| The successful use of modern genetic breeding and feeding techniques has led to a tremendous increase in feed efficiency and performance of broilers,but the excessive feed efficiency and growth rate has put the broiler intestinal health under great stress.Intact mucosal morphology,stable microbiota,physical and chemical barriers and mucosal immunity are important markers of intestinal health.Numerous studies have pointed out that when the intestinal barrier function of broiler chickens is damaged,it triggers a series of immune responses in the body.Studies have shown that the intestinal barrier and systemic immune function of broiler chickens can be improved by means of nutritional modulation.In the modern broiler industry,corn is the most commonly used cereal ingredient in diets,and corn starch provides about 60% of the apparent metabolic energy of poultry diets.There is a certain proportion of resistant starch in corn starch.Currently,resistant starch has fully demonstrated its health benefits as a prebiotic in humans and mammals,with enhanced intestinal barrier and immune function.However,the effects of resistant starch on intestinal barrier and systemic immune function in poultry and its mechanism of action are not fully understood.Therefore,in this study,we used corn resistant starch diets to feed broiler chickens in order to clarify the effects of resistant starch on the changes of cecum microbiota,mucosal morphology,mucosal barrier,mucosal immunity and systemic immune function of broilers,and the potential relationship between microbial changes and intestinal mucosal barrier and systemic immune state of broilers was also studied.The results of the study can provide a theoretical basis for clarifying the regulatory effects of corn resistant starch on intestinal barrier and systemic immune function.1.Effect of corn resistant starch on growth performance,cecal microbiota and bacterial metabolites of broilers.In Experiment 1,320 one-day-old male broilers were randomly assigned into five dietary treatments: one normal corn-soybean(NC)diet,one corn-soybean based diet supplementation with 20% corn starch(CS),Diets for the 4%,8% and 12% resistant starch groups(4%RS,8%RS and 12%RS)were produced by replacing equal amounts of corn starch in the CS group with 6.67%,13.33% and 20.00% corn resistant starch(Hi-Maize260?,60% RS purity),respectively.The experiment lasted 42 days.The results showed that the CS,4%RS,8%RS,and 12%RS treatments decreased the average daily gain(ADG)and average daily feed intake(ADFI)(P<0.05)in 1-21 d,21-42 d,and 1-42 d,while increasing feed/gain ratio(F/G)(P <0.05)in 1-21 d,compared with the NC group.In addition,with the increasing dietary RS concentration,there was a linear increase in ADFI and F/G in 1-21 d,ADG and ADFI in 21-42 d,and 1-42d(P <0.05),compared to the CS group.Microbiota composition results showed that resistant starch altered the microbiota of the broiler in cecum,mainly by increasing the abundance of short-chain fatty acid-producing bacteria: the 4%RS,8%RS and 12%RS treatments significantly increased Faecalibacterium at 21 d,and the 8%RS and 12%RS treatments significantly increased Faecalibacterium and Bacteroides at 42d(P <0.05).Reducing the abundance of mucin-degrading bacteria: the 4%RS,8%RS and 12%RS treatments reduced Akkermansia at 42d(P <0.05).Increasing the abundance of some pathogenic bacteria: The 8%RS and12%RS treatments increased Alistipes at 21 d,4%RS and 12%RS treatments increased Ruminococcus torques group at 21 d,and 4%RS,8%RS and 12%RS treatments increased Desulfovibrio at 42d(P <0.05).The measurement of short-chain fatty acids concentration showed that the 8%RS and 12%RS treatments increased the concentrations of acetic acid,propionic acid and butyric acid at 21 d and 42d(P <0.05),and the 12%RS treatment increased the concentrations of acetic acid at 42d(P <0.05).With the increasing RS concentration,there was a linear trend to increase the concentrations of acetic acid,propionic acid and butyric acid at 42d(P <0.05).2.Effect of corn resistant starch on intestinal