Messenger RNA sequencing and pathway analysis provide novel insights into the biological basis of chickens’ feed efficiency
© Zhou et al.; licensee BioMed Central. 2015
Received: 31 July 2014
Accepted: 20 February 2015
Published: 17 March 2015
Advanced selection technologies have been developed and continually optimized to improve traits of agricultural importance; however, these methods have been primarily applied without knowledge of underlying biological changes that may be induced by selection. This study aims to characterize the biological basis of differences between chickens with low and high feed efficiency (FE) with a long-term goal of improving the ability to select for FE.
High-throughput RNA sequencing was performed on 23 breast muscle samples from commercial broiler chickens with extremely high (n = 10) and low (n = 13) FE. An average of 34 million paired-end reads (75 bp) were produced for each sample, 80% of which were properly mapped to the chicken reference genome (Ensembl Galgal4). Differential expression analysis identified 1,059 genes (FDR < 0.05) that significantly divergently expressed in breast muscle between the high- and low-FE chickens. Gene function analysis revealed that genes involved in muscle remodeling, inflammatory response and free radical scavenging were mostly up-regulated in the high-FE birds. Additionally, growth hormone and IGFs/PI3K/Akt signaling pathways were enriched in differentially expressed genes, which might contribute to the high breast muscle yield in high-FE birds and partly explain the FE advantage of high-FE chickens.
This study provides novel insights into transcriptional differences in breast muscle between high- and low-FE broiler chickens. Our results show that feed efficiency is associated with breast muscle growth in these birds; furthermore, some physiological changes, e.g., inflammatory response and oxidative stress, may occur in the breast muscle of the high-FE chickens, which may be of concern for continued selection for both of these traits together in modern broiler chickens.
KeywordsRNA-seq Differential expression analysis Chicken feed efficiency Breast muscle Muscle remodeling IGFs/PI3K/Akt signaling pathway
Genetic selection has tremendously improved livestock and plant production over the past 50 years [1,2]. Advanced selection technologies have been developed and continually optimized to genetically improve traits of agricultural importance [1,3,4]. However, these methods have been primarily applied without knowledge of underlying biological changes that may be induced by selection [5,6]. Previous studies reported possible association between selection for improved performance and increased rate of physiological and metabolic disorders in modern breeds [7-9]. For example, chickens and turkeys selected for high growth rate have shown increased incidence of muscle disorders, heart failure syndrome and ascites [10-12]. A detailed characterization of traits of breeding interest may help to anticipate unfavorable consequences of long-term selection programs and adjust or perhaps redefine breeding objectives accordingly.
One of the most important traits in broiler chicken production is feed efficiency (FE), which defines the chicken’s ability to convert feed into body weight. As feed cost represents nearly 70% of the total cost in poultry production, improving FE has been an important goal in broiler chicken breeding programs . Selection for FE in broiler chickens can be accomplished using different measurements and procedures. A widely used measure of FE in broiler chickens is residual feed consumption (RFC), which is defined as the difference between an animal’s actual feed intake and expected feed intake on the basis of body weight and growth . Although moderate heritability, ranging from 0.42 to 0.45, for RFC has been reported in a previous study using more diverse chicken populations , to our knowledge this trait exhibits lower heritability (~0.2) in the commercial pure lines, possibly explaining the relatively slow progress in improving FE in commercial breeding programs. Insights into the biological basis of differences in chicken FE are required to develop more efficient and sustainable selection strategies.
Previous studies have revealed a link between mitochondrial function and FE in broiler chickens. Lower electron transport chain coupling and greater hydrogen peroxide (H2O2) production were observed in mitochondria of low-FE birds . A microarray gene expression analysis of breast muscle samples from high- and low-FE broiler chickens identified 782 differentially expressed genes [16,17]. Most of the genes up-regulated in high-FE chickens were related to anabolic metabolism, whereas genes up-regulated in low-FE chickens were associated with muscle fiber development, muscle function, cytoskeletal organization and stress response . With the rapid development of next-generation sequencing technologies, RNA sequencing (RNA-seq) has been replacing microarray technology for transcriptome-wide gene expression analysis. Avoiding technical issues inherent to microarray such as cross-hybridization and narrow ranges of signal detection, RNA-seq can provide more accurate and comprehensive information regarding changes in gene expression between different conditions or different phenotypes [18-21]. Therefore, a global gene expression study using RNA-seq is required for better understanding the molecular basis of FE in broiler chickens.
