| The health state of weaned piglets has an enormous impact on their subsequent performance in adaptation to nutritional, psychological and environmental stressors. These stressors may result in low feed intake, low weight gain and poor health, and thus antibiotics are usually used to improve animal growth and health. However, recent studies have found the horizontal transfer of antibiotic resistance genes between bacteria from farm animals, human food and humans. Concerns about transference of antibiotic resistance genes from animals to humang led to withdraw approval for antibiotics as growth promoters in the European Union in2006. Currently, more countries are making a greater effort to ban on the use of antibiotics as animal growth promoters.Probiotics are defined as live microorganisms that produce a health benefit when administered in adequate amounts to animals, including humans. They are potential alternatives to growth promoting antibiotics which can affect the health of pigs. Furthermore, it is increasingly realized that the intestinal microflora has been suggested to play an important role in the growth of weaned piglets, and the intestinal microbiota balance of pigs can be modulated by dietary intervention, such as by supplementing probiotics. Probiotics are mainly lactic acid bacteria (LAB) and Bacillus species. In this study, firstly, the effects of Bacillus subtilis KN-42, Enterococcus faecalis LAB31, Clostridium butyricum B1and Bacillus coagulans F5on growth performance, diarrhea incidence and bacterial diversity of weaned piglets were investigated individually. Then, the compound probiotics were studied about their effects on growth of weaned piglets.16S rDNA gene-based methods were applied for bacterial composition analysis of the fresh fecal samples. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to characterize the bacterial composition and diversity of fecal samples, and the molecular microbial ecology methods were combined to analysis the effects of probiotics on the growth of weaned piglets.(1)B. subtilis KN-42A total of360piglets (initial body weight7.14±0.63kg) weaned at26±2days of age were randomly allotted to5treatment groups (4pens per treatment with18pigs per pen) for a28-day trial. Dietary treatments were basal diet without any antimicrobial (negative control; NC), basal diet supplemented with120mg/kg feed of neomycin sulfate (positive control; PC) and basal diet supplemented with2×109(L),4×109(M) and20x109(H) CFU/kg feed of B. subtilis KN-42. During the overall period, average daily gain (ADG) and feed efficiency (G/F) of piglets were higher in groups PC, M, and H than those in group NC (P﹤0.05), and all probiotics and antibiotics groups had a lower diarrhea index than group NC (P﹤0.05). The fecal samples were collected on day28. The result of denaturing gradient gel electrophoresis (DGGE) analysis showed that supplementation of B. subtilis KN-42to the diet changed the bacterial communities, with a higher bacterial diversity and band number in group M than in the other four groups. Real-time PCR analysis showed that the relative number of Lactobacillus was higher in groups PC and H than in group NC (P﹤0.05), and the supplemented B. subtilis KN-42to the diet also reduced the relative number of E. coli (P﹤0.05). These results suggest that dietary addition of B. subtilis KN-42can improve the growth performance and gastrointestinal health of piglets.(2) Enterococcus faecalis LAB31A total of360piglets weaned at26±2days of age were randomly allotted to5groups (20pens, with4pens for each group) for a trial of28days: group N (negative control, without antibiotics or probiotics); group PC (Neomycin sulfate,100mg/kg feed); groups L, M and H (supplemented with E. faecalis LAB310.5×109,1.0×109, and2.5×109CFU/kg feed, respectively). ADG and G/F were found to be higher in group H than in group N, and they were significantly different between group H and group PC (P﹤0.05). Furthermore, groups H and PC had a lower diarrhea index than the other three groups (P﹤0.05). Denaturing gradient gel electrophoresis (DGGE) showed that the application of probiotics to the diet changed the bacterial community, with a higher bacterial diversity in group M than in the other four groups. Real-time PCR revealed that the relative number of Lactobacillus increased by addition of probiotics, and was higher in group H than in group NC (P﹤0.05). However, group-specific PCR-DGGE showed no obvious difference among the five groups in Lactobacillus composition and diversity. Therefore, the dietary addition of E. faecalis LAB31can improve growth performance, reduce diarrhea, and increase the relative number of Lactobacillus in feces of weaned piglets.