CN106213052A - A kind of bream feed additive and preparation method thereof - Google Patents
A kind of bream feed additive and preparation method thereof Download PDFInfo
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- CN106213052A CN106213052A CN201610626334.9A CN201610626334A CN106213052A CN 106213052 A CN106213052 A CN 106213052A CN 201610626334 A CN201610626334 A CN 201610626334A CN 106213052 A CN106213052 A CN 106213052A
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
Abstract
The present invention provides a kind of bream feed additive and preparation method thereof, and described additive includes following raw material components: spirulina, magnesium acetate, Herba Taraxaci, Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii, Rhizoma Phragmitis, carambola, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven, Radix Glycyrrhizae, Mel and blackberry pollen.The invention has the beneficial effects as follows: its composition for having medicinal and edible dual function concurrently, can antibacterial, antiinflammatory, improve the immunity of bream, simultaneously can effectively treat and prevent bream gas bubble disease.Bream premunition after edible additive of the present invention is strong, and fast growth, meat are nutritious, and the economic benefit of raiser has been significantly greatly increased, have that formula is simple, science, rationally, fully natural green noresidue, practical, have no side effect, the significant advantage of effect.
Description
Technical field
The present invention relates to field of feed additive technology, particularly to a kind of bream feed additive and preparation method thereof.
Background technology
Bream, has another name called bream, the longest body bream, bream flower, oily bream；Ancient name raft head bream, contracting item bream.In China, bream is also three
Angle triangular bream, the general designation of Megalobrama amblycephala (blunt snout bream).Body length 40 cm, ratio is well suited to hydrostatic sexual life.Bream is the good water nourished
Product food, not only delicious flavour, and high nutritive value.Its protein content is the twice of Carnis Sus domestica, and belongs to high-quality protein,
Human absorptivity is high.Rich in abundant thiamine, riboflavin, nicotinic acid, vitamin D and a certain amount of calcium, phosphorus, ferrum etc. in bream
Mineral.Though fat content is low in bream meat, but fatty acid therein is proved blood sugar lowering, protects the effect of the heart and anti-cancer.Bream
Vitamin D in meat, calcium, phosphorus, can the most pre-anti-osteoporosis.Can defying age, skin care, beneficially blood circulation, appetizing,
Nourishing, prevents tumor.
The delicious meat of bream is tender, is of high nutritive value, it has also become one of aquatic products that people are best.The end of spring and the beginning of summer, fry and fish
Planting easy gassing sick, the hazardness to fry especially is relatively big, and bream is most sensitive to oxygen saturation, is susceptible to suffer from this sick.Fry or fish
Plant intestinal and bubble occurs；Or the many minute bubbles of attachment on body surface, the gill, the gill turns white, and between the gill filament, mucus increases, and makes fish body float or trip
Dynamic disequilibrium, travelling slow, unable, can not normally sink, cause and can not normally ingest, exhaust because of power eventually, can cause time serious
Mortality.The feedstuff of commercial type is all general fish meal at present, does not treat the feedstuff of the gas bubble disease of bream, does not more have
There is the feed additive strengthening its immunity.
Summary of the invention
The technical problem to be solved is, it is provided that a kind of bream feed additive and preparation method thereof, can carry
The immunity of high bream, effectively treats gas bubble disease simultaneously.Bream premunition after edible additive of the present invention is strong, the speed of growth
Hurry up, meat nutritious, the economic benefit of raiser has been significantly greatly increased, has had that formula is simple, a science, rationally, fully natural green
Noresidue, practical, have no side effect, the significant advantage of effect.
For solving above-mentioned technical problem, the present invention provides a kind of bream feed additive, and described additive includes following group
Point: spirulina, magnesium acetate, Herba Taraxaci, Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii, Rhizoma Phragmitis, carambola, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower,
Button seven, Radix Glycyrrhizae, Mel and blackberry pollen.
In described additive, the parts by weight of each raw material are: spirulina 5-10 part, magnesium acetate 0.02-0.06 part, Herba Taraxaci
12-21 part, Fructus Sechii edulis 14-24 part, Semen Pisi sativi 4-11 part, Bulbus Allii 15-27 part, Rhizoma Phragmitis 7-13 part, carambola 6-17 part, Radix Isatidis 10-
20 parts, Radix Notoginseng 3-13 part, flexuose bittercress herb and seed 0.4-1.1 part, villous amomum flower 0.3-0.8 part, button seven 0.2-0.7 part, Radix Glycyrrhizae 5-12 part, honeybee
Honey 17-28 part and blackberry pollen 11-22 part.
In described additive, the parts by weight of each raw material are: spirulina 7-9 part, magnesium acetate 0.03-0.05 part, Herba Taraxaci
15-19 part, Fructus Sechii edulis 17-20 part, Semen Pisi sativi 6-9 part, Bulbus Allii 18-24 part, Rhizoma Phragmitis 9-11 part, carambola 9-14 part, Radix Isatidis 13-17
Part, Radix Notoginseng 6-10 part, flexuose bittercress herb and seed 0.4-1.0 part, villous amomum flower 0.3-0.7 part, button seven 0.2-0.5 part, Radix Glycyrrhizae 5-11 part, Mel
20-24 part and blackberry pollen 14-19 part.
In described additive, the parts by weight of each raw material are: spirulina 9 parts, magnesium acetate 0.03 part, Herba Taraxaci 17 parts, Fructus Citri Sarcodactylis
20 parts of melon, Semen Pisi sativi 9 parts, 24 parts of Bulbus Allii, Rhizoma Phragmitis 10 parts, carambola 9 parts, Radix Isatidis 17 parts, Radix Notoginseng 6 parts, flexuose bittercress herb and seed 0.8 part, Fructus Amomi
Spend 0.6 part, 7 0.2 parts of button, 11 parts of Radix Glycyrrhizae, Mel 24 parts and blackberry pollen 16 parts.
In described additive, the parts by weight of each raw material are: spirulina 7 parts, magnesium acetate 0.04 part, Herba Taraxaci 15 parts, Fructus Citri Sarcodactylis
17 parts of melon, Semen Pisi sativi 8 parts, 18 parts of Bulbus Allii, Rhizoma Phragmitis 11 parts, carambola 14 parts, Radix Isatidis 15 parts, Radix Notoginseng 8 parts, flexuose bittercress herb and seed 1.0 parts, Fructus Amomi
Spend 0.3 part, 7 0.5 parts of button, 8 parts of Radix Glycyrrhizae, Mel 20 parts and blackberry pollen 19 parts.
In described additive, the parts by weight of each raw material are: spirulina 8 parts, magnesium acetate 0.05 part, Herba Taraxaci 19 parts, Fructus Citri Sarcodactylis
18 parts of melon, Semen Pisi sativi 6 parts, 21 parts of Bulbus Allii, Rhizoma Phragmitis 9 parts, carambola 12 parts, Radix Isatidis 13 parts, Radix Notoginseng 10 parts, flexuose bittercress herb and seed 0.4 part, Fructus Amomi
Spend 0.7 part, 7 0.4 parts of button, 5 parts of Radix Glycyrrhizae, Mel 22 parts and blackberry pollen 14 parts.
