RNA-Seq Analysis Reveals Spatial and Sex Differences in Pectoralis Major Muscle of Broiler Chickens Contributing to Difference in Susceptibility to Wooden Breast Disease

Abstract

Wooden Breast Disease (WBD) is a novel myopathy affecting the pectoralis major muscle of modern broiler chickens. The etiology of WBD is not currently known, but has been linked to increased feed efficiency, growth rate, and muscle yield in broiler chickens. Differential effect of WBD has been detected between regions of the P. major and between sexes of broilers-male birds and the cranial aspect of the muscle tend to be more severely affected by the disease than females and the caudal aspect. This study aimed to characterize biological differences in the P. major between regions of the muscle and sexes of birds. Samples were taken from the cranial and caudal aspects of P. major muscles of 3-week-old, unaffected male and female birds for RNA sequencing. RNA was extracted and used for preparation of cDNA libraries, which were sequenced by the Delaware Biotechnology Institute (DBI) using HiSeq2500. Sequence reads were aligned to the chicken reference genome with HISAT, and genes were analyzed for differential expression between regions of the breast muscle and sexes of birds using CuffDiff. Functional analysis was performed on differentially expressed genes (DEGs) between sex groups using DAVID and Ingenuity Pathway Analysis (IPA). There were 12 DEGs between cranial and caudal samples, and 260 between male and female birds. Out of the 260 genes differentially expressed between sexes, 189 were upregulated in males. Of this subset, 103 genes (55%) were located on the Z-chromosome. There was increased expression of genes involved in fat metabolism and oxidative stress responses in the cranial region of the P. major muscle, as well as increased expression of fat metabolism, oxidative stress response, antiangiogenesis, and connective tissue proliferation genes in male broilers. These results support the hypothesis that there are biological characteristics in male broilers and the cranial region of the breast muscle that may make them more susceptible to WBD, as well as raising the possibility of a metabolic switch in modern broiler chickens that may be more prominent in males.

Keywords: RNA sequencing; broiler; chicken; gene expression; metabolism; myopathy; wooden breast.

FIGURE 1

Comparison of weekly body weight by sex. Day 1 refers to the day of hatch. Male broilers have significantly higher average body weights than females at ages 7, 14, 21, 28, 35, 42, 49, and 56 days (P ≤ 0.0001).

FIGURE 2

Histological sections of P. major sample showing the various stages of pathology associated with WB: (A) unaffected muscle sample; (B) mildly affected muscle tissue exhibiting multifocal myodegeneration (arrowhead) and focal infiltration by inflammatory cells (arrow); (C) moderately affected sample as shown by diffuse myofiber degeneration multifocal inflammatory cell infiltration and fibrosis (asterisk); (D) severely/markedly affected muscle sample as indicated by diffuse myodegeneration, diffuse myofiber inflammation, lipid infiltration and phlebitis (open arrowhead).

FIGURE 3

Activation network diagrams for PPARA (left) and palmitic acid (right). Pink and green shapes represent genes upregulated or downregulated in males, respectively. Orange arrows represent IPA’s prediction that the upstream regulator activates the corresponding genes. Yellow arrows represent disagreement between the direction of the gene expression (i.e., up or downregulation) and IPA’s prediction of the relationship between the upstream regulator and the corresponding gene. Gray arrows mean that IPA predicts a relationship between the upstream regulator and the corresponding gene but cannot predict the exact effect. Image produced using IPA.

FIGURE 4

Activation network diagram for NFE2L2. Pink shapes represent genes that were upregulated in the dataset. Orange arrows demonstrate that IPA predicts NFE2L2 activates the corresponding genes. The yellow line to CTGF means that the fold change for CTGF is not consistent with IPA’s predicted relationship between NFE2L2 and CTGF. Image produced using IPA.

FIGURE 5

Log2 fold-change of differentially expressed genes separated by whether they are located on an autosome or the Z chromosome. Notice the consistency in log2-fold-change values of genes on the Z chromosome.