(3) Clostridium butyricum B1A total of360piglets (initial body weight6.91±0.55kg) weaned at26±2days of age were randomly allotted to5treatment groups (4pens per treatment with18pigs per pen) for a28-day trial. Dietary treatments were basal diet without any antimicrobial (negative control; N), basal diet supplemented with100mg/kg feed of neomycin sulfate and75mg/kg quinocetone (positive control; P) and basal diet supplemented with0.5×108(L),1×108(M) and5×108(H) CFU/kg feed of C. butyricum B1. During the overall period, ADG and G/F of piglets were higher in groups PC and L than those in group NC (P﹤0.05), and all probiotics and antibiotics groups had a lower diarrhea index than group NC C (P<0.05). On day27, the fasting blood samples were analysed and the results showed that the leukocyte count, erythrocyte count, hemoglobin, hematocrit value, platelet count, mean corpuscular hemoglobin concentration (MCHC), lymphocyte and neutrophil concentrations were not affected by diet types (P>0.05). On day14and28, the fresh fecal samples were collected for bacterial composition analysis, and there were4lanes (replicates) in each treatment. The results of DGGE for total bacteria and group specific DGGE for Lactobacillus showed that considerable variations were found between individuals. The most common bacteria were Clostridium species, and the most common Lactobacillus specie was L. reuteri.(4) Bacillus coagulans F5In experiment I: A total of120piglets (initial body weight8.0±0.64kg) weaned at28±2days of age were allotted to4treatment groups (4pens per treatment with10piglets per pen) for a14-day trial. Dietary treatments were basal diet supplemented with80mg/kg feed of chlorotetracycline (positive control; PC) and basal diet supplemented with0.5×108(L),2.5×108(M) and12.5×108(H) CFU/kg feed of B. coagulans F5. The results showed that ADG and G/F of piglets were higher in group PC, M, and H than those in group L, and no significant differences were observed in the diarrhea among4groups.In experiment Ⅱ:A total of360piglets (initial body weight7.69±0.46kg) weaned at26±2days of age were randomly allotted to5treatment groups (4pens per treatment with18pigs per pen) for a21-day trial. Dietary treatments were basal diet without any antimicrobial (negative control; NC), basal diet supplemented with80mg/kg feed of chlorotetracycline (positive control; PC) and basal diet supplemented with5×108(L),10×108(M) and25×108(H) CFU/kg feed of B. coagulans F5. During the overall period, ADG and G/F of piglets were higher in group M than those in group NC (P﹤0.05), G/F of piglets was higher in group PC than that in group NC, and groups PC, M, and H had a lower diarrhea index than group NC (P<0.05). On day21, the fresh fecal samples were collected for bacterial composition analysis, and there were4lanes (replicates) in each treatment. The results of DGGE and bacterial diversity analysis showed that the Shannon’s index H’of samples was considerably higher in groups L, and M than those in group NC, and P, however, no significant differences were found among them due to the individual differences.(5) Compound ProbioticsA total of360piglets (initial body weight7.08±0.82kg) weaned at26±2days of age were randomly allotted to5treatment groups (4pens per treatment with18pigs per pen) for a28-day trial. Dietary treatments were basal diet without any antimicrobial (negative control; NC), basal diet supplemented with80mg/kg feed of chlorotetracycline (positive control; PC) and basal diet supplemented with compound probiotics Ml (B. subtilis20×108, E. faecalis5×108, E.faecium5x108, C. butyricum0.3×108CFU/kg feed) and M2(B. subtilis20×108, E. faecalis12.5×108, B. coagulans5×108, C. butyricum0.3×108CFU/kg feed).During the overall period, ADG of piglets was higher in groups M1and M2than those in group PC (P﹤0.05), G/F of piglets was higher in groups Ml and M2than those in group NC (P<0.05), and compound probiotics groups had a lower diarrhea index than group NC and PC (P<0.05). The fecal samples were collected on day14(phase I) and day28(phase II), respectively. The results showed that no significant differences were observed in acetic acid, propionic acid, butyric acid and total volatile fatty acids among the5groups or day11, however, acetic acid and propionic acid of fecal samples were higher in group NC than those of in groups PC, M1and M2, butyric acid were lower in group PC than group NC, and the total volatile fatty acids were higher in group NC than PC. The result of DGGE analysis showed that supplementation of compound probiotics to the diet changed the bacterial communities, with a higher bacterial diversity in groups Ml and M2than in groups NC and PC on day14, whereas, the bacterial diversity of samples from high to low are PC, M2, M1and NC. Therefore, the dietary addition of compound probiotics can improve growth performance, and reduce diarrhea and the content of fecal acetic acid and propionic acid. |
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