For solving above-mentioned technical problem, the present invention also provides for the preparation method of a kind of bream feed additive, its preparation side
Method comprises the following steps:
(1) Fructus Sechii edulis of described mass fraction, Semen Pisi sativi, Bulbus Allii and carambola are put into pulverizer to pulverize, by described mass parts
Number takes Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and the carambola after spirulina and magnesium acetate, pulverizing, and placing 15-18h after mix homogeneously must mix
Thing one；
(2) in described mass fraction ratio take Herba Taraxaci, Rhizoma Phragmitis, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven,
Radix Glycyrrhizae mixes, and adds relative to mixture 10～the ethanol that alcohol volumetric concentration is 90%～95% of 12 times, is heated to seething with excitement back
Flow 3～5 hours, filter, collect filtrate, be evaporated to when 40～50 DEG C relative subsequently under vacuum 0.05～0.08Mpa
Density is the mastic of 1.00～1.04, is spray-dried, the inlet temperature 160 of spray dryer～175 DEG C, leaving air temp 80～85
DEG C, it is ground into powder subsequently, makes dried cream powder；
(3) mixture one that described mass parts Mel, blackberry pollen and step (1) obtain, the dry cream that step (2) obtains are taken
Powder mix homogeneously obtains bream feed additive.
The technical scheme that the embodiment of the present invention provides has the benefit that its composition is medicinal and edible dual for having concurrently
Effect, can antibacterial, antiinflammatory, improve the immunity of bream, simultaneously can effectively treat and prevent bream bubble.The edible present invention adds
Bream premunition after agent is strong, and fast growth, meat are nutritious, the economic benefit of raiser has been significantly greatly increased, has had and join
Side is simple, science, rationally, and fully natural green noresidue is practical, has no side effect, the significant advantage of effect.
Detailed description of the invention
The invention provides a kind of bream feed additive and preparation method, have including raw material: spirulina, magnesium acetate, Pu
Herba Taraxaci, Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii, Rhizoma Phragmitis, carambola, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven, Radix Glycyrrhizae, Mel and
Blackberry pollen.
Herba Taraxaci: another name Herba crotalariae albidae, Po Poding.This product is feverfew Herba Taraxaci, alkali ground Herba Taraxaci or belongs to several together
The dry herb of plant.Spend spring to autumn and excavate when just opening, remove impurity, clean, dry.Bitter, sweet, cold.Return liver, stomach warp.Clearly
Thermal detoxification, dispersing swelling and dissipating binds, inducing diuresis for treating stranguria syndrome.
Semen Pisi sativi: another name bean, cold bean, Bi Dou, avenges bean.Seed for leguminous plant Semen Pisi sativi.Sweet, flat.Regulating middle-JIAO and making the adverse QI downward, profit is little
Just, sore is solved.
Rhizoma Phragmitis: another name reed root, reed head.This product is the fresh of grass phragmites communis or dry rhizome.The whole year all can excavate,
Remove bud, fibrous root and membranaceous leaf, using fresh herb or dry.Sweet, cold.Attach to the lung and stomach meridians.Clearing away heat and promoting production of body fluid, relieving restlessness, preventing or arresting vomiting, diuresis.
Radix Isatidis: another name great Lan root, Radix Clerodenri Cyrtophylli.This product is the dry root of cruciferae isatis.Autumn excavates, and removes
Silt, dries.Hardship, cold.GUIXIN, stomach warp.Heat-clearing and toxic substances removing, removing heat from blood sore-throat relieving.
Radix Notoginseng: this product is the dry root of panax araliaceae plant.Hardship sweet, micro-, temperature.Return liver, stomach warp.Dissipating blood stasis stops blooding, detumescence
Analgesic therapy.For spitting blood, spit blood, epistaxis, to have blood in stool, metrorrhagia, traumatic hemorrhage, chest and abdomen twinge, tumbling and swelling.
Flexuose bittercress herb and seed: another name passeris montani saturati dish, wild foster dish, popped rice perfume (or spice) Herba Capsellae.For crucifer Hairy Bittercress and bending Hairy Bittercress
Herb.Sweet in the mouth；Light；Cool in nature.Clearing away heat-damp and promoting diuresis；Calm the nerves；Hemostasis.Main damp-heat dysentery；Pyretic stranguria；Leucorrhea；Cardiopalmus；Insomnia；Asthenic fire tooth
Bitterly；Infantile malnutrition；Spit blood；Have blood in stool；Furuncle.
Villous amomum flower: for the dry flower of zingiberaceous plant Fructus Amomi, green shell sand or SEMEN AMOMI LONGILIGULA.Acrid in the mouth, warm in nature.Return spleen, stomach, kidney
Warp.Removing dampness is whetted the appetite, warming spleen and stopping diarrha, regulates the flow of vital energy antiabortive.For stagnation of QI in spleen and stomach, distension and fullness in the abdomen, vomiting and nausea.
Button seven: another name Rhizoma Panacis Majoris, panax japonicus c.a.mey.var.major (burk.) c.y.wu et k.m.feng, panax japonicus, Pestalotia funera, pimple seven, dish seven.Araliaceae Panax plant
Thing panax japonicus c.a.mey.var.major (burk.) c.y.wu et k.m.feng, is used as medicine with root stock.Autumn gathers, and cleans and dries.Bitter in the mouth, micro-sweet, warm in nature.Promoting tissue regeneration by removing blood stasis, analgesic hemostatic.With
In traumatic injury, rheumatic arthritis, stomachache；Traumatic hemorrhage is controlled in external.
Radix Glycyrrhizae: sweet, flat.GUIXIN, lung, spleen, stomach warp.Invigorating the spleen and replenishing QI, heat-clearing and toxic substances removing, expelling phlegm for arresting cough, relieving spasm to stop pain, it is in harmonious proportion all
Medicine.For weakness of the spleen and stomach, fatigue and weakness, shortness of breath and palpitation, cough with copious phlegm, gastral cavity abdomen, extremity contraction urgency pain, carbuncle sore tumefacting virus, abirritant
Thing toxicity, strong.
Embodiment is below used to describe embodiments of the present invention in detail, whereby to the present invention how application technology means
Solve technical problem, and the process that realizes reaching technique effect can fully understand and implement according to this.
Embodiment 1 additive 1
A kind of bream feed additive, wherein additive includes: spirulina 9g, magnesium acetate 0.03g, Herba Taraxaci 17g, Fructus Citri Sarcodactylis
Melon 20g, Semen Pisi sativi 9g, Bulbus Allii 24g, Rhizoma Phragmitis 10g, carambola 9g, Radix Isatidis 17g, Radix Notoginseng 6g, flexuose bittercress herb and seed 0.8g, villous amomum flower 0.6g, button
Son seven 0.2g, Radix Glycyrrhizae 11g, Mel 24g and blackberry pollen 16g.
The preparation method of additive comprises the following steps:
(1) Fructus Sechii edulis of above-mentioned mass parts, Semen Pisi sativi, Bulbus Allii and carambola are put into pulverizer to pulverize, by described mass fraction
Take Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and the carambola after spirulina and magnesium acetate, pulverizing, place 15-18h after mix homogeneously and obtain mixture
One；
(2) in described mass fraction ratio take Herba Taraxaci, Rhizoma Phragmitis, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven,
Radix Glycyrrhizae mixes, and adds relative to mixture 10～the ethanol that alcohol volumetric concentration is 90%～95% of 12 times, is heated to seething with excitement back
Flow 3～5 hours, filter, collect filtrate, be evaporated to when 40～50 DEG C relative subsequently under vacuum 0.05～0.08Mpa
Density is the mastic of 1.00～1.04, is spray-dried, the inlet temperature 160 of spray dryer～175 DEG C, leaving air temp 80～85
DEG C, it is ground into powder subsequently, makes dried cream powder；
(3) mixture one that described mass parts Mel, blackberry pollen and step (1) obtain, the dry cream that step (2) obtains are taken
Powder mix homogeneously obtains bream feed additive.