morphology and barrier function of broilersThe experimental design was the same as experiment 1 and the results show that: 1)Feeding RS diets inhibited the development of mucosal morphology.The jejunal villus height(VH)and the ratio of villus height to crypt depth(VH/CD)were lower in the CS,4%RS,8%RS,and 12%RS groups than those in the NC group(P <0.05).2)Feeding the RS diets inhibited the development of goblet cells.The 4%RS,8%RS and 12%RS treatments reduced goblet cells density(GCD)of duodenum at 21 d and 42 d as well as ileum at 42d(P<0.05),and also there was a reduction trend of jejunal GCD at 21d(P =0.061).With the increasing dietary resistant starch concentration,there was a linear decrease in jejunal and ileal GCD compared to the CS group(P <0.05).3)Feeding RS diets depleted the physical and chemical barriers function of broilers.The mRNA expression of mucin1 and claudin-1at 21 d,as well as the mucin2 at 42 d in 8%RS and 12%RS groups were lower than that in NC group(P <0.05).And with the increase of dietary RS concentration,the expression of mucin1 gene decreased linearly at 21 d compared to the CS group(P <0.05).4)Correlation analysis of mucosal barrier indicators and microbial abundance showed that Alistipes,Bacteroides,Ruminococcustorques group,Desulfovibrio and mucin2 were negatively correlated.Akermansia and GCD as well as mucin2 were positively correlated.3.Effect of corn resistant starch on systematic immune function of broilers.The experimental design is the same as the experiment 1 and the results showed that 1)RS can improved the immune organ index.Compared to the NC,the 4%RS,8%RS,and12%RS treatments increased the relative weights of bursa at 21 d and spleen at 42d(P<0.05).The relative weights of spleen,thymus,and bursa of 21 d increased linearly with increasing dietary resistant starch concentration compared to the CS group(P <0.05).2)RS could increase the density of intraepithelial lymphocytes of 21-day-old broilers.8%RS group had more mucosal lymphocytes than the NC group at 21d(P <0.05).3)RS could increase the density of SIg A-positive cells of broilers.The density of jejunal SIg A-positive cells of the CS,4%RS,and 8%RS groups was higher than that of the NC group at 21d(P<0.05).And there was a linear trend to increase with the increasing dietary RS concentration compared to the CS group(P =0.052).4)RS could increase the level of immune cytokine in plasma.The levels of IFN-γ in the CS,4%RS,8%RS,and 12%RS groups at 21 d,and IL-4 in 8%RS at 21 d,as well as IL-2 in 8%RS at 42 d were higher than those in NC group(P <0.05),and RS linearly increase with the increasing dietary RS concentration compared to the CS group(P <0.05).5)RS could increase the NOS activity and NO concentration of plasma.The NO concentration in the 4%RS,8%RS,and 12%RS groups at 21 d and TNOS and i NOS activity in the 12%RS groups at 42 d were higher than those in NC group(P <0.05).and TNOS and i NOS activity linearly increased with the increasing dietary RS concentration compared to the CS group(P <0.05).6)RS could increase the mRNA expression of jejunal mucosal inflammatory cytokine.IL-10 in 4% RS group at 21 d,TNF-α in 8% RS group at 21 d,and IL-8,IL-10,and TNF-α in 4%RS group at42 d were all higher than those in NC group(P <0.05).7)Correlation analysis of mucosal immune parameters and microbial abundance showed that Desulfovibrio was positively correlated with IL-8,IL-10,and TNF-α mRNA expression;Akkermansia was negatively correlated with IL-8,and Lachnoclostridium was negatively correlated with IL-8,IL-10,and TNF-α mRNA expressions.4.Effect of corn resistant starch in pellet feed on broiler growth performance,intestinal barrier function and cecal microbiota.In experiment 2,the NC and 4% RS diets in experiment 1 were fed to broilers in pellet form to exclude the interference of the feed intake factor and to further clarify the effects of resistant starch on broiler growth performance,intestinal barrier function and cecal microbiota.96 one-day-old male broilers were randomly assigned into two treatments.Each group had six replicates with eight