Broiler gut microbiota and expressions of gut barrier genes affected by cereal type and phytogenic inclusion
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摘要
The present study assessed the effects of cereal type and the inclusion level of a phytogenic feed additive(PFA) on broiler ileal and cecal gut microbiota composition, volatile fatty acids(VFA) and gene expression of toll like receptors(TLR), tight junction proteins, mucin 2(MUC2) and secretory immunoglobulin A(sIgA). Depending on cereal type(i.e. maize or wheat) and PFA inclusion level(i.e. 0, 100 and 150 mg/kg diet), 450 one-day-old male broilers were allocated in 6 treatments according to a 2 × 3 factorial arrangement with 5 replicates of 15 broilers each, for 42 d. Significant interactions(P 0.05) between cereal type and PFA were shown for cecal digesta Bacteroides and Clostridium cluster XIVa, ileal digesta propionic and branched VFA, ileal s IgA gene expression, as well as cecal digesta branched and other VFA molar ratios. Cereal type affected the cecal microbiota composition. In particular, wheat-fed broilers had higher levels of mucosa-associated Lactobacillus(P_(CT)= 0.007) and digesta Bifidobacterium(P_(CT)< 0.001),as well as lower levels of total bacteria(P_(CT)= 0.004) and Clostridia clusters I, IV and XIVa(P_(CT) 0.05),compared with maize-fed ones. In addition, cereal type gave differences in fermentation intensity(P_(CT)= 0.021) and in certain individual VFA molar ratios. Wheat-fed broilers had higher(P 0.05) ileal zonula occluden 2(ZO-2) and lower ileal and cecal TLR2 and sIgA levels, compared with maize-fed broilers. On the other hand, PFA inclusion at 150 mg/kg had a stimulating effect on microbial fermentation at ileum and a retarding effect in ceca with additional variable VFA molar patterns. In addition, PFA inclusion at 100 mg/kg increased the ileal mucosa expression of claudin 5(CLDN5)(PPFA= 0.023) and MUC2(PPFA= 0.001) genes, and at 150 mg/kg decreased cecal TLR2(PPFA= 0.022) gene expression compared with the un-supplemented controls. In conclusion, cereal type and PFA affected in combination and independently broiler gut microbiota composition and metabolic activity as well as the expression of critical gut barrier genes including TLR2. Further exploitation of these properties in cases of stressor challenges is warranted.
The present study assessed the effects of cereal type and the inclusion level of a phytogenic feed additive(PFA) on broiler ileal and cecal gut microbiota composition, volatile fatty acids(VFA) and gene expression of toll like receptors(TLR), tight junction proteins, mucin 2(MUC2) and secretory immunoglobulin A(sIgA). Depending on cereal type(i.e. maize or wheat) and PFA inclusion level(i.e. 0, 100 and 150 mg/kg diet), 450 one-day-old male broilers were allocated in 6 treatments according to a 2 × 3 factorial arrangement with 5 replicates of 15 broilers each, for 42 d. Significant interactions(P 0.05) between cereal type and PFA were shown for cecal digesta Bacteroides and Clostridium cluster XIVa, ileal digesta propionic and branched VFA, ileal s IgA gene expression, as well as cecal digesta branched and other VFA molar ratios. Cereal type affected the cecal microbiota composition. In particular, wheat-fed broilers had higher levels of mucosa-associated Lactobacillus(P_(CT)= 0.007) and digesta Bifidobacterium(P_(CT)< 0.001),as well as lower levels of total bacteria(P_(CT)= 0.004) and Clostridia clusters I, IV and XIVa(P_(CT) 0.05),compared with maize-fed ones. In addition, cereal type gave differences in fermentation intensity(P_(CT)= 0.021) and in certain individual VFA molar ratios. Wheat-fed broilers had higher(P 0.05) ileal zonula occluden 2(ZO-2) and lower ileal and cecal TLR2 and sIgA levels, compared with maize-fed broilers. On the other hand, PFA inclusion at 150 mg/kg had a stimulating effect on microbial fermentation at ileum and a retarding effect in ceca with additional variable VFA molar patterns. In addition, PFA inclusion at 100 mg/kg increased the ileal mucosa expression of claudin 5(CLDN5)(PPFA= 0.023) and MUC2(PPFA= 0.001) genes, and at 150 mg/kg decreased cecal TLR2(PPFA= 0.022) gene expression compared with the un-supplemented controls. In conclusion, cereal type and PFA affected in combination and independently broiler gut microbiota composition and metabolic activity as well as the expression of critical gut barrier genes including TLR2. Further exploitation of these properties in cases of stressor challenges is warranted.
