RNA-sequencing analysis of Wooden Breast disease: characterization of a novel muscle disease in chickens through differential gene expression and pathway analysis

Abasht, BehnamImprovements in poultry production within the past 50 years have led to increased muscle yield and growth rate, which may in turn be contributing to an increased rate and development of new muscle disorders in chickens. Previously reported muscle disorders and conditions are generally associated with poor meat quality traits and have a significant negative economic impact on the poultry industry. Recently, a novel myopathy phenotype has emerged which is characterized by palpably "hard" or tough breast muscle. The objective of this study is to identify the underlying biological mechanisms that contribute to this emerging muscle disorder colloquially referred to as "Wooden Breast", through the use of RNA-sequencing technology. We constructed cDNA libraries from five affected and six unaffected breast muscle samples from a line of commercial broiler chickens. After paired-end sequencing of samples using the Illumina Hiseq platform, we used Tophat to align the resulting sequence reads to the chicken reference genome and then used Cufflink to find significant changes in gene transcript expression between each group. By comparing our gene list to previously published histology findings for this disorder and using Ingenuity Pathways Analysis (IPA ® ), we aimed to develop a characteristic gene expression profile for this novel disorder through analyzing genes, gene families, and predicted biological pathways. There is convincing evidence by RNA-seq analysis to support localized hypoxia, oxidative stress, increased intracellular calcium, as well as the possible presence of muscle fiber-type switching, as key features of Wooden Breast Disease, which are supported by reported microscopic lesions of the disease. We also conducted a large-scale phenotypic study of over 2,500 commercial broiler chickens raised under commercial conditions for 29 days in which they were placed into experimental stations to measure individual feed efficiency until market-age at 47 days. This experimental study was designed to understand the incidence rates and phenotypic trait correlation with Wooden Breast disease. This newly described myopathy has been reported to severely impact as much as 10% (per flock) of commercial bred chickens. During the feed efficiency trial, chickens were housed in an experimental station to measure individual feed intake, and ultimately scored for Wooden Breast disease by manual palpation and visual observation of macroscopic lesions under the groups: "hard", "normal", and "soft". At the conclusion of this study, multiple phenotypic characteristics such as: initial body weight, final body weight, final breast muscle weight, feed consumption, final abdominal fat weight, and pH were recorded to use for statistical analyses. Records of initial and final body weights and feed consumption were used to estimate each bird's residual feed consumption as a measure of feed efficiency. These traits were analyzed statically by Tukey's HSD test to determine differences in mean phenotypic values using each phenotypic measurement as the independent variable and breast muscle hardness score as the dependent variable. From our data, we find evidence to suggest that hardness of the breast muscle is significantly associated with high performing birds that exhibit increased body weight, breast muscle weight, and feed efficiency, as well as lower abdominal fat weight. We also can conclude that breast muscle hardness is also associated with breast muscle that had higher ultimate pH values.University of Delaware, Department of Animal and Food SciencesM.S