CN112913975A

CN112913975A - Feed additive for improving immunity of Chinese softshell turtles

Info

Publication number: CN112913975A
Application number: CN202110230675.5A
Authority: CN
Inventors: 包宇航; 徐洁皓; 钱国英; 汪财生
Original assignee: Zhejiang Wanli University
Current assignee: Zhejiang Wanli University
Priority date: 2021-03-02
Filing date: 2021-03-02
Publication date: 2021-06-08

Abstract

The invention provides a feed additive for improving the immunity of Chinese soft-shelled turtles, which can enhance the immunity and disease resistance of the Chinese soft-shelled turtles and simultaneously solve the problems of drug resistance enhancement of pathogenic agents and antibiotic residue in animal bodies caused by the problem of large abuse of the traditional medicine feed additives such as antibiotics and chemicals. The Chinese herbal medicine mixture comprises the following raw material components in parts by mass: 10 parts of astragalus membranaceus, 10 parts of houttuynia cordata, 10 parts of gallnut, 10 parts of honeysuckle, 10 parts of Chinese pulsatilla root, 10 parts of pomegranate bark, 5 parts of coptis chinensis, 15 parts of hawthorn, 10 parts of folium artemisiae argyi and 10 parts of sargassum fusiforme polysaccharide. The feed additive provided by the invention has the functions of regulating the intestinal flora structure, stopping bleeding and attracting food; the immunopotentiator has scientific compatibility, easily obtained materials, low cost and simple preparation method, can be used for a long time, can replace antibiotics, and has the effects of preventing bacterial diseases and enhancing immunity.

Description

Feed additive for improving immunity of Chinese softshell turtles

Technical Field

The invention belongs to the technical field of feed additives, and particularly relates to a feed additive for improving the immunity of Chinese softshell turtles.

Background

At present, the cultivation of Chinese softshell turtles in China is developed to an industrialized cultivation road, the cultivation intensification degree is continuously improved, the cultivation scale is continuously enlarged, and the cultivation density is also continuously increased. With the expansion of the culture scale, diseases of the Chinese soft-shelled turtles during the culture period also frequently occur. More than 30 diseases of the Chinese softshell turtles are known, wherein the skin rot, hemorrhagic enteritis, costalitis and the like are diseases with high morbidity, and the immunity of the Chinese softshell turtles is reduced to cause various diseases to burst, mainly because the immunity is reduced to cause the weakening of the defense mechanism of the organism, bacteria and viruses are easy to invade the organism, so a series of diseases are caused. The morbidity of greenhouse cultivation is high, the morbidity area of the greenhouse cultivated Chinese softshell turtles is about 30-50% according to statistics, and the mortality rate is about 20-50%. Therefore, the disease causes the annual economic loss of the turtle raising industry to reach hundreds of millions of yuan, and seriously threatens the healthy development of the turtle raising industry in China.

The aquatic medicament antibiotics and chemicals play an important role in preventing and treating the diseases of the aquatic animals, and become one of important means for preventing and treating the diseases of the aquatic animals due to the characteristics of obvious curative effect, simple and convenient production and use, low cost and the like. However, the use of antibiotics and chemicals in a large amount not only can cause drug residues in aquatic products and harm human health, but also can cause the problems of microbial drug resistance, water environment pollution and the like.

The Chinese herbal medicine serving as a natural medicine has the advantages of small toxic and side effects, less residue, unobvious drug resistance and the like in the aspect of preventing and treating aquatic animal diseases, so that the Chinese herbal medicine has the drug property in preventing and treating the diseases, and can play the purposes of preventing and treating the diseases through the nutritional effect, the immune enhancement effect, the hormone-like effect and the like of the aquatic animals. At present, the research on the effect of Chinese herbal medicines on improving the immunity of the Chinese soft-shelled turtle is still relatively deficient, and if the Chinese herbal medicines used by other animals are directly adopted, the Chinese soft-shelled turtle has poor treatment effect on diseases or has treatment effect, but the growth of the Chinese soft-shelled turtle is seriously influenced.

Disclosure of Invention

The invention aims to provide a feed additive for improving the immunity of Chinese soft-shelled turtles, which can enhance the immunity and disease resistance of the Chinese soft-shelled turtles and simultaneously solve the problems of drug resistance enhancement of pathogenic agents and antibiotic residue in animal bodies caused by the wide abuse of the traditional medicine feed additives such as antibiotics and chemicals.