The present study assessed the effects of cereal type and the inclusion level of a phytogenic feed additive(PFA) on broiler ileal and cecal gut microbiota composition, volatile fatty acids(VFA) and gene expression of toll like receptors(TLR), tight junction proteins, mucin 2(MUC2) and secretory immunoglobulin A(sIgA). Depending on cereal type(i.e. maize or wheat) and PFA inclusion level(i.e. 0, 100 and 150 mg/kg diet), 450 one-day-old male broilers were allocated in 6 treatments according to a 2 × 3 factorial arrangement with 5 replicates of 15 broilers each, for 42 d. Significant interactions(P 0.05) between cereal type and PFA were shown for cecal digesta Bacteroides and Clostridium cluster XIVa, ileal digesta propionic and branched VFA, ileal s IgA gene expression, as well as cecal digesta branched and other VFA molar ratios. Cereal type affected the cecal microbiota composition. In particular, wheat-fed broilers had higher levels of mucosa-associated Lactobacillus(P_(CT)= 0.007) and digesta Bifidobacterium(P_(CT)< 0.001),as well as lower levels of total bacteria(P_(CT)= 0.004) and Clostridia clusters I, IV and XIVa(P_(CT) 0.05),compared with maize-fed ones. In addition, cereal type gave differences in fermentation intensity(P_(CT)= 0.021) and in certain individual VFA molar ratios. Wheat-fed broilers had higher(P 0.05) ileal zonula occluden 2(ZO-2) and lower ileal and cecal TLR2 and sIgA levels, compared with maize-fed broilers. On the other hand, PFA inclusion at 150 mg/kg had a stimulating effect on microbial fermentation at ileum and a retarding effect in ceca with additional variable VFA molar patterns. In addition, PFA inclusion at 100 mg/kg increased the ileal mucosa expression of claudin 5(CLDN5)(PPFA= 0.023) and MUC2(PPFA= 0.001) genes, and at 150 mg/kg decreased cecal TLR2(PPFA= 0.022) gene expression compared with the un-supplemented controls. In conclusion, cereal type and PFA affected in combination and independently broiler gut microbiota composition and metabolic activity as well as the expression of critical gut barrier genes including TLR2. Further exploitation of these properties in cases of stressor challenges is warranted.
引文
Amerah AM.Interactions between wheat characteristics and feed enzyme supplementation in broiler diets.Anim Feed Sci Technol 2015;199:1-9.
Apajalahti J,Kettunen A,Graham H.Characteristics of the gastrointestinal microbial communities,with special reference to the chicken.Worlds Poultry Sci2004;60:223-32.
Boroojeni FG,Vahjen W,Mader A,Knorr F,Ruhnke I,Rohe I,Hafeez A,Villodre C,Manner K,Zentek J.The effects of different thermal treatments and organic acid levels in feed on microbial composition and activity in gastrointestinal tract of broilers.Poultry Sci 2014;93:1440-52.
Brenes A,Roura E.Essential oils in poultry nutrition:main effects and modes of action.Anim Feed Sci Technol 2010;158:1-14.
Cao PH,Li FD,Li YF,Ru YJ,Peron A,Schulze H,Bento H.Effect of essential oils and feed enzymes on performance and nutrient utilization in broilers fed a corn/soy-based diet.Int J Poultry Sci 2010;9:749-55.
