| Summary: | 博士 === 國立中興大學 === 動物科學系所 === 107 === The objective of this study was to investigate effects of dietary supplement of multi-strain lactic acid bacteria (Lactobacillus acidophilus LAP5, L. fermentum P2, L. caseiL21, Pediococcus acidophilus LS) on intestinal health of chicken. The study includes five parts; An in vitro test was conducted to evaluate anti-microbial ability of multi-strain lactic acid bacteria. Four in vivo experiments were conducted to study the effects of multi-strain lactic acid bacteria on intestine health of broiler chickens, native chickens, Salmonella-challenged specific-pathogen-free (SPF) chickens, and Salmonella-challenged broiler chickens, by evaluating different biomarkers of gastrointestinal functionality. In the first study, anti-microbial ability of lactic acid bacteria was measured by the agar well diffusion assay. The in vitro results showed that the multi-strain probiotics enhanced antimicrobial ability compared to the single strain, inhibiting pathogen likes Salmonella enterica subsp. enterica ST19, Escherichia coli BCRC 10675T, and Clostridium perfringens ATCC 13124T. The second study was conducted to evaluate effects of multi-strain lactic acid bacteria on intestinal microflora, volatile fatty acid, intestinal morphology, blood characteristic and growth performance. Three hundreds of one-day-old native chickens were allocated into three groups, including the control, 0.1% multi-strain lactic acid bacteria (107 cfu/g) (P), and 10 ppm antibiotic (avilamycin) treatment (A). The results showed that there was no significant difference in the growth performance among all treatments. The multi-strain lactic acid bacteria and antibiotic treatents decreased C. perfringens and Bacteroidetes in the ileum, increased Bifidobacterium sp. in the ceca, and increased Lactobacillus sp. in the ileum compared with the control group. Supplementation of the P and A in diets increased total volatile fatty acids, acetate and propionate in the ceca and increased villi height and villi height / crypt depth ratio in the jejunum and ileum of the chickens compared to the control treatment. In addition, supplementation of multi-strain lactic acid bacteria and antibiotic increased serum high-density lipoprotein, and decreased serum cholesterol compared to the control treatment. In the third part, apart from the intestinal biomarkers mention above, we determined denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (qPCR) to evaluate the effects of combination of multi-strain lactic acid bacteria and herbal medicine Gardeniae fructus on the intestinal health broiler chickens. A total of 400 1-day-old broiler chickens were divided into four groups, including the control (C), 10 ppm antibiotic (avilamycin) treatment (A), 0.1% multi-strain lactic acid bacteria (107 cfu/g) (P), and 0.1% multi-strain lactic acid bacteria combined with 0.05 % herbal medicine G. fructus (PH). The results showed no significant differences in growth performance across all groups. The denaturing gradient gel electrophoresis analysis indicated that the groups PH, P, and A exhibited an increase in the similarity coefficients of their intestinal microbial populations. The real-time polymerase chain reaction (PCR) analysis showed that the relative concentrations of cecal Firmicutes and Lactobacillus sp. and ileal Bifidobacterium sp. were higher in the PH, P, and A than group C. Moreover the multi-strain lactic acid bacteria combined with G. fructus decreased the concentrations of cecal Escherichia sp. and C. perfringens. The broilers in PH group significantly increased cecal total SCFA, acetic acid, and butyric acid compared to the other groups. The A, P, and PH treatments increased the villi height and height/crypt depth ratio in the ileum of broilers. In the fourth chapter, we studied the effect of multi-strain lactic acid bacteria on Salmonella infection in SPF chickens. Next Generation Sequencing was used to analysis intestinal microbiota, and immune responses were measured by real-time quantitative polymerase chain. SPF chickens were randomly assigned to three treatment groups: control (Cont) group, Salmonella‐infected (Sal) group, and Salmonella‐infected group treated with multi‐strain lactic acid bacteria (ProSal) group. All chickens except those in the Cont group were gavaged with 1 × 108 cfu/ml of S. enterica subsp. enterica ST19 on day 4 post hatching. The results showed that Salmonella counts in the liver, spleen, and feces were decreased in the multi‐strain lactic acid bacteria group. The Proteobacteria in the ceca were also decreased in ProSal group. At the genus level, chickens in ProSal group exhibited increasing numbers of Lactobacillus and Oscillospira compared with those in the other groups. In the Cont group, LEfSe showed enrichment of Clostridiales, Clostridia, and Firmicutes, whereas an increase presence of the Bacilli class was founded in the ProSal treatment group. Furthermore, the relative abundance of the Proteobacteria phylum was higher in the Sal group than other groups. Chickens in the ProSal group exhibited a significant increase of cecal SCFAs compared with the Sal group. Chickens in the ProSal group increased gene expression of anti‐inflammatory cytokines, IL‐10 and TGF‐β4, and decreased proinflammatory cytokine, IFN‐γ, in the cecal tonsils compared with those in the Sal group. Based on the results from chapter 4, the intestinal biomarker and mRNA expression of intestinal tight junction protein were determined in experiment 5. The 5th study was conducted to evaluate the effect of multi-strain lactic acid bacteria on Salmonella infection in broiler chickens. A total of 240 1-day-old Ross 308 broiler chickens were used in the similar experimental design as fourth part, but adding a 0.1% multi-strain lactic acid bacteria (Pro) treatment group. The Salmonella challenge significant decreased body weight, body weight gain and increased feed conversion ratio of broiler chickens. Chickens fed with multi‐strain lactic acid bacteria showed no significant difference in the body weight, body weight gain and feed intake, but significantly increased feed conversion ratio. Chickens challenged with Salmonella decreased cecal microbial diversity. Furthermore, chickens in the Sal group exhibited abundant Proteobacteria and Enterobacteriaceae levels than those in the Cont, Pro, and ProSal groups. Pro group also increased numbers of Lactobacillaceae compared with other groups. The results of the MetagenomeSeq showed that the Pro treatment group significantly reduced the harmful microbial levels such as Gammaproteobacteria, Enterobacteriales and Escherichia/shigella. Chickens fed with multi‐strain lactic acid bacteria (Pro and ProSal) treatment increased numbers of Lactobacillus (L. casei, L. fermentum, and L. salivarius). Regarding to the gene expression of tight junction protein, results showed that Salmonella infection decreased Occludin and Claudin gene expression in the jejunum and ileum. Moreover, supplementation with lactic acid bacteria (Pro and ProSal) increased the mRNA expression of Occludin, ZO1 and Mucin 2, in the ileum. In the results of immune responses, the Salmonella infection increased gene expression of interferon-γ (IFN-γ), interleukin-6 (IL-6), IL-1β, and LPS-induced TNF-alpha factor (LITAF), and also reduced levels of TGF-β4 and IL-10 gene expression. In addition, probiotic supplementation downregulated the mRNA levels of IFN-γ and LITAF and upregulated IL-10 expression in the cecal tonsils. In summary, by evaluating different intestinal biomarkers, we can conclude that dietary supplementation of specific screened multi-strain lactic acid bacteria or combining with specific phytogenic (G. fructus) improved intestinal health of chickens. The administration of multi-strain lactic acid bacteria can be a potential alternative to antibiotics in broiler diets by protecting chickens from Salmonella infection, increasing gene expression of tight junction proteins, and enhancing immunomodulatory activity.
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