| This experiment was conducted to study the effects of dietary coated sodium butyrate(CSB)on the growth performance and histological structure and function of immune organs of the broilers subjected to cyclic heat stress,and to analyze the expression changes of DAMPs,cytokines,genes associated with innate immunity and inflammatory signaling pathways.The mechanism of sodium butyrate alleviating heat stress induced immune tissue damage of broilers was preliminarily revealed.A total of 500 one-day-old AA broilers were selected for this experiment.After routine immunization and feeding to 21 days of age,a total of 450 healthy broilers with similar body weight were selected and randomly divided into 5 treatment groups with 6 replicates per treatment and 15 broilers per replicate.The design of the 5 treatment groups is as follows: 1)Normal temperature group(22±2℃): broilers feed a basal diet;2)Heat stress treatment groups(HS):broilers fed a basal diet;3)HS + 300 mg/kg CSB: broilers feed a basal diet supplemented with 300 mg/kg CSB;4)HS + 600 mg CSB/kg: broilers feed a basal diet supplemented with 600 mg/kg CSB;5)HS + 900 mg/kg CSB: broilers feed a basal diet supplemented with 900 mg/kg CSB.The heat-stress broilers were reared in a diurnal high temperature condition(28℃-36℃-28℃).The experiment lasted for 14 days.Samples were collected at 1 and 14 days after heat stress treatment for detection and analysis.The results are as follows:(1)Heat stress reduced the growth performance of broilers.Compared with NT group,heat stress significantly reduced the average daily feed intake and average daily gain of the broilers subjected to heat stress(P<0.01),and the ratio of feed to gain showed an increasing trend(0.05<P<0.1).Dietary supplementation with sodium butyrate could significantly increase average daily gain and average daily feed intake of broilers under heat stress(P<0.05),among them,the effect of promoting growth was the best in the group supplemented with 900 mg CSB in this experiment.(2)Heat stress induced oxidative stress in broilers,and dietary supplementation with CSB can improve the antioxidant capacity of the broilers subjected to heat stress.Compared with NT group,heat stress significantly or extremely significantly decreased the activity of antioxidant enzyme CAT and the level of T-AOC,significantly or extremely significantly increased the activities of LDH and CK and the contents of MDA,α1-AGP、ET and TBARS in serum of the broilers subjected to heat stress for 1 day(P<0.05 or P<0.01),and the activity of antioxidant enzyme GSH-Px showed a decreasing trend(0.05<P<0.1).Dietary supplementation with sodium butyrate could significantly increase the activity of antioxidant enzyme SOD and the level of T-AOC(P<0.05),extremely significantly decrease the activity of LDH in serum of the broilers subjected to heat stress,and the activity of CK and the contents of MDA,α1-AGP and TBARS in serum showed a decreasing trend(0.05<P<0.1).Compared with NT group,heat stress significantly or extremely significantly decreased the activities of GSH-Px,CAT and SOD and the level of T-AOC,significantly or extremely significantly increased the contents of MDA,α1-AGP,ET and TBARS in serum of the broilers subjected to heat stress for 14 days(P<0.05 or P<0.01).Dietary supplementation with sodium butyrate could significantly improved the activities of GSH-Px,CAT and SOD and the level of T-AOC,significantly or extremely significantly decreased the activity of LDH and the contents of α1-AGP,ET and TBARS in serum of the broilers subjected to heat stress,the antioxidant capacity of the broilers in the group fed dietary supplementation with 900 mg/kg CSB was the best in this experiment.(3)Heat stress induced inflammatory response in broilers.Compared with NT group,heat stress significantly increased the contents of pro-inflammatory cytokines IL-6 and IL-8 in serum of the broilers subjected to heat stress for 1 day(P<0.05),and the content of anti-flammatory cytokines IL-10 in serum showed a decreasing trend(0.05<P<0.1).Dietary supplementation with sodium butyrate could significantly decreased the content of IL-6(P<0.01),and significantly or extremely significantly increased the contents of anti-flammatory cytokines IL-10 and TGF-β in serum of the broilers subjected to heat stress(P<0.05 or P<0.01),and the contents of pro-inflammatory cytokines of IL-1β and IL-8 showed a decreasing trend(0.05<P<0.1).Compared with NT group,heat stress extremely significantly increased the contents of IL-6,TNF-α,IL-1β and IL-8(P<0.01).Dietary supplementation with sodium butyrate could significantly or extremely significantly decreased the contents of IL-6,TNF-α,IL-1β and IL-8(P<0.05 or P<0.01),significantly increased the content of TGF-β(P<0.05).The anti-flammatory effect of the broilers in the group fed dietary supplemented with 900 mg/kg CSB was the best in this experiment.