Celi P,Cowieson AJ,Fru-Nji F,Steinert RE,Kluenter AM,Verlhac V.Gastrointestinal functionality in animal nutrition and health:new opportunities for sustainable animal production.Anim Feed Sci Technol 2017;234:88-100.
Chen J,Tellez G,Richards JD,Escobar J.Identification of potential biomarkers for gut barrier failure in broiler chickens.Front Vet Sci 2015;2:2-10.
Cho JH,Kim HJ,Kim IH.Effects of phytogenic feed additive on growthperformance,digestibility,blood metabolites,intestinal microbiota,meat color and relative organ weight after oral challenge with Clostridium perfringens in broilers.Livest Sci 2014;160:82-8.
Choct M,Hughes RJ,Wang J,Bedford MR,Morgan AJ,Annison G.Increased small intestine fermentation is partly responsible for the antinutritive activity on non-starch polysaccharides in chickens.Br Poultry Sci 1996;37:609-21.
Choct M.Managing gut health through nutrition.Br Poultry Sci 2009;50:9-15.
Clifford RJ,Milillo M,Prestwood J,Quintero R,Zurawski DV,Kwak YI,Waterman PE,Lesho EP,Mc Gann P.Detection of bacterial 16S rRNA and identification of four clinically important bacteria by Real-Time PCR.PLoS One 2012;7:1-6.
Cross DE,McDevitt RM,Acamovic T.Herbs,thyme essential oil and condensed tannin extracts as dietary supplements for broilers,and their effects on performance,digestibility,volatile fatty acids and organoleptic properties.Br Poultry Sci 2011;52:227-37.
Cummings JH,Macfarlane GT.The control and consequences of bacterial fermentation in the human colon.J Appl Microbiol 1991;70:443-59.
Delroisse JM,Boulvin AL,Parmentier I,Dauphin RD,Vandenbol M,Portetelle D.Quantification of Bifidobacterium spp.and Lactobacillus spp.in rat fecal samples by real-time PCR.Microbiol Res 2008;163:663-70.
Du E,Wang W,Gan L,Li Z,Guo S,Guo Y.Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens.J Anim Sci Biotechnol 2016;7:2-10.
Ducatelle R,Goossens E,Meyer FD,Eeckhaut V,Antonissen G,Haesebrouck F,Immersee FV.Biomarkers for monitoring intestinal health in poultry:present status and future perspectives.Vet Res 2018;49:43.
EC.43.Council Directive of 28 June 2007 laying down minimum rules for the protection of chickens kept for meat production.O J E U 2007;182:19-28.
EU.63.Directive of the European Parliament and of the Council of 22 September2010 on the protection of animals used for scientific purposes.O J E U 2010;276:33-79.
Franciosini MP,Casagrande-Proietti P,Forte C,Beghelli D,Acuti G,Zanichelli D,Bosco A,Castellini C,Trabalza-Marinucci M.Effects of oregano(Origanum vulgare L.)and rosemary(Rosmarinus officinalis L.)aqueous extracts on broiler performance,immune function and intestinal microbial population.J Appl Anim Res 2015;44:474-9.
Hashemipour H,Kermanshahi H,Golian A,Veldkamp T.Effect of thymol and carvacrol feed supplementation on performance,antioxidant enzyme activities,fatty acid composition,digestive enzyme activities,and immune response in broiler chickens.Poultry Sci 2013;92:2059-69.
Hashemipour H,Khaksar V,Rubio LA,Veldkamp T,Van Krimpen MM.Effect of feed supplementation with a thymol plus carvacrolmixture,in combination or not with an NSP-degrading enzyme,on productive and physiological parameters ofbroilers fed on wheat-based diets.Anim Feed Sci Technol 2016;211:117-31.