The objective of this study is to characterize the biological basis of differences between high- and low-FE chickens through breast muscle RNA-seq analysis. Using tissue samples from extreme high- and low-FE broiler chickens, the present study identifies genes and pathways differentially regulated in breast muscle between these two groups of chickens, providing important information toward understanding the biological basis of variation in FE in broiler chickens.
Animals and sample collection
where FC represents the feed consumption of each bird; Level represents the fixed effects of row location (top or bottom level) on FC; Row (Level) represents the fixed effects of row nested within row location; BW29 is the initial (29-day) body weight; BW46 is the ending (46-day) body weight; c is the intercept; and b1 and b2 are the partial regression coefficients of FC on BW29 and BW46.
After excluding outliers and erroneous data (3.3%) and data from birds with defects (1.2%; leg and wings problem, etc.), samples from clinically healthy chickens exhibiting the highest (n = 12) and lowest (n = 13) RFC from the six groups of 400 birds were selected for cDNA library preparation. Two samples from the high-FE group did not produce enough cDNA libraries, so samples from 23 birds, 10 high- and 13 low-FE, were used for further analysis. The protocols were submitted to, and the use of the collected data and samples for research was approved by, the University of Delaware Agricultural Animal Care and Use Committee.
The frozen breast muscle samples were smashed into pieces by hammering. Pulverized tissues were stored at -80°C until RNA extraction. The total RNA was isolated from 70-100 mg of fragmented breast muscle tissues using a mirVana™ miRNA isolation kit (Ambion®; Austin, TX), according to the manufacturer’s protocol. RNA quantity and quality were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Wilmington, DE) and Agilent 2100 bioanalyzer (Agilent Technologies; Santa Clara, CA). The RNA integrity numbers of all the RNA samples were above 8.0.
RNA-seq library preparation and sequencing
In total, 23 cDNA libraries were constructed using an Illumina Truseq stranded RNA sample preparation kit following the manufacturer’s instruction (Illumina Inc.; San Diego, CA). Briefly, polyA containing mRNA molecules were purified by oligo (dT) magnetic beads and subsequently fragmented. The purified RNA fragments were reversely transcribed into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen™; Austin, TX). The second-strand cDNA was synthesized using dUTP instead of dTTP, as a result, the second-strand cDNA was not amplified during PCR because the polymerase can’t add nucleotide to dUTP. The double-strand cDNA was adenylated at the 3’ end and ligated to the Illumina indexing adapters. After PCR enrichment, cDNA quantity and quality were assessed using a NanoDrop ND-1000 spectrophotometer and Agilent 2100 bioanalyzer. The averaged size of synthesized cDNA fragments was approximately 260 bp. cDNA libraries were normalized to 10 nm for each sample and then pooled together and sequenced on four lanes of an Illumina Hiseq 2000 sequencer at Delaware biotechnology institute, University of Delaware. Approximate 68 million fragments per sample were sequenced by 75-bp from both ends.
Mapping reads to the chicken reference genome
Before read alignment, the quality of raw sequence reads was checked using the FastQC program, and nucleotide calls with a quality score of 28 or higher were considered very good quality . Sequencing reads from each sample were mapped to the chicken reference genome [Ensembl Galgal4 (GCA_000002315.2)] using the TopHat program . Because only the strand generated during the first-strand synthesis was sequenced, “-library-type fr-firststrand” was applied as one of the parameters in our read alignment using TopHat. Only one alignment for a given read was allowed in our analysis (i.e., -g 1), and both reads from a single sequence fragment were required to be mapped to the reference genome in a concordant manner (i.e., --no-discordant and --no-mixed). To summarize the alignments statistics, the resulting alignment files (SAM files) statistics were analyzed using SAMtools .
Differential expression analysis
Cuffdiff, a companion tool of Cufflinks (v 2.1.1), was used to quantify the gene expression levels and to perform a differential expression test . The fragment counts were normalized via a geometric method, as described previously . Genes with a false discovery rate of less than 5% (i.e., FDR < 0.05) were considered significant.