The invention firstly provides a Chinese herbal medicine mixture which comprises the following raw material components in parts by mass: 10 parts of astragalus membranaceus, 10 parts of houttuynia cordata, 10 parts of gallnut, 10 parts of honeysuckle, 10 parts of Chinese pulsatilla root, 10 parts of pomegranate bark, 5 parts of coptis chinensis, 15 parts of hawthorn, 10 parts of folium artemisiae argyi and 10 parts of sargassum fusiforme polysaccharide.

The Chinese herbal medicine mixture provided by the invention is used for preparing a feed additive;

the feed additive is a turtle feed additive;

the invention also provides a turtle feed, wherein the feed is added with a feed additive prepared from the Chinese herbal medicine raw materials;

preferably, the feed additive is added into feed in an amount of 1-2%.

The feed additive provided by the invention has the functions of regulating the intestinal flora structure, stopping bleeding and attracting food; the immunopotentiator has scientific compatibility, easily obtained materials, low cost and simple preparation method, can be used for a long time, can replace antibiotics, and has the effects of preventing bacterial diseases and enhancing immunity.

Drawings

FIG. 1 is a dilution curve of the results of high throughput sequencing of various intestinal samples according to the present invention;

FIG. 2 is a graph showing a comparison of the diversity of samples in the intestine according to the present invention;

FIG. 3 is a graph showing the correlation analysis of bacterial diversity in various samples of the intestine according to the present invention (the figure is the number of OTUs in each sample);

FIG. 4 is a visual comparison of sample abundance;

figure 5 is a graph of similarity and difference in composition of multiple samples at each classification level.

Detailed Description

According to the invention, through scientific compatibility, a compound of a plurality of Chinese herbal medicines is used as a Chinese herbal medicine feed additive and is added into the powder for the conventional soft-shelled turtles, and the continuous feeding test result shows that the activity of each immunoenzyme of the Chinese soft-shelled turtles in the test group added with the Chinese herbal medicines is enhanced, the death rate during the test period is obviously lower than that of the control group, the body weight of the test group is slightly higher than that of the control group, and the difference change of the immunoenzymes in the blood serum of the two groups of Chinese soft-shelled turtles is.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1: preparation of Chinese soft-shelled turtle feed additive

Mixing 10 parts of dried astragalus membranaceus, 10 parts of houttuynia cordata, 10 parts of gallnut, 10 parts of honeysuckle, 10 parts of Chinese pulsatilla root, 10 parts of pomegranate rind, 5 parts of coptis chinensis, 15 parts of hawthorn, 10 parts of folium artemisiae argyi and 10 parts of sargassum fusiforme polysaccharide, preparing the mixture into coarse powder by using a common pulverizer, performing micro-powder processing by using a super-micro pulverizer, enabling the crushed powder to pass through a 200-mesh sieve with the granularity of 80%, and storing the processed Chinese herbal medicine compound powder in a cool and dry place for later use as a feed additive.

Example 2: external bacteriostasis experiment of water extract liquid of Chinese herbal medicine additive

(1) Preparing a Chinese herbal medicine liquid extract:

respectively drying compound Chinese herbal medicines and 10 antibacterial Chinese herbal medicines (folium Artemisiae Argyi, radix Pulsatillae, Coptidis rhizoma, radix astragali, flos Lonicerae, pericarpium Granati, fructus crataegi, Galla chinensis, herba Houttuyniae, and Cyrtymenia Sparsa polysaccharide) in 50 deg.C constant temperature drying oven to constant weight, and grinding into powder. Weighing 10g of the single Chinese herbal medicine powder and the compound powder respectively, and adding distilled water according to the proportion of 1:10 to soak for 30 min. Decocting in an electromagnetic oven until boiling, decocting with slow fire for 30min, filtering with 4 layers of gauze, and collecting filtrate. Decocting the filtered Chinese medicinal materials with water, and filtering for 2 times. Mixing the filtrates, decocting, and concentrating to 10mL to obtain medicinal liquid with crude drug content of 1 g/mL. Pouring the water extract into a 50ml centrifuge tube, autoclaving, and storing at 4 ℃ for later use.

(1) The Chinese herbal medicine liquid extract in vitro bacteriostasis test:

1. and (3) purifying and amplifying strains:

test strains for in vitro bacteriostatic testing include: pseudomonas hydrophila, Bacillus thuringiensis, Staphylococcus aureus, pneumococcus, and Chryseobacterium. The 5 bacteria to be tested were inoculated into 10ml of LB broth for enrichment, and cultured at the optimum temperature for 12H.