Hong JC,Steiner T,Aufy A,Lien TF.Effects of supplemental essential oil on growth performance,lipid metabolites and immunity,intestinal characteristics,microbiota and carcass traits in broilers.Livest Sci 2012;144:253-62.
Hu CH,Gu LY,Luan ZS,Song J,Zhu K.Effects of montmorilloniteezinc oxide hybrid on performance,diarrhea,intestinal permeability and morphology of weanling pigs.Anim Feed Sci Technol 2012;177:108-15.
Hubener K,Vahjen W,Simon O.Bacterial responses to different dietary cereal types and xylanase supplementation in the intestine of broiler chicken.Arch Anim Nutr 2002;56:167-87.
Kaldhusdal M,Hofshagen M.Barley inclusion and avoparcin supplementation in broiler diets.2.Clinical,pathological,and bacteriological findings in a mild form of Necrotic Enteritis 1992;71:1145-53.
Kawai T,Akira S.TLR signaling.Semin Immunol 2007;19:24-32.
Keestra AM,Zoete MR,Bouwman LI,Vaezirad MM,Van Putten JPM.Unique features of chicken Toll-like receptors.Dev Comp Immunol 2013;41:316-23.
Kiarie E,Romero LF,Ravindran V.Growth performance,nutrient utilization,and digesta characteristics in broiler chickens fed corn or wheat diets without or with supplemental xylanase.Poultry Sci 2014;93:1186-96.
Lan Y,Verstegen MWA,Tamminga S,Williams BA.The role of the commensal gut microbial community in broiler chickens.Worlds Poultry Sci 2004;61:95-104.
Lee KW,Everts H,Beynen AC.Essential oils in broiler nutrition.Int J Poultry Sci2004;3:738-52.
Lee SA,Apajalahti J,Vienola K,Gonzalez-Ortiz G,Fontes CMGA,Bedford MR.Age and dietary xylanase supplementation affects ileal sugar residues and short chain fatty acid concentration in the ileum and caecum of broiler chickens.Anim Feed Sci Technol 2017;234:29-42.
Lillehoj HS,Lee KW.Immune modulation of innate immunity as alternatives-toantibiotics strategies to mitigate the use of drugs in poultry production.Poultry Sci 2012;91:1286-91.
Liu D,Guo S,Guo Y.Xylanase supplementation to a wheat-based diet alleviated the intestinal mucosal barrier impairment of broiler chickens challenged by Clostridium perfringens.Avian Pathol 2012;41:292-8.
Lu H,Adedokun SA,Adeola L,Ajuwon KM.2014.Anti-Inflammatory effects of nonantibiotic alternatives in Coccidia challenged broiler chickens.J Poultry Sci2014;51:14-21.
Lu J,Idris U,Harmon B,Hofacre C,Maurer JJ,Lee MD.Diversity and succession of the intestinal bacterial community of the maturing broiler chicken.Appl.Environ.Microbiol.2003;69:6816-24.
Matsuki T,Watanabe K,Fujimoto J,Takada T,Tanaka R.2004.Use of 16S r RNA genetargeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces.Appl Environ Microbiol 2004;70:7220-8.
Michiels J,Missotten J,Dierick NI,Fremaut D,Maene P,De Smet S.In vitro degradation and in vivo passage kinetics of carvacrol,thymol,eugenol and transcinnamaldehyde along the gastrointestinal of piglets.J Sci Food Agric 2008;88:2371-81.
Mountzouris KC,Tsirtsikos P,Kalamara E,Nitsch S,Schatzmayr G,Fegeros K.Evaluation of the efficacy of a probiotic containing Lactobacillus,Bifidobacterium,Enterococcus,and Pediococcus strains in promoting broiler performance and modulating cecal microflora composition and metabolic activities.Poultry Sci2007;86:309-17.
Mountzouris KC,Paraskeuas V,Tsirtsikos P,Palamidi I,Steiner T,Schatzmayr G,et al.Assessment of a phytogenic feed additive effect on broiler growth performance,nutrient digestibility and caecal microflora composition.Anim Feed Sci Technol2011;168:223-31.