broilers per replicate.The experiment lasted 42 days.The results showed that RS treatment reduced ADFI(P <0.05)in 1-21 d,increased F/G in 21-42 d and 1-42d(P <0.05),reduced jejunal VH and CD at 21d(P <0.05),ZO-2 and mucin1 mRNA expression at 21 d,and claudin-1,ocludin,ZO-2,mucin1 and mucin2 mRNA expression at 42d(P <0.05).The analysis results of the cecal microbiota showed that the RS treatment increased the abundance of Bacteroides,Alistipes,Synergistes and Eubacterium＿coprooligenes＿group at both 21 d and 42 d as well as the abundance of Desulfovibrio and Ruminococcus＿torques＿group at 42 d,while decreased Akkermansia,Faecalibacterium,Ruminococcaceae＿UCg-014 and Lactobacillus.Among them,the changes of Bacteroides,Alistipes,Desulfovibrio and Ruminococcus＿torques＿group are consistent with the results of the experiment 1.5.Effects of corn resistant starch in pellet feed on systematic immune and antioxidant functions of broilersThe experimental design was the same as that in experiment 2.The NC and 4%RS diets were fed broilers in form of pellet to exclude the interference of feed intake factors in experiment 1,and further clarify the effects of RS on the systematic immune and antioxidant functions of broilers.The results showed that RS treatment improved the relative weight of spleen and bursa at 21 d and 42 d,compared with the NC(P <0.05).Plasma NO concentration and TNOS activity were increased at 21d(P<0.05),and plasma TNOS activity was increased at 42d(P <0.05).The mRNA expression of IL-6 and TNF-α at21 d and IL-6,IL-8 as well as IL-10 at 42 d were up-regulated(P <0.05).The enzyme activities of T-SOD at 21 d and CAT at 42 d in jejunal mucosa were increased(P <0.05),and the enzyme activities of CAT,T-SOD at 21 d and T-AOC at 42 d of plasma were also increased(P <0.05).As stated above,the conclusions are as follow:(1)Compared to corn-soybean meal-based diets,corn-resistant starch diets can alter the cecal microbiota,increased short-chain fatty acid-producing bacteria and some pathogenic bacteria,decreased mucin-degrading bacteria,and increased short-chain fatty acid production,but significantly decreased the growth performance and carcass traits.(2)Compared to corn-soybean meal-based diets,corn-resistant starch diets hindered the development of goblet cell,resulting in a decrease in the expression of intestinal barrier function-related genes such as tight junctions and mucins,and weakened the physical and chemical intestinal barrier function.Combined with correlation analysis of microbiota and intestinal physical and chemical barrier function indicators,it was found that the decreased abundance of the mucin-degrading bacterium Akkermansia and increased abundance of the pathogenic bacteria Alistipes,Ruminococcus torques group,and Desulfovibrio may be the main bacteria responsible for the weakened of intestinal physical and chemical barrier functions.(3)Compared to corn-soybean meal-based diets,corn-resistant starch diets increased the relative weight of lymphoid organs and the density of lymphocytes in the intestinal epithelium,upregulated mRNA expression of jejunal mucosal cytokines and some immunoreactive substances in plasma,and activated mucosal and systematic immune status.Combined with the correlation analysis of microbiota and immune indicators,it suggested that microorganisms have a potential role in the regulation of mucosal immunity.The abundance of pro-inflammatory bacteria,Alistipes,Desulfovibrio and Bacteroides showed a positive correlation with IL-8 and TNF-α mRNA expression,while Alistipes showed a negative correlation with SIg A cell density.(4)Corn resistant starch diets fed broilers in form of pellet feed,excluding the interference of feed intake factors in previous studies,which further proved that resistant starch does alter the cecal microbiota,increase the production of short-chain fatty acids,weaken the intestinal physical,chemical and microbial barrier functions,and activated the intestinal mucosa and systematic immune function of broiler chickens. |
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