(4)Heat stress inhibited the immune organ development.Compared with NT group,heat stress significantly decreased the immune organ indexes of bursa of Fabricius,spleen and thymus of the broilers subjected to heat stress for 14 days(P<0.05).Dietary supplementation with sodium butyrate significantly increased the immune organ indexes of the broilers subjected to heat stress(P<0.05).Heat stress damaged the structure of liver and immune organs of the broilers subjected to heat stress for 1 day and 14 days.It damaged the portal areas of the liver,significantly widened the dirichlet gap and the liver cells showed steatosis.Spleen hyperemia,and the boundaries were vague between red and white pulps.The lymphatic follicles of bursa of Fabricius were atrophied,the boundaries of the cortex and medulla were not clear,and had many cavity-like structures in it.Dietary supplementation with sodium butyratecould reduce the damage of structure of liver and immune organs caused by heat stress,and the improvement effects were the best in the group supplemented with 900 mg/kg CSB in this experiment.(5)Heat stress increased the expression of DAMPs.Compared with NT group,heat stress significantly increased the immune reactivities,m RNA expressions and protein of HMGB1,HSP70 and HSP90 in bursa of Fabricius,spleen and liver of the broilers subjected to heat stress for 1 day and 14days(P<0.05 or P<0.01).Dietary supplementation with sodium butyratesignificantly decreased the immune reactivities,m RNA expressions and protein of HMGB1,HSP70 and HSP90 in bursa of Fabricius,spleen and liver of the broilers subjected to heat stress for 1 day and 14days(P<0.05 or P<0.01),and the expression levels of DAMPs of the broilers in the group supplemented with 900 mg/kg CSB subjected to heat stress were decreased more than the other groups.(6)Heat stress changed Toll receptor pattern expressions,but there were differences in different tissues.Compared with NT group,heat stress significantly or extremely significantly decreased the immune reactivities,m RNA expressions and protein of TLR2 in bursa of Fabricius(P<0.05 or P<0.01),significantly or extremely significantly increased the immune reactivities,m RNA expressions and protein of TLR4,TLR5 and TLR15 in bursa of Fabricius,spleen and liver of the broilers subjected to heat stress for 1day and 14days(P<0.05 or P<0.01).Dietary supplementation with sodium butyratesignificantly or extremely significantly increased the immune reactivities,m RNA expressions and protein of TLR2 in bursa of Fabricius,spleen and liver(P<0.05 or P<0.01),significantly or extremely significantly decreased the immune reactivities,m RNA expressions and protein of TLR4,TLR5 and TLR15 in bursa of Fabricius,spleen and liver of the broilers subjected to heat stress for 1 day and 14days(P<0.05 or P<0.01).(7)Heat stress increased the downstream gene expressions of Toll receptor.Compared with NT group,heat stress significantly or extremely significantly increased the m RNA expressions of My DD88 in bursa of Fabricius and liver,TRAF6 and TRIF in spleen,My DD88,TRAF6 and TRIF in thymus of the broilers subjected to heat stress for 1day(P<0.05 or P<0.01),and the m RNA expressions of TRAF6 and TRIF in bursa of Fabricius and TRIF in liver showed an increasing trend(0.05<P<0.1).Dietary supplementation with sodium butyrate significantly decreased the m RNA expressions of My DD88 in bursa of Fabricius and liver,TRIF in spleen,My DD88 and TRAF6 in thymus of the broilers subjected to heat stress for 1 day(P<0.05),and the m RNA expressions of TRAF6 in bursa of Fabricius and spleen and TRIF in thymus showed a decreasing trend(0.05<P<0.1).Compared with NT group,heat stress extremely significantly increased the m RNA expressions of My DD88,TRAF6 and TRIF in bursa of Fabricius,spleen,thymus and liver of the broilers subjected to heat stress for 14 days(P<0.05 or P<0.01).Dietary supplementation with sodium butyrate significantly the m RNA expressions of My DD88,TRAF6 and TRIF in bursa of Fabricius,spleen,thymus and liver of the broilers subjected to heat stress for 14 days(P<0.05),and the suppressing effects of downstream gene expressions of Toll receptor were better in the group supplemented with 900 mg/kg CSB than others subjected to heat stress in this experiment.(8)Heat stress activated inflammatory signaling pathways,but the dietary supplementation with sodium butyrate restrained inflammatory signaling pathways.Compared with NT group,heat stress significantly or extremely significantly increased the phosphorylation levels of ERK1/2,p38 and p65 in bursa of Fabricius,ERK1/2,JNK,p38 and p65 in spleen and thymus,and p38 and p65 in liver of the broilers subjected to heat stress for 1 day(P<0.05 or P<0.01),the phosphorylation level of JNK in liver showed a decreasing trend(0.05<P<0.1).Dietary supplementation with sodium butyrate significantly or extremely significantly decreased the phosphorylation level of ERK1/2,p38 and p65 in bursa of Fabricius,ERK1/2,JNK,p38 and p65 in spleen,JNK,p38 and p65 in thymus and p38 and p65 in liver of the broilers subjected to heat