Preparation Of Bovine Microecological Agents And Evaluation Of Its Clinical Application

Microecological agents can not only prevent diseases and promote the growth and development of animal organisms,but also have the advantages of being green,safe and residue-free,so they are widely used in the livestock breeding industry.It was found that,due to the species specific adhesion of probiotics,developing bovine microecological agents using the probiotics from cattle has good application value and application prospects.However,the reports of microecological agents for cattle are not enough.Therefore,the aim of this study was to further explore the bovine microecological agents,and evaluate their clinical effects.Probiotic potential strains were isolated from the six segments of the intestinal tract of Dabieshan cattle,and were effectively screened by methods such as morphological detection,Gram stain,and microscopic examination;the screened strains were sequenced for 16 S r DNA gene,and the results were compared with the Gen Bank database for homology;acid tolerance test and bile salt tolerance test were carried out on the strains respectively,and two strains with high survival rate under acid and bile salt environment were selected;the bacterial strain was expanded by culture and prepare the compound microecological agents for subsequent clinical feeding experiments.Twelve healthy weaned Dabieshan calves with similar body size were selected and randomly divided into three groups by random number table method: control group(CG),Bacillus group(BG),experimental group(EG).The CG was fed a basic diet,the BG was a fed basal diet plus bacillus subtilis and the EG was a fed basal diet plus compound microecological agents.The experiment was conducted for 30 days.Blood samples were collected from all calves via jugular vein on day 0,15 and 30,and serums were separated to detect the biochemical indexes,immune indexes,cytokines and oxidation and antioxidant indexes.On the 30 th day of the experiment,cow manure was collected for high-throughput sequencing to detect the abundance and structural changes of its flora.The results were as follows:1.A total of 23 strains were isolated from the intestine of Dabieshan cattle,and 12 strains were excluded by morphological examination and staining microscopy.The remaining 11 strains were sequenced with 16 S r DNA gene.After identification,the 11 strains contained 6 strains of Bacillus and 5 strains of Lactobacillus.2.The acid resistance results of Bacillus showed that the four Bacillus strains B2,B3,B5 and B6 were able to tolerate the p H 2 PBS solution,and the survival rates were 48.59%,73.89%,84.91% and 54.20% respectively.After that,the four Bacillus strains were tested for bile salt tolerance,the results showed that B2,B3 and B5 were able to tolerate 0.6% of bile salt solution,and the survival rates were 23.44%,27.44% and 68.16% respectively.The acid resistance results of Lactobacillus showed that L1,L3,L4 and L5 were able to tolerate the p H 2 PBS solution,and the survival rates were 40.55%,70.23%,42.69% and 58.99% respectively.Then,the four strains were tested for bile salt tolerance,and the results showed that L3 and L4 were able to tolerate 0.6% of bovine bile salt solution,and the survival rates were 50.57% and 39.10% respectively.Considering that the strains with high survival rate in both acid and bile salt can achieve better results as microecological agents,B5 and L3 were chose.3.The test results of serum biochemical parameters showed that,compared with CG group,there was no significant difference on ALT,AST,TP,ALB,GLB,UREA in BG group and EG group.The increase of ALP content was extremely significant(P <0.01).4.The test results of immune indicators showed that,compared with CG group,the increase of Ig G content in BG group and EG group was extremely significant(P <0.01).And at day 30,the Ig G content in EG group was significantly higher than that in BG group(P <0.05).The Ig M content in EG group was significantly higher than that in CG group and BG group to the extreme(P <0.01).The Ig A content in BG group and EG group was not significantly different from that in CG group.5.Compared with CG group,the concentrations of IL-1 and TNF-? in BG group and EG group were not significantly different.The IL-2 content in EG group was significantly higher than that in CG group(P <0.05),but was not significantly different from that in BG group.However,with the extension of feeding time,the IL-2 content in BG group showed an upward trend.The IL-6 content in BG group and EG group was significantly higher than that in CG group(P <0.05).The IFN-? content in BG group and EG group was significantly higher than that in CG group(P <0.05).6.Compared with CG group,there was a significant increase in all the three indicators of T-AOC,GSH-Px and CAT(P <0.05)in BG group and EG group,and there was no significant difference between BG group and EG group.The content of MDA in BG group and EG group was significantly lower than that of CG group.The activity of SOD in EG group was significantly higher than that of CG group to the extreme(P <0.01),and the activity of SOD in BG group was significantly higher than that of CG group(P <0.05),the difference between BG group and EG group was not significant.7.Intestinal high-throughput sequencing results showed that,there was no significant change in the abundance of intestinal flora between groups,but the proportion of Bacteroidetes microorganisms in EG group and BG group increased,and the increase in EG group was more significant than that in BG group.Meanwhile,the proportion of Proteobacteria microorganisms in EG group and BG group decreased,and the decrease was more significant in EG group than that of BG group.In conclusion,the addition of bovine-derived microecological agents to the diet of weaned Dabieshan cattle can promote their growth and development,enhance the immune function and antioxidant level,and improve disease resistance of organism.Meanwhile,the compound microecological agent can improve the intestinal flora structure of the Dabieshan cattle,increase the number of bacterial flora,decrease the number of harmful bacterial flora,and its effect on the body is better than that of single microecological agents.