Alterations Of Gut-Lung Microbiota In Sodium Butyrate Alleviating Heat Stress-Induced Lung Injury Of Broilers

Heat stress is a non-specific stressor that seriously threatens broiler health.During heat stress,broiler panting and oxygen consumption increase,which puts a lot of pressure on the lungs.However,heat stress-induced broiler lung injury and its effect on the gut-lung microbiota have been less studied.Sodium butyrate(SB)has a wide range of biological functions,including inhibiting pathogenic bacteria,improving immunity and enhancing antioxidant capacity.The purpose of this study was to investigate the effect of dietary supplementation of SB on heat stress-induced lung injury in broilers,and to elucidate the changes and contributions of the gut-lung microbiota during this process.The results consist of the following 3 parts:Experiment 1: Dietary supplementation of SB alleviates heat-stress broilers lung injuryIn this experiment,100 1-day-old,unvaccinated,healthy mixed-sex AA broilers were randomly divided into 5 groups,namely normal temperature group(NT),heat stress group(HS),heat stress group with SB(HS+SB),heat stress group with AB(HS+AB),and heat stress group with AB and SB(HS+AB+SB).At the age of 26 days,HS+SB and HS+AB+SB broilers were orally administered with SB daily,and other groups were orally administered with the same amount of normal saline.At the age of 28 days,NT broilers were fed in a constant temperature(24±1°C)environment,and the other groups were treated with high temperature(32±1°C),and sacrificed 14 days later for sampling.The test results of the samples showed that heat stress led to a decrease in survival rate,eventually weight loss and abnormal immune organ indexes of broilers.In addition,heat stress resulted in increased plasma levels of creatine kinase(CK)and lactate dehydrogenase(LDH),as well as increased expression of heat shock protein 60(HSP60),interleukin 10(IL-10),and tumor necrosis factor alpha(TNF-α)in the lungs of broilers.The results of HE staining of lung tissue further confirmed that heat stress induced broiler lung injury.SB effectively alleviated heat stress-induced lung injury in broilers.However,when antibiotics disrupt the gut microbial homeostasis,the effective effect of SB was lost.This suggests that the effects of SB depend on the gut microbial homeostasis.Experiment 2: Effects of SB on gut microbiota of heat-stressed broilersOn the basis of Experiment 1,the effects of heat stress on the gut microbiota of broilers and the regulation of SB were explored.The samples were randomly selected from the cecal contents of 6 broilers in each group of NT,HS,HS+SB and HS+AB+SB,and 16 S r RNA sequencing was performed.The results showed that heat stress under normal diet feeding did not change the alpha-diversity and beta-diversity of gut microbiota,while antibiotics significantly affected the diversity and structural composition of gut microbiota.Heat stress induced a decrease in the relative abundance of p＿Firmicutes and an increase in the relative abundance of p＿Bacteroidetes.Heat stress also reduced the relative abundance of bacteria genera such as g＿Faecalibacterium and g＿Eubacterium＿hallii＿group in the gut.Dietary supplementation of SB effectively regulated the changes in gut microbiota induced by heat stress.Based on this,this study further analyzed the potential functional changes of gut microbiota.Correlation analysis showed that the changes in gut microbiota induced by heat stress were closely associated with injury,and the gut microbiota regulated by SB were associated with alleviating the injury.Prediction of gut microbial function shows that stress response could accelerate amino acid and vitamin metabolism and affect signal transduction.Pathways significantly enriched in gut microbiota of broilers supplemented of SB were related to maintaining cell integrity,promoting water reabsorption and regulating body homeostasis.Experiment 3: Effects of SB on lung microbiota of heat-stressed broilersOn the basis of Experiment 2,the effects of heat stress on the lung microbiota of broilers and the regulation of SB were explored.The samples of this experiment were selected from the bronchoalveolar lavage fluid(BALF)of 24 broilers selected in NT,HS,HS+SB and HS+AB+SB in the experiment 2,and 16 S r RNA sequencing was performed.The results showed that heat stress and gut microbial disturbance affected the diversity and structural composition of lung microbiota.Heat stress enriched p＿Actinobacteriota and p＿Proteobacteria in the lung.The microbiota was commonly found in the oropharyngeal and nasal cavities,and their enrichment has been associated with wheezing in broilers.Heat stress also increased the relative abundance of g＿Staphylococcus in the lungs.SB effectively alleviated these effects,and significantly up-regulated the relative abundance of beneficial bacteria such as g＿Lachnospiraceae＿unclassified and g＿Lactococcus.Likewise,the regulatory effect of SB on the lung microbiota was lost when gut microbiota was perturbed.Based on this,the study further analyzed the potential functional changes of the lung microbiota.Correlation analysis showed that the lung microbiota genera that played an important role under the influence of heat stress and gut microbial disturbance were similar,and these changes were associated with lung injury,and dietary supplementation of SB relieved them.Likewise,functional predictions of the lung microbiota indicated that SB regulated cell motility,nervous system,environmental adaptation,excretory system,endocrine system,and immune system pathways of lung microbiota were at active levels,which was related to stimulating bacterial behavior,regulating mental state,enhancing the body’s environmental adaptability and enhancing immunity.In conclusion,this study demonstrates that dietary supplementation of SB alleviated the heat stress-induced lung injury of broilers,and the gut-lung microbiota regulated by SB might be play an important role in this process.