Nanostring nCounter® gene expression assay
The gene expression data was verified by NanoString nCounter® technology, as described previously . Briefly, 23 RNA samples were submitted to NanoString, Inc. (Seattle, WA USA) for gene expression assay. With 12 housekeeping genes, 192 endogenous transcripts were selected across multiple on-going RNA-seq projects in our laboratory as target sequences to be measured. Designs of specific probes for target sequences were provided by NanoString  and were screened to avoid areas of high SNP density. A total of 100 ng of each RNA sample were hybridized to the CodeSet®, which was composed of both capture and reporter probes . After 16 hours incubation, the samples were transferred to the nCounter® Prep Station and Digital Analyzer for transcript quantification. Positive control normalization factors and reference genes were used to normalize the raw data for biological analysis . Log2 ratios of gene expression levels between high- and low-FE groups were calculated to compare with the corresponding log2 ratio values from RNA-seq analysis.
Ingenuity pathway analysis
Genes differentially expressed (FDR < 0.05) between high- and low-FE birds were included in pathway and function analysis using Ingenuity pathway analysis (IPA; Ingenuity® Systems, http://www.ingenuity.com). The functional and canonical pathway analysis was used to identify the significant biological functions and pathways. Functions and pathways with P-value < 0.05 (Fischer’s exact test) were considered to be statistically significant. IPA’s upstream regulator analysis function was used to identify potential transcriptional regulators that could explain the observed changes in gene expression between high- and low-FE chickens. The activation z-score was calculated to predict activation or inhibition of transcriptional regulators based on published findings accessible through the Ingenuity knowledge base. Regulators with z-score greater than 2 or less than -2 were considered to be significantly activated or inhibited.
Results and discussion
Statistics of the measurements from high- and low-feed efficiency (FE) chickens
High-FE birds (n = 10)
Low-FE birds (n = 13)
Bird weight (Kg), 29-d
1.316 ± 0.140
1.345 ± 0.169
Bird weight (Kg), 46-d
3.093 ± 0.283
2.960 ± 0.176
Weight gain (Kg), 29- to 46-d
1.778 ± 0.188*
1.615 ± 0.099*
Feed consumption (Kg), 29- to 46-d
2.874 ± 0.249**
3.325 ± 0.136**
Feed conversion ratioa
1.620 ± 0.054**
2.063 ± 0.085**
Residual feed consumption (Kg)b
-0.276 ± 0.040**
0.356 ± 0.048**
Breast muscle weight (%BW), 47-d
23.2 ± 01.6*
21.6 ± 1.4*
Breast muscle weight (Kg), 47-d
0.721 ± 0.100*
0.648 ± 0.071*
Transcriptional profile of chicken breast muscle
A total of 23 cDNA libraries were constructed using RNA samples of breast muscle tissues from 10 high- and 13 low-FE chickens and sequenced for 75 cycles from both ends on four lanes. In total, about 1.573 billion of 75-base sequence reads are obtained with an average of 393 million raw reads per lane. No significant difference in the number of reads between these four lanes is observed. The total number of reads for one sample ranges from 50 million to 88 million, with an average of 68 million reads per sample. Based on quality check reports, the averaged quality score of sequence reads is high, approximately 38, with the average GC content ranging from 49% to 51%. On average, 80% of the paired-end reads are properly mapped to the chicken reference genome (Ensembl Galgal4). The summary of alignment for all samples is shown in Additional file 1. The relative expression of a gene is normalized as fragments per kilobase of transcript per million mapped fragments (FPKM), which is proportional to the number of cDNA fragments originated from the gene transcript. The lowest limit of gene expression value is set to be 0.1 FPKM in at least one of the 23 samples. According to this limit, 14,148 genes are identified as being expressed in the breast muscle tissues. To assess the consistency of the gene expression levels between different samples, the Pearson’s correlation coefficient was calculated for each pairwise combination of samples . The averaged pairwise correlation coefficient between samples is 0.794, reflecting pretty consistent gene expression profiles.
Gene differential expression analysis
Differentially expressed genes were detected by Cuffdiff, an internal program of Cufflinks. Of 17,107 genes in the Ensemble database (Ensembl Galgal4), 1,059 were identified as significant genes with different expression levels between high- and low-FE chickens (q-value < 0.05) (Additional files 2 and 3). All of this group of 1,059 genes have a fold change greater than 1.3, and 642 genes (60.6%) have a fold change above 1.5. Among the 1,059 differentially expressed genes, 327 and 732 genes are down- and up-regulated in high-FE birds, respectively (Additional file 4). This relative imbalance in the number of down- and up-regulated genes is likely due to the increased breast muscle regeneration and inflammatory response in the high-FE chickens (discussed below). Since muscle development and inflammatory response require higher levels of activators such as growth factors, hormones and cytokines, the gene expression may be positively regulated by these activators in the breast muscle of the high-FE birds.
