Copyright Statement This copy of the thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the author’s prior consent. 1 The effects of probiotics, prebiotics and synbiotics on gut flora, immune function and blood characteristics of broilers By Rebin Aswad Mirza Akoy A thesis submitted to the University of Plymouth in partial fulfilment for the degree of DOCTOR OF PHILOSOPHY School of Biological Sciences Faculty of Science and Engineering (July 2015) 2 The effects of probiotics, prebiotics and synbiotics on gut flora, immune function and blood characteristics of broilers Rebin Aswad Mirza Akoy Abstract The microbial populations in the gastrointestinal tracts of poultry play an important role in normal digestive processes and in maintaining animal health. The purpose of this study was to evaluate the effects of probiotics, prebiotics and synbiotics on the growth parameters, gut ecosystem, histology and immune function. In this study, four experiments one in vitro and three in vivo were conducted using specific pathogen free (SPF) and Hubbard broiler chickens. The first experiment was designed to determine the influence of inulin as an effective prebiotic on lactic acid bacteria (LAB) strains, and to screen LAB for selection as a source of chicken probiotic. Eight strains of LAB were isolated from chicken caeca and three strains from the Plymouth University culture collection were screened for potential probiotic properties for growth in inulin from Jerusalem artichoke (Helianthus tuberosus) and commercial inulin (Frutafit® HD, Netherlands). Lactobacillus animalis JCM 8692 strain isolated from chicken caeca showed the highest auto-aggregation and co-aggregation ability, resistance to acidity and bile salts, strong suppression of pathogens and ability to adhere to epithelial cells compared with other isolated strains. The second experiment was conducted to investigate the influence of commercial inulin and Jerusalem artichoke tubers as prebiotic supplementation on the diversity of the caecal microflora, jejunum histology and immune organ of SPF chickens. This investigation has found that inulin which was extracted from JA had a similar result when compared with commercial inulin and could be a suitable candidate for an inulin source in broiler diets. The third experiment was conducted to investigate the influence of Bactocell® (PRO1) and Lb. animalis (PRO2) as probiotic supplements on broiler chickens. EPEF was significantly increased in probiotic1 and probiotic2 compared with control (311.03, 309.87 and 260.06) respectively. Both types of probiotics supported the growth of chicks healthy and could be a suitable candidate as a source of probiotic in broiler diet. The fourth experiment was conducted to investigate the influence of dietary supplementation of a probiotic (Lb. animalis), a prebiotic JA tuber and a combination of both (Synbiotic) in broiler chickens. Growth performance was improved in all additive supplementation compared with the control group. EPEF was increased in probiotic, prebiotic and synbiotic compared with control (290.8±11.8, 300.9±3.86, 322.1±7.09 and 262.3±5.94) respectively. Beneficial bacteria in the guts of chicks fed probiotic, prebiotic and synbiotic was increased compared with chicks fed control diet. The diversity of microbial population in the gastrointestinal tract of chickens improved due to additives. The intestinal villus lengths and microvilli density was improved in all additives supplementation in comparison with control. Overall, it was concluded that probiotic, prebiotic and synbiotics can positively affect production performance and can improve the gut health. i Contents Abstract ........................................................................................................................... i List of figures ................................................................................................................ vi List of Tables ................................................................................................................ vii List of abbreviations ...................................................................................................... x Acknowledgments ....................................................................................................... xiii Dedication .................................................................................................................... xiv Author’s Declaration .................................................................................................. xvii CHAPTER ONE: Literature review ................................................................................. 1 1.1 Introduction ............................................................................................................. 1 1.2 The concept of probiotic .......................................................................................... 4 1.3 The concept of prebiotic .......................................................................................... 7 1.4 The concept of synbiotic ......................................................................................... 8 1.5 Probiotics and prebiotics in poultry diet ................................................................. 10 1.6 Ecology of microflora in the chicken gastrointestinal tract ..................................... 11 1.7 Histology of the bird small intestine ....................................................................... 13 1.8 Action of probiotics in the gastrointestinal tract of chicken..................................... 15 1.8.1 Competitive exclusion ..................................................................................... 16 1.8.2 Bacterial antagonism ...................................................................................... 18 1.8.3 Immune modulation ........................................................................................ 