2. And (3) diluting the bacteria concentration:

adding a proper amount of LB liquid into a conical flask according to the proportion of 1:100, shaking the bacteria for 3-4h, and measuring the OD value of the bacteria by using an ultraviolet spectrophotometer. Diluting the bacterial concentration to 1.5x10 according to the standard curve of the corresponding bacterial strain⁸About one/ml, and pouring into a 50ml centrifuge tube for later use.

3. Coating and placing an oxford cup:

the diluted bacterial liquid is spread on an LB broth culture medium, and each plate contains 100 mu L of the bacterial liquid. 3 sterilized Oxford cups are placed in each plate, and 100 mu L of Chinese herbal medicine extract is added into the Oxford cups. After culturing for 24h at the optimum temperature of each bacterium, measuring the size of the inhibition zone on the plate, and performing 3 parallel repeated experiments on each Chinese herbal medicine. And calculating the average value of the diameters of the inhibition zones and the standard deviation of the sample data to be used as the measurement standard of the antibacterial abilities of different Chinese herbal medicines to different strains.

(3) Analyzing the result of the bacteriostatic experiment:

the in-vitro bacteriostatic effect of 10 single prescriptions in the Chinese herbal medicine compound and the formula on 5 common aquatic pathogenic bacteria is determined by an oxford cup method. The results show that different strains have certain differences in sensitivity to different Chinese herbal medicines, and in the whole, the 4 traditional Chinese medicines of pomegranate bark, hawthorn, Chinese gall and coptis chinensis have the most obvious bacteriostatic effect on 5 test bacteria, and the average bacteriostatic diameter of the four medicines on 5 bacteria is larger than 18 mm; secondly, the three traditional Chinese medicines have certain bacteriostatic effects, and the average bacteriostatic diameter of the three traditional Chinese medicines to 5 bacteria is about 14 mm; and some Chinese herbal medicines have weak bacteriostatic effect, such as honeysuckle and sargassum fusiforme polysaccharide, and the average bacteriostatic diameter of the Chinese herbal medicines to 5 kinds of bacteria is about 10 mm. In addition, the astragalus has no obvious bacteriostatic effect on 5 bacteria; pulsatilla chinensis has no bacteriostatic effect on staphylococcus aureus and pseudomonas hydrophila, but has weak bacteriostatic effect on bacillus thuringiensis, pneumococcus and chryseobacterium (see table 1).

Table 1: bacteriostatic circle (diameter mm) of Chinese herbal medicine liquid extract to common pathogenic bacteria

The relationship between the diameter of the inhibition zone of the Oxford cup method and the inhibition capacity is as follows: (diameter of zone of inhibition D)

D is less than 10mm, and has no antibacterial effect

Has a D of 10mm to 15mm and has a mild antibacterial effect

D is less than 20mm and has moderate antibacterial effect

D is greater than 20mm, and has high antibacterial effect

For the 5 pathogenic bacteria, pomegranate rind, hawthorn, Chinese gall and coptis have strong bacteriostatic ability, and then houttuynia cordata, folium artemisiae argyi and a compound preparation are used, so that honeysuckle and sargassum fusiforme polysaccharide have weak bacteriostatic ability, astragalus membranaceus has no bacteriostatic action, and the Chinese pulsatilla has a certain pertinence to strains. The compound Chinese herbal medicine preparation of the invention has obvious bacteriostatic action on most of the common pathogenic bacteria.

Example 3: detection of influence of Chinese herbal medicine feed additive on intestinal flora of Chinese soft-shelled turtles

(1) Grouping and processing experimental animals: 103 Chinese three months old are purchased from a certain culture farm in Huzhou, the average weight is 45.53g, the posture is good, the mental activity is good, 100 Chinese medicinal herbs are selected, the Chinese medicinal herbs. The first 1-5 groups were experimental groups, the second 6-7 groups were control groups, the mean body weight of the treated groups was 48.46g, and the body weight of the control groups was 41.88 g. The Chinese soft-shelled turtles in the test are cultured in water tanks in the same environment, the water temperature is maintained at 28 +/-2 ℃, and water is changed every 2-3 days. Wherein, the experimental group is mixed with the feed and fed with the Chinese herbal medicine additive, 20g (2%) of the Chinese herbal medicine compound preparation is added to each kg of the compound feed powder, the control group is only fed with the compound feed powder, the feeding is carried out for 1 time every day, and the feeding is continuously carried out for 37 days.