Confirmation of RNA-seq data
Overview of IPA analysis
To fully interpret the biological implications of the results from the differential expression analysis, all significant genes with their respective log2 fold-change were submitted for Ingenuity® Pathway Analysis. The top 10 up-regulated and top 10 down-regulated genes in high-FE chickens are listed in Additional file 5.
Top biological functions and pathways enriched by differentially expressed genes between high- and low-FE chickens 1
Top molecular and cellular functions
Cellular function and maintenance
Cell-to-cell signaling and interaction
Cellular growth and proliferation
Cell death and survival
Top canonical pathways
Hepatic fibrosis/hepatic stellate cell activation
Fcg receptor-mediated phagocytosis in macrophages and monocytes
Leukocyte extravasation signaling
Role of tissue factor in cancer
PI3K signaling in B lymphocytes
Top functions enriched by genes up-regulated or down-regulated in high- FE chickens
Genes down-regulated in high-FE chickens
Small molecule biochemistry
Nucleic acid metabolism
Genes up-regulated in high-FE chickens
Cellular function and maintenance
Cellular growth and proliferation
Cell-to-cell signaling and interaction
Cell death and survival
Upstream regulator analysis through IPA predicted the cascade of upstream transcriptional regulators that can potentially explain the differences in gene expression profile between high- and low-FE chickens. A summary of the upstream regulators identified by IPA is presented in Additional file 6. A total of 27 transcriptional regulators are predicted to be activated or inhibited (24 activated and 3 inhibited) in the high-FE chickens, of which 24 regulators are considered to be significant with P-value < 0.05 (21 activated and 3 inhibited).
Increased muscle growth and remodeling in high-FE chickens
Of all differentially expressed genes, 32 are associated with muscle development (Additional file 7), supporting the increased breast muscle weight in the high-FE birds. Among them, both hepatocyte growth factor (HGF) and insulin-like growth factor 2 (IGF2) encode key growth factors that have autocrine or paracrine effects on chicken skeletal muscle development and regeneration . HGF can not only activate the proliferation of quiescent muscle satellite cells, it also can induce the migration of activated satellite cells to the injured sites . IGF2 acts as a crucial regulator in muscle regeneration by stimulating muscle cell differentiation as well as inducing muscle cell hypertrophy . Other muscle growth-related genes that are up-regulated in the high-FE chickens include myogenin (MYOG), cysteine and glycine-rich protein 3 (CSRP3), myoferlin (MYOF), glypican 1 (GPC1), protein tyrosine phosphatase, receptor type, A (PTPRA) and gap junction protein (GJA1). As a member of myogenic regulatory factors (MRFs), MYOG is essential for the fusion of myoblasts into myotubes during muscle growth and regeneration . The CSRP3 gene encodes muscle LIM protein, which is able to increase the activity of MRFs and plays a critical role in enhancing myogenesis . The MYOF-encoded protein is a fundamental modulator for myoblast fusion, highly expressed during muscle repair and regeneration . The stimulatory effects of GPC1 on muscle satellite cell differentiation and myotube formation was reported in turkeys . The protein encoded by the PTPRA gene is a signaling molecule that was found to increase myogenesis of rat muscle L6 cells . The GJA1-encoded protein is a major component in gap junctions and required for myogenesis and regeneration . Collectively, the up-regulation of genes that can positively regulate muscle growth indicates that muscle growth and development is elevated in the high-FE chickens.
