Oxidative Stress and Metabolic Perturbations in Wooden Breast Disorder in Chickens

Abstract

This study was conducted to characterize metabolic features of the breast muscle (pectoralis major) in chickens affected with the Wooden Breast myopathy. Live birds from two purebred chicken lines and one crossbred commercial broiler population were clinically examined by manual palpation of the breast muscle (pectoralis major) at 47-48 days of age. Metabolite abundance was determined by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using breast muscle tissue samples from 16 affected and 16 unaffected chickens. Muscle glycogen content was also quantified in breast muscle tissue samples from affected and unaffected chickens. In total, levels of 140 biochemicals were significantly different (FDR<0.1 and fold-change A/U>1.3 or <0.77) between affected and unaffected chickens. Glycogen content measurements were considerably lower (1.7-fold) in samples taken from Wooden Breast affected birds when compared with samples from unaffected birds. Affected tissues exhibited biomarkers related to increased oxidative stress, elevated protein levels, muscle degradation, and altered glucose utilization. Affected muscle also showed elevated levels of hypoxanthine, xanthine, and urate molecules, the generation of which can contribute to altered redox homeostasis. In conclusion, our findings show that Wooden Breast affected tissues possess a unique metabolic signature. This unique profile may identify candidate biomarkers for diagnostic utilization and provide mechanistic insight into altered biochemical processes contributing to tissue hardening associated with the Wooden Breast myopathy in commercial chickens.

Fig 1. Top 30 Metabolites Based on their Importance for Separating the Groups (Affected and Unaffected Tissues).

Random forest analysis (RFA) was utilized to determine which variables (biochemicals) had the largest contribution on classification. “Mean Decrease Accuracy” (MDA) was used as a measure of “variable importance”.

Fig 2. Histidine Metabolism in Affected Tissues.

Levels (median-scaled and log-transformed) of histidine and other related metabolites in breast muscle tissue samples obtained from affected (A) and unaffected (U) chickens that were sampled from two genetically distinct purebred lines (lines 1 and 2) and a commercial broiler (CB) population. Elevated histidine levels in affected tissue were accompanied by an accumulation of histamine, 1-methylhistidine, and 3-methylhistidine, which may reflect skeletal muscle degeneration and oxidative stress. In contrast, the histidine derived antioxidants carnosine and anserine, were depleted in affected breast muscle tissue suggesting altered redox homeostasis.

Fig 3. Glutathione Metabolism in Affected Tissues.

Levels (median-scaled and log-transformed) of glutathione and other related metabolites in breast muscle tissue samples obtained from affected (A) and unaffected (U) chickens that were sampled from two genetically distinct purebred lines (lines 1 and 2) and a commercial broiler (CB) population. Higher levels of the cysteine derived metabolites glutathione (GSH) and oxidized glutathione (GSSG) may reflect free radical exposure. These tissues also possessed higher levels of cysteine-glutathione disulfide (historically a marker of oxidative stress), the gamma-glutamyl amino acid catabolite 5-oxoproline that may suggest import of extracellular glutathione, and tripeptide ophthalmate that is an analogue of glutathione often produced in response to increased ROS and glutathione depletion.

Fig 4. Deregulated Lipid Metabolism in Affected Tissues.

Levels (median-scaled and log-transformed) of lipid and lipid metabolism related metabolites in breast muscle tissue samples obtained from affected (A) and unaffected (U) chickens that were sampled from two genetically distinct purebred lines (lines 1 and 2) and a commercial broiler (CB) population. Multiple long chain fatty acids including palmitate, palmitoleate, stearate, and oleate accumulated in affected muscle. Increased lipid availability may support mitochondrial β-oxidation as lower levels of free carnitine may reflect utilization for long chain fatty acid transport into the mitochondria. Furthermore, elevated levels of the ketone body 3-hydroxybutyrate (BHBA) have historically been a marker of lipid oxidation. Increased lipid availability may also consequently facilitate the generation of lipid peroxidation products such as 13-HODE and 9-HODE. These metabolites are often indicative of free radical exposure. The eicosanoids 15-HEPE, 15-HETE, and 15-KETE were also elevated in affected tissues and may represent a compensatory mechanism to balance inflammation since these lipid mediators have been shown to exhibit anti-inflammatory properties.

Fig 5. Altered Carbohydrate Metabolism in Wooden Breast Disease.

The affected tissues exhibited evidence of altered glycogen metabolism and glucose utilization that resulted in the accumulation of multiple intermediates of the pentose phosphate, glucosamine and glucuronic acid pathways. These changes collectively indicate a rerouting of carbohydrate flux from glycolysis to these metabolic pathways in order to combat oxidative stressors and support detoxification, regeneration and excessive remodeling in the affected tissues. Metabolites (or glycogen) presented in the orange and green boxes were at significantly higher and lower levels in Wooden Breast, respectively. The numeric values under the colored boxes represent the fold-change (FC) differences in metabolite levels or glycogen content between affected (A) and unaffected (U) tissues (A / U). The tick arrows represent the potentially most active biochemical reactions and pathways.