Muscle Metabolome Profiles in Woody Breast-(un)Affected Broilers: Effects of Quantum Blue Phytase-Enriched Diet

Abstract

Woody breast (WB) myopathy is significantly impacting modern broilers and is imposing a huge economic burden on the poultry industry worldwide. Yet, its etiology is not fully defined. In a previous study, we have shown that hypoxia and the activation of its upstream mediators (AKT/PI3K/mTOR) played a key role in WB myopathy, and supplementation of quantum blue (QB) can help to reduce WB severity via modulation of hypoxia-related pathways. To gain further insights, we undertook here a metabolomics approach to identify key metabolite signatures and outline their most enriched biological functions. Ultra performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS) identified a total of 108 known metabolites. Of these, mean intensity differences at P < 0.05 were found in 60 metabolites with 42 higher and 18 lower in WB-affected compared to unaffected muscles. Multivariate analysis and Partial Least Squares Discriminant analysis (PLS-DA) scores plot displayed different clusters when comparing metabolites profile from affected and unaffected tissues and from moderate (MOD) and severe (SEV) WB muscles indicating that unique metabolite profiles are present for the WB-affected and unaffected muscles. To gain biologically related molecule networks, a stringent pathway analyses was conducted using IPA knowledge-base. The top 10 canonical pathways generated, using a fold-change -1.5 and 1.5 cutoff, with the 50 differentially abundant-metabolites were purine nucleotide degradation and de novo biosynthesis, sirtuin signaling pathway, citrulline-nitric oxide cycle, salvage pathways of pyrimidine DNA, IL-1 signaling, iNOS, Angiogenesis, PI3K/AKT signaling, and oxidative phosphorylation. The top altered bio-functions in term of molecular and cellular functions in WB-affected tissues included cellular development, cellular growth and proliferation, cellular death and survival, small molecular biochemistry, inflammatory response, free radical scavenging, cell signaling and cell-to-cell interaction, cell cycles, and lipid, carbohydrate, amino acid, and nucleic acid metabolisms. The top disorder functions identified were organismal injury and abnormalities, cancer, skeletal and muscular disorders, connective tissue disorders, and inflammatory diseases. Breast tissues from birds fed with high dose (2,000 FTU) of QB phytase exhibited 22 metabolites with significantly different levels compared to the control group with a clear cluster using PLS-DA analysis. Of these 22 metabolites, 9 were differentially abundant between WB-affected and unaffected muscles. Taken together, this study determined many metabolic signatures and disordered pathways, which could be regarded as new routes for discovering potential mechanisms of WB myopathy.

Keywords: IPA; broilers; metabolomics; quantum blue; woody breast.

Figure 1

Woody breast categories and sampling locations. Birds were processed at d56, and breast filets were macroscopically scored. NORM, normal breast without harden area; MOD, moderate woody breast with a score between 0.5 and 1.5; SEV, severe woody breast with a score between 2 and 3. S1, cranial surface; S2, caudal surface; WN, woody normal; WW, woody woody.

Figure 2

Partial Least Squares Discriminate Analysis (PLS-DA) scores plots. PLS-DA was constructed using the statistical package DiscriMiner in R version 3.6.1 and displayed different clusters when comparing metabolite profiles between WB-affected and unaffected birds (A), MOD and SEV WB (B), WN-MOD, and WN-SEV (C), and all the groups together (D).

Figure 3

The top two networks (A,B) built with IPA program from metabolomics data that was determined on WB-affected muscles. Metabolites in red were upregulated and metabolites in green were down-regulated in WB-affected tissues. Networks constructed from the IPA knowledgebase by connecting the altered molecules are not limited by canonical pathway boundaries. EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase.

Figure 4

Partial Least Squares Discriminate Analysis (PLS-DA) scores plots and heat maps of relative levels of most significant metabolites modulated by high dose quantum blue-enriched diets. (A) PLS-DA was constructed using the statistical package DiscriMiner in R version 3.6.1 and displayed different clusters when comparing metabolite profiles between all diets, (B) heat maps was build using log2 ratio and VIP score and comparing each diet to the PC group.

Figure 5

Interconnected altered metabolic pathways in WB-affected muscles. Altered metabolites from the top canonical pathway analysis along with their cross-independence in KEGG pathways. Red text metabolites represent elevated mean intensity in WB-affected, whereas green text represent reduced intensity. The figure is representative and not for scale.