19 1.9 Action of prebiotics in the gastrointestinal tract ..................................................... 20 1.10 Jerusalem artichoke as prebiotic ......................................................................... 22 1.10.1 General characteristics of Jerusalem artichoke ............................................ 22 1.10.2 Biological value of Jerusalem artichoke ........................................................ 23 1.10.3 The methods of determination of inulin ......................................................... 27 1.11 Selection probiotic properties .............................................................................. 28 1.11.1 Aggregation ability ........................................................................................ 29 1.11.2 Antagonistic activity ...................................................................................... 30 1.11.3 Resistance to acidity and bile salts ............................................................... 30 1.11.4 Adhesion of LAB strains to intestinal mucosa ............................................... 31 1.11.5 Cell surface hydrophobicity ........................................................................... 33 1.12 Effects of probiotics, prebiotics and synbiotics on broiler chickens ...................... 34 1.12.1 Performance parameters .............................................................................. 34 1.12.2 Intestinal microflora ...................................................................................... 37 1.12.3 Histology of intestine .................................................................................... 38 ii 1.12.4 Haematological parameters and cholesterol content .................................... 39 1.12.5 Meat quality .................................................................................................. 42 1.13 The aims of this study: ........................................................................................ 44 CHAPTER TWO ............................................................................................................ 46 Preparation of prebiotics, probiotics and its application in vitro ............................. 46 2.1 Introduction ........................................................................................................... 46 2.2 Material and Methods and Results ........................................................................ 47 2.2.1 Preparation of Jerusalem artichoke tubers and extraction of inulin ................. 47 2.2.2 Determination of inulin content from Jerusalem artichoke ............................... 48 2.2.3 Selection of bacteria strain as probiotics......................................................... 50 2.2.4 Isolation of microorganisms ............................................................................ 51 2.2.5 Screening of LAB strains ................................................................................ 52 2.3 Statistical analysis ................................................................................................ 75 2.4 Discussion ............................................................................................................ 76 2.5 Conclusion ............................................................................................................ 82 CHAPTER THREE ......................................................................................................... 83 The effect of dietary inulin supplementation on intestinal microflora, immune functions and blood characteristics of SPF chicks ................................................... 83 3.1 Introduction ........................................................................................................... 83 3.2 Material and Methods ........................................................................................... 84 3.2.1 Ethical approval .............................................................................................. 84 3.2.2 Experimental design and treatments .............................................................. 84 3.2.3 Diets ............................................................................................................... 87 3.2.4 Measurement of pH value of the digestive tract .............................................. 89 3.2.5 Short-Chain fatty acids and lactic acid analysis .............................................. 90 3.2.6 Conventional culture-based techniques .......................................................... 91 3.2.7 Molecular microbial techniques ...................................................................... 92 3.2.8 Gut Histology .................................................................................................. 98 3.2.9 Histology of Bursa of Fabricius ..................................................................... 100 3.2.10 Haematology .............................................................................................. 101 3.3 Statistical analysis .............................................................................................. 102 3.4 Results................................................................................................................ 103 3.4.1 Feed composition ......................................................................................... 103 3.4.2 Live body weight ........................................................................................... 103 3.4.3 The measurement of pH value ..................................................................... 104 3.4.4 Changes in the Short-Chain Fatty Acids and Lactate Concentrations ........... 105 iii 3.4.5 Microbial enumeration of the caecum ........................................................... 106 3.4.6 PCR-DGGE of caecum digesta .................................................................... 