Mountzouris KC,Tsirtsikos P,Papadomichelakis G,Schatzmayr G,Fegeros K.Evaluation of the efficacy of sequential or continuous administration of probiotics and phytogenics in broiler diets.Anim Prod Sci 2015;55:720-8.
Paraskeuas V,Fegeros K,Hunger C,Theodorou G,Mountzouris KC.Dietary inclusion level effects of a phytogenic characterised by menthol and anethole on broiler growth performance,biochemical parameters including total antioxidant capacity and gene expression of immune-related biomarkers.Anim Prod Sci2016;57:33-41.
Paraskeuas V,Fegeros K,Palamidi I,Hunger C,Mountzouris KC.2017.Growth performance,nutrient digestibility,antioxidant capacity,blood biochemical biomarkers and cytokines expression in broiler chickens fed different phytogenic levels.Ani Nu 2017;3:114-20.
Pathak M,Mandal G,Patra AK,Samanta I,Pradhan S,Haldar S.Effects of dietary supplementation of cinnamaldehyde and formic acid on growth performance,intestinal microbiota and immune response in broiler chickens.Anim Prod Sci2016;57:821-7.
Peinado MJ,Ruiz R,Echavarri A,Aranda-Olmedo I,Rubio LA.Garlic derivative PTS-Omodulates intestinal microbiota composition and improves digestibility in growing broiler chickens.Anim Feed Sci Technol 2013;181:87-92.
Pfaffl MW.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res 2001;29:45.
Rodriguez ML,Rebole A,Velasco S,Ortiz LT,Trevino J,Alzueta C.Wheat-and barleybased diets with or without additives influence broiler chicken performance,nutrient digestibility and intestinal microflora.J Sci Food Agric 2012;92:184-90.
Sacranie A,Svihus B,Denstadli V,Moen B,Iji PA,Choct M.The effect of insoluble fiber and intermittent feeding on gizzard development,gut motility,and performance of broiler chickens.Poultry Sci 2012;91:693-700.
Schwiertz A,Taras D,Sch€afer K,Beijer S,Bos NA,Donus C,Hardt PD.Microbiota and SCFA in lean and overweight healthy subjects.Obes Res 2010;18:190-5.
Silkie SS,Nelson KL.Concentrations of host-specific and generic fecal markers measured by quantitative PCR in raw sewage and fresh animal feces.Water Res2009;43:4860-71.
Song J,Xiao K,Ke YL,Jiao LF,Hu CH,Diao QY,Shi B,Zou XT.Effect of a probiotic mixture on intestinal microflora,morphology,and barrier integrity of broilers subjected to heat stress.Poultry Sci 2014;93:581-8.
Suzuki T,Hara H.Role of flavonoids in intestinal tight junction regulation.J Nutr Biochem 2011;22:401-8.
Svihus BM,Choct M,Classen HL.Function and nutritional roles of the avian caeca:a review.World’s Poultry Sci J 2013;69:249-64.
Tsirtsikos P,Fegeros K,Kominakis A,Balaskas C,Mountzouris KC.Modulation of intestinal mucin composition and mucosal morphology by dietary phytogenic inclusion level in broilers.Animal 2012;6:1049-57.
Van Der Wielen PWJJ,Biesterveld S,Notermans S,Hofstra H,Urlings BAP,Van Knapen F.Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth.Appl Environ Microbiol 2000;66:2536-40.
Windisch WM,Schedle K,Plitzner C,Kroismayr A.Use of phytogenic products as feed additives for swine and poultry.J Anim Sci 2008;86:140-8.
Zou Y,Xiang Q,Wang J,Peng J,Wei H.Oregano essential oil improves intestinal morphology and expression of tight junction proteins associated with modulation of selected intestinal bacteria and immune status in a pig,model.BioMed Res Int 2016;2016:1-11.