Embodiment 2 additive 2
A kind of bream feed additive, wherein additive includes: spirulina 7g, magnesium acetate 0.04g, Herba Taraxaci 15g, Fructus Citri Sarcodactylis
Melon 17g, Semen Pisi sativi 8g, Bulbus Allii 18g, Rhizoma Phragmitis 11g, carambola 14g, Radix Isatidis 15g, Radix Notoginseng 8g, flexuose bittercress herb and seed 1.0g, villous amomum flower 0.3g,
Button seven 0.5g, Radix Glycyrrhizae 8g, Mel 20g and blackberry pollen 19g.
The preparation method of additive comprises the following steps:
(1) Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and carambola are put into pulverizer to pulverize, take spirulina and vinegar by described mass fraction
Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and carambola after acid magnesium, pulverizing, places 15-18h and obtains mixture one after mix homogeneously；
(2) in described mass fraction ratio take Herba Taraxaci, Rhizoma Phragmitis, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven,
Radix Glycyrrhizae mixes, and adds relative to mixture 10～the ethanol that alcohol volumetric concentration is 90%～95% of 12 times, is heated to seething with excitement back
Flow 3～5 hours, filter, collect filtrate, be evaporated to when 40～50 DEG C relative subsequently under vacuum 0.05～0.08Mpa
Density is the mastic of 1.00～1.04, is spray-dried, the inlet temperature 160 of spray dryer～175 DEG C, leaving air temp 80～85
DEG C, it is ground into powder subsequently, makes dried cream powder；
(3) mixture one that described mass parts Mel, blackberry pollen and step (1) obtain, the dry cream that step (2) obtains are taken
Powder mix homogeneously obtains bream feed additive.
Embodiment 3 additive 3
A kind of bream feed additive, wherein additive includes: spirulina 8g, magnesium acetate 0.05g, Herba Taraxaci 19g, Fructus Citri Sarcodactylis
Melon 18g, Semen Pisi sativi 6g, Bulbus Allii 21g, Rhizoma Phragmitis 9g, carambola 12g, Radix Isatidis 13g, Radix Notoginseng 10g, flexuose bittercress herb and seed 0.4g, villous amomum flower 0.7g,
Button seven 0.4g, Radix Glycyrrhizae 5g, Mel 22g and blackberry pollen 14g.
The preparation method of additive comprises the following steps:
(1) Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and carambola are put into pulverizer to pulverize, take spirulina and vinegar by described mass fraction
Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and carambola after acid magnesium, pulverizing, places 15-18h and obtains mixture one after mix homogeneously；
(2) in described mass fraction ratio take Herba Taraxaci, Rhizoma Phragmitis, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven,
Radix Glycyrrhizae mixes, and adds relative to mixture 10～the ethanol that alcohol volumetric concentration is 90%～95% of 12 times, is heated to seething with excitement back
Flow 3～5 hours, filter, collect filtrate, be evaporated to when 40～50 DEG C relative subsequently under vacuum 0.05～0.08Mpa
Density is the mastic of 1.00～1.04, is spray-dried, the inlet temperature 160 of spray dryer～175 DEG C, leaving air temp 80～85
DEG C, it is ground into powder subsequently, makes dried cream powder；
(3) mixture one that described mass parts Mel, blackberry pollen and step (1) obtain, the dry cream that step (2) obtains are taken
Powder mix homogeneously obtains bream feed additive.
Correlation test
1, acute toxicity test
(1) experimental animal, feedstuff and raising
Cleaning grade Kun Ming mice, body weight 18-22g, 20, male and female half and half.Limited by Shanghai Si Laike laboratory animal
Company provides.Full nutrition growth feedstuff is provided by Guangzhou Datainong Feed Co., Ltd..Artificial circadian rhythm, temperature: 24 ± 2
DEG C, humidity: 45 ± 5%.
(2) test reagent
Carboxymethyl cellulose (analytical pure), is configured to 0.5% suspension with distilled water stand-by.
(3) test method
Test method selects maximum tolerated dose method.Taking 20 body weight is cleaning grade Kun Ming mice healthy for 18-22g,
Each 10 of male and female.Fasting 16h (overnight) before test, can't help water.Take the additive 1 in the 5g embodiment of the present invention 1, use 0.4% carboxylic
Methylcellulose is diluted to 18mL.Dividing upper and lower noon every bis-per os gavages of 4h, after gavage, 2h takes food for the second time, total gavage agent
Amount is 10g (kg bw)-1.Continuous Observation 14d, record poisoning manifestations and death condition.
2, mutagenicity test
2.1 test strain
TA97, TA98, TA100, TA102 tetra-strain Salmonella typhimurium.Thered is provided by Center for Disease Control (CDC) of Heilongjiang Province.
2.2 experimental animals and reagent
2.2.1Ames test
The preparation of table 1 nutrient broth medium
| Reagent | Dosage |
| Carnis Bovis seu Bubali cream | 2.4g |
| Peptone | 5.0g |
| Sodium chloride | 2.4g |
| Dipotassium hydrogen phosphate (K2HPO4 3H2O) | 1.2g |
| Distilled water | 500mL |
Heating for dissolving, by mentioned reagent, adjusts pH to 7.4, subpackage, sterilizing (0.101MPa, 20min), and general refrigerator preserves standby
With (less than half a year).
Table 2 phosphate stock solution is prepared
| Reagent | Dosage |
| Dibastic sodium phosphate ammonia (NaNH4HPO4 4H2O) | 17.4g |
| Citric acid (C6H8O7 H2O) | 10.0g |
| Dipotassium hydrogen phosphate (K2HPO4) | 50.0g |
| Magnesium sulfate (MgSO4 7H2O) | 1.0g |
Magnesium sulfate is the most slowly put into after other reagent are completely dissolved so that it is continue to dissolve (otherwise can separate out precipitation).
Table 3 1.5% agar culture medium is prepared
| Reagent | Dosage dose |
| Agar | 6.0g |
| Instill distilled water extremely | 400mL |
0.102MPa sterilizing 25min after mentioned reagent thawing.
Table 4 bottom culture medium is prepared
| Reagent | Dosage |
| Sterilizing agar culture medium (80 DEG C) | 400mL |
| Phosphate stock solution | 7mL |
| 40% glucose solution | 20mL |
It is sequentially added into mentioned reagent in a reservoir, fully mixes, when temperature is down to about 80 DEG C, be down flat ware, every ware
25mL, removes moisture while 37 DEG C of overnight incubation, then check for polluting.
Table 5 top agar is prepared
| Reagent | Dosage |
| Agar | 3.0g |
| Sodium chloride | 2.5g |
| Instill distilled water extremely | 500Ml |
Table 6 0.5mmol/L histidine-biotin solution preparation
| Reagent | Dosage |
| Bio (molecular weight 244) | 30.4mg |
| L-Histidine (molecular weight 155) | 19.3mg |
| Instill distilled water extremely | 250mL |
The preparation of table 7 10%S-9 mixed liquor
| Reagent | Dosage |
| Phosphate buffer (0.2mol/L, pH7.4) | 6mL |
| Klorvess Liquid (1.65mol/L) | 0.2mL |
| Magnesium chloride solution n (0.4mol/L) | 0.2mL |
| G6Pna saline solution (0.05mol/L) | 1.0mL |
| Coenzyme-II solution (0.0025mol/L) | 1.5mL |
| Liver S-9 liquid | 1.0mL |
Mixing, faces used time preparation, puts in water-bath stand-by.
