L, chlorine residue＜0.05mg/L.Water pH value is about 6.9.
Hybridized prussian carp then postlarva obtains from Guan Qiao plant of Inst. of Hydrobiology, Chinese Academy of Sciences, and before formal test, the test fish is supported temporarily in cultivating system, and each cylinder is put 100 tail fishes.Supporting the phase temporarily is one month, and during this period, every day, twice (09:00 and 15:00) used control feed feeding experiment fish.When on-test, neat specification is chosen in all fish fasting one day at random, and 45 tail fishes of apparent health (approximately 6.7g/ tail) are put into each cylinder, totally 6 test cylinder (jar)s.Duration of test is satiated with food and throws something and feeds twice (09:00 and 15:00) every day, and whole culture experiment continues 65 days.
Challenge test: tarda (Edwardsiella tarda) strain is applied to dull and stereotyped 37 ℃ of TSB and cultivates 12h.Use the PBS washing plate 3 times, collect bacterium liquid and use the centrifugal 10min collection of centrifuge 500g bacterium.Bacterial concentration is adjusted to 2.76 * 10
6Cfu/mL.
That carries out in test carried out challenge test on the 66th day.After sampling in 66 days, remaining 20 tail fishes are put back to continuation raising in the former cylinder after adopting lumbar injection tarda liquid 0.3mL in every cylinder.The lethality of adding up 7 days continuously.
The result shows: compare with control group, feed 0.6% Ganodenna Lucidum P.E feed 65 days can improve the leucocyte respiratory burst vigor 15.58% of fish body, improves leukocytes phagocytic vigor 5.28%, improve serum myeloperoxidase enzyme activity 29.19%, increase the grazing rate 2.94% of fish body.Therefore, this Ganodenna Lucidum P.E can improve the immune vigor such as the leukocytes phagocytic vigor, respiratory burst vigor, myeloperoxidase enzyme activity of fish body, can promote the ingestion ability of fish body.
Other step is identical with embodiment 1.
Embodiment 4:
The purposes of a kind of Ganodenna Lucidum P.E in strengthening cultured fishes immunity the steps include:
Test feed: control feed is not added Ganodenna Lucidum P.E, and control feed is the large aquatic products manufactured feed (production number 112) in sea, Guangdong.Test feed is to reduce on the bentonitic basis of 0.6% filler (total usage amount of control feed is 99.4%) in control feed, adds 0.6% Ganodenna Lucidum P.E again, keeps toatl proportion 100% constant.Test feed uses minitype fodder-pelleting machine (SLR-150, fishery machinery research institute of Chinese aquatic science institute) to make the heavy property particle of diameter 2mm, and 60 ℃ of oven dry also are stored in-20 ℃.
Described Ganodenna Lucidum P.E main component is 30% GL-B, is provided by Wuhan Yuancheng Technology Development Co., Ltd..
Test processed group: throw something and feed control feed 65 days of two processed group of growth test design, control group; And the test feed after 21 days that experimental group is thrown something and fed and added 0.6% Ganodenna Lucidum P.E, the control feed of throwing something and feeding again 44 days; 3 tests of each test processed group are parallel.
Growth test: growth test carries out in the flowing water system that contains 6 glass fibre cylinders (diameter 80cm, water volume is 300L), and this flowing water system is furnished with an aeration basin of filling zeolite and activated carbon, and replenish fresh tap water and account for 1/6 of system's Total Water every day.The water inlet flow velocity of each cylinder is 2.5L/min.Duration of test by aeration, keeps the dissolved oxygen 6mg/L in the water, and water temperature maintains 25 ℃ to 29 ℃ (27 ± 2 ℃), and periodicity of illumination is illumination (08:00-20:00) in 12 hours, and water surface light intensity is between 120lx to 160lx.Ammonia nitrogen and chlorine residue are monitored weekly once, ammonia nitrogen＜0.1mg/L, chlorine residue＜0.05mg/L.Water pH value is about 6.9.
Hybridized prussian carp then postlarva obtains from Guan Qiao plant of Inst. of Hydrobiology, Chinese Academy of Sciences, and before formal test, the test fish is supported temporarily in cultivating system, and each cylinder is put 100 tail fishes.Supporting the phase temporarily is one month, and during this period, every day, twice (09:00 and 15:00) used control feed feeding experiment fish.When on-test, neat specification is chosen in all fish fasting one day at random, and 45 tail fishes of apparent health (approximately 6.7g/ tail) are put into each cylinder, totally 6 test cylinder (jar)s.Duration of test is satiated with food and throws something and feeds twice (09:00 and 15:00) every day, and whole culture experiment continues 65 days.
Challenge test: tarda (Edwardsiella tarda) strain is applied to dull and stereotyped 37 ℃ of TSB and cultivates 12h.Use the PBS washing plate 3 times, collect bacterium liquid and use the centrifugal 10min collection of centrifuge 500g bacterium.Bacterial concentration is adjusted to 2.76 * 10
6Cfu/mL.
That carries out in test carried out challenge test on the 66th day.After sampling in 66 days, remaining 20 tail fishes are put back to continuation raising in the former cylinder after adopting lumbar injection tarda liquid 0.3mL in every cylinder.The lethality of adding up 7 days continuously.
The result shows: compare with control group, feed 0.6% Ganodenna Lucidum P.E feed after 21 days, the control feed of throwing something and feeding again 44 days, can improve the leucocyte respiratory burst vigor 24.03% of fish body, improve leukocytes phagocytic vigor 8.46%, improve serum myeloperoxidase enzyme activity 22.22%, increase the grazing rate 5.88% of fish body.Therefore, this Ganodenna Lucidum P.E can improve the immune vigor such as the leucocyte respiratory burst vigor, phagocytic activity, myeloperoxidase enzyme activity of fish body, can promote the ingestion ability of fish body.
Other step is identical with embodiment 1.
Claims (1)
1. the application of Ganodenna Lucidum P.E in strengthening cultured fishes immunity;
Described cultured fishes are wherein any of crucian, grass carp, carp, black carp, bream.
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