In addition to genes involved in muscle development, genes associated with muscle hypertrophy, including F-box protein 32 (FBXO32; fold change = −1.879), F-box protein 40 (FBXO40; fold change = −1.879), F-box protein 9 (FBXO9; fold change = −1.347), forkhead box O3 (FOXO3; fold change = −1.540) and myotrophin (MTPN; fold change = 1.426), are found differentially expressed in the breast muscle of chickens with high versus low FE. MTPN, a well-known positive growth factor in promoting muscle growth , is up-regulated in high-FE chickens. The increased MTPN expression may indicate that myocyte growth and protein synthesis are augmented in the breast muscle of high-FE birds, accordingly, contributing to breast muscle hypertrophy in these chickens. Furthermore, the down-regulation of FOXO3 and F-box family proteins in high-FE chickens further explains muscle growth differences between high- and low-FE chickens. Protein encoded by FOXO3 is a master regulator of both autophagy-lysosome and ubiquitin-proteasomal pathways, promoting protein degradation and thereby contributing to muscle atrophy . Proteins from the F-box family mediate the interaction between substrates and ubiquitin-conjugating enzymes, which facilitate proteolysis in diverse tissues . Of them, the FBXO32-encoded protein, known as atrogin 1, is a well-recognized muscle-specific ubiquitin ligase leading to muscle atrophy in a wide range of diseases [41-43]. The FBXO40-encoded protein also has been proposed to play a role in muscle atrophy in mammals . Thus, the decreased expression of atrophy-related genes in breast muscle of high-FE birds suggests that muscle protein loss is reduced in high-FE chickens in contrast to low-FE birds. The transcription of these genes is regulated by the PI3K/Akt signaling pathway, which will be discussed later. Taken together, the up-regulation of MTPN and down-regulation of FOXO3 and FBXO family genes in the high-FE chickens suggest that birds with high FE may have elevated protein synthesis and decreased protein degradation in their breast muscle.
Genes associated with extracellular matrix (ECM) remodeling are also up-regulated in the high-FE birds. The ECM of skeletal muscle serves as a scaffold for maintaining the structure of muscle and guiding new fiber formation . Muscle regeneration is frequently accompanied by the degradation of ECM because it facilitates satellite cell migration to specific sites for proliferation and fusion into myotubes [32,46]. Therefore, the up-regulation of genes involved in ECM remodeling implies that muscle remodeling is increased in the breast muscle of high-FE chickens. Matrix metalloproteinases (MMPs) are the main endopeptidases responsible for degrading all kinds of ECM, consequently, playing an important role in mediating muscle cell migration and regeneration [47,48]. As presented in Additional file 8, six genes from the MMP family are differentially expressed in our study, all of which are up-regulated in high-FE birds. Of the proteins encoded by these genes, MMP1 and MMP13 belong to MMP collagenases that are capable of cleaving interstitial collagen types I, II and III . Through an in vitro wound-healing assay, MMP1 was able to promote myoblast migration and differentiation by increasing the expression of N-cadherin and β-catenin or pre-MMP-2 and TIMP . MMP13 also plays a role in muscle regeneration, expressing in all muscle cells during muscle regeneration, and its expression level is positively correlated with the extent of muscle damage . MMP9 is a gelatinase that also relates to muscle regeneration . Evidence showed that the expression levels of MMP9 were greatly increased in response to inflammation and the activation of satellite cells in injured muscle [52-54]. However, contrary to the positive function of MMP1 on muscle regeneration, a recent study revealed that MMP9 could lead to muscle cell necrosis, inflammation and fibrosis . Collectively, the up-regulation of MMPs in the high-FE birds suggests an augmented muscle remodeling in these birds compared with the low-FE chickens.
Apart from JunB, a main transcriptional factor in the formation of mature sarcomeres, myocyte-specific enhancer factor 2C (MEF2C), is predicted as being activated in breast muscle of high-FE birds [58,59]. Protein encoded by MEF2C is a member of the myocyte enhancer factor 2 (MEF2) family, which directly cooperates with MRFs and enhances skeletal muscle development . In the present study, MEF2C is predicted to be an activated upstream regulator that increases the transcription of GJA1, MMP13, MYOG, myozenin 2 (MYOZ2; fold change = 2.400) and ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (ATP2A2) (fold change = 1.390) (Figure 3B). GJA1, MMP13 and MYOG are all involved in myogenesis and exert positive effects on skeletal muscle growth and regeneration, which has been discussed above. Thus, MEF2C’s activation may augment the muscle development in high-FE birds. Moreover, MYOZ2 is also predicted as being up-regulated by MEF2C. The MYOZ2-encoded protein belongs to a family of calcineurin-interacting proteins that modulates specific skeletal muscle signaling pathways through suppressing calcineurin . It has been shown that MYOZ2 plays a role in regulating myocyte differentiation and promoting slow-oxidative fibers growth . Collectively, MYOZ2 may be more active in breast muscle of high-FE chickens and, consequently, mediates some biological pathways and leads to muscle remodeling in these birds.
