Morphological and molecular characterization of wooden breast myopathy in commercial broiler chickens
Date
2019
Authors
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Publisher
University of Delaware
Abstract
The development of commercial broiler chickens through artificial genetic selection has been one of the greatest milestones in the poultry industry on the quest for a food secure world. Indeed, modern broiler chickens have high genetic merit for economically important production traits such as fast-growth rate, high muscle yield and high feed efficiency. The possession of these traits by the genetically selected broiler chickens has contributed immensely to a steady increase of chicken meat production in the U.S and around the world for the past 50 years. However, the development of modern broiler chickens has also been accompanied by the emergence of myodegenerative disorders targeting the fast-growing and high-feed efficient broiler birds. One of these disorders is Wooden Breast (WB) myopathy. ☐ Wooden Breast myopathy is a relatively novel myopathy that frequently affects the pectoralis (P.) major muscles of modern broiler chickens resulting in their extreme firmness and microscopic changes in the muscle tissue thereby compromising meat quality. While significant knowledge about the gross, microscopic and molecular changes of WB in commercial broiler chickens has been gained through several studies on chickens around market age, information about time of onset, early pathogenesis of the myopathy including pathological and molecular changes in the P. major muscle is scarce. Consequently, the disorder continues to cause significant economic losses in the poultry industry unabated. To allow for development of mitigation strategies and prevention of losses attributed to WB in the poultry industry, it was imperative that a comprehensive understanding of the onset and progression ofthe myopathy throughout the growth phase of broiler chickens be undertaken. This, therefore, formed the main objective of the present study project, that is to gain an indepth understanding of the pathology and pathogenesis of WB in modern broiler chickens in commercial settings. To accomplish this major objective, three-pronged systematic studies aimed at assessing clinical/phenotypic, macroscopic, microscopic and molecular perturbations in pectoral muscles associated with the onset and development of WB in broiler chickens was conducted. ☐ The first study involved a time-series assessment of the clinical/phenotypic features coupled with gross and microscopic changes of the pectoral muscles affected with WB in chickens raised from day-old to week 7 post-hatch. This study utilized chickens belonging to a high-breast-muscle-yield, purebred commercial broiler line, raised in standards that were similar to those in commercial settings. Upon weekly evaluation of chickens for phenotypic, gross and microscopic changes, it was established that WB exhibits an earlier onset than when detectable by clinical examination. Further, this study showed that the disease assumes a progressive course with acute vasculitis limited to small-caliber veins, lipid infiltration and deposition, and myodegeneration occurring in the earlier stages, followed by a chronic fibrotic phase. ☐ The second study involved molecular evaluations focusing on the global gene expression in the P. major muscles between affected and unaffected chickens at week 2, 3 and 4 of age. The specific aim of this study was to identify the main genes that were differentially expressed between WB-affected and unaffected chickens, and subsequent establishment of their biological relevance to the pathogenesis of the myopathy. This particular study used the same experimental chicken samples as that of the first study. However, unlike the first study which used necropsy samples, this study used muscle biopsy samples harvested from the cranial and caudal ends of the P. major muscles from selected chickens at the 3-time points and then processed for RNA-sequencing. The findings from this study revealed existence of molecular perturbations involving energy metabolism associated with lipid and carbohydrate metabolism, vasculature and extracellular matrix, as well as response to inflammation as being pertinent to the onset and early pathogenesis of WB in commercial meat-type chickens. ☐ The third study, essentially a continuation of the second one, was based on our previous knowledge from transcriptomic studies that indicated possible evidence of perturbations in lipid metabolism and presence of slow myofiber-phenotype in the typically fast myofiber-type of P. major muscles in affected chickens. The objective of this study was, therefore, to evaluate and confirm the occurrence of altered lipid metabolism and occurrence of slow myofiber-phenotype during the development of WB in commercial broiler chickens. Specifically, the aim was to localize the cellular expression of specific lipid metabolism-related and slow muscle-related genes in the P. major muscle tissue in affected and unaffected chickens. To accomplish this objective, P. major muscle tissue samples from two chicken lines, namely slow growing Legacy chickens (not known to be affected by WB), and fast-growing Ross chickens (frequently affected by the muscle disorder), were harvested at around 3 weeks of age and subsequently processed for RNA in situ hybridization targeting the expression of specific genes. The specific genes examined included lipoprotein lipase (LPL) and perilipin 1 (PLIN1) for lipid-related genes, and myosin binding protein C slow-type (MYBPC1), and cysteine and glycine rich protein 3 (CSRP3) for slow-type muscle-related genes. Concomitant to this, the global transcriptomic profile (RNAseq) of 10 genes related to lipid metabolism and 7 genes related to slow-myofiber-type muscle genes were examined. The RNA-seq expression data was generated from a comparison between WB-affected and unaffected chickens at 3 weeks post-hatch (early stage of the myopathy) and 7 weeks post-hatch (late stage of the myopathy). From this study, LPL was revealed to be expressed in the vascular endothelial cells of capillaries and small-caliber veins of all chickens. As such, this became the first study to reveal the expression of LPL from the endothelial cells in chickens, a feature that is in stark contrast to that of mammals. Further, this study confirmed presence of dysregulation of lipid metabolism between the early and late stages of WB. On the other hand, cellular expression of slow myofiber-type genes were enhanced in mature myofibers of affected chickens suggesting the existence of appearance of slow myofiber-type phenotype in the face of WB. Similarly, analysis of global gene expression related to slow myofiber-type indicates a concomitant increase of the rate of expression of slow myofiber-type isoform genes with the severity of the disease. ☐ In conclusion, the findings from the three studies have shown that WB is a complex myopathy that begins early in the life of chickens, with the initial sites or processes to be affected being the vasculature, energy homeostasis involving lipids and carbohydrates, and extracellular matrix. As a response to the homeostatic imbalance, recruitment of inflammatory cells and fibroblasts, together with the emergence of slow-myofiber phenotype occurs resulting in the various gross and microscopic changes that characterize WB.