Differential expression of miRNAs associated with pectoral myopathies in young broilers: insights from a comparative transcriptome analysis.

INTRODUCTION: White Striping (WS) and Wooden Breast (WB) pectoral myopathies are relevant disorders for contemporary broiler production worldwide. Several studies aimed to elucidate the genetic components associated with the occurrence of these myopathies. However, epigenetic factors that trigger or differentiate these two conditions are still unclear. The aim of this study was to identify miRNAs differentially expressed (DE) between normal and WS and WB-affected broilers, and to verify the possible role of these miRNAs in metabolic pathways related to the manifestation of these pectoral myopathies in 28-day-old broilers. RESULTS: Five miRNAs were DE in the WS vs control (gga-miR-375, gga-miR-200b-3p, gga-miR-429-3p, gga-miR-1769-5p, gga-miR-200a-3p), 82 between WB vs control and 62 between WB vs WS. Several known miRNAs were associated with WB, such as gga-miR-155, gga-miR-146b, gga-miR-222, gga-miR-146-5p, gga-miR- 29, gga-miR-21-5p, gga-miR-133a-3p and gga-miR-133b. Most of them had not previously been associated with the development of this myopathy in broilers. We also have predicted 17 new miRNAs expressed in the broilers pectoral muscle. DE miRNA target gene ontology analysis enriched 6 common pathways for WS and WB compared to control: autophagy, insulin signaling, FoxO signaling, endocytosis, and metabolic pathways. The WS vs control contrast had two unique pathways, ERBB signaling and the mTOR signaling, while WB vs control had 14 unique pathways, with ubiquitin-mediated proteolysis and endoplasmic reticulum protein processing being the most significant. CONCLUSIONS: We found miRNAs DE between normal broilers and those affected with breast myopathies at 28 days of age. Our results also provide novel evidence of the miRNAs role on the regulation of WS and in the differentiation of both WS and WB myopathies. Overall, our study provides insights into miRNA-mediated and pathways involved in the occurrence of WS and WB helping to better understand these chicken growth disorders in an early age. These findings can help developing new approaches to reduce these complex issues in poultry production possibly by adjustments in nutrition and management conditions. Moreover, the miRNAs and target genes associated with the initial stages of WS and WB development could be potential biomarkers to be used in selection to reduce the occurrence of these myopathies in broiler production.

SAP30 Gene Is a Probable Regulator of Muscle Hypertrophy in Chickens.

Animals with muscle hypertrophy phenotype are targeted by the broiler industry to increase the meat production and the quality of the final product. Studies characterizing the molecular machinery involved with these processes, such as quantitative trait loci studies, have been carried out identifying several candidate genes related to this trait; however, validation studies of these candidate genes in cell culture is scarce. The aim of this study was to evaluate SAP30 as a candidate gene for muscle development and to validate its function in cell culture in vitro. The SAP30 gene was downregulated in C2C12 muscle cell culture using siRNA technology to evaluate its impact on morphometric traits and gene expression by RNA-seq analysis. Modulation of SAP30 expression increased C2C12 myotube area, indicating a role in muscle hypertrophy. RNA-seq analysis identified several upregulated genes annotated in muscle development in treated cells (SAP30-knockdown), corroborating the role of SAP30 gene in muscle development regulation. Here, we provide experimental evidence of the involvement of SAP30 gene as a regulator of muscle cell hypertrophy.

Differential Expression of Myogenic and Calcium Signaling-Related Genes in Broilers Affected With White Striping.