108 3.4.7 Histology of Jejunum .................................................................................... 115 3.4.8 Relative weight of Bursa of Fabricius ............................................................ 118 3.4.9 Histology of Bursa of Fabricius ..................................................................... 119 3.4.10 Haematology .............................................................................................. 122 3.5 Discussion .......................................................................................................... 124 3.6 Conclusions ........................................................................................................ 130 CHAPTER FOUR ......................................................................................................... 131 The effects of dietary probiotic supplementation on gut microflora, histology and immune functions of broiler chickens. ..................................................................... 131 4.1 Introduction ......................................................................................................... 131 4.2 Material and Methods ......................................................................................... 132 4.2.1 Experimental design and treatments ............................................................ 132 4.2.2 Freeze drying Lactobacillus animalis ............................................................ 134 4.2.3 Characteristics studied ................................................................................. 134 4.3 Statistical analysis .............................................................................................. 137 4.4 Results................................................................................................................ 138 4.4.1 Freeze dried Lactobacillus animalis .............................................................. 138 4.4.2 Effects of probiotic on performance parameters ........................................... 138 4.4.3 Effects of probiotics on pH value .................................................................. 142 4.4.4 Changes in the Short-Chain Fatty Acids and Lactate Concentrations ........... 143 4.4.5 Effects of probiotics on microflora ................................................................. 144 4.4.6 Effects of probiotic on jejunum histology ....................................................... 163 4.4.7 Relative weight of Bursa of Fabricius ............................................................ 164 4.4.8 Histology of Bursa of Fabricius ..................................................................... 165 4.5 Discussion .......................................................................................................... 166 4.6 Conclusion .......................................................................................................... 170 CHAPTER FIVE: .......................................................................................................... 171 The influences of probiotic, prebiotic and synbiotic on gut microflora, immune function, blood characteristics and meat quality of broiler chickens .................... 171 5.1 Introduction ......................................................................................................... 171 5.2 Material and Methods ......................................................................................... 172 5.2.1 Experimental design and treatments ............................................................ 172 5.2.2 Characteristics studied ................................................................................. 174 4.2.2.1 Production performance ............................................................................ 174 iv 5.2.2.2 Gut microflora analysis .............................................................................. 174 4.2.2.3 Length of GI tract ....................................................................................... 174 5.2.2.4 Histomorphology ....................................................................................... 175 5.2.2.5 Haematological parameters ....................................................................... 176 5.2.2.6 Meat quality ............................................................................................... 179 5.3 Statistics analysis ............................................................................................... 184 5.4 Results................................................................................................................ 185 5.4.1 Performance parameters .............................................................................. 185 5.4.2 The pH value of intestinal tract ..................................................................... 191 5.4.3 Changes in the Short-Chain Fatty Acids and Lactate Concentrations ........... 193 5.4.4 Length of digestive tract ............................................................................... 195 5.4.5 Weight of Caecum ........................................................................................ 197 5.4.6 Effects of probiotics, prebiotics and synbiotics on gut microflora .................. 198 5.4.7 Jejunum Histomorphology ............................................................................ 217 5.4.8 Relative weight of Bursa of Fabricius ............................................................ 229 5.4.9 Histology of Bursa of Fabricius ..................................................................... 230 5.4.10 Haematological and biochemical traits ....................................................... 232 5.4.11 Chemical composition of breast and leg ..................................................... 234 5.4.12 Colour and pH of meat ............................................................................... 