(2) Sampling and high throughput sequencing analysis: after the experiment feeding is finished, respectively taking 10 Chinese soft-shelled turtles from an experimental group and a control group which are fed with Chinese herbal medicine compound micro-powder at random, placing the Chinese soft-shelled turtles in an anatomical plate, wiping the body surfaces of the Chinese soft-shelled turtles with 75% alcohol cotton balls, dissecting the bodies of the soft-shelled turtles with sterilized scissors and tweezers, stripping off intestinal tracts, shearing midgut tissues with scissors, extruding contents into a sterile centrifugal tube, and respectively taking 20 samples which are respectively a turtle body intestinal tract sample (CPG1-10) which is fed with Chinese herbal medicine compound preparations and a turtle body intestinal tract sample (CG1-10) which is fed with a common feed control group. Total bacterial DNA was extracted from the samples using the QIA amp DNA pool Mini Kic (Qiagen). The primers 5'-CCTACGGGNGGCWGCAG-3' and 5'-GACTACHVGGGTATCTAATCC-3' are used for PCR amplification of the bacterial 16S rDNA V3-V4 region sequence in the sample. The total volume of the reaction system for PCR was 25. mu.L, containing

Hot Start Flex 2X Master Mixart version 12.5. mu.L, Forward Primer (1. mu.M) 2.5. mu.L, Reverse Primer (1. mu.M) 2.5. mu.L, Template DNA 50ng, sterile deionized water was added to 25. mu.L. And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 45s for 35 cycles; finally, extension was carried out at 72 ℃ for 10 min. The amplified DNA sequence and the PCR product were confirmed by 2% agarose gel electrophoresis. Ultrapure water was used instead of the sample solution throughout the DNA extraction process to exclude the possibility of false positive PCR results as negative controls, PCR products were purified by AMPure XT bead (BeckmanCoulter Genomics, Danvers, MA, USA), and quantit (Invitrogen, USA) was quantified. The amplicon pool was used for sequencing and the size and number of amplicon pools were evaluated on a library quantification kit from an Agilent2100 bioanalyzer (Agilent, usa) Illumina (KapaBiosciences, Woburn, MA, usa), respectively. The libraries are ordered on the NovaSeqPE250 platform.

(3) Data analysis flow: samples were sequenced on the Illumina NovaSeq platform according to the manufacturer's recommendations, supplied by LC-Bio. The paired end sequences were assigned to the samples according to their unique barcodes, and the barcode and primer sequences introduced by the pooling were removed. And using FLASH to merge the reading of the matching end. The raw read data is quality filtered under specific filtering conditions to obtain a high quality clean label according to fqtrm (v 0.94). The chimeric sequence was filtered using Vsearch software (v2.3.4). Demodulation is performed using DADA2 to obtain a feature table and a feature sequence. Diversity and diversity were calculated by normalizing to the same random sequence. The feature abundances are then normalized by the relative abundance of each sample according to the SILVA (release 132) classifier. Alpha diversity is used to analyze the complexity of sample species diversity by 5 indices including Chao1, Observed, species, Goods coverage, Shannon, Simpson, all of which are calculated using QIIME2 in our samples. Beta diversity was calculated by QIIME2, plotted in R-package. Sequence alignment was performed using Blast, and each representative sequence was annotated with the SILVA database for the signature sequences.

(4) Quality analysis of sequencing results: the high throughput sequencing data for the 4 samples were counted (see table two). The results show that the number of the high-quality sequences obtained by each sample is more than 4 ten thousand, the high-quality sequences are kept at a corresponding level, the ratio of effective data is more than 90%, the proportion of data with the data quality of more than or equal to Q20 in the effective data is more than 95%, the proportion of data with the data quality of more than or equal to Q30 in the effective data is more than 90%, and the GC content of the data in the effective data is also kept more than 50%. And (5) the sampling depth is proved to be credible.