Liver S-9 liquid is provided by Center for Disease Control (CDC) of Heilongjiang Province.Standard mutagenesis agent is respectively fenaminosulf, sodium azide, 2-second
Acylamino-fluorenes, 1,8-istizin, Center for Disease Control (CDC) of Heilongjiang Province provide.
2.2.2 micronucleus test
50 cleaning grade Kun Ming mice, body weight 25-30g, male and female half and half.By the Shanghai limited public affairs of Si Laike laboratory animal
Department provides.Full nutrition growth feedstuff is provided by Guangzhou Datainong Feed Co., Ltd..Artificial circadian rhythm, temperature: 24 ± 2
DEG C, humidity: 45 ± 5%.
Calf serum: calf serum is put in water bath with thermostatic control after considering bacterium, 56 DEG C of inactivation 1h.4 DEG C it are stored at after inactivation
Refrigerator saves backup.
Jim Sa (Giemsa) dye liquor: weigh Giemsa3.8g, adds 375ml methanol (analytical pure) and grinds, treat the most molten
Xie Hou, adds 125ml glycerol.It is placed in 37 DEG C of calorstat 48h to shake for several times.Filter, stand two Zhou Houyong.
Jim Sa (Giemsa) application liquid: take a Giemsa dye liquor and 6 parts of phosphate buffers mix.Face the used time
Preparation.
Table 8 1/15moL/L phosphate buffer (pH6.8) is prepared
| Reagent | Dosage |
| Potassium dihydrogen phosphate (KH2PO4) | 4.50g |
| Sodium dihydrogen phosphate (Na2HPO4 12H2O) | 11.78g |
| Instill distilled water extremely | 1000mL |
All reagent is in addition to indicating, and is analytical pure, and test water is distilled water.
2.2.3 sperm malformation test
50 cleaning grade Kunming kind male white mouses, body weight 25-35g.Carried by Shanghai Slac Experimental Animal Co., Ltd.
Supply.Full nutrition growth feedstuff is provided by Guangzhou Datainong Feed Co., Ltd..Artificial circadian rhythm, temperature: 24 ± 2 DEG C, wet
Degree: 45 ± 5%.
Methanol.1%-2% eosin stains liquid: weigh Yihong 1-2g, is dissolved in 100mL distilled water standby.All reagent is except mark
Outside bright, being analytical pure, test water is distilled water.
2.3 test method
2.3.1Ames test
(1) principle: saltant type (i.e. histidine deficient) bacterial strain of Salmonella typhimurium is under the conditions of having histidine
Can not be able to grow in the culture medium existed without histidine with normal growth in culture medium.If but having mutagen to be present in nothing
Time in the culture medium of histidine, then mutant salmonella type can back mutation be wild type (Phenotype), thus can be grown on
Without in the culture medium of histidine, so can be that standard judges that tested material causes the power of prominent property according to bacterium colony quantity of formation.Some
It is wild type that special tested material needs just to make mutant salmonella type back mutation after metabolism activation system processes, generation
Thank to activation system and use S-9 mixed liquor (preparation method: utilize Polychlorinated biphenyls (Polychlorinated biphenyl, PCB) to lure
Lead rat liver homogenate (S-9)).
(2) test strain: use TA97, TA98, TA100, TA102 tetra-strain Salmonella typhimurium saltant,
TA97 and TA98 can detect various frame shift type mutagenic agent；TA100 can detect the mutagenic agent causing base pair replacement；TA102 energy
Detect some mutagenic agent that other test strain can not detect or seldom detect.
Additive 1 dosage in (3) 5 embodiment of the present invention 1 is respectively 5000,1000,200,40 and 8 μ g/ wares.
(4) increase bacterium training and take nutrient broth medium 5ml, add in aseptic little triangular flask or sterile test tube, freezing is protected
The inoculation deposited is in nutrient broth medium, and 37 DEG C of vibrations (100 times/min) cultivate 10h to increased logarithmic phase, every milliliter
Viable count is more than 1 × 109～2 × 109Individual, wrap up in culture bottle with black paper bag, irradiated by light with bacteriological protection.
(5) several bottom culture medium plate is prepared.
(6) top layer culture medium melted and be sub-packed in aseptic small test tube, often pipe 2ml, being incubated in 45 DEG C of water-baths.
(7) fresh for the test strain of 0.1ml enrichment liquid is sequentially added in the top layer culture medium of insulation, mixing；Then drip
Enter 0.1ml tested material (separately adding 0.5ml 10%S-9 mixed liquor during activation), then mix, be quickly poured in bottom culture medium, and
Making it be uniformly distributed on bottom, keep flat solidification, in sterile board, (37 DEG C) cultivate 48h observed result.
(8) separately do solvent control (i.e. negative control, distilled water 0.1ml/ ware) and positive control (is respectively adopted sodium azide
1.5 μ g/ wares, fenaminosulf 50 μ g/ ware, 2-acetamidofluorene 10 μ g/ ware, 1,8-istizin 50 μ g/ ware).Solvent control adds goes out
Bacterium distilled water；Positive control is not added with tested material, only adds standard mutagenic agent；Additive method is ibid.It is repeated twice.
2.3.2 micronucleus test
Table 9 micronucleus test design (n=10)
| Dosage group | Dosage (g (kg bw d)-1) |
| Negative group | 0.5% carboxymethyl cellulose |
| Low dose group | 0.5 |
| Middle dosage group | 5 |
| High dose group | 9 |
| Positive group | 0.04 |
It is administered to tested material method by 30h, positive controls cycli phosphate amine once abdominal cavity injection 0.04g (kg bw)-1,
Remaining respectively organizes gavage, 2 dosing interval 24h, and after being administered for the 2nd time, 6h puts to death animal.
Film-making: after being administered 6h at the 2nd time, mice is taken off cervical vertebra and puts to death, take mice vertebra, remove muscle, cut off the bone of one end
Vertebra, drips in calf serum (0.05mL is placed on microscope slide) with needle-nose pliers extrusion bone marrow.
Push jack: after mixing, push jack several, standing and drying.
Fixing: with methanol solution, dry smear being fixed 5～10min, taking-up is dried.The same day, achromophil smear also should
Preserve after Gu Ding.
Dyeing: the smear 15～30min fixed with Jim Sa-phosphate buffer (pH is 6.4) dyeing of 1:10.With steaming
Distilled water is rinsed, the most to be checked.Every white mice counts 1 000 PCE (Poiychromatic erythrocytes, PCE),
Micronuclear rates represents with ‰；It addition, when counting PCE, count RBC (Red blood cell count, RBC) number, calculate simultaneously
PCE/RBC value.
2.3.3 sperm malformation test
Table 10 sperm malformation test design (n=10)
| Dosage group | Dosage (g (kg bw d)-1) |
| Negative group Negative control | 0.5% carboxymethyl cellulose |
| Low dose group | 0.5 |
| Middle dosage group | 5 |
| High dose group | 10 |
| Positive group | 0.04 |
Every day contaminates once, and continuous 5d records body weight, food-intake and absorption additive capacity every day.Positive controls uses
0.04g·(kg·bw·d)-1Cyclophosphamide carries out lumbar injection, and the administering mode of the embodiment of the present invention 1 additive 1 is gavage.