Growth hormone (GH) and IGFs/PI3K/Akt signaling pathway over-represented in the differentially expressed genes
Inflammatory response in the breast muscle of high-FE chickens
In the present study, a large number of the differentially expressed genes (136 genes) are involved in inflammatory response. Most of these genes (124 genes), including genes encoding for interleukin 8 (IL-8) and chemokine (C-X-C motif) ligand 14 (CXCL14), are expressed greater in the high-FE chickens. Although the cellular source of IL-8 and CXCL14 remains unknown in the current study, both not only exert direct effects on immune cell recruitment but also act as paracrine or endocrine factors in skeletal muscle. IL-8 has been recently classified as a myokine that can promote angiogenesis within the muscle [75,76]. CXCL14 is encoded by an obesity-induced gene in mice that inhibits the insulin-induced glucose uptake in cultured myocytes . In addition, the gene encoding for corticotropin-releasing hormone (CRH) is also up-regulated in the high-FE birds (fold change = 2.824). Previous studies have demonstrated that CRH is secreted from nerve terminals and epithelial cells at inflammation sites and has a local proinflammatory effect on resident immune cells . Therefore, it is likely that the elevated transcription of CRH functions to augment an immune response in the breast muscle of high-FE chickens. Apart from its immunomodulatory role, an increase in CRH may have a positive impact on thermogenesis of skeletal muscle in high-FE birds .
A series of genes encoding for cytokine receptors are also up-regulated in the high-FE chickens, further indicating an augmented immune response in the breast muscle of the high-FE chickens. These genes include chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), interleukin 17 receptor A (IL17RA), interleukin 18 receptor 1(IL18R1), interleukin 1 receptor, type I (IL1R1), interleukin 1 receptor, type II (IL1R2), interleukin 1 receptor-like 1 (IL1RL1), interleukin 2 receptor, gamma (IL2RG) and interleukin 5 receptor, alpha (IL5RA). Among them, CCR2 was found to be expressed in infiltrating macrophages and playing a crucial role in muscle regeneration . This gene can recruit macrophages to injured muscle, which then produces a high level of IGF-I to promote muscle regeneration . Therefore, the up-regulation of CCR2 suggests that, compared with the low-FE chickens, macrophage infiltration and muscle regeneration are increased in the breast muscle of the high-FE birds.
The IPA canonical pathway analysis also supports our hypothesis regarding augmented immune response and active recruitment of immune cells to the breast muscle of the high-FE chickens. Several over-represented pathways involved in inflammatory response are identified in our analysis (Additional file 9). Given that nearly all genes mapped to these pathways are up-regulated in the high-FE chickens, we conclude that these immune-related pathways are activated in the breast muscle of the high-FE chickens. According to the predictions from IPA, a number of transcription factors associated with inflammatory response are also activated in the breast muscle of the high-FE chickens: v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1), spleen focus forming virus (SFFV), spi-1 proto-oncogene (SPI1), X-box binding protein 1 (XBP1) and runt-related transcription factor 1(RUNX1).
Free radical scavenging enriched in the differentially expressed genes between high- and low-FE chickens
Several differentially expressed genes in our dataset are involved in the production of ROS. Genes encoding for ROS-generating enzymes, including cytochrome b-245, beta polypeptide (CYBB) [fold-change = 2.08] and NADPH oxidase organizer 1 (NOXO1) [fold-change = 2.38], are all up-regulated in the high-FE birds, suggesting that ROS production is increased in the breast muscle of these birds compared with the low-FE birds. CYBB, also known as NADPH oxidase 2 (NOX2), is a major enzyme responsible for superoxide production in the sarcoplasmic reticulum . NOXO1, a positive mediator of NOX1 and NOX3, initiates the activity of NOX1 and NOX3 for generating ROS . Moreover, the down-regulation of uncoupling protein 3 (UCP3) [fold-change = -1.67] in the high-FE birds may indicate that mitochondria from the breast muscle of the high-FE chickens have higher electron transport chain coupling compared with that from low-FE chickens. This assumption is consistent with previous findings [15,89]. Because UCP3-mediated uncoupling can attenuate the production of ROS , the down-regulation of UCP3 in the high-FE birds may also suggest a higher production of ROS from the mitochondria of the breast muscle of these birds. Collectively, our data suggest that, compared with the low-FE birds, ROS is produced at a higher level in the breast muscle of the high-FE chickens.
