Investigating differential gene expression in the pectoral muscles of broiler chickens affected by wooden breast condition

Nowadays, poultry represents the primary driver in global meat production. This has been possible due to the remarkable advancements in poultry selection, nutrition, housing, and meat processing. As a downside, the outstanding progress in this field also determined the occurrence of challenges. Among these, the manifestation of meat quality defects, commonly referred to as “growth-related myopathies”, particularly affecting the Pectoralis major muscles (PMs) of the modern fast-growing broilers, remains a major area of research. Within this context, the present study was developed within the PRIN 2022 PACHOL4 project, which aims to investigate the molecular basis of wooden breast (WB), a major quality defect in broilers, with a specific focus on characterizing the transcriptomic profile of WB-affected breast muscles. Given the importance of accurately classifying PMs to properly investigate factors associated with the onset of these abnormalities and to avoid misleading results, a total of 180 breast fillets were collected at a commercial processing plant and classified as WB-affected or normal (N) based on their macroscopic appearance. After validating their macroscopic classification through multivariate analysis of meat quality data, 10 samples/group (WB and N) were selected and used for transcriptomic analysis. Briefly, total RNA extracted from each sample was checked for quality and used for RNA-seq using NovaSeq X-Plus technology. Raw-reads quality check, trimming, alignment to the reference genome, and count were performed on the High-Performance Computing platform provided by ELIXIR-IIB HPC@CINECA. After raw-reads processing, differential analysis was performed in R using DESeq2 R and limma packages, while functional categories significantly enriched by the DEGs were detected by using DAVID Knowledgebase v2024q4 and considering a Benjamini–Hochberg adjusted p-value of 0.05 as significance threshold. Considering a log2fold-change>|1| and a q value<0.05, a total of 565 differentially expressed genes (DEGs) were found. Among those, 483 and 82 were respectively up- and down-regulated in WB compared to NB. In accordance with the literature, functional analysis of up-regulated DEGs evidenced genes involved in PI3K-Akt and cytokine-mediated signaling pathways (P<0.001), as well as cellular components related to collagen trimer composition and ECM-receptor interaction (P<0.001), while functional terms related to carbon metabolism, glycolysis, and biosynthesis of amino acids were found enriched by the down-regulated DEGs. Notably, MYBPC3, TNNT2, and ACTC1, which are involved in the dilated and hypertrophic cardiomyopathies in humans and generally transiently expressed in embryonic chick skeletal muscles, were found among the up-regulated DEGs in WB (P<0.05). Therefore, the present results not only corroborate part of the existing literature but also highlight the dysregulation of several genes involved in cardiac-related pathways in PMs affected by WB. Even though a direct association with cardiac hypertrophy in WB-affected birds cannot be speculated, similarities in the underlying biological and molecular mechanisms between these two conditions, as well as the occurrence of gene expression patterns resembling the early stage of muscle development in WB muscles, have been hypothesized. This research was supported by NextGenerationEU, National Grant PRIN2022 (Prot. n. 2022EPWEPW).