Proteomics And Metabolomics Analysis On The Molecular Mechanism Of Heat Stress In Broilers

Heat stress is a common and key concern in the modern poultry industry and its exact molecular mechanism is not fully clear. Currently, most researchers focus on the function of individual gene or single pathway. In this study, in order to investigate the key proteins and sensitive indicators which global responses to heat stress, and to reveal the relevant regulatory networks of broilers under heat exposure, high-throughput and proteomics and metabolomics technologies were exploited to analyze the effects of heat stress on global protein and small molecular metabolite composition in broiler chicken(Gallus gallus). The main results are summarized as follows:1) Establishment of broilers heat response models: 144 21 d healthy AA male broilers were randomly divided into two groups: control group(26±1℃) and continuous heating group(32±1℃), with 72 male broilers per group. The body temperature, productive performance and blood biochemical indices were determined. The results revealed that after heat exposure(32℃±1℃) for 3 weeks from 21 to 42 d in broilers, heat stress(HS) significantly reduced ADG and ADFI by 60%(P<0.01) and 42%(P<0.01) at 42 d of age, respectively. HS significantly improved the feed:gain by 46%(P<0.01) at 42 d of age. Compared with control group, the HS group had lower blood TG(P<0.05) and higher core body temperature(P<0.05), LDL-C and GLU(P<0.05) levels after 72 hours continuous exposure at 32 ℃. The core body temperature(P<0.05), blood AST(P<0.05), LDL-C(P<0.05), GLU(P<0.01) and HDL-C(P<0.01) levels were significantly improved after 1 week continuous exposure.2) Study on broiler liver proteome analysis methods based on mass spectrometer: 18 AA male broilers were randomly selected at 72 h. Then livers of 9 broilers from each group were analyzed for proteome and SWATH quantification. The number of identified proteins were increased by acetone-extracting, extending the elution time and mass "grading" in chicken liver. Using these methods, the numbers of identified proteins and peptides increased to 4271 and 36073, respectively, at a 1% FDR in 165 minutes. Over 50% of the proteins matched more than five peptides. These identified proteins were related to 328 signing pathways, including those for carbohydrate metabolism and lipid metabolism. Our work provides good method basis for broiler liver proteome quantitative analysis in the future.3) Label-free quantitative techniques reveal heat response mechanisms of broiler liver proteins: We identified 257 differentially expressed proteins in liver tissues after heat treatments using the high-throughput, label-free quantitative strategy of SWATH. These proteins, which were mainly involved in protein translation, the oxidation-reduction process, protein folding, signal transduction and the negative regulation of apoptotic processes, were involved in energy metabolism, amino acid metabolism, lipid metabolism and the regulation of immune pathways. The identities of GSTA2, NDUFA8, CYP3A7, FADS1 and HSPA5 proteins were also verified by MRM and western blotting. The results showed that these five proteins trends consistently with SWATH analysis and confirmed the reliability of quantitative proteomics using the strategy of SWATH.4) Molecular mechanism of broiler heat response based on metabolomics: At 71-73 h, 10 AA male broilers were randomly selected from each group, whose fecal samples were collected for metabolomics analysis. We established fecal metabolomics analysis techniques based on UPLC/MS, and investigated fecal metabolic variations using these techniques.. We found 10 metabolic potential biomarkers which differ greatly between the heat stress group and the control group, involving glycolysis, gluconeogenesis, and lipid/amino acid metabolism.Thorough analysis of above results and the progress of heat response in broilers, we found that the broiler may inhibit ERK1/2 signaling pathway to regulate the cell proliferation, differentiation and apoptosis of thermal stress damage repair, reduce the ACOT1 and ACOT8 expression which were reduced fatty acid synthesis. They can also increase the BHMT and decrease GAMT expression which were promoted methionine synthesis and removed the stress-generated ROS, increase the P2RX1 and decrease KIT expression to promote ceramide synthesis, increase the DDC and PAH and reduce COMT expression to promote secretion of catecholamine, which were increased neuronal excitability in order to enhance the ability of anti-stress. Meanwhile, they can increase HSP90AA1 and LUM expression, induce liver cell inflammatory and immune responses to enhance the phagocytic ability of macrophages.Our study revealed the key proteins involved in heat response regulation, and the indicators to evaluate the heat response. We also uncovered the regulation networks of broiler heat response, and explored the related mechanisms. Our study provides a foundation for early warning in the environmental stress, evaluation of the degree of comfort, and development of strategies to ameliorate the negative impacts of heat on broilers production and welfare.