Summary of the invention
At the deficiencies in the prior art part, the invention provides a kind of cost low, can improve the animal immune ability, improve the animal immunity potentiator of its growth performance.
The present invention is for reaching above purpose, be to realize: a kind of animal immunity potentiator is provided by such technical scheme, the recipe ingredient of this reinforcing agent is: 5~10 parts of 10~20 parts of the Rhizoma Atractylodis Macrocephalaes, 20~30 parts of Fructus Crataegis, 10~20 parts in Fructus Hordei Germinatus and Rhizoma Atractylodis, above component all by weight.
Animal immunity potentiator of the present invention, what select for use all is common raw material, therefore can control production cost to greatest extent.Particularly the Rhizoma Atractylodis Macrocephalae of one of raw material has characteristics abundant, cheap in china natural resources, safety non-toxic; Use as a kind of feed additive with its animal immunity potentiator that is raw material is made, can improve the animal immune function, but therefore also supplement feed albumen effect when improving the animal intestinal microecological balance can improve the growth of animal performance.Animal immunity potentiator of the present invention, suitable animal is taken for a long time, and animal is developed immunity to drugs, can be all or most of antibiotic that substitutes in the feedstuff.
Animal immunity potentiator of the present invention can prove by following experimental technique to the enhancing of animal immune ability with to the function of improving of animal intestinal microecological balance.
1, choose 60 store pigs that body weight is close, be divided into 2 groups (matched group and test group) at random, every group of 3 repetitions, each repeats 10.Added the animal immunity potentiator of the present invention of 1% weight ratio in the feedstuff that test group ate, matched group eats normal diet.Test was raised 7 days in advance, was just trying 30 days phases, write down feed intake every day.
2, butcher sampling during off-test, weigh respectively internal organs and immune organ calculate organ coefficient; Blood sample collection prepares serum, adopts immune turbidimetry kit measurement serum antibody IgA, IgG, IgM, complement C3, C4, and ELISA method kit measurement Cytokine of Serum IL-1, IL-2 content are measured on the CHEM-5 semi-automatic biochemical analyzer; Get test pig venous blood 10ml, adding is added with in the centrifuge tube of 1% aseptic heparin in advance, takes back laboratory and carries out cell culture and NBT detection.
3, gather fresh blood and spleen, adopt mtt assay to carry out peripheral blood lymphocyte and cultivate and the spleen lymphocyte proliferation test, measure T/B lymphopoiesis effect:
1) gets the aseptic anticoagulation of 2.0ml and mix the dilute suspension cell with equivalent Hanks liquid.Cell suspension carefully slowly is added on the lymphocyte separation medium with blood aliquot (2.0ml), blood is overlapped on the layering liquid, the centrifugal 2000rpm 20min of room temperature level.Remove uppermost blood plasma, with capillary tube will be between blood plasma and the layering liquid thin but more clearly milky buffy coat be drawn in the test tube that contains 5ml Hanks liquid, the centrifugal 10min of 1000r/min behind the mixing cleans twice, the lymphocyte separation medium of flush away remnants.Detect cell viability>95% and counting with trypan blue dyeing.With the cell culture fluid that contains 20% hyclone cell is adjusted into 2 * 10
6Individual cell/ml.
2) the every hole of 96 porocyte culture plates adds single cell suspension 100ul and 100ul PHA, and every group is provided with 5 repeating holes, and the culture fluid blank is set simultaneously, puts 5%C0
2, 37 ℃ of cell culture incubators leave standstill and cultivate 48h, stop cultivating every hole adding 5mg/ml tetramethyl azo azoles salt (MTT) solution 20ul continuation cultivation in preceding 4 hours and add DMSO solution 100ul again in every hole after 4 hours, dissolve black-and-blue Jia Za precipitation, use enzyme-linked immunosorbent assay instrument (BIO-RAD) to detect every hole OD570nm afterwards.Result of the test is represented with stimulation index SI:
The blank OD570 of SI=test group OD570/
4, spleen cell culture experiment ConA (Sigma):
1) the aseptic pig spleen of getting, place serum-free RPMI-1640 culture fluid (containing penicillin and streptomycin) plate, flushing repeatedly, carefully spleen is crushed with pin bucket bolt, filter through 400 order stainless steel filtering nets, clean twice (1000rpm/min, 5 minutes) with culture fluid, make cell suspension with the RPMI-1640 culture fluid that contains 20% hyclone.Adjusting the splenocyte number with the RPMI-1640 culture fluid that contains 20% hyclone is 6 * 10
6Individual cell/ml.
2) cell inoculation: cultivate with 96 holes are dull and stereotyped, every hole adds above-mentioned cell suspension 100ul, adds ConA or PHA in addition, and every group is provided with 5 repeating holes, and the culture fluid blank is set simultaneously, puts 5%CO
2, 37 ℃ of cell culture incubators leave standstill and cultivate 48h.