Apajalahti J,Kettunen A,Graham H.Characteristics of the gastrointestinal microbial communities,with special reference to the chicken.Worlds Poultry Sci2004;60:223-32.
Boroojeni FG,Vahjen W,Mader A,Knorr F,Ruhnke I,Rohe I,Hafeez A,Villodre C,Manner K,Zentek J.The effects of different thermal treatments and organic acid levels in feed on microbial composition and activity in gastrointestinal tract of broilers.Poultry Sci 2014;93:1440-52.
Brenes A,Roura E.Essential oils in poultry nutrition:main effects and modes of action.Anim Feed Sci Technol 2010;158:1-14.
Cao PH,Li FD,Li YF,Ru YJ,Peron A,Schulze H,Bento H.Effect of essential oils and feed enzymes on performance and nutrient utilization in broilers fed a corn/soy-based diet.Int J Poultry Sci 2010;9:749-55.
Celi P,Cowieson AJ,Fru-Nji F,Steinert RE,Kluenter AM,Verlhac V.Gastrointestinal functionality in animal nutrition and health:new opportunities for sustainable animal production.Anim Feed Sci Technol 2017;234:88-100.
Chen J,Tellez G,Richards JD,Escobar J.Identification of potential biomarkers for gut barrier failure in broiler chickens.Front Vet Sci 2015;2:2-10.
Cho JH,Kim HJ,Kim IH.Effects of phytogenic feed additive on growthperformance,digestibility,blood metabolites,intestinal microbiota,meat color and relative organ weight after oral challenge with Clostridium perfringens in broilers.Livest Sci 2014;160:82-8.
Choct M,Hughes RJ,Wang J,Bedford MR,Morgan AJ,Annison G.Increased small intestine fermentation is partly responsible for the antinutritive activity on non-starch polysaccharides in chickens.Br Poultry Sci 1996;37:609-21.
Choct M.Managing gut health through nutrition.Br Poultry Sci 2009;50:9-15.
Clifford RJ,Milillo M,Prestwood J,Quintero R,Zurawski DV,Kwak YI,Waterman PE,Lesho EP,Mc Gann P.Detection of bacterial 16S rRNA and identification of four clinically important bacteria by Real-Time PCR.PLoS One 2012;7:1-6.
Cross DE,McDevitt RM,Acamovic T.Herbs,thyme essential oil and condensed tannin extracts as dietary supplements for broilers,and their effects on performance,digestibility,volatile fatty acids and organoleptic properties.Br Poultry Sci 2011;52:227-37.
Cummings JH,Macfarlane GT.The control and consequences of bacterial fermentation in the human colon.J Appl Microbiol 1991;70:443-59.
Delroisse JM,Boulvin AL,Parmentier I,Dauphin RD,Vandenbol M,Portetelle D.Quantification of Bifidobacterium spp.and Lactobacillus spp.in rat fecal samples by real-time PCR.Microbiol Res 2008;163:663-70.
Du E,Wang W,Gan L,Li Z,Guo S,Guo Y.Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens.J Anim Sci Biotechnol 2016;7:2-10.
Ducatelle R,Goossens E,Meyer FD,Eeckhaut V,Antonissen G,Haesebrouck F,Immersee FV.Biomarkers for monitoring intestinal health in poultry:present status and future perspectives.Vet Res 2018;49:43.
EC.43.Council Directive of 28 June 2007 laying down minimum rules for the protection of chickens kept for meat production.O J E U 2007;182:19-28.
EU.63.Directive of the European Parliament and of the Council of 22 September2010 on the protection of animals used for scientific purposes.O J E U 2010;276:33-79.
Franciosini MP,Casagrande-Proietti P,Forte C,Beghelli D,Acuti G,Zanichelli D,Bosco A,Castellini C,Trabalza-Marinucci M.Effects of oregano(Origanum vulgare L.)and rosemary(Rosmarinus officinalis L.)aqueous extracts on broiler performance,immune function and intestinal microbial population.J Appl Anim Res 2015;44:474-9.