Alpha diversity refers to the diversity within a particular environment or ecosystem, primarily to reflect species abundance and uniformity and sequencing depth (see Table 3). Alpha diversity reflects richness and uniformity primarily through indexes of Chao1, underlying scenes, Goods _ coverage, shannon, Simpson, and the like. Chao1, the supplemented species index mainly reflects the species abundance information of the sample; the experimental group is slightly more abundant than the control group. Wherein, Chao1 and observed _ species (see figures 1-3 and 1-4) mainly estimate the number of species contained in the community, Goods coverage (see figure 1-1) refers to the coverage rate of microorganisms, the higher the value of the coverage rate is, the lower the probability that a new species is not detected in a sample is, and the index actually reflects whether the sequencing result represents the true condition of the sample or not; the Shannon index (see fig. 1-2) is derived from the entropy of information, and the larger the Shannon index, the larger the uncertainty. The larger the uncertainty is, the more unknown factors in the community are represented, namely the diversity is high; simpson (see FIGS. 1-5) ranges from 0 to 1, where Simpson is a minimum of 0 when there is only one species in the population, and is also the smallest diversity we intuitively understand. When the species are infinite (highest abundance) and the number of species are consistent (highest uniformity), the Simpson value is at most 1. The method is characterized in that the diversity index value of the microbial community in the intestinal contents of an experimental group is slightly higher than the community diversity of a control group, QIIME software is used for randomly sampling the total number of sequences of each sample in an OTU abundance matrix at different depths, a sparse curve (shown in figure 1) is drawn according to the number of the sequences extracted at each depth and the number of the OTUs corresponding to the number of the sequences, and as the randomly extracted number of the sequences is further increased, the curve of each sample gradually becomes gentle, so that the effective sequencing number can better cover the diversity of the strains, the sampling depth is credible, and the sequencing result can truly reflect the number relation of dominant bacteria in the sample.

Table 2: effective data statistical table

Table 3: alpha diversity analysis Table

(5) Analysis of bacterial diversity and abundance of dominant OTU species in each sample: making a Rank-abundance graph (see fig. 2) by using R language tool statistics and mapping software, wherein each broken line represents OTU abundance distribution of a sample, the length of the broken line on a horizontal axis reflects the number of OTUs in the sample and represents the abundance of a community, and the longer the broken line is, the more the number of OTUs in the sample is; the gentle degree of broken line has then reflected the degree of consistency that the community is constituteed, and the more gentle broken line, the degree of consistency that the community is constituteed is higher, and the broken line is steeper, and the abundance difference between each OTU in the community is bigger, and the degree of consistency is lower.

The results show that the colony enrichment degree of the intestinal Content (CPG) sample after the Chinese herbal medicine feeding is obviously higher than that of the intestinal Content (CG) sample before the control group is fed, and the colony composition uniformity is also higher than that of the sample before the feeding. The correlation of bacterial diversity was analyzed for each sample and a VENN plot was constructed (see fig. 3). 324 OTUs are contained in intestinal contents of the Chinese softshell turtles after the Chinese herbal medicines are fed. The Chinese herbal medicine Chinese soft-shelled turtle community enrichment degree is higher than that of a control group Chinese soft-shelled turtle, and the Chinese herbal medicine is fed with 403 different OUT.

And further carrying out statistics on bacterial phyla and relative abundance in different samples, selecting 30 species classes with the highest abundance by default according to a species abundance table and a species annotation table, and displaying the relative abundance of each sample/group in different forms. The histograms are presented in stacked histogram form (see fig. 4) to facilitate more intuitive comparison of sample abundances.

In each level, the expression of the dominant bacteria and the change trend in different treatments can be visually seen. The results shown in FIG. 4-1 show that Proteobacteria (Proteobacteria), Firmicutes (Firmicutes), Thielavia (Tenericutes), Bacteroides (Bacteroides), Actinomycetes (Actinobacillus), Acidobacterium (Acidobacterium), Cyanobacterium (Cyanobacterium) and Fusobacterium (Fusobacteria) are the dominant phylum of each sample. Wherein the bacterial flora of Proteobacteria and Thelephora accounts for more than 75% of the total flora. Further statistical results of dominant bacteria in each sample show that, as shown in fig. 4-2, the dominant bacteria mainly include Rhizobiales (Rhizobiales), lactobacilli (lactobacilli), bacilli (bacilliales), Burkholderiales (Burkholderiales), cladoniales (mycoplasmates), Clostridiales (Clostridiales), pseudomonales (pseudomonales), Enterobacteriales (Enterobacteriales), wherein the ratio of lactobacilli to bacilli in the intestinal content samples of the experimental group is slightly increased compared with that of the control group, while the ratio of bacteria in the cladoniales is significantly decreased, and the ratio of bacteria in other objects is not significantly different and changed.