3) mtt assay is surveyed cell proliferation: add 5mg/mlMTT solution 20ul in stopping cultivating preceding 4 hours every holes, continue to put the incubator cultivation and add DMSO solution 100ul again in every hole after 4 hours, concussion is 10 minutes on miniature oscillator, uses afterwards and detects every hole OD570 value under enzyme-linked immunosorbent assay instrument (BIO-RAD) the 570nm wavelength.Result of the test is represented with the cell proliferation rate percentage rate:
The rate of increase (%)=(the blank OD570 of test group OD570-)/blank OD570 * 100%
5, select fresh blood to adopt the NBT reducing process to measure the neutrophil reducing power: get above-mentioned anticoagulation 0.1ml, the NBT that adds equivalent uses liquid, puts into 12 * 75mm plastic tube mixing, and on mouthpiece cover, test tube can be put 37 degree incubators, and jolting therebetween once.The aforesaid liquid taking-up is shaken up, draw 1 end in slide with capillary burette and do push jack, require to release afterbody, push jack wants thickness suitable, dry up with hair-dryer immediately, methanol is 1-2min fixedly, dries up, with 1% husky yellow fluid solution-dyed 5min, the tap water flushing, oily sem observation, counting.
Result of the test is as shown in the table:
Table 1 is the influence of the present invention to DLY ablactational baby pig daily gain, material anharmonic ratio and diarrhea rate;
Table 2 is the influence of the present invention to DLY ablactational baby pig spleen and thymus relative weight;
Table 3 is the influence of the present invention to DLY ablactational baby pig peripheral blood lymphocyte conversion ratio;
Table 4 is the influence of the present invention to DLY ablactational baby pig spleen lymphocyte propagation;
Table 5 for the present invention to DLY ablactational baby pig serum IgG, IgA and IgM level influence;
Table 6 is the influence of the present invention to C3 and C4 level in the DLY ablactational baby pig serum;
Table 7 is the influence of the present invention to IL-1 and IL-2 level in the DLY ablactational baby pig serum;
Table 1
|
Matched group |
Test group |
Eventually heavy (kg) daily gain of starting weight (kg) (g) material anharmonic ratio diarrhea rate (%) |
14.80±1.17 38.89±1.81 535±15.5 1.78±0.08 9.47±1.31 |
14.83±1.22 41.46±1.80 584±13.6 1.61±0.11 4.50±1.34 |
Compare test group daily gain 9.16% (p<0.01), material anharmonic ratio 9.55% (p<0.05), diarrhea rate 52.48% (p<0.01) with matched group
Table 2,
|
Matched group |
Test group |
Spleen (g/kg) thymus (g/kg) |
1.59±0.02 0.54±0.09 |
2.21±0.11 0.62±0.09 |
Compare with matched group, the test group thymus index has raise 38.99%, index and spleen index 14.81% (p<0.01) that raise.
Table 3
|
Matched group |
Test group |
Con induces (SI) LPS to induce (SI) |
1.789±0.09 1.518±0.11 |
2.631±0.156 2.201±0.134 |
Compare with matched group, test group T-lymhocyte transformation rate under Con induces has improved 46.92% (p<0.01) respectively.Induce the B-lymhocyte transformation rate to improve 44.74% (p<0.01) at Lps.
Table 4
|
Matched group |
Test group |
Con induces (SI) LPS to induce (SI) |
1.637±0.09 1.435±0.03 |
2.234±0.229 1.99±0.27 |
Compare with matched group, test group spleen cell conversion ratio under Con induces has improved 35.98% (p<0.01) respectively.Inducing down at LPS, the spleen cell conversion ratio has improved 38.19% (p<0.01).
Table 5
|
Matched group |
Test group |
IgG(g/l) IgA(g/l) IgM(g/l) |
4.25±0.62 0.38±0.02 0.83±0.0 |
5.75±0.43 0.47±0.04 0.86±0.04 |
Compare with matched group, test group porcine blood serum IgG level has improved 35.29% (p<0.01), and the IgA level has improved 23.68% (p<0.01) respectively, and the IgM level difference is not remarkable.
Table 6
|
Matched group |
Test group |
C3(g/l) C4(g/l) |
0.177±0.015 0.046±0.006 |
0.198±0.026 0.055±0.005 |
Compare with matched group, the C3 level has improved 11.86% in the test group serum, and difference is not remarkable.The C4 level has improved 19.57% (p<0.05) in the serum.
Table 7
|
Matched group |
Test group |
IL-1(pg/ml) IL-2(pg/ml) |
2.06±0.2275 16.84±2.198 |
2.756±0.579 22.24±2.011 |
Compare with matched group, the IL-1 level has improved 33.78% in the test group serum, significant difference.The IL-2 level has improved 32.07% (p<0.05) in the serum.
The specific embodiment
Embodiment 1, a kind of animal immunity potentiator take by weighing raw material (kg) by following proportioning:
The Rhizoma Atractylodis Macrocephalae 15, Fructus Crataegi 25, Fructus Hordei Germinatus 15, Rhizoma Atractylodis 8.
Above-mentioned raw materials is simply mixed.
Embodiment 2, a kind of animal immunity potentiator take by weighing raw material (kg) by following proportioning:
The Rhizoma Atractylodis Macrocephalae 10, Fructus Crataegi 30, Fructus Hordei Germinatus 20, Rhizoma Atractylodis 5.
Above-mentioned raw materials is simply mixed.
Embodiment 3, a kind of animal immunity potentiator take by weighing raw material (kg) by following proportioning:
The Rhizoma Atractylodis Macrocephalae 20, Fructus Crataegi 20, Fructus Hordei Germinatus 10, Rhizoma Atractylodis 10.
Above-mentioned raw materials is simply mixed.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.