Hashemipour H,Kermanshahi H,Golian A,Veldkamp T.Effect of thymol and carvacrol feed supplementation on performance,antioxidant enzyme activities,fatty acid composition,digestive enzyme activities,and immune response in broiler chickens.Poultry Sci 2013;92:2059-69.
Hashemipour H,Khaksar V,Rubio LA,Veldkamp T,Van Krimpen MM.Effect of feed supplementation with a thymol plus carvacrolmixture,in combination or not with an NSP-degrading enzyme,on productive and physiological parameters ofbroilers fed on wheat-based diets.Anim Feed Sci Technol 2016;211:117-31.
Hong JC,Steiner T,Aufy A,Lien TF.Effects of supplemental essential oil on growth performance,lipid metabolites and immunity,intestinal characteristics,microbiota and carcass traits in broilers.Livest Sci 2012;144:253-62.
Hu CH,Gu LY,Luan ZS,Song J,Zhu K.Effects of montmorilloniteezinc oxide hybrid on performance,diarrhea,intestinal permeability and morphology of weanling pigs.Anim Feed Sci Technol 2012;177:108-15.
Hubener K,Vahjen W,Simon O.Bacterial responses to different dietary cereal types and xylanase supplementation in the intestine of broiler chicken.Arch Anim Nutr 2002;56:167-87.
Kaldhusdal M,Hofshagen M.Barley inclusion and avoparcin supplementation in broiler diets.2.Clinical,pathological,and bacteriological findings in a mild form of Necrotic Enteritis 1992;71:1145-53.
Kawai T,Akira S.TLR signaling.Semin Immunol 2007;19:24-32.
Keestra AM,Zoete MR,Bouwman LI,Vaezirad MM,Van Putten JPM.Unique features of chicken Toll-like receptors.Dev Comp Immunol 2013;41:316-23.
Kiarie E,Romero LF,Ravindran V.Growth performance,nutrient utilization,and digesta characteristics in broiler chickens fed corn or wheat diets without or with supplemental xylanase.Poultry Sci 2014;93:1186-96.
Lan Y,Verstegen MWA,Tamminga S,Williams BA.The role of the commensal gut microbial community in broiler chickens.Worlds Poultry Sci 2004;61:95-104.
Lee KW,Everts H,Beynen AC.Essential oils in broiler nutrition.Int J Poultry Sci2004;3:738-52.
Lee SA,Apajalahti J,Vienola K,Gonzalez-Ortiz G,Fontes CMGA,Bedford MR.Age and dietary xylanase supplementation affects ileal sugar residues and short chain fatty acid concentration in the ileum and caecum of broiler chickens.Anim Feed Sci Technol 2017;234:29-42.
Lillehoj HS,Lee KW.Immune modulation of innate immunity as alternatives-toantibiotics strategies to mitigate the use of drugs in poultry production.Poultry Sci 2012;91:1286-91.
Liu D,Guo S,Guo Y.Xylanase supplementation to a wheat-based diet alleviated the intestinal mucosal barrier impairment of broiler chickens challenged by Clostridium perfringens.Avian Pathol 2012;41:292-8.
Lu H,Adedokun SA,Adeola L,Ajuwon KM.2014.Anti-Inflammatory effects of nonantibiotic alternatives in Coccidia challenged broiler chickens.J Poultry Sci2014;51:14-21.
Lu J,Idris U,Harmon B,Hofacre C,Maurer JJ,Lee MD.Diversity and succession of the intestinal bacterial community of the maturing broiler chicken.Appl.Environ.Microbiol.2003;69:6816-24.
Matsuki T,Watanabe K,Fujimoto J,Takada T,Tanaka R.2004.Use of 16S r RNA genetargeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces.Appl Environ Microbiol 2004;70:7220-8.