According to the relative abundance table of the samples, the community composition data with the highest 30 relative abundances of each classification level is clustered according to the abundance distribution of the classification units or the similarity degree among the samples, the classification units and the samples are respectively sorted according to the clustering result, and are presented through a heat map (see figure 5). Through clustering, the high-abundance and low-abundance classification units can be distinguished, and the similarity and the difference of the compositions of a plurality of samples at each classification level are reflected by color gradient and similarity degree. The gradual change from blue to red in the graph reflects the change of the abundance from low to high, and the closer to blue, the lower the abundance is, and the closer to red, the higher the abundance is. The abundance of the Chinese herbal medicine feeding experimental group is higher than that of the control group.

The analysis result of high-throughput sequencing data shows that Proteobacteria (Proteobacteria) and Firmicutes (Firmicutes) occupy the dominant positions in the intestinal tracts of cultured Chinese soft-shelled turtles, which indicates that the Proteobacteria and the Firmicutes are the inherent bacterial phyla of the intestinal tracts of the soft-shelled turtles, but the roles of the Proteobacteria and the Firmicutes in the intestinal tracts of different soft-shelled turtles are still unclear at present. The statistical result based on the dominant flora ratio shows that the ratio change of the microbial flora in the intestinal tract of the Chinese soft-shelled turtle fed with the Chinese herbal medicines is slightly larger than that of the Chinese soft-shelled turtle in a control group. The analysis result of the VENN graph also shows that the difference OTU quantity of the intestinal contents of the experimental group and the control group is larger than that of the intestinal wall sample, which indicates that the flora of the intestinal contents is more easily influenced by external factors, and the compound Chinese herbal medicine feed additive has certain favorable influence on the intestinal tract of the Chinese soft-shelled turtle.

Early studies indicate that an intestinal micro-ecosystem constructed by intestinal microorganisms and a host plays a crucial role in the processes of host digestive metabolism, intestinal development, homeostasis, immune response, disease resistance and the like, so that the intestinal microorganisms are also called as external organs of the host by many scholars, a normally balanced intestinal microorganism can positively affect the intestinal immune regulatory function of the host, and a disproportionate microorganism can cause the immune regulatory function disorder of the host and increase the occurrence probability of diseases. With the acceptance of Chinese herbal medicines by more and more people, research on the influence and action mechanism of the Chinese herbal medicines on intestinal flora is also carried out to a certain extent. The invention shows that the colony enrichment degree of the intestinal tract content sample after the Chinese herbal medicine is fed is higher than that of the control group sample, and the colony composition uniformity is higher than that of the control group. The pseudomonas and mycoplasma flora suggest that there is a certain correlation with enteritis, and the ratio of bacteria of the order lactobacillales and bacillales in the intestinal tract is increased after Chinese herbal medicine feeding treatment, which is beneficial to intestinal health.

Example 4 detection of immune effect of Chinese herbal medicine feed additive on Chinese soft-shelled turtles

(1) Sample treatment: the blood serum collection adopts a carotid blood collection method, and the specific steps are that the feeding is stopped for 24 hours after the test is finished for 37 days. Randomly taking 18 Chinese medicinal soft-shelled turtles (divided into 3 groups, each group comprises 6) from the experimental group and the control group, and injecting into the abdominal cavity with 10 injections⁷CFU/ml Aeromonas hydrophila 1ml, blood collection in 12h, 24h and 72h respectively, and then 6 blood collection from the control group and the experimental group respectively as 0h control. Feeding Chinese herbal medicine 10⁷The CFU/ml aeromonas hydrophila challenge groups are named as CPG0h, CPG12h, CPG24h and CPG72h, 6 in each group, and the control group 10⁷The CFU/ml aeromonas hydrophila challenge groups are named as CG0h, CG12h, CG24h and CG72 h.

(2) Collecting samples: weighing two groups at the time points specified in the test, exposing the neck of the Chinese soft-shelled turtle, fixing with hemostatic forceps, cutting carotid artery with a scalpel, discharging blood, collecting with a collection tube prepared in advance and sterilized with the specification of 5ml, centrifuging for 1min at 8000r/min in a centrifuge, and separating serum and blood cells. The whole blood sampling process needs to be rapid to prevent blood from solidifying.

(3) Non-specific immune index determination: determination of SOD (superoxide dismutase) and LZM (lysozyme) ACP (acid phosphatase) AGP (alkaline phosphatase), the treated serum is subjected to determination of various indexes according to the kit instruction (the kit is purchased from Nanjing to build a bioengineering research institute).