Require during off-test often to organize at least 5 surviving animals.
Sperm sampling and microscopy: after giving tested material first, mice cervical dislocation is put to death by the 5th week (35d), opens
Abdominal cavity, wins both sides epididymis, puts in little plate and (fills about 2ml normal saline).Being shredded with eye scissors by epididymis (can not be too
Broken).With four layers, fragment of tissue is wiped paper filter off.Filtrate is centrifuged 5min (1000～1500r/min).Retain about 0.5ml liquid, its
Remaining supernatant reject, after itself and precipitate being shaken up, drips 1 on clean microscope slide and carries out smear (typically every mice is cooked 4
～5 smears).Smear is dried in atmosphere, uses methanol solution to fix 5min.With 2% Yihong solution at its natural drying
Poststaining 1h, gently rushes with water, is dried to be checked.
Under low power lens, find that sperm overlap is less, the part of clear background, with high power lens sequential search sperm, and count
Number.All imperfect, profile is unclear, or overlapping, or obvious genus does not artificially shred person and do not calculates.One sperm is only counted the brightest
Aobvious a kind of deformity.
Secondly the deformity of sperm is mainly manifested in head, and at afterbody, deformity type has banana-shaped, amorphous, Wugou, fat
Head, double end, double tail, tail folding etc..The sperm count of recording exceptional and Exception Type, and calculate the sperm composition ratio of deformity type.
Every 1000 complete sperms of Mus meter, abnormal rate represents with ‰.
Rate of teratosperm (‰)=sperm deformity sum/check sperm sum × 1 000
3, chronic and subchronic test
3.1 experimental animals, feedstuff and raising
3.1.1 rat 30d feeding trial
Cleaning grade Wistar rat, body weight 150-180g, 80, male and female half and half.Had by Shanghai Si Laike laboratory animal
Limit company provides.Full nutrition growth feedstuff by Beijing Australia pull together feed corporation,Ltd provide.II grade of Animal House, saves the most round the clock
Rule, temperature: 24 ± 2 DEG C, humidity: 45 ± 5%.
3.1.2 laying hen 56d feeding trial
29 week old sea match laying hens 240, are provided by Harbin benefit agriculture fowl industry.Basic drawing is reached feed factory by Harbin China and carries
Supply.Layer breeding agriculture university's practice base Laying House northeastward.
3.2 test reagent
General chemistry reagent: formaldehyde, paraffin, ethanol, dimethylbenzene, hematoxylin, Yihong, acetone, phosphate buffer etc..
Biochemical Indexes test kit: glutamic oxaloacetic transaminase, GOT (Glutamic-oxalacetic transaminease, GOT),
Glutamate pyruvate transaminase (Glutamic-pyruvic transaminase, GPT), blood urea nitrogen (Urea nitrogen, BUN), creatinine
(Creatinine, CRE), cholesterol (Cholesterol, CHO), blood glucose (Glucose, GLU), TG, albumin (Albumin,
ALB), total protein (Total protein, TP) test kit is purchased from middle raw north control (product batch number: 100731)
Cellanalyzer test agent: stain (STROMATOLYSER-4DS FFS-800A), haemproteins detection examination
Agent (SULFOLYSERSLS-210A1015), basophilic granulocyte and white blood cell detection reagent (STROMATOLYSER-
FBR1039), diluent (PK-30L G2109).All reagent is analytical pure, and test water is distilled water.
3.3 test method
3.3.1 rat 30d feeding trial
Table 11 rat 30d feeding trial design (n=20)
| Group | Process |
| Matched group | Complete feed |
| Low dose group | Complete feed+0.4% embodiment of the present invention 1 additive 1 |
| Middle dosage group | Complete feed+2% embodiment of the present invention 1 additive 1 |
| High dose group | Complete feed+10% embodiment of the present invention 1 additive 1 |
Animal is bought after isolation is fed 1 week and is for experiment, often group male and female half and half.Experimental animal free choice feeding, freely drink water.
(1) physiochemical indice:
General index: observe the searching for food of rat, drink water, fall ill and the situation such as death, every day twice sooner or later, body weight with raise
Material is each weekly to be claimed once, and calculates average weight gain, feed intake, starting weight, end weight and efficiency of feed utilization.
Hematological indices: 30d feeds after terminating, blood sampling sample, mensuration erythrocyte (Red blood cell count,
RBC), leukocyte (White blood cell count, WBC), hemoglobin (Hemoglobin, HGB), lymphocyte number
(Lymphocyte, LYM) and leukocyte differential count.
Biochemical indicator: 30d feed terminate after, gather Rat blood samples centrifuging and taking serum, measure GOT, GPT, BUN,
CRE、CHO、GLU、TG、ALB、TP。
(2) pathological examination:
Postmortem: observing and record the naked eyes change of each system organ, tissue, typical cytopathic is taken pictures.
Organ coefficient: the tissues such as heart, liver, kidney, spleen, testis or ovary and body weight are weighed, and calculate internal organs system
Number.
Histological examination: while above-mentioned Dissection test animal changes with perusal, core dirty, kidney, spleen, liver
The organs and tissues 1 such as dirty, stomach, testis or ovary～2 pieces, fix with formalin, paraffin embedding, section, HE (H Ematine,
HE) dyeing, basis of microscopic observation record organization change.
3.3.2 laying hen 56d feeding trial
Table 12 laying hen 56d feeding trial design (n=60)
Animal starts test after adapting to 7 days under this experimental condition after buying again.Experimental animal free choice feeding, freely drink
Water.
(1) physiochemical indice:
General index: observe the searching for food of laying hen, drink water, fall ill and the situation such as death, every day twice sooner or later, to egg size with
Feedstuff respectively claims once every day, and calculates feedstuff-egg ratio, laying rate.
Hematological indices: after nursing in 56 days terminates, carries out blood sampling and measures RBC, WBC, HGB, LYM laying hen.
Biochemical indicator: 56 days feed terminate after, laying hen is acquired centrifugal blood take determination of serum GOT, GPT,
BUN、CRE、CHO、GLU、TG、ALB、TP。
(2) pathological examination:
Postmortem: perusal also records the change of each system organ, tissue, and typical cytopathic is taken pictures.
Organ coefficient: the tissue weight such as satisfactory dirty, liver, lungs, kidney, spleen, pancreas, glandular stomach, muscular stomach and body weight.
Histological examination: while above-mentioned Dissection test animal changes with perusal, take liver, spleen, kidney, gland
The organs and tissues such as stomach 1～2 pieces are fixed with formalin, paraffin embedding, section, and HE dyes, basis of microscopic observation record organization
Learn change.
3.4Data process
Result mean+SD represents, uses SPSS 17.0 to carry out statistical analysis.Acute toxicity test, DABAI
Mus 30d feeding trial and laying hen 56d feeding trial result carry out single factor test variance (one-wayANOVA) and analyze；Salmonella reversion test is tied
Fruit and mouse marrow cell micro nuclear test result all carry out X 2 test；Mouse inbred strain result carries out single factor test variance
Analyze and Duncan ' s method multiple comparisons.P < 0.05 is significance of difference criterion, and P < 0.01 significantly judges mark for difference
Accurate.
