Identification Of Probiotics And Effects Of Probiotics On Weaned Piglets, Calves And The Gastrointestinal Microbiate

In this study, two different strains of microorganisms were isolated from soil and identified by16SrRNA molecular technology, and the properties of these strains were evaluated in vitro. The in vivoeffects of these microbes as probiotics in weaned piglets and pre-and post-weaning calves wereconducted. The present study included6trials which described as follows.Experiment1: Isolation and identification of Lactobacillus sp. GF103and assessment of it asa potential probioticA Lactobacillus sp. strain GF103which inhibited the growth of pathogenic bacteria E.coli andStaphylococcus aureus was isolated from soil nearby Beijing Daxing Breeding pig farm. It wasidentified by16S rRNA sequence analysis as Lactobacillus plantarum (GenBank accession number:JN560899). In vitro assessments showed that a large amount of the strain GF103survived under thecondition of pH=3. The strain GF103had tolerated0.3%of bile salt. The viable counts of strain GF103did not change after0.5h of growing in simulated gastric fluid and decreased1log value after3h. Theviable counts of strain GF103did not decrease in simulated small intestinal fluid after3h. The strainlikely survived and propagated in the animal’s intestinal tract. These results suggested that the strainGF103possessed probiotic properties and should be further studied its application in animals as feedadditives.Experiment2: Isolation and identification of Bacillus subtilis B27and assessment it aspotential probioticsThirty-six Bacillus sp. were isolated from soil and the strain B27inhibited the growth ofpathogenic bacteria E. coli, staphylococcus aureus and salmonella. By16S rRNA sequence analysis itwas identified as Bacillus subtilis (GenBank accession number: JQ673431). In vitro assessment showedthat72%of the strain B27survived under the condition of pH=3. It tolerated0.3%of bile salt. Theviable counts of strain GF103in simulated gastric and small intestinal fluid after3h were87.7and96.7%, respectively. This strain likely survived and propagated in the animal’s intestinal tract. Theseresults indicated that Bacillus subtilis B27had probiotic properties and could be used as feed additives.Experiment3: Effects of dietary probiotics on growth performance, fecal microbiota andserum profiles in weaned pigletsOne hundred and forty-four piglets of Large White×Landrace weaned at35-37days of age wereselected and divided into four groups, and the piglets from each group were assigned randomly to sixpens (replicates) with6animals each. Each group was fed one of four diets for5weeks: a basal dietwithout antibiotics and probiotics (control), or the basal diet supplemented with Lactobacillusplantarum GF103, Bacillus subtilis B27, or a mixture of Lactobacillus plantarum GF103and Bacillussubtilis B27. During the first two weeks of the trial, the piglets supplemented with probiotics had lower(P<0.05) average daily feed intake (ADFI) than control. The feed conversion ratio (FCR) was improved(P<0.05) in probiotic-supplemented groups when compared with that of control. The counts of E. coli in feces of the piglets supplemented with Lactobacillus plantarum GF103was lower (P<0.05) than that ofcontrol. On day14, dietary supplementation of the combination of Lactobacillus plantarum GF103andBacillus subtilis B27increased (P<0.05) the serum concentration of total protein, globulin, andcreatinine, but decreased (P<0.05) the ratio of serum albumin to serum globulin when compared withcontrol. On the same day, probiotic-supplemented piglets had increased (P<0.05) serum IgMconcentrations compared with control animals. Supplementation of Bacillus subtilis B27or thecombination of Lactobacillus plantarum GF103and Bacillus subtilis B27increased (P<0.05) the serumIgA concentrations at the end of the trial. These results indicated that dietary probiotics improvedgrowth performance and enhanced immune responses at the early stage of the post-weaning period inpiglets.Experiment4: Changes of intestinal bacteria in weaned piglets supplemented with probioticsThe experiment design was the same as experiment3. The trial period lasted for35d. On14and35d,4piglets from each treatment (one from each pen, totally16piglets in each period) wereslaughtered for sample collection. The intestinal tissues were taken for histological masurements and thecontents of intestinal were collected for analysis of bacteria. The results showed that, during the firsttwo weeks after weaning, the piglets supplemented with probiotics had lower pH in gastral fluids thanthat in control (P<0.05), the piglets supplemented with complex probiotics also had lower pH induodenal fluids (P<0.05). There were no significant difference between on intestinal samples of pigletssupplemented with probiotics and control group (P>0.05). Compared with the first two weeks, thespecies of intestinal microbiota became more diverse in the last three weeks. The dietary supplementedwith probiotics was more effective at the early stage of the post-weaning period than that at the laterstage in piglets. Although the number of total bacteria, lactic acid bacteria and E.coli in the digestivetract of piglets was not influenced by probiotics, the piglets supplemented with probiotics tended tohave a decreased population of E.coli in the intestinal tract. These results indicated that the probioticsimproved intestinal bacterial community at the early stage. The species of intestinal bacteria becamemore diverse, and the microbiota in the intestinal tract became balanced.Experiment5: Effects of probiotics on growth performance, serum pararmeter,rumenfermentation and ruminal microflora in the pre-weaned calvesTwenty-four newborn Holstein calves were randomly divided into3groups (with4males and4females each). A basal diet without antibiotics and probiotics was used as control (CT group), and theother two diets were supplemented with Lactobacillus plantarum GF103(1.7×1010CFU/h.d) as LBgroup or with a complex of Lactobacillus plantarum GF103(8.6×109CFU/h.d) and Bacillus subtilisB27(2.0×108CFU/h.d) as LBS group. The trial lasted for8weeks. The results showed that there wereno significant difference on growth performance (P>0.05), but feed conversion efficiency was improvedby probiotics (P<0.05). The fecal scores of calves supplemented with probiotics were improved and thediarrhea incidence was decreased. Dietary probiotics did not affect serum parameters of calves (P>0.05).The calves supplemented with a complex of Lactobacillus plantarum and Bacillus subtilis had highervalerate in rumen fluids (P>0.05), but the other VFA were not affected by probiotics (P>0.05). The rumen microbial community of calves supplemented with probiotics was not affected as indicated byPCR/DGGE or RT-PCR analysis. Besides, the probiotics bacteria were not found in the rumen. Theseresults suggested that supplementation with probiotics decreased the incidence of diarrhea, but theprobiotics did not colonize in the rumen.Experiment6: Effects of probiotics on growth performance, serum pararmeter,rumenfermentation and ruminal microflora in the post-weaned calvesTwenty-four post-weaned calves were randomly divided into3groups with4males and4femaleseach. A basal diet without antibiotics or probiotics was used as control, and the other two group werefed the control diet supplemented with Lactobacillus plantarum GF103(1.7×1010CFU/h.d) as LBgroup or with a complex of Lactobacillus plantarum GF103(8.6×109CFU/h.d) and Bacillus subtilisB27(2.0×108CFU/h.d) as LBS group. The trial lasted for4weeks. The results showed that the feedconversion ratio (FCR) was improved (P<0.05) in probiotic-supplemented groups when compared withthat of control during the first two weeks and the entire trial. But average daily gains (ADG) and feedintake (FI) were not affected by probiotics (P>0.05). There were no significant differences in serumparameters and rumen fermentation (P>0.05) among the treatments. The probiotics bacteria were notfound in rumen fluids, but the microflora polymerized together with the same treatments and formed abranch by DGGE analysis. The counts of Ruminococcus flavefaciens and Ruminobacter albus in therumen of calves supplemented with the complex probiotics were lower than those in control. Theseresults showed that probiotics improved performance and FCR in post-weaned calves at the early stage.