However, in contrast to our findings, Bottje et al. (2002) reported higher amounts of ROS produced in the breast muscle of their low-FE birds . This inconsistency is likely caused by the difference in broiler chickens between two studies. Male breeders, presumably with relatively low breast muscle yield, were studied in the Bottje et al. (2002) research , whereas we study broiler chickens from a commercial cross with high breast muscle yield. The ancestors of this cross have been intensively selected for the disproportionate growth of breast muscle, and the resulting higher levels of variation in breast muscle in the broiler cross may be responsible for a significant part of the variation in FE in this cross compared to the male breeder strain in the study by Bottje et al. . In regard to broiler chickens in the current study, intensive inflammatory response is possibly a major source of increased ROS in the breast muscle of the high-FE chickens. ROS-generating enzymes, such as NOX in muscle cells, can be stimulated through extracellular inflammatory cytokines including interleukin (IL)-1, IL-6 and IL-8 in a ligand-induced pattern [99,100]. Furthermore, the implied infiltrating immune cells in the breast muscle of high-FE birds may be another cause for increased ROS. It is well known that immune cells produce a large amount of ROS to support their functions during inflammation . Hence, in our study, strong indications for elevated ROS production in the breast muscle of the high-FE chickens are likely due to augmented inflammatory response, whereas the higher level of ROS observed in the study by Bottje et al. (2002) is possibly from mitochondria of breast muscle cells. Further study of genes associated with free radical scavenging may support our assumption. Indeed, in our study, a large part of these genes (Additional file 10) are also related to inflammatory response (P-value = 1.03E-23--5.15E-06), suggesting that production of ROS in the high-FE birds is closely associated with an increased immune response in the breast muscle.
Notably, growth factors including HGF, IGF-1 and fibroblast growth factor (FGF)-2 are also found to be able to induce intracellular generation of ROS in different types of cells . As mentioned above, the breast muscle of high-FE birds have higher expression of HGF and IGF-2, which may play a role in stimulating ROS production in these birds. Moreover, such generated ROS exerts insulin-mimicking effects on the insulin/IGFs signaling pathway, which has shown to be a second messenger in insulin/IGFs signal transduction under physiological conditions . Therefore, in the breast muscle of the high-FE birds, the insulin/IGFs receptor signaling pathway may be activated, in part, because of increased ROS production.
Higher ROS production may also lead to an increase in intracellular calcium concentration. It has been found that ROS mediates the influx of extracellular Ca2+ and mobilization of intracellular Ca2+ stores [103-105]. In the present study, genes involved in calcium transport [solute carrier family 8, member B1 (SLC8B1), phospholipase C, beta 2 (PLCB2) and ATPase, Ca ++ transporting, cardiac muscle, slow twitch 2 (ATP2A2)] are all up-regulated in the high-FE birds, indicating increased calcium mobilization in the breast muscle of these birds. ATP2A2 encodes sarcoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2), which is an important pump responsible for muscle relaxation through transporting Ca2+ from the cytosol into the sarcoplasmic reticulum lumen in muscle cells . Because more SERCA2 are needed to maintain calcium homeostasis when high Ca2+ levels are present in cytosols, the up-regulation of ATP2A2 in the high-FE birds may imply a high level of cytosolic Ca2+ in the breast muscle of these chickens compared with the low-FE birds.
Transcriptional regulation of hypoxia-inducible factor-1α (HIF1α)
Moreover, the HIF1α signaling pathway is over-represented among significantly differentially expressed genes (P-value = 7.58E-04; ratio = 1.39E-01). In response to hypoxia or a variety of peptide stimulators under normoxic conditions, PI3K/Akt and MAPK signaling pathways are activated to induce the accumulation of HIF1α in human cells [108,109]. Consequently, the accumulated HIF1α is translocated to the nucleus to modulate the transcription of genes involved in angiogenesis, glucose metabolism, matrix metabolism, erythropoiesis and apoptosis . In our study, with increased expression of PIK3CB, PIK3CD, PIK3R5 and muscle RAS oncogene homolog (MRAS), both the Akt/PI3K and MAPK signaling pathways are predicted to be activated in the high-FE chickens. The activated Akt/PI3K and MAPK signaling pathways may stimulate the induction of HIF1α, as reflected by the up-regulation of HIF1α and its downstream genes [glucose transporter type 3 (GLUT3), glucose transporter-like protein 5 (GLUT5), matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 13 (MMP13), matrix metallopeptidase 27 (MMP27), matrix metallopeptidase 28 (MMP28) and lactate dehydrogenase B (LDHB)] in the high-FE birds. Based on the gene expression profile, we conclude that, compared with the low-FE birds, the activity of HIF1α signaling pathway is increased in the breast muscle of the high-FE birds.