4, result and analysis
4.1 acute toxicity
Table 13 acute toxicity
From table 13, after off-test, all mices are the most without exception during testing, and drink water, search for food and feces is equal
Normally, all survive；The weight gain of two kinds of sex mices has no significant difference (P > 0.05).To be the least after off-test
Mus takes off neck and puts to death, and dissects, and the internal organs such as the perusal heart, liver, spleen, lung, stomach, kidney, thymus all do not occur that abnormal pathologic changes.Result
Show that the LD50 > 10mg (kg bw)-1 of the embodiment of the present invention 1 additive 1 belongs to actual non-toxic type material.
4.2 mutagenicity
4.2.1Ames test
From table 14,15,16,17, compared with negative control group, adding and be not added with twice Salmonella reversion test in the case of S-9
Positive controls recovery mutation colony number significantly increases (P < 0.01), and each dosage group of the embodiment of the present invention 1 additive 1 is replied prominent
Become clump count without significant difference (P > 0.05), without dose-response relationship, i.e. under this dosage the embodiment of the present invention 1 additive 1 to Mus
Salmonella typhi is that mutagenesis is negative.
Table 14 embodiment of the present invention 1 additive 1 impact (test ,-S9 for the first time) on recovery mutation colony number
Note: in same column, * represents that difference is extremely notable (P < 0.01).Table 15,16,17 is same
Table 15 embodiment of the present invention 1 additive 1 impact (test ,+S9 for the first time) on recovery mutation colony number
Table 16 embodiment of the present invention 1 additive 1 impact (second time test ,-S9) on recovery mutation colony number
Table 17 embodiment of the present invention 1 additive 1 impact (second time test ,+S9) on recovery mutation colony number
4.2.2 mouse marrow cell micro nuclear test
Table 18 mouse marrow cell micro nuclear test (n=10)
Note: in same column, shoulder mark * person represents that difference is extremely notable (P < 0.01)
From table 18, compared with negative control group, positive controls micronuclear rates pole significantly improves (P < 0.01), female, male
The mice embodiment of the present invention 1 additive 1 respectively organizes micronuclear rates all without significant difference (P > 0.05), illustrates that the present invention is real under this dosage
Execute example 1 additive 1 not make significant difference Micronucleus, i.e. this result of the test is negative.Additionally, except positive control
Outside group, the embodiment of the present invention 1 additive 1 each group PCE/RBC compared with negative control group, without significant difference (P > 0.05), illustrates this
Under dosage, the embodiment of the present invention 1 additive 1 has no cytotoxic effect to experimental animal.
4.2.3 mouse inbred strain
From table 19, each group mice all has a number of teratospermia to occur, compared with negative control group, positive right
Significantly improve (P<0.01) according to group rate of teratosperm pole, the embodiment of the present invention 1 additive 1 respectively organize difference not significantly (P>
0.05), without dose-response relationship, illustrate that Sperm Abnormalities of Mice is not had by the embodiment of the present invention 1 additive 1 under this dosage
Have an impact.
Table 19 mouse inbred strain (n=10)
| Group | Sperm deformity number (individual) | Rate of teratosperm (‰) |
| Negative group | 31 | 6.2 |
| Low dose group | 31 | 6.2 |
| Middle dosage group | 40 | 8.0 |
| High dose group | 46 | 9.2 |
| Positive group control | 513* | 102.6* |
Note: in same column, shoulder mark * person represents that difference is extremely notable (P < 0.01)
4.3 is chronic chronic with Asia
4.3.1 rat 30d feeding trial
4.3.1.1 general index
Table 20 rat 30d feeding trial weight gain, food-intake, food utilization (n=10)
From table 20, weight gain, total food-intake and food after the embodiment of the present invention 1 additive 1 feed rat 30d
Utilization rate compared with matched group all without significant difference (P > 0.05).
4.3.1.2 hematological indices
Table 21 rat 30d feeding trial hematological indices (1) (n=10)
Table 22 rat 30d feeding trial hematological indices (2) (n=10)
From table 21 and 22, compared with matched group, the embodiment of the present invention 1 additive 1 feeds male and female rat after rat 30d
Leukocyte, erythrocyte, hemoglobin and differential blood count difference the most notable (P > 0.05).
4.3.1.3 blood parameters
Table 23 rat 30d feeding trial blood parameters (1) (n=10)
Table 24 rat 30d feeding trial blood parameters (2) (n=10)
From table 23 and 24, compared with matched group, the embodiment of the present invention 1 additive 1 feeds male and female rat after rat 30d
9 blood parameters differences the most notable (P > 0.05).
4.3.1.4 pathological examination
(1) gross examination of skeletal muscle
Dissect each group of rat after off-test and carry out gross necropsy.Respectively organize rat trachea, heart, thoracic aorta, liver, kidney,
Adrenal gland, spleen, stomach, caecum, colon, rectum, pancreas, part mesenteric lymph node, mammary gland, duodenum, jejunum, prostate,
The tissue such as testis, epididymis seminal vesicle or ovary, uterus, vagina, sciatic nerve, bladder, skin is showed no obvious abnormalities.
Table 25 rat 30d feeding trial organ weight ratio (1) (n=10)
Table 26 rat 30d feeding trial organ weight ratio (2) (n=10)
From table 25,26, the embodiment of the present invention 1 additive 1 feeds the internal organs after rat 30d with matched group male and female rat
It is the most not notable (P > 0.05) that weight ratio compares difference.
(2) organs and tissues pathologic finding
After off-test, all rat hearts, liver, spleen, kidney, stomach, testis or ovary are carried out HE dyeing.Under microscope
Observing, each dosage group has no any obvious exception compared with matched group.
4.3.2 laying hen 56d feeding trial
4.3.2.1 general index
From table 27, compared with matched group, laying hen after the 56d embodiment of the present invention 1 additive 1 feeds daily ingestion amount,
Average egg weight difference is not the most notable (P > 0.05)；Average egg production significantly improves, and feedstuff-egg ratio significantly reduces (P < 0.05), shows to fit
When the dosage embodiment of the present invention 1 additive 1 can improve the production performance of laying hen.
Table 27 laying hen feeding trial generality index (n=60)
Note: in colleague, shoulder upper letter difference person represents significant difference (P < 0.05)
4.3.2.2 hematological indices
From table 28, compared with matched group, laying hen leukocyte, red after the 56d embodiment of the present invention 1 additive 1 feeds
Cell, hemoglobin, hematocrit value, mean corpuscular volume, mean corpuscular hematochrome, erythrocyte average protein concentration,
Platelet difference is not the most notable (P > 0.05).
Table 28 laying hen feeding trial hematological indices (n=60)
4.3.2.3 blood parameters
Table 29 laying hen feeding trial blood parameters (n=60)
Note: in colleague, shoulder upper mark * person represents significant difference (P < 0.05)
From table 29, compared with matched group, high dose group laying hen blood BUN level significantly reduces (P < 0.05), other
Index is all without significant difference (P > 0.05).
4.3.2.4 pathological examination
(1) gross examination of skeletal muscle
After off-test, each group of laying hen is dissected, carries out gross necropsy.Each group main organs trachea of laying hen, heart,
Liver, kidney, adrenal gland, spleen, stomach, caecum, colon, rectum, pancreas, part mesenteric lymph node, duodenum, ovary, uterus, skin
Skins etc. by the naked eye, show no obvious abnormalities.