(4) Data processing: ONE-WAY analysis of variance (ONE-WAY ANOVA) was performed using SPSS19.0 software, and multiple comparisons were tested for significance of differences using Duncan's and results are expressed as mean. + -. standard error (x. + -. SE).

(5) And (4) analyzing results: as shown in Table 4, the activities of superoxide dismutase (SOD), Lysozyme (LZM) and alkaline phosphatase (AGP) in the serum of the Chinese soft-shelled turtle fed to the Chinese herbal medicine experimental group, except for acid phosphatase (ACP), are all significantly different from those of the Chinese soft-shelled turtle in the control group. There were no significant differences within each group, following 10⁷The toxicity attacking time of CFU/ml aeromonas hydrophila is increased, the immunoenzyme activities in the serum of the Chinese softshell turtle in CPG12h, CPG24h, CPG72h, CG12h, CG24h and CG72h groups are increased along with the increase of the toxicity attacking time, the immunoenzyme activities in the experimental group and the control group after the experiment is finished (CPG0h and CG0h) are shown by the non-toxicity attacking experimental group and the control group, and the immunoenzyme activity in the Chinese herbal medicine feeding experimental group is obviously higher than that in the experimental group.

Table 4: influence table of compound Chinese herbal medicine on immune enzyme indexes of Chinese softshell turtles

Adjacent letters indicate significant difference (p <0.05) alternate alphabets indicate very significant difference (p <0.01) the same letter indicates no significant difference.

Example 5 detection of growth index of Chinese softshell turtle by compound Chinese herbal medicine

(1) Growth indexes are as follows: after the Chinese herbal medicine test is finished for 37 days, and the feeding is stopped for 24 hours, weighing the Chinese soft-shelled turtles in the experimental group and the Chinese soft-shelled turtles in the control group respectively, recording, and calculating the weight gain rate, the death rate, the feed coefficient and the like. The calculation formula is as follows:

weight gain ratio (CR) (test end mass-test initial mass) (g)/test initial mass (g)

Mortality rate is the number of deaths/initial population tested

Feed Factor (FCR) ═ total feed intake (g)/(test end mass-test initial mass) (g)

(2) Results and analysis: as can be seen from table 5, after the experiment, the growth indexes of the experimental groups fed with the compound Chinese herbal medicines are all higher than those of the control group, and the weight average of the experimental trionyx sinensis bodies of each group is increased, wherein the weight gain of the experimental groups is higher than that of the control group, and the weight gain rate of the experimental groups is slightly higher than that of the control group, (the experimental period is short, and results of insignificant differences of growth indexes are generated for animals with long growth periods such as the trionyx sinensis) the feed coefficient experimental groups are obviously lower than that of the control group, which indicates that the absorption utilization rate of the feed by the experimental groups fed with the compound Chinese herbal medicines is higher than that of the control group, and the mortality rate is low.

Table 5: influence of compound Chinese herbal medicine on growth indexes of Chinese softshell turtles

To summarize: a plurality of results of the experiment show that the enrichment degree, the activity and the growth index of the intestinal flora of the experimental group fed with Chinese herbal medicines are higher than those of the control group. The results show that the compound Chinese herbal medicine additive has important influence on the immunity of the Chinese softshell turtles.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. The Chinese herbal medicine mixture is characterized by comprising the following raw material components in parts by mass: 10 parts of astragalus membranaceus, 10 parts of houttuynia cordata, 10 parts of gallnut, 10 parts of honeysuckle, 10 parts of Chinese pulsatilla root, 10 parts of pomegranate bark, 5 parts of coptis chinensis, 15 parts of hawthorn, 10 parts of folium artemisiae argyi and 10 parts of sargassum fusiforme polysaccharide.

2. Use of a herbal mixture according to claim 1 in the preparation of a feed additive.

3. The use of claim 2, wherein the feed additive is a turtle feed additive.

4. The use of claim 3, wherein the turtles are Chinese soft-shelled turtles.

5. A feed for terrapin, which is characterized in that the Chinese herbal medicine mixture of claim 1 is added into the feed for terrapin.

6. A feed as claimed in claim 5, wherein the amount of the herbal mixture is 1-2% by weight.

7. A turtle feed according to claim 5 or 6, wherein the turtles are Chinese turtles.