Gene Expression Essential for Myostatin Signaling and Skeletal Muscle Development Is Associated With Divergent Feed Efficiency in Pedigree Male Broilers.

Background: Feed efficiency (FE, gain to feed) is an important genetic trait as 70% of the cost of raising animals is due to feed costs. The objective of this study was to determine mRNA expression of genes involved in muscle development and hypertrophy, and the insulin receptor-signaling pathway in breast muscle associated with the phenotypic expression of FE. Methods: Breast muscle samples were obtained from Pedigree Male (PedM) broilers (8 to 10 week old) that had been individually phenotyped for FE between 6 and 7 week of age. The high FE group gained more weight but consumed the same amount of feed compared to the low FE group. Total RNA was extracted from breast muscle (n = 6 per group) and mRNA expression of target genes was determined by real-time quantitative PCR. Results: Targeted gene expression analysis in breast muscle of the high FE phenotype revealed that muscle development may be fostered in the high FE PedM phenotype by down-regulation several components of the myostatin signaling pathway genes combined with upregulation of genes that enhance muscle formation and growth. There was also evidence of genetic architecture that would foster muscle protein synthesis in the high FE phenotype. A clear indication of differences in insulin signaling between high and low FE phenotypes was not apparent in this study. Conclusion: These findings indicate that a gene expression architecture is present in breast muscle of PedM broilers exhibiting high FE that would support enhanced muscle development-differentiation as well as protein synthesis compared to PedM broilers exhibiting low FE.

Enrichment of Autophagy and Proteosome Pathways in Breast Muscle of Feed Efficient Pedigree Male Broilers.

Background: Feed efficiency (FE) is an important genetic trait in poultry and livestock. Autophagy (self-eating) and proteosomes are cellular processes that remove damaged cell components (e.g., proteins, organelles). As evidence of extensive protein oxidation was observed in Pedigree Male (PedM) broilers exhibiting a low FE (LFE) phenotype compared to a high FE (HFE) phenotype, the main goal of this study was to assess gene and protein expression of the autophagy and proteosome pathways in breast muscle obtained in PedM broilers exhibiting HFE and LFE phenotypes. Methods: Feed efficiency was calculated as weight gain divided by feed intake gain in individual PedM broilers that were measured between 6 and 7 weeks of age. Targeted gene expression was conducted on breast muscle using quantitative real-time polymerase chain reaction (qPCR) to determine mRNA expression of genes associated with the autophagy pathway; AMP-activated protein kinase alpha 1 (AMPKα1), mammalian target of rapamycin (mTOR), Beclin 1, and autophagy genes (Atg) 3, Atg7, and Atg16L1. Binomial distribution analysis was conducted on transcriptomic and data obtained by RNAseq and shotgun proteomics, respectively on the same set of tissues for genes associated with autophagy, vacuole formation, and proteosome expression. Results: Greater efficiency was attained in the HFE PedM broilers by greater weight gain on the same amount of feed consumed resulting in FEs of 0.65 ± 0.01 and 0.46 ± 0.01 in the HFE and LFE phenotypes, respectively. Targeted mRNA expression analysis revealed significant (P < 0.05) elevations in AMPKa1, mTOR, Atg16L1, and Atg7 and a marginal (P = 0.07) elevation in Beclin1. Binomial distribution analysis transcriptomic and proteomic data revealed significant skews favoring autophagy-, vacuole-, and proteosome-related genes in the HFE phenotype. These results indicate that the autophagy and proteosome expression is enhanced in the HFE compared to the LFE pedigree male broiler phenotype suggesting that protein and organelle quality control may be enhanced in high feed efficiency.

Proteogenomics Reveals Enriched Ribosome Assembly and Protein Translation in Pectoralis major of High Feed Efficiency Pedigree Broiler Males.