236 6.4.13 Cooking losses and shearing force ............................................................. 238 5.5 Discussion .......................................................................................................... 239 5.6 Conclusion .......................................................................................................... 257 CHAPTER SIX: General conclusion and future work ............................................... 259 6.1 General conclusion ............................................................................................. 259 6.2 Future work ......................................................................................................... 265 References .................................................................................................................. 266 Appendices ................................................................................................................. 296 v List of figures Figure 1.1: Major types of surveyed bacteria along the gastrointestinal .............. 12 Figure 1.2: Histological structure of small intestine consist of four layers ............ 14 Figure 1.3: The beneficial bacteria when added to diet of poultry ........................ 17 Figure 1.4: Inhibition of pathogenic bacteria by the antagonistic ......................... 18 Figure 1.5: Blocking bacterial attachment and thus inhibiting host colonization... 21 Figure 1.6: The tubers of Jerusalem artichoke ..................................................... 22 Figure 1.7: Chemical structure of Inulin ............................................................... 25 Figure 1.8: The principle of megazyme assay of enzymatic fructan .................... 28 Figure 1.9: Sections of the digestive system of the chicken with the pH .............. 31 Figure 2.1: The process of prebiotic production from Jerusalem artichoke .......... 47 Figure 2.2: Layout of the screening and selection process .................................. 50 Figure 2.3: Growth curves of 11 probiotic LAB strains in basal MRS medium ..... 57 Figure 2.4: The level of mucus binding of five LAB strains .................................. 65 Figure 2.5: The location of sample of epithelial cells from ileum of chicken ......... 67 Figure 2.6: Adhesion of LAB strains to the intestinal epithelial cells of chicken ... 69 Figure 3.1: Layout of the feed trial. ...................................................................... 85 Figure 3.2: Chicks House located at University of Plymouth animal housing ...... 86 Figure 3.3: Schematic representation of the principal steps of the denaturation . 97 Figure 3.4: Major steps of the tissues sectioning for histological studies ............. 99 Figure 3.5: The bursa of Fabricius in chickens .................................................. 100 Figure 3.6: DGGE fingerprints of caecum digesta of treated and control group . 109 Figure 3.7: (A) Cluster analysis (B and C) non-metric multidimensional scaling 112 Figure 3.8: Haematoxylin and eosin stained section of jejunum of chicks ......... 116 Figure 3.9: Haematoxylin and eosin stained section of jejunum of chicks ......... 117 Figure 3.10: Effect of inulin on relative weight of BF .......................................... 118 Figure 3.11: The effects of inulin on diameter of follicles of Fabricius in ............ 119 Figure 3.12: Bursa of Fabricius in chicks fed diets containing inulin at 14 days. 120 Figure 3.13: Bursa of Fabricius in chicks fed diets containing inulin at 21 days. 121 Figure 3.14 Blood film of chicken showed red blood cells (RBCs) ..................... 123 Figure 3.15: Blood film of chicken showed the red blood cells have a nucleus.. 123 Figure 4.1: Effect of probiotic supplementation on European Production Effic .. 141 Figure 4.2: PCR amplified product of DNA templates of the Caecum (A) and ... 147 Figure 4.3: DGGE fingerprints of ileum digesta of treated and control group..... 149 Figure 4.4: (Top) Cluster analysis (Bottom) non-metric multidimensional sca ... 151 Figure 4.5: DGGE fingerprints of caecum digesta of treated and control group . 157 Figure 4.6: (Top) Cluster analysis (Bottom) non-metric multidimensional .......... 159 Figure 4.7: Effect of probiotic supplementation on Bursa of Fabricius weight .... 164 Figure 4.8: Effect of probiotic supplementation on diameter of Bursa of Fabri... 165 Figure 5.1: Layout of the feed trial ..................................................................... 173 Figure 5.2: Scanning electron microbiology unit at the University of Plymouth .. 176 Figure 5.3: Accutrent® GC meter for cholesterol determination ........................ 178 Figure 5.4: Soxhlet system operated in the nutrition laboratory of the Universi . 180 vi Figure 5.5: Left is computerized digestion block and right is distillation unit ...... 182 Figure 5.6: Texture analyser unit at the University of Plymouth ......................... 184 Figure 5.7: PCR amplified product of DNA templates of the Caecum ................ 202 Figure 5.8: DGGE fingerprints of caecum digesta of treated and control ........... 204 Figure 5.9: (Top) Cluster analysis (Bottom) non-metric multidimensional .......... 206 Figure 5.10: DGGE fingerprints of ileum digesta of treated and control group... 211 Figure 5.11: (Top) Cluster analysis (Bottom) non-metric multidimensional ........ 213 Figure 5.12: Haematoxylin and eosin stained section of jejunum of broilers fed 220 Figure 5.13: Alcian blue and PAS stained section of jejunum of broilers fed ..... 221 Figure 5.14: SEM micrograph of control chicken jejunum showed the length .... 223 Figure 5.15: SEM micrograph of top side view of the intestinal villi of control .... 223 Figure 5.16: SEM micrograph of top side view of the intestinal villi of probioti ... 224 Figure 5.17: SEM micrograph of top side view of the intestinal villi of prebiotic . 224 Figure 5.18: SEM micrograph of top side view of the intestinal villi of synbiotic. 