Although it is unclear from our results whether hypoxia and/or mediators such as IGFs induced HIF1α activation in the breast muscle of the high-FE birds, we would like to speculate here about potential mechanisms underlying this activation. It is widely accepted that inflammation and hypoxia are closely interdependent in a wide array of physiological and pathological conditions [110-114]. Inflammation is frequently accompanied with hypoxia because of the high oxygen consumption of infiltrating immune cells . Assuming an increased inflammatory response in the breast muscle of the high-FE birds, we speculate that the up-regulation of HIF1α is partly caused by an inflammation-induced hypoxia. Alternatively, the up-regulation of HIF1α may be caused by excessive muscle remodeling, which may be the result of selection for breast muscle proportion. Elevated muscle growth and rearrangements may have led to the reconstruction of vasculature, consequently reducing the blood flow and resulting in oxygen deficiency in the breast muscle of the high-FE birds . Furthermore, insulin and IGFs have shown to be modulators of HIF1α induction during both normoxia and hypoxia . Given that IGF2 is up-regulated in the breast muscle of the high-FE birds, this growth factor may also have contributed to the activation of HIF1α.
Finally, the activation of HIF1α may also be partly due to a higher production of ROS in the breast muscle of the high-FE chickens. Studies have found that ROS are essential for the stabilization of HIF1-DNA, thereby triggering HIF1α-induced transcription [116,117]. It was also proposed that cellular ROS-producing proteins could sense changes in cellular oxygen concentration . Evidence indicated that low oxygen tension inhibited mitochondrial electron transport and therefore increased ROS production. The generated ROS then acted as a second messenger that contributed to HIF1α activation . Thus, the ROS production may have been increased in the breast muscle of the high-FE chickens partly because of a relatively low oxygen concentration within this tissue, which in turn may have played a role in HIF1α activation.
The current study provides a global view of gene expression differences in the breast muscle of broiler chickens with extremely high and low FE from a population of a modern commercial high-meat-yield broiler cross. To our knowledge, this study reports for first time the RNA-seq analysis of a trait of selection and breeding importance in chickens. We identify 1,059 genes significantly differentially expressed in the breast muscle between high- and low-FE chickens based on the RNA-seq experiment. Furthermore, we achieve a large-scale validation of our RNA-seq experiment by quantifying the expression of a large number of target genes (192 transcripts + 12 house-keeping genes) using a high-sensitive non-PCR-based method, i.e. NanoString nCounter® Technology . Function and pathway analysis of the differentially expressed genes sheds light on some of the underlying mechanisms that regulate chicken FE. Birds with high FE exhibit higher expression of genes involved in muscle growth, development and remodeling, which may explain why these birds have more breast muscle than do the low-FE chickens. Pathway analysis shows that anabolic pathways, including growth hormone signaling and IGFs/PI3K/Akt signaling pathways, are more activated in the high-FE birds, which may have not only led to the increased muscle growth in the high-FE chickens but also contributed to the feed conversion advantages of these birds. Our results also suggest that transcriptional factors JunB and MEF2C play crucial roles in regulating muscle growth and remodeling in high-FE chickens.
Furthermore, most of the genes up-regulated in the high-FE birds are associated with inflammatory response and oxidative stress, suggesting augmented inflammation and oxidative stress in the breast muscle of these birds. Our results also show increased activity of HIF1α, which may be caused by a lower oxygen environment in the breast muscle of high-FE chickens. Although no clinical symptoms of sickness or muscle damage were observed in the birds used in the current study, some of the molecular changes in the high-FE chickens may be hypothesized to lead to recently reported muscle quality issues in modern broiler chickens such as white striping and wooden breast [120-122]. These disorders have been reported to be more frequent in birds with high breast muscle weight and high FE, suggesting that the susceptibility may be primarily induced by breeding for these traits. Further investigation (e.g., histological and protein analysis) would be helpful for examining inflammation and oxidative stress in the breast muscle of high-FE and high-breast-muscle-yield birds.
Availability of supporting data
The data supporting the results of this article are included within the article and its additional files. Readers may contact the corresponding author for additional information.
This work was partly funded by Delaware Bioscience Center for Advanced Technology and Heritage Breeders, LLC. Thanks to Brewster F. Kingham in Delaware Biotechnology Institute (DBI) for performing RNA sequencing.
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