Background: In production animal agriculture, the cost of feed represents 60-70% of the total cost of raising an animal to market weight. Thus, development of viable biomarkers for feed efficiency (FE, g gain/g feed) to assist in genetic selection of breeding stock remains an important goal in commercial breeding programs. Methods: Global gene (cDNA microarray, RNAseq) and protein expression (shotgun proteomics) analyses have been conducted on breast muscle samples obtained from pedigree broiler males (PedM) exhibiting high and low FE phenotypes. Using the entire datasets (i.e., no cutoffs for significance or fold difference in expression) the number of genes or proteins that were expressed numerically higher or lower in the high FE compared to the low FE phenotype for key terms or functions, e.g., ribosomal, mitochondrial ribosomal, tRNA, RNA binding motif, RNA polymerase, small nuclear ribonucleoprotein, and protein tyrosine phosphatase, were determined. Bionomial distribution analysis (exact) was then conducted on these datasets to determine significance between numerically up or down expression. Results: Processes associated with mitochondrial proteome expression (e.g., mitochondrial ribosomal proteins, mitochondrial transcription, mitochondrial tRNA, and translation) were enriched in breast muscle from the high FE compared to the low FE pedigree male broiler phenotype. Furthermore, the high FE phenotype exhibited enrichment of ribosome assembly (e.g., RNA polymerase, mitochondrial and cytosolic ribosomes, small, and heterogeneous nuclear ribonucleoproteins), as well as nuclear transport and protein translation processes compared to the low FE phenotype. Quality control processes (proteosomes and autophagy) were also enriched in the high FE phenotype. In contrast, the low FE phenotype exhibited enrichment of cytoskeletal proteins, protein tyrosine phosphatases, and tyrosine kinases compared to the high FE phenotype. These results suggest that processes of mitochondrial and cytosolic ribosomal construction, activity, and protein translation would be enhanced in high FE breast muscle, and that phosphorylation of tyrosine moieties of proteins could be prolonged in the high compared to low FE phenotype. The results indicate the presence of a proteogenomic architecture that could enhance ribosome construction, protein translation, and quality control processes and contribute to the phenotypic expression of feed efficiency in this PedM broiler model.

Correction: Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria.

[This corrects the article DOI: 10.1371/journal.pone.0155679.].

Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria.

As feed represents 60 to 70% of the cost of raising an animal to market weight, feed efficiency (the amount of dry weight intake to amount of wet weight gain) remains an important genetic trait in animal agriculture. To gain greater understanding of cellular mechanisms of feed efficiency (FE), shotgun proteomics was conducted using in-gel trypsin digestion and tandem mass spectrometry on breast muscle samples obtained from pedigree male (PedM) broilers exhibiting high feed efficiency (FE) or low FE phenotypes (n = 4 per group). The high FE group had greater body weight gain (P = 0.004) but consumed the same amount of feed (P = 0.30) from 6 to 7 wk resulting in higher FE (P < 0.001). Over 1800 proteins were identified, of which 152 were different (P < 0.05) by at least 1.3 fold and ≤ 15 fold between the high and low FE phenotypes. Data were analyzed for a modified differential expression (DE) metric (Phenotypic Impact Factors or PIF) and interpretation of protein expression data facilitated using the Ingenuity Pathway Analysis (IPA) program. In the entire data set, 228 mitochondrial proteins were identified whose collective expression indicates a higher mitochondrial expression in the high FE phenotype (binomial probability P < 0.00001). Within the top up and down 5% PIF molecules in the dataset, there were 15 mitoproteome proteins up-regulated and only 5 down-regulated in the high FE phenotype. Pathway enrichment analysis also identified mitochondrial dysfunction and oxidative phosphorylation as the number 1 and 5 differentially expressed canonical pathways (up-regulated in high FE) in the proteomic dataset. Upstream analysis (based on DE of downstream molecules) predicted that insulin receptor, insulin like growth receptor 1, nuclear factor, erythroid 2-like 2, AMP activated protein kinase (α subunit), progesterone and triiodothyronine would be activated in the high FE phenotype whereas rapamycin independent companion of target of rapamycin, mitogen activated protein kinase 4, and serum response factor would be inhibited in the high FE phenotype. The results provide additional insight into the fundamental molecular landscape of feed efficiency in breast muscle of broilers as well as further support for a role of mitochondria in the phenotypic expression of FE. Funding provided by USDA-NIFA (#2013-01953), Arkansas Biosciences Institute (Little Rock, AR), McMaster Fellowship (AUS to WB) and the Agricultural Experiment Station (Univ. of Arkansas, Fayetteville).