| The heat stress response in chickens is a complex biological process that causes changes in blood biochemical indices and leads to decreased production performance and reduced welfare of chickens,and the mechanisms related to the heat stress response in chickens are still unclear.In this study,we first measured the blood biochemical indexes of Xinhua hens before and after acute heat stress(36℃,6 hours),and made paraffin sections of heart,liver,spleen,lung and kidney tissues for HE staining and observation to determine the effects of acute heat stress on metabolism and tissue damage in chickens;afterwards,transcriptome sequencing of five visceral organs before and after heat stress was performed to elucidate the potential mechanisms by which heat stress affects visceral metabolism and damage through differential expression analysis and gene function enrichment analysis;screening of key genes for acute heat stress response by weighted gene co-expression network analysis and verification of m RNA expression levels of key genes in tissues by q PCR;finally,a heat stress model of chicken embryonic primary hepatocytes was established,and overexpression vectors of AGMAT gene and MAT structural domain of FASN gene were constructed and transfected into chicken embryonic primary hepatocytes to further verify the role of key genes in the heat stress response.The main findings are as follows:(1)Acute heat stress(36℃,6 hours)treatment was performed on Xinhua laying hens,and it was found that acute heat stress caused significant increases in BT,ALT,AST,LDH,CK and GLU,and significant decreases in TP,TG,TCHO and CO2.Paraffin sections of heart,liver,spleen,lung and kidney tissues were made and observed before and after acute heat stress,and it was found that heat stress caused damage to heart,liver,spleen,lung and kidney tissues.(2)Transcriptome sequencing was performed on heart,liver,spleen,lung and kidney tissues of four chickens before and after acute heat stress,and differential expression analysis was performed with padj≤0.05 and |log2Fold Change|≥1.0.552,519,27,103 and213 differentially expressed genes were screened in heart,liver,spleen,lung and kidney tissues,respectively,and the number of differentially expressed genes shared by different tissues The number of differentially expressed genes shared by different tissues was low and the number of unique differential genes was high.(3)WGCNA analysis was performed on 1093 differentially expressed genes screened in 5 tissues to screen for key gene modules and key genes in the tissue heat stress response,ultimately identifying 12 genes in the heart(including USP13,ANOS1,ADCYP1,COL6A1,MYOCD,PKP2,KLHL34,etc.),42 genes in the liver(including AGMAT,FASN,ELOVL6,GRHPR,PRDX4,SCCPDH,HSD17B12,etc.),and 2 genes in the lung(DLL4,ENSGALG00000045238)were key genes for heat stress response;some key genes were detected by q PCR in the liver and heart of another 5 heat-stressed chickens and 5 non-heatstressed chickens The expression of some key genes was detected by q PCR in the liver and heart of five other heat-stressed chickens and five non-heat-stressed chickens,demonstrating that the expression of key genes did differ before and after heat stress.(4)Through functional enrichment analysis of differentially expressed genes and heat stress key gene module genes in different tissues,we found that acute heat stress affects glucose,lipid and protein metabolism in the liver,such as glycine and serine metabolism,pyruvate metabolism,and fatty acid chain extension;the expression of genes related to extracellular matrix,energy supply,and myocardial contraction in the heart;the expression of genes related to steroid biosynthesis and tryptophan metabolism in the kidney;the expression of genes related to vascular development and gas transport in the lung;and the expression of genes related to coenzyme A biosynthesis in the spleen.(5)A heat stress model of chicken embryonic primary hepatocytes was established,and overexpression vectors of AGMAT gene and MAT structural domain of FASN gene were constructed and transfected into chicken embryonic primary hepatocytes for heat stress treatment,and it was found that overexpression of AGMAT gene and MAT structural domain of FASN gene could rescue the decrease of TG in cell supernatant caused by heat stress,but could not alleviate the increase of LDH caused by heat stress.In conclusion,acute heat stress(36°C,6h)causes changes in the heart,liver,spleen,lung and kidney transcriptome of laying hens,resulting in metabolic disorders in the organism;the liver is a key organ in response to acute heat stress;acute heat stress decreases fatty acid synthesis and extension-related gene expression in the liver,which may be one of the reasons for the decrease in TG in serum due to heat stress;increased expression of genes related to fatty acid degradation in the heart,increased energy supply and enhanced myocardial contraction;increased expression of genes related to vascular development in the lung,inhibited expression of genes related to coagulation and affected gas exchange in the lung;inhibited expression of genes related to vitamin D3 synthesis in the kidney;and promoted expression of genes related to coenzyme A biosynthesis in the spleen.The above results will improve the knowledge of acute heat stress response and adaptation in laying hens. |
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