Transcriptome-based screening of intracellular pathways and angiogenesis related genes at different stages of thiram induced tibial lesions in broiler chickens.

ABSTRACT

BACKGROUND: The Tibial dyschondroplasia (TD) in fast-growing chickens is mainly caused by improper blood circulation. The exact mechanism underlying angiogenesis and vascularization in tibial growth plate of broiler chickens remains unclear. Therefore, this research attempts to study genes involved in the regulation of angiogenesis in chicken red blood cells. Twenty-four broiler chickens were allotted into a control and thiram (Tetramethyl thiuram disulfide) group. Blood samples were collected on day 2, 6 (8- and 14-days old chickens) and 15 (23 days old chickens). RESULTS: Histopathology and hematoxylin and eosin (H&E) results showed that angiogenesis decreased on the 6th day of the experiment but started to recover on the 15th day of the experiment. Immunohistochemistry (IHC) results confirmed the expressions of integrin alpha-v precursor (ITGAV) and clusterin precursor (CLU). Transcriptome sequencing analysis evaluated 293 differentially expressed genes (DEGs), of which 103 up-regulated genes and 190 down-regulated genes were enriched in the pathways of neuroactive ligand receptor interaction, mitogen-activated protein kinase (MAPK), ribosome, regulation of actin cytoskeleton, focal adhesion, natural killer cell mediated cytotoxicity and the notch signalling pathways. DEGs (n = 20) related to angiogenesis of chicken erythrocytes in the enriched pathways were thromboxane A2 receptor (TBXA2R), interleukin-1 receptor type 1 precursor (IL1R1), ribosomal protein L17 (RPL17), integrin beta-3 precursor (ITGB3), ITGAV, integrin beta-2 precursor (ITGB2), ras-related C3 botulinum toxin substrate 2 (RAC2), integrin alpha-2 (ITGA2), IQ motif containing GTPase activating protein 2 (IQGAP2), ARF GTPase-activating protein (GIT1), proto-oncogene vav (VAV1), integrin alpha-IIb-like (ITGA5), ras-related protein Rap-1b precursor (RAP1B), tyrosine protein kinase Fyn-like (FYN), tyrosine-protein phosphatase non-receptor type 11 (PTPN11), protein patched homolog 1 (PTCH1), nuclear receptor corepressor 2 (NCOR2) and mastermind like protein 3 (MAML3) selected for further confirmation with qPCR. However, commonly DEGs were sarcoplasmic/endoplasmic reticulum calcium ATPase 3 (ATP2A3), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), centriole cilia and spindle-associated protein (CCSAP), coagulation factor XIII A chain protein (F13A1), shroom 2 isoform X6 (SHROOM2), ras GTPase-activating protein 3 (RASA3) and CLU. CONCLUSION: We have found potential therapeutic genes concerned to erythrocytes and blood regulation, which regulated the angiogenesis in thiram induced TD chickens. This study also revealed the potential functions of erythrocytes. 1. Tibial dyschondroplasia (TD) in chickens were more on day 6, which started recovering on day 15. 2. The enriched pathway observed in TD chickens on day 6 was ribosome pathway, on day 15 were regulation of actin cytoskeleton and focal adhesion pathway. 3. The genes involved in the ribosome pathways was ribosomal protein L17 (RPL17). regulation of actin cytoskeleton pathway were Ras-related C3 botulinum toxin substrate 2 (RAC2), Ras-related protein Rap-1b precursor (RAP1B), ARF GTPase-activating protein (GIT1), IQ motif containing GTPase activating protein 2 (IQGAP2), Integrin alpha-v precursor (ITGAV), Integrin alpha-2 (ITGA2), Integrin beta-2 precursor (ITGB2), Integrin beta-3 precursor (ITGB3), Integrin alpha-IIb-like (ITGA5). Focal adhesion Proto-oncogene vav (Vav-like), Tyrosine-protein kinase Fyn-like (FYN).