Muscle disorder or metabolic disorder: genomic, transcriptomic, and metabolomic insights into the pathogenesis of wooden breast and white striping in commercial broiler chickens
Date
2021
Authors
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Publisher
University of Delaware
Abstract
Commercial broilers represent a remarkable feat in the optimization of meat production, principally due to many generations of intensive artificial selection for economically important production traits such as rapid growth, high breast muscle yield, and low feed utilization. One of the unintended consequences of such selection among modern broiler chickens has been the relatively recent emergence of several muscle disorders mainly affecting the pectoralis major muscle and severely impacting meat quality. Two such myopathies, wooden breast and white striping, are the focus of this dissertation due to their high prevalence and economic importance. Severe wooden breast presents grossly as pale, enlarged, and palpably firm pectoralis major muscle, with visible signs of inflammation such as petechial hemorrhages and tissue edema. White striping, which often co-occurs with wooden breast and is considered by many to be part of the same disease spectrum, presents as fatty white striations running parallel to the muscle fiber. ☐ The overarching objective of this dissertation was to improve our understanding of the pathogenesis of wooden breast and white striping in modern commercial broiler chickens primarily using ‘omics technologies and techniques. However, the first section eschews primary research and instead presents a novel hypothesis on the etiology and pathogenesis of these myopathies founded in existing literature, unpublished data from our lab, and an examination of the intricacies of avian glucose transport and metabolism. It purports that evident dysfunction of glucose and lipid metabolism in wooden breast closely aligns with similar changes associated with type 2 diabetes and metabolic syndrome in mammals, and that reliance on insulin-independent glucose transport in the skeletal muscle of chickens may produce the wooden breast phenotype in lieu of increased plasma glucose levels. The remaining studies investigated hematologic, metabolomic, transcriptomic, and genomic changes associated with development of the wooden breast phenotype in a crossbred commercial broiler population that was raised to 7 weeks of age and scored for both wooden breast and white striping. ☐ The first study assessed the association of wooden breast with perturbations in various venous blood parameters – blood gases, electrolytes, etc. – ¬acquired through a rapid blood test and also examined changes in body weight distribution and lung morphology. Chickens with wooden breast were found to exhibit blood gas disturbances potentially indicative of insufficient respiratory gas exchange and elevated metabolism, including higher potassium and total carbon dioxide and lower oxygen saturation and pH. A microscopic comparison of lung tissues did not provide any evidence that lung morphology was a contributing factor, but affected broilers possessed a significantly heavier pectoralis major muscle and whole feathered wing compared to unaffected broilers, which could contribute to increased metabolic demand or impaired respiration. ☐ The second study involved untargeted metabolomic profiling of blood plasma in 250 male broilers to find metabolites associated with wooden breast and white striping and to identify plasma biomarkers that could be used as a diagnostic tool for future research. Two statistical approaches were employed to achieve the two distinct goals of this study ¬– mixed linear modeling and stepwise feature selection for a support vector machine. The findings from this study revealed changes in histidine metabolism, lipid metabolism, and nucleotide metabolism associated with wooden breast and found that metabolites associated with white striping were almost entirely a subset of those associated with wooden breast. The metabolite 3-methylhistidine, which is often used as an index of myofibrillar breakdown in skeletal muscle and is increased in plasma of individuals with uncontrolled diabetes mellitus, was the top metabolite for both wooden breast and white striping in our mixed linear model and was also the metabolite with highest marginal prediction accuracy (82%) for wooden breast in our support vector machine. The final support vector machine achieved a prediction accuracy of 94% using only 6 metabolites, supporting the use of plasma biomarkers as an objective and quantitative measure of wooden breast severity for future research. ☐ The third study looked at early changes in gene expression in the pectoralis major muscle associated with later development of wooden breast at market age. Biopsy samples of the left pectoralis major muscle were collected from birds at 14 days of age, and birds were subsequently raised to 7 weeks of age to allow sample selection based on the wooden breast phenotype at market age. RNA sequencing was performed on 5 unaffected and 8 affected female chicken samples, and differential expression analysis found wooden breast status at market age to be associated mainly with increased lipid metabolism and altered calcium homeostasis at 2 weeks of age. The genes peroxisome proliferator-activated receptor gamma (PPARG) and lipoprotein lipase (LPL) were two of many lipid metabolism genes expressed higher in wooden breast affected birds, and encode proteins that function respectively as an important transcription factor for lipid uptake and adipogenesis and as the rate-limiting catalyst for hydrolysis of triglycerides in circulating lipoproteins. A large proportion of differentially expressed genes were also found to be linked to type 2 diabetes in humans, with Ras related glycolysis inhibitor and calcium channel regulator (RRAD) perhaps the best example of this. This gene was originally named Ras-related associated with diabetes because it was identified via subtraction cloning as the only gene out of 4000 cDNA clones that was overexpressed in skeletal muscle of type 2 diabetic individuals compared to non-diabetic or type 1 diabetic individuals. ☐ The fourth and final study was a genome-wide association study of nearly 1200 broilers to investigate the genetic basis of wooden breast and white striping and identify candidate genes using genomic marker data. Heritability was estimated at 0.5 for both wooden breast and white striping with high genetic correlation between them (0.9). The majority of quantitative trait loci identified for both myopathies were located in a ~8 Mb region of chicken chromosome 5, which has highly conserved synteny with a portion of human chromosome 11 containing a cluster of imprinted genes associated with growth and metabolic disorders such as type 2 diabetes and Beckwith-Wiedemann syndrome. Top candidate genes include potassium voltage-gated channel subfamily Q member 1 (KCNQ1), involved in insulin secretion and cardiac electrical activity, lymphocyte-specific protein 1 (LSP1), involved in inflammation and immune response, and dynamin 2 (DNM2), involved in biphasic insulin secretion and associated with two congenital neuromuscular diseases in humans. ☐ Collectively, this dissertation frames the pathogenesis of wooden breast and white striping as closely related metabolic muscle disorders involving and potentially resulting from extensive dysregulation of glucose and lipid metabolism. Most importantly, this work emphasizes the systemic nature of these breast muscle disorders and highlights the need for future research on other organs that likely contribute to myopathy development, including the pancreas, liver, and adipose tissue.
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Keywords
Broiler, Chicken, Diabetes, Meat quality, White striping, Wooden breast