225 Figure 5.19: SEM micrograph of bacterial colonisation in the jejunum of the..... 225 Figure 5.20: SEM image of the jejunum of chicken fed synbiotic showed the.... 226 Figure 5.21: SEM image of the jejunum of chicken fed synbiotic showed the.... 226 Figure 5.22: SEM image of the control jejunum chicken group showed the ...... 227 Figure 5.23: SEM image of the control jejunum chicken group showed the ...... 227 Figure 5.24: Comparative SEM micrographs of microvilli density ...................... 228 Figure 5.25: Follicles of Bursa of Fabricius in broilers fed diets containing ....... 231 List of Tables Table 1.1: List of probiotics studied for application in animal feed. ........................ 5 Table 1.2: A number of probiotic products are available commercially for use ...... 6 Table 1.3: Major oligosaccharide candidates for prebiotics ................................... 7 Table 1.4: The scientific classification of Jerusalem artichoke ............................. 22 Table 1.5: Safety criteria and characteristics of probiotics as a health-promotin..29 Table 1.6: General effects of probiotics, prebiotics and synbiotics in poultry ....... 36 Table 2.1: Procedure of fructan measurement in the Jerusalem artichoke tube .. 49 Table 2.2: The preparation of MRS broth by diluting these amounts of comp ..... 53 Table 2.3: Growth (OD ± SD) of LAB strains on Frutafit inulin ......................... 55 620 Table 2.4: Growth (OD ± SD) of LAB 10 CFU ml-1 on Frutafit inulin ............... 55 595 5 Table 2.5: Different auto-aggregation times of six LAB strains ............................ 59 Table 2.6: Co-aggregation activity of LAB strains isolated from chicken caeca ... 60 Table 2.7: Antagonistic activity of LAB strains isolated from chicken caecal ....... 62 Table 2.8: Number of LAB strains (Log CFU ml-1) after incubation at various ... 63 10 Table 2.9: Number of LAB strains (Log CFU ml-1) after incubation at different . 64 10 Table 2.10: Adhesion of LAB strains to mucin from epithelial chicken intestinal .. 65 Table 2.11: Adhesion of LAB strains to chicken intestinal epithelial cells ............ 68 Table 2.12: Cell surface hydrophobicity of four selected LAB strains to toluene.. 71 vii Table 3.1: The composition of standard diet of starter (1-21 days) which was .... 88 Table 3.2: The nutritional information of Frutafit® HD commercial inulin and ....... 89 Table 3.3: Chemical composition analysed of each broiler diet samples ........... 103 Table 3.4: Effect of commercial and JA inulin on weekly live body weight of ..... 104 Table 3.5: Effect of dietary inulin supplementation on the pH value of ileum .... 105 Table 3.6: Concentration (mmol/L) of short-chain fatty acids in caecum dige .... 106 Table 3.7: Bacterial counts (Log CFU mL-1) at 14, 18 and 21 days of age in .. 107 10 Table 3.8: The half matrix similarity of bacterial population of DGGE ................ 110 Table 3.9: Band numbers of bacterial community based on the PCR-DGGE .... 112 Table 3.10: Diversity index of bacterial community in caecal digesta based...... 113 Table 3.11: Summary results of sequencing analysis bands of PCR-DGGE ..... 114 Table 3.12: Effects of dietary inulin supplementation on villus height (μm) ........ 115 Table 3.13: Results of WBC’s counts and heterophil/lymphocyte ratio .............. 122 Table 4.1: The composition of standard broiler diets which was used for the .... 133 Table 4.2: Effect of probiotic supplementation on weekly and final live body .... 140 Table 4.3: Effect of probiotic supplementation on weekly and final body weig .. 140 Table 4.4: Effect of probiotic supplementation on weekly and accumulative ..... 140 Table 4.5: Effect of probiotic supplementation on weekly and feed conversion . 141 Table 4.6: Effect of probiotic supplementation on pH value in ileum and caec .. 142 Table 4.7: Influence of supplementation of probiotic on the short-chain fatty .... 143 Table 4.8: Bacterial counts (Log CFU mL-1) at 14 and 28 days of age in il ..... 145 10 Table 4.9: Bacterial counts (Log CFU mL-1) at 14 and 28 days of age in cae . 146 10 Table 4.10: The half matrix similarity of bacterial population of DGGE fingerp .. 150 Table 4.11: Band numbers of ileum bacterial community based on the PCR .... 152 Table 4.12: Diversity index of bacterial community in ileum digesta based on .. 153 Table 4.13: Summary results of sequencing analysis bands of PCR-DGGE ..... 155 Table 4.14: The half matrix similarity of bacterial population of DGGE fingerp .. 158 Table 4.15: Band numbers of caecum bacterial community based on the PCR 160 Table 4.16: Diversity index of bacterial community in caecum digesta based ... 161 Table 4.17: Summary results of sequencing analysis bands of PCR-DGGE ..... 162 Table 4.18: Effect of probiotic supplementation on villus height (μm) ................ 163 Table 5.1: Effect of probiotic, prebiotic and synbiotic on weekly and final live ... 187 Table 5.2: Estimated coefficients for weekly live body weight (g) ...................... 187 Table 5.3: Effect of probiotic, prebiotic and synbiotic on weekly and final ......... 188 Table 5.4: Estimated coefficients for weekly and final body weight gain ............ 188 Table 5.5: Effect of probiotic, prebiotic and synbiotic on weekly and accumul ... 189 Table 5.6: Estimated coefficients for weekly and accumulative feed intake ....... 189 Table 5.7: Effect of probiotic, prebiotic and synbiotic on weekly and feed ......... 190 Table 5.8: Estimated coefficients for weekly and final feed conversion ratio ..... 190 Table 5.9: Effect of probiotic, prebiotic and synbiotic on production index ......... 191 Table 5.10: Estimated coefficients for probiotic, prebiotic and synbiotic on. ...... 191 Table 5.11: Effect of probiotic, prebiotic and synbiotic on pH value ................... 192 Table 5.12: Estimated coefficients for probiotic, prebiotic and synbiotic on pH . 192 Table 5.13: Influence of supplementation of probiotic, prebiotic and synbiotic . 194 viii