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.

Glucolipotoxicity: A Proposed Etiology for Wooden Breast and Related Myopathies in Commercial Broiler Chickens

“…The greater role of insulinindependent glucose transport in cardiac and smooth muscle allows glucose uptake even when glycolysis and glycogenesis are impaired. The importance of insulin-insensitive glucose transport in development of diabetic retinopathy (Kumagai, 1999), atherosclerosis (Wall et al, 2018), diabetic nephropathy (Brosius and Heilig, 2005), pulmonary fibrosis (Cho et al, 2017), diabetic myocardial fibrosis (Asbun and Villarreal, 2006;Gorski et al, 2019) and non-alcoholic fatty liver disease (Nanji et al, 1995) has been demonstrated and is in support of our hypothesis that wooden breast shares substantial etiological factors with type 2 diabetes in mammals.…”

“…Increased expression of LPL is a consistent and early signal of the overprovision of lipids to the pectoralis major in affected birds. LPL is upregulated in 2-and 3-week-old broilers that later develop wooden breast (Papah et al, 2018;Lake et al, 2019), it has been proposed as a contributor to sex-linked differences in wooden breast incidence rate and severity (Brothers et al, 2019), and its increased expression in affected birds has been localized to the site where disease is first microscopically apparent -the endothelium of veins undergoing phlebitis .…”

Evaluation via Supervised Machine Learning of the Broiler Pectoralis Major and Liver Transcriptome in Association With the Muscle Myopathy Wooden Breast

“…Other molecules identified in IPA with changes in expression due to WB were JAKMIP2, which is invovlved in microtubule binding and CAPSL which is involved in calcium ion binding. This IPA pathway has also been detected when evaluating the PM of differential gene experssion between male and females birds with WB (Brothers et al, 2019).…”

“…Research covering muscle myopathies in broilers reveals that myopathies have increased in recent years and have been correlated with the selection of larger breast muscle (Petracci et al, 2015). The cause of WB is currently unknown, but it is more prevalent in older, heavier male broilers than young birds (Kuttappan et al, 2017;Brothers et al, 2019). Larger breast muscle has been associated with hypertrophied muscle fibers which is thought to impact blood supply and number of satellite cells that are needed in muscle regeneration (Daughtry et al, 2017;Malila et al, 2019).…”

“…This was achieved through the use of algorithms to identify genes within an extensive RNA sequencing dataset whose expression can be used to correctly distinguish normal tissue apart from severe/moderate WB. Previous gene expression datasets have characterized many molecular relationships present in birds with WB (Mutryn et al, 2015;Papah et al, 2018;Brothers et al, 2019). This study uses a different mechanism to evaluate gene expression data in the hopes for a more concise evaluation of the WB myopathy.…”