White Striping (WS) has been one of the main issues in poultry production in the last years since it affects meat quality. Studies have been conducted to understand WS and other myopathies in chickens, and some biological pathways have been associated to the prevalence of these conditions, such as extracellular calcium level, oxidative stress, localized hypoxia, possible fiber-type switching, and cellular repairing. Therefore, to understand the genetic mechanisms involved in WS, 15 functional candidate genes were chosen to be analyzed by quantitative PCR (qPCR) in breast muscle of normal and WS-affected chickens. To this, the pectoral major muscle (PMM) of 16 normal and 16 WS-affected broilers were collected at 42 days of age and submitted to qRT-PCR analysis. Out of the 15 genes studied, six were differentially expressed between groups. The CA2, CSRP3, and PLIN1 were upregulated, while CALM2, DNASE1L3, and MYLK2 genes were downregulated in the WS-affected when compared to the normal broilers. These findings highlight that the disruption on muscle and calcium signaling pathways can possibly be triggering WS in chickens. Improving our understanding on the genetic basis involved with this myopathy might contribute for reducing WS in poultry production.

Identification of selection signatures involved in performance traits in a paternal broiler line.

BACKGROUND: Natural and artificial selection leads to changes in certain regions of the genome resulting in selection signatures that can reveal genes associated with the selected traits. Selection signatures may be identified using different methodologies, of which some are based on detecting contiguous sequences of homozygous identical-by-descent haplotypes, called runs of homozygosity (ROH), or estimating fixation index (FST) of genomic windows that indicates genetic differentiation. This study aimed to identify selection signatures in a paternal broiler TT line at generations 7th and 16th of selection and to investigate the genes annotated in these regions as well as the biological pathways involved. For such purpose, ROH and FST-based analysis were performed using whole genome sequence of twenty-eight chickens from two different generations. RESULTS: ROH analysis identified homozygous regions of short and moderate size. Analysis of ROH patterns revealed regions commonly shared among animals and changes in ROH abundance and size between the two generations. Results also suggest that whole genome sequencing (WGS) outperforms SNPchip data avoiding overestimation of ROH size and underestimation of ROH number; however, sequencing costs can limited the number of animals analyzed. FST-based analysis revealed genetic differentiation in several genomic windows. Annotation of the consensus regions of ROH and FST windows revealed new and previously identified genes associated with traits of economic interest, such as APOB, IGF1, IGFBP2, POMC, PPARG, and ZNF423. Over-representation analysis of the genes resulted in biological terms of skeletal muscle, matrilin proteins, adipose tissue, hyperglycemia, diabetes, Salmonella infections and tyrosine. CONCLUSIONS: Identification of ROH and FST-based analyses revealed selection signatures in TT line and genes that have important role in traits of economic interest. Changes in the genome of the chickens were observed between the 7th and 16th generations showing that ancient and recent selection in TT line may have acted over genomic regions affecting diseases and performance traits.

Proximal femoral head transcriptome reveals novel candidate genes related to epiphysiolysis in broiler chickens.

BACKGROUND: The proximal femoral head separation (FHS) or epiphysiolysis is a prevalent disorder affecting the chicken femur epiphysis, being considered a risk factor to infection which can cause bacterial chondronecrosis with osteomyelitis in broilers. To identify the genetic mechanisms involved in epiphysiolysis, differentially expressed (DE) genes in the femur of normal and FHS-affected broilers were identified using RNA-Seq technology. Femoral growth plate (GP) samples from 35-day-old commercial male broilers were collected from 4 healthy and 4 FHS-affected broilers. Sequencing was performed using an Illumina paired-end protocol. Differentially expressed genes were obtained using the edgeR package based on the False Discovery Rate (FDR < 0.05). RESULTS: Approximately 16 million reads/sample were generated with 2 × 100 bp paired-end reads. After data quality control, approximately 12 million reads/sample were mapped to the reference chicken genome (Galgal5). A total of 12,645 genes were expressed in the femur GP. Out of those, 314 were DE between groups, being 154 upregulated and 160 downregulated in FHS-affected broilers. In the functional analyses, several biological processes (BP) were overrepresented. Among them, those related to cell adhesion, extracellular matrix (ECM), bone development, blood circulation and lipid metabolism, which are more related to chicken growth, are possibly involved with the onset of FHS. On the other hand, BP associated to apoptosis or cell death and immune response, which were also found in our study, could be related to the consequence of the FHS. CONCLUSIONS: Genes with potential role in the epiphysiolysis were identified through the femur head transcriptome analysis, providing