Michiels J,Missotten J,Dierick NI,Fremaut D,Maene P,De Smet S.In vitro degradation and in vivo passage kinetics of carvacrol,thymol,eugenol and transcinnamaldehyde along the gastrointestinal of piglets.J Sci Food Agric 2008;88:2371-81.
Mountzouris KC,Tsirtsikos P,Kalamara E,Nitsch S,Schatzmayr G,Fegeros K.Evaluation of the efficacy of a probiotic containing Lactobacillus,Bifidobacterium,Enterococcus,and Pediococcus strains in promoting broiler performance and modulating cecal microflora composition and metabolic activities.Poultry Sci2007;86:309-17.
Mountzouris KC,Paraskeuas V,Tsirtsikos P,Palamidi I,Steiner T,Schatzmayr G,et al.Assessment of a phytogenic feed additive effect on broiler growth performance,nutrient digestibility and caecal microflora composition.Anim Feed Sci Technol2011;168:223-31.
Mountzouris KC,Tsirtsikos P,Papadomichelakis G,Schatzmayr G,Fegeros K.Evaluation of the efficacy of sequential or continuous administration of probiotics and phytogenics in broiler diets.Anim Prod Sci 2015;55:720-8.
Paraskeuas V,Fegeros K,Hunger C,Theodorou G,Mountzouris KC.Dietary inclusion level effects of a phytogenic characterised by menthol and anethole on broiler growth performance,biochemical parameters including total antioxidant capacity and gene expression of immune-related biomarkers.Anim Prod Sci2016;57:33-41.
Paraskeuas V,Fegeros K,Palamidi I,Hunger C,Mountzouris KC.2017.Growth performance,nutrient digestibility,antioxidant capacity,blood biochemical biomarkers and cytokines expression in broiler chickens fed different phytogenic levels.Ani Nu 2017;3:114-20.
Pathak M,Mandal G,Patra AK,Samanta I,Pradhan S,Haldar S.Effects of dietary supplementation of cinnamaldehyde and formic acid on growth performance,intestinal microbiota and immune response in broiler chickens.Anim Prod Sci2016;57:821-7.
Peinado MJ,Ruiz R,Echavarri A,Aranda-Olmedo I,Rubio LA.Garlic derivative PTS-Omodulates intestinal microbiota composition and improves digestibility in growing broiler chickens.Anim Feed Sci Technol 2013;181:87-92.
Pfaffl MW.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res 2001;29:45.
Rodriguez ML,Rebole A,Velasco S,Ortiz LT,Trevino J,Alzueta C.Wheat-and barleybased diets with or without additives influence broiler chicken performance,nutrient digestibility and intestinal microflora.J Sci Food Agric 2012;92:184-90.
Sacranie A,Svihus B,Denstadli V,Moen B,Iji PA,Choct M.The effect of insoluble fiber and intermittent feeding on gizzard development,gut motility,and performance of broiler chickens.Poultry Sci 2012;91:693-700.
Schwiertz A,Taras D,Sch€afer K,Beijer S,Bos NA,Donus C,Hardt PD.Microbiota and SCFA in lean and overweight healthy subjects.Obes Res 2010;18:190-5.
Silkie SS,Nelson KL.Concentrations of host-specific and generic fecal markers measured by quantitative PCR in raw sewage and fresh animal feces.Water Res2009;43:4860-71.
Song J,Xiao K,Ke YL,Jiao LF,Hu CH,Diao QY,Shi B,Zou XT.Effect of a probiotic mixture on intestinal microflora,morphology,and barrier integrity of broilers subjected to heat stress.Poultry Sci 2014;93:581-8.
Suzuki T,Hara H.Role of flavonoids in intestinal tight junction regulation.J Nutr Biochem 2011;22:401-8.
Svihus BM,Choct M,Classen HL.Function and nutritional roles of the avian caeca:a review.World’s Poultry Sci J 2013;69:249-64.
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