stress for 1 day(P<0.05 or P<0.01).Compared with NT group,heat stress significantly or extremely significantly increased the phosphorylation levels of ERK1/2,JNK,p38 and p65 in bursa of Fabricius,spleen thymus and liver of the broilers subjected to heat stress for 14 day(P<0.05 or P<0.01).Dietary supplementation with sodium butyrate significantly or extremely significantly decreased the phosphorylation levels of ERK1/2,JNK,p38 and p65 in bursa of Fabricius and spleen,ERK1/2,JNK and p65 in thymus and JNK,p38 and p65 in liver of the broilers subjected to heat stress for 14 day(P<0.05 or P<0.01),the phosphorylation level of p38 in thymus showed a decreasing trend(0.05<P<0.1),the restraining effect that inflammatory signaling pathways were the best in the group supplemented with 900 mg/kg CSB in this experiment.(9)Heat stress increased the expressions of pro-inflammatory cytokines but decreased the expressions of anti-inflammatory cytokines.Dietary supplementation with sodium butyrate could reverse these changes.Compared with NT group,heat stress significantly or extremely significantly increased the m RNA expressions of pro-inflammatory cytokines IL-1β,IL-6,IL-8 and TNF-α in bursa of Fabricius,spleen thymus and liver of the broilers subjected to heat stress for 1 day and 14 days(P<0.05 or P<0.01),and significantly increased the m RNA expressions of anti-inflammatory cytokines IL-10 in bursa of Fabricius,IL-4 and IL-10 in spleen and TGF-β in thymus(P<0.05).Dietary supplementation with sodium butyrate significantly decreased the m RNA expressions of IL-1β,IL-6,IL-8 and TNF-α in bursa of Fabricius,IL-6 and IL-8 in spleen,IL-6,IL-8 and TNF-α in thymus and liver of the broilers subjected to heat stress for 1 day(P<0.05),and the m RNA expressions of IL-1β in spleen,thymus and liver showed a decreasing trend(0.05<P<0.1),significantly or extremely significantly increased the m RNA expressions of anti-inflammatory IL-4 and IL-10 in bursa of Fabricius and IL-4,IL-10 and TGF-β in spleen,thymus and liver of the broilers subjected to heat stress(P<0.05 or P<0.01).Dietary supplementation with sodium butyrate significantly or extremely significantly decreased the m RNA expressions of IL-1β,IL-6,IL-8 and TNF-α in bursa of Fabricius,spleen,thymus and liver of the broilers subjected to heat stress for 14 days(P<0.05 or P<0.01),significantly decreased the m RNA expressions of IL-4 and IL-10 in bursa of Fabricius,TGF-β in spleen and liver and IL-10 and TGF-β in thymus(P<0.05),and the m RNA expressions of IL-4 in thymus and liver showed a decreasing trend(0.05<P<0.1),the decreasing effects of m RNA expressions of pro-inflammatory cytokines were the best in the group supplemented with 900 mg/kg CSB in this experiment.(10)Heat stress had different effects on Toll receptors negative regulatory factors expressions in different tissues.Compared with NT group,heat stress significantly increased the m RNA expressions of SIGIRR in bursa of Fabricius,SIGIRR,SOCS1 and Tollip in spleen,A20 and SIGIRR in thymus and A20,SIGIRR and SOCS1 in liver of the broilers in group subjected to heat stress for 1 day(P<0.05 or P<0.01).Dietary supplementation with sodium butyrate significantly or extremely significantly increased the m RNA expressions of A20 and SIGIRR in bursa of Fabricius,SIGIRR,SOCS1 and Tollip in spleen,A20,SIGIRR,SOCS1 and Tollip in thymus and SIGIRR,SOCS1 and Tollip in liver the broilers in group subjected to heat stress(P<0.05 or P<0.01).Compared with NT group,heat stress had no effects on the m RNA expressions of Toll receptors negative regulatory factors of liver and immune organs of the broilers in group subjected to heat stress for 14 days.Dietary supplementation with sodium butyrate significantly decreased the m RNA expressions of A20 and Tollip in bursa of Fabricius and Tollip in liver of the broilers in group subjected to heat stress for 14 days(P<0.05),the m RNA expressions of A20 in liver showed a decreasing trend(0.05<P<0.1),significantly or extremely significantly increased the m RNA expressions of A20,SIGIRR and Tollip in spleen and thymus and SIGIRR and SOCS1 in liver of the broilers in group subjected to heat stress for 14 days(P<0.05 or P<0.01).In conclusion,heat stress activated the DAMPs-TLRs-MAPKs/NF-κB mediated inflammatory signaling pathway,increased the expressions of pro-inflammatory cytokines,decreased the expression of anti-inflammatory cytokines and negative regulatory factors of TLRs signaling pathway,resulted in damages to the structure and function of immune tissues and organs,and ultimately reduced the performance of broilers.Dietary supplementation with sodium butyrate could reverse the above changes,alleviated the immune organ damage caused by heat stress and improved the production performance.Sodium butyrate might mitigate the damaging effects on structure and function of immune organs of the broilers by regulating DAMPs-TLRS-MAPKS/NF-κB signaling pathway. |
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