Table 30 laying hen feeding trial organ weight ratio (n=60)
| Project | Matched group | Low dose group | Middle dosage group | High dose group |
| Heart weight ratio | 3.79±0.51 | 4.12±0.56 | 2.59±0.13 | 3.72±0.59 |
| Liver weight ratio | 23.89±4.88 | 27.28±3.98 | 23.51±3.38 | 28.09±6.01 |
| Kidney weight ratio | 6.03±1.03 | 7.01±0.41 | 6.33±0.58 | 7.25±0.37 |
| Spleen weight ratio | 1.21±1.02 | 0.94±0.12 | 1.02±0.12 | 0.81±0.72 |
| Fabricius bursa weight ratio | 1.19±0.49 | 0.95±0.18 | 0.69±0.50 | 0.61±0.32 |
| Glandular stomach weight ratio | 4.19±0.42 | 3.91±0.31 | 3.40±0.71 | 4.29±0.28 |
| Muscular stomach weight ratio | 16.24±1.14 | 15.93±1.25 | 14.28±2.98 | 18.05±2.18 |
From table 30, compared with matched group, the embodiment of the present invention 1 additive 1 respectively organizes laying hen internal organs weight differences the most not
Significantly (P > 0.05).
(2) organs and tissues pathologic finding
Liver of Laying Hens, spleen, kidney, glandular stomach through HE dye, examine under a microscope, each dosage group compared with matched group the most not
See any obvious exception)
To sum up experimental result:
1. the median lethal dose(LD 50) of the embodiment of the present invention 1 additive 1 is more than 10g (kg bw)-1, belongs to actual non-toxic type
Material.
2., under this experimental condition, the embodiment of the present invention 1 additive 1 does not has mutagenicity.
3., under this experimental condition, the embodiment of the present invention 1 additive 1 does not has chronic and subchronic toxicity to rat and laying hen,
And the production performance of laying hen can be improved.
In sum, this result of the test is pointed out, and under this experimental condition, the embodiment of the present invention 1 additive 1 had not both had acute poison
Property, mutagenicity, do not have chronic or subchronic toxicity yet, and the embodiment of the present invention 1 additive 1 is the most permissible under suitable dosage
As a kind of green, natural, safe functional feedstuff additive, and it is applied in husbandry sector.
The additive of embodiments of the invention 2 and embodiment 3 has carried out above-mentioned test equally, is also demonstrated that the present invention implements
The additive of example 2 and embodiment 3 had not both had acute toxicity, mutagenicity, did not had chronic or subchronic toxicity yet, in suitable agent
As a kind of green, natural, safe functional feedstuff additive, and can be applied in husbandry sector completely under amount.
Contrast test:
Choose certain bream plant.Select that outward appearance is normal, body constitution is healthy and strong, bream of uniform size, by random for 320 tail breams
Being divided into 4 groups, often organize 80 tails, 4 groups respectively feed the embodiment of the present invention 1 additive 1 group, feed the embodiment of the present invention 2 additive 2
Group, feed the embodiment of the present invention 3 additive 3 groups and feed the contrast groups of normal diet.Additive quality accounts for feedstuff gross weight
0.05%.Putting in a suitable place to breed respectively in the net cage of internal diameter 1.0m*1.0m*1.0m by 4 groups of breams, test water is that natural sea-water is through sand
Filter gained, salinity is 31-33, and water temperature is 23 DEG C-26 DEG C, and the depth of water is 0.6m, process of the test dissolved oxygen be maintained at 6.0mg/L with
On.Experimental period is 90 days.On-test, at the end of, to test net cage in bream count, measure body weight, sampling measure
Body is long.The method ground of the work aquaculture managements routinely such as feeding and management daily between experimental period, disease control.Number of times of throwing something and feeding is 2
Secondary d-1, the bait throwing in time is 7:30～8:00,17:00～17:30.Feeding rate controls 1%～1.5%, and daily ration, feeding quantity is according to gas
The situation of ingesting of time, water temperature, water quality and test fish does respective change.Examine the active situation of bream in net cage every day, in detail
Carefully record the actual feeding volume of each net cage, if fish has death, make a record, and timing measure water temperature, every 3d measure 1 time molten
Solve oxygen.
1, index determining
At the end of feeding experiment, 10 tail breams randomly drawed by each net cage, measure body weight, body length, internal organs weight, liver
Weight.
Tail absolute gain/g=test fish is terminated cabrage-test fish and starts cabrage
Body weight increase rate/%=(tail absolute gain/on-test cabrage) * 100%
Feed coefficient=total daily ration, feeding quantity/total augment weight
Survival rate/%=(off-test fish tail number/on-test fish tail number) * 100%
Coefficient of condition K=fish body weight (g) * 100/ [fish length (cm)]3
Dirty body ratio/%=(internal organs weight/fish body weight) %100%
Liver body ratio/%=(liver weight/fish body weight) * 100%
2, the detection of hemocyte phagocytic activity
With the Candida albicans inclined-plane of the flushed activation of physiological saline solution, through coming, the bacterium making 1.4*107/ml alive is hanged
Liquid.Carry out inoculation test by feeding above-mentioned 4 groups of breams of 90 days, often organize and take 20 tail breams at random, carry out muscle in bream back
The bacteria suspension of injection inoculation 0.6ml.With 1ml blood collection needle making blood smear slide, dyeing, inspection according to a conventional method after 1h
Survey 100 hemocytees, calculate phagocytic rate and phagocytic index.Computing formula is as follows:
Percentage phagocytosis (PP)=100 hemocyte is joined the cell number/100*100% of phagocytosis
Phagocytic index (PI)=100 participates in intracellular total number of fungi/100 of phagocytosis
Experimental result is as follows:
31 4 groups of bream growing states of table
| Growth indexes | Additive 1 group | Additive 2 groups | Additive 3 groups | Contrast groups |
| Rate of body weight gain/% | 52.32±1.03b | 51.49±0.43b | 51.28±0.38b | 34.12±0.54a |
| Feed coefficient | 1.62±0.06b | 1.63±0.05b | 1.63±0.10b | 1.91±0.04a |
| Dirty body ratio/% | 10.02±1.04b | 10.01±0.48b | 9.98±1.06b | 9.06±0.51a |
| Liver body ratio/% | 1.80±0.42b | 1.81±0.73b | 1.83±0.51b | 2.41±0.65a |
| Coefficient of condition K | 2.42±0.82b | 2.40±0.43b | 2.38±0.37b | 2.03±0.26a |
| Survival rate/% | 100 | 100 | 100 | 91 |
Note: same column letter is identical to be indicated without significant difference (P > 0.05)；Letter is different, then there were significant differences (P < 0.05).
Embodiment of the present invention gained additive is compared with normal diet as can be seen from the above data, and gaining effect is obvious,
Rapidly, survival rate is high in growth.
The detection of hemocyte phagocytic activity before and after table 32 test
| Group | Phagocytic index | Phagocytic rate |
| Additive 1 group | 4.322±0.365b | 43.21±1.35b |
| Additive 2 groups | 4.246±0.476b | 42.79±1.24b |
| Additive 3 groups | 4.187±0.311b | 41.26±0.79b |
| Matched group | 1.537±0.687a | 16.73±1.64a |
Note: colleague's letter is identical to be indicated without significant difference (P > 0.05)；Letter is different, then there were significant differences (P < 0.05).
Embodiment of the present invention gained additive is compared with normal diet as can be seen from the above data, and immunity has obtained pole
Big raising.
3, choose sick fish 80 tail suffering from gas bubble disease in this bream plant, be divided into 4 groups, often organize 20 tails.Respectively raise for 4 groups
Feed the embodiment of the present invention 1 additive 1 group, feed the embodiment of the present invention 2 additive 2 groups, feed the embodiment of the present invention 3 additive 3
Group and feed the contrast groups of common additives group.The addition of additive is the 0.1% of feedstuff gross weight.By 4 groups of breams respectively
Putting in a suitable place to breed in the net cage of 1.0m*1.0m*0.5m, experimental period is 7 days.The work such as daily feeding and management, disease control are routinely
The method ground of aquaculture management.Number of times of throwing something and feeding is 2 d-1, the bait throwing in time is 7:30～8:00,17:00～17:30.Feeding rate control
System is 1%～1.5%, and daily ration, feeding quantity does respective change according to the situation of ingesting of weather, water temperature, water quality and test fish.
Symptom:
There is bubble in the fish intestinal that causes a disease；Or the many minute bubbles of attachment on body surface, the gill, the gill turns white, and between the gill filament, mucus increases, and makes
Fish body floats or disequilibrium of moving about, travelling slow, unable, can not normally sink, cause and can not normally ingest, exhaust because of power eventually,
Mortality can be caused time serious.
Efficacy determination:
Recovery from illness: appetite and activity are normal, bubble collapse on body surface, the gill, the gill is grown normal；
Effective: appetite and movable improvement, on body surface, the gill, bubble symptom alleviates；
Invalid: dead.
Experimental result:
1, after 80 sick breams being fed 4 days, experimental result is shown in Table 33:
After the sick bream of table 33 feeds 4 days, experimental result
Above experimental result, can be notable it can be seen that embodiment of the present invention gained additive is compared with common additives
Improve immunity and the resistance against diseases of bream, quite notable to the gas bubble disease effect for the treatment of bream, there is the instant effect of uniqueness, treatment
The advantage that journey is short, have no side effect.
To sum up experimental result, gained bream additive of the present invention, relative to common additives, can significantly improve bream
Immunity, greatly improves the bream speed of growth, can effectively treat bream gas bubble disease simultaneously, improve the economic worth of bream.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (7)
1. a bream feed additive, it is characterised in that described additive includes following components: spirulina, magnesium acetate, Pu Gong
English, Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii, Rhizoma Phragmitis, carambola, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven, Radix Glycyrrhizae, Mel and black
Certain kind of berries pollen.
2. bream feed additive as claimed in claim 1, it is characterised in that the parts by weight of each raw material in described additive
For: spirulina 5-10 part, magnesium acetate 0.02-0.06 part, Herba Taraxaci 12-21 part, Fructus Sechii edulis 14-24 part, Semen Pisi sativi 4-11 part, Bulbus Allii
15-27 part, Rhizoma Phragmitis 7-13 part, carambola 6-17 part, Radix Isatidis 10-20 part, Radix Notoginseng 3-13 part, flexuose bittercress herb and seed 0.4-1.1 part, villous amomum flower
0.3-0.8 part, button seven 0.2-0.7 part, Radix Glycyrrhizae 5-12 part, Mel 17-28 part and blackberry pollen 11-22 part.
3. bream feed additive as claimed in claim 1 or 2, it is characterised in that the weight of each raw material in described additive
Number is: spirulina 7-9 part, magnesium acetate 0.03-0.05 part, Herba Taraxaci 15-19 part, Fructus Sechii edulis 17-20 part, Semen Pisi sativi 6-9 part, big
Bulbus Allii 18-24 part, Rhizoma Phragmitis 9-11 part, carambola 9-14 part, Radix Isatidis 13-17 part, Radix Notoginseng 6-10 part, flexuose bittercress herb and seed 0.4-1.0 part, Fructus Amomi
Flower 0.3-0.7 part, button seven 0.2-0.5 part, Radix Glycyrrhizae 5-11 part, Mel 20-24 part and blackberry pollen 14-19 part.
4. the bream feed additive as described in claims 1 to 3 is arbitrary, it is characterised in that each raw material in described additive
Parts by weight are: spirulina 9 parts, magnesium acetate 0.03 part, Herba Taraxaci 17 parts, Fructus Sechii edulis 20 parts, Semen Pisi sativi 9 parts, 24 parts of Bulbus Allii, Rhizoma Phragmitis
10 parts, carambola 9 parts, Radix Isatidis 17 parts, Radix Notoginseng 6 parts, flexuose bittercress herb and seed 0.8 part, villous amomum flower 0.6 part, 7 0.2 parts of button, 11 parts of Radix Glycyrrhizae,
Mel 24 parts and blackberry pollen 16 parts.
5. the bream feed additive as described in Claims 1-4 is arbitrary, it is characterised in that each raw material in described additive
Parts by weight are: spirulina 7 parts, magnesium acetate 0.04 part, Herba Taraxaci 15 parts, Fructus Sechii edulis 17 parts, Semen Pisi sativi 8 parts, 18 parts of Bulbus Allii, Rhizoma Phragmitis
11 parts, carambola 14 parts, Radix Isatidis 15 parts, Radix Notoginseng 8 parts, flexuose bittercress herb and seed 1.0 parts, villous amomum flower 0.3 part, 7 0.5 parts of button, 8 parts of Radix Glycyrrhizae,
Mel 20 parts and blackberry pollen 19 parts.
6. the bream feed additive as described in claim 1 to 5 is arbitrary, it is characterised in that each raw material in described additive
Parts by weight are: spirulina 8 parts, magnesium acetate 0.05 part, Herba Taraxaci 19 parts, Fructus Sechii edulis 18 parts, Semen Pisi sativi 6 parts, 21 parts of Bulbus Allii, Rhizoma Phragmitis
9 parts, carambola 12 parts, Radix Isatidis 13 parts, Radix Notoginseng 10 parts, flexuose bittercress herb and seed 0.4 part, villous amomum flower 0.7 part, 7 0.4 parts of button, 5 parts of Radix Glycyrrhizae,
Mel 22 parts and blackberry pollen 14 parts.
7. the preparation method of the holothurian feed additive as described in claim 1 to 6 is arbitrary, it is characterised in that described system
Preparation Method comprises the following steps:
(1) described mass fraction Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and carambola are put into pulverizer to pulverize, take spiral shell by described mass fraction
Revolve Fructus Sechii edulis, Semen Pisi sativi, Bulbus Allii and the carambola after algae and magnesium acetate, pulverizing, place 15-18h after mix homogeneously and obtain mixture one；
(2) Herba Taraxaci, Rhizoma Phragmitis, Radix Isatidis, Radix Notoginseng, flexuose bittercress herb and seed, villous amomum flower, button seven, Radix Glycyrrhizae are taken in described mass fraction ratio
Mixing, adds relative to mixture 10～the ethanol that alcohol volumetric concentration is 90%～95% of 12 times, is heated to boiling reflux 3～5
Hour, to filter, collect filtrate, when being evaporated to 40～50 DEG C subsequently under vacuum 0.05～0.08Mpa, relative density is
The mastic of 1.00～1.04, is spray-dried, the inlet temperature 160 of spray dryer～175 DEG C, leaving air temp 80～85 DEG C, with
After be ground into powder, make dried cream powder；
(3) taking the mixture one that described mass parts Mel, blackberry pollen and step (1) obtain, the dried cream powder that step (2) obtains mixes
Close and uniformly obtain bream feed additive.
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Application publication date: 20161214 |