Effects Of Ammonia Exposure On Production Performance And Tracheal Immune Function Of Broilers

Ammonia（NH₃）is a colorless and pungent alkaline gas widely found in poultry sheds,which is mainly produced in a variety of metabolic processes in poultry and livestock and excreted with excretion and emitted into the air.It will seriously endanger the health of poultry and livestock with exceedingly high concentrations.In many centralized farms in my country,due to large-scale supervision but not very standardized and other reasons,many livestock raised in sheds,especially broilers and other animals that are more sensitive to NH₃stimulation,will be affected by NH₃,which will cause diseases in breathing system,digestive system,nutritional and metabolic process and other symptoms and pathological changes that seriously damage the life safety and fanatic value of animals,resulting in unnecessary waste of resources.The trachea is a vital part of the respiratory tract,whose epithelial mucosa has an immune barrier function and can be used as a research object for the damage of inhaled toxic gases such as NH₃to the animal body.At present,the specific regulatory mechanism of the effects of NH₃exposure on the tracheal tissue of broilers is still unclear.In order to evaluate the effect of ammonia exposure on broiler performance,as well as the effect and mechanism of ammonia on the immune function of tracheal tissue,this experiment uses broilers as the research subjects,using group comparison method to establish an ammonia exposure model.The ammonia in the environmental control chamber of the control group was controlled below 5 mg/m³,and the ammonia concentration in the environmental control chamber of the exposure group was 20 mg/m³at 0-3 weeks of age and 45mg/m³at 4-6 weeks old.Conventional methods was used to assess the impact of ammonia on production performance,optical microscope and transmission electron microscope were used to observe the structural changes of tracheal tissue,transcriptome sequencing technology was used to sequence and screen the proteome of tracheal tissue differentially expressed proteins,q RT-PCR and western blot were used to confirm the differentially expressed genes and proteins in immune function,oxidative stress and mitochondria and endoplasmic reticulum stress-ralated cell apoptosis pathway.The specific test results show that:（1）The survival rate,average body weight and european index of the test broilers were negatively correlated with the NH₃concentration,confirming that NH₃exposure reduced broiler performance.（2）NH₃exposure can cause down-regulation of the mRNA expression of antimicrobial peptide-related genes AVBD1,AVBD6,AVBD7,AVBD9,AVBD10 and AVBD12 in broiler tracheal tissue;the mRNA expression of cytokines IL-1β,IL-6 and TGF-β,and that of IL-2,IL-4,IL-10 and IFN-γwas down-regulated;the expression of mucosal immunoglobulin-related genes Ig A,Ig G and Ig M showed a decrease,indicating that NH₃exposure can cause a decrease in tracheal immune function.（3）The results of microstructure observation showed that the NH₃exposure caused obvious pathological changes in epithelial tissue,including shedding of epithelial cells and severe loss of ciliated cells.Observation of transmission electron microscope showed that volume of broiler tracheal epithelial cells decreased,the cytoplasm was concentrated,the nuclear chromatin was marginalized,the mitochondria swelled and ruptured,and the endoplasmic reticulum expanded and swelled into vesicles,indicating that NH₃exposure can cause cell apoptosis in tracheal tissues.（4）The results of proteomics analysis showed that NH₃exposure caused 106 protein differential expressions in broiler tracheal tissues,of which 95 proteins were up-regulated and 11proteins were down-regulated.The differential expression threshold was p<0.05,and the multiple of change was≥1.2;GO rich analysis showed that compared with the control group,the NH₃exposure group had significant differences in lipid metabolism,mitochondrial complex,transmembrane transport,and organelle-related functional protein expression;the KEGG pathway analysis showed that there were significant differences in protein expression in oxidative phosphorylation,metabolic pathways,TGF-βsignaling pathways,and cytokine-receptor interactions between NH₃exposure group and the control group.The results consistently indicate that ammonia exposure may induce mitochondrial pathways and endoplasmic reticulum stress-related cell apoptosis by causing oxidative stress in broiler tracheal tissue.（5）Detection of oxidative stress-related indicators by q RT-PCR found that the mRNA expression of GSH-Px,SOD,and CAT was down-regulated,indicating that excessive ammonia exposure caused oxidative stress in broiler tracheal tissue.（6）The detection of mitochondrial function and mitochondrial apoptosis-reelated pathway genes and proteins revealed that the expression of mitochondrial complex-related factors COX2,ATP5O and mitochondrial apoptosis pathway factors Caspase3,Caspase9,Bax and Bak were significantly up-regulated,and the expression of mitochondrial apoptosis inhibitors Bcl-2 and Bcl-xl decreased.The test results of endoplasmic reticulum stress-related factors showed that the expression of IRE1,GRP78,XBP1,CHOP,and SERCA were all significantly up-regulated,and the expression of the specific factor existing in endoplasmic reticulum stress-induced apoptosis Caspase12 also increased,indicating that NH₃exposure can affect mitochondrial function and apoptosis pathway-related factors as well as cause endoplasmic reticulum stress and induce cell apoptosis in broiler tracheal tissue.In summary,NH₃exposure can reduce broiler performance and cause oxidative stress in broiler tracheal tissue.This response affects mitochondrial function,triggers endoplasmic reticulum stress,and further induces the activation of related apoptosis pathways,resulting in a large number of cell apoptosis,causing immune dysfunction of the broiler trachea.This research provides new insights for understanding the toxicological mechanism of NH₃respiratory tract,and direction for the improvement of the immunity of breeding broilers and the prevention and treatment of respiratory diseases in production practice.