Gene expression profiling of the early pathogenesis of wooden breast disease in commercial broiler chickens using RNA-sequencing

Abstract

Wooden Breast Disease (WBD), a myopathy in commercial broiler chickens characterized by abnormally firm consistency of the pectoral muscle, impacts the poultry industry negatively due to severe reduction in meat quality traits. To unravel the molecular profile associated with the onset and early development of WBD in broiler chickens, we compared time-series gene expression profiles of Pectoralis (P.) major muscles between unaffected and affected birds from a high-breast-muscle-yield, purebred broiler line. P. major biopsy samples were collected from the cranial and caudal aspects of the muscle belly in birds that were raised up to 7 weeks of age (i.e. market age). Three subsets of biopsy samples comprising 6 unaffected (U) and 10 affected (A) from week 2 (cranial) and 4 (caudal), and 4U and 11A from week 3 (cranial) were processed for RNA-sequencing analysis. Sequence reads generated were processed using a suite of bioinformatics programs producing differentially expressed (DE) genes for each dataset at fold-change (A/U or U/A) >1.3 and False Discovery Ratio (FDR) <0.05 (week 2: 41 genes; week 3: 618 genes and week 4: 39 genes). Functional analysis of DE genes using literature mining, BioDBnet and IPA revealed several biological processes and pathways associated with onset and progress of WBD. Top among them were dysregulation of energy metabolism, response to inflammation, vascular disease and remodeling of extracellular matrix. This study reveals that presence of molecular perturbations involving the vasculature, extracellular matrix and metabolism are pertinent to the onset and early pathogenesis of WBD in commercial meat-type chickens.

Fig 1. Overlapping differentially expressed genes across 3-week period.

Differentially expressed genes between Wooden Breast Disease affected and unaffected pectoral muscle showing overlap across broiler chickens at 2, 3 and 4 weeks of age. Only 5 DE genes were found in common at all time points.

Fig 2. Top canonical pathways (Z-scores >1.8) as predicted by IPA for pectoral muscles at week 3 of age.

Activated pathways are represented by orange-colored bars (color shade increasing with strength of activation) while inhibited pathways are shown by blue-colored bars (color shade increasing with the strength of inhibition).

Fig 3. Disease and functional characterization of DE genes from the pectoral muscles at week 3 of age as predicted by IPA.

Activated terms have (Z-scores ≥ 2) while inhibited terms have (Z-scores ≤ -2). Each biological term has cluster annotation(s) associated with it. In addition, the main genes associated with the cluster annotation(s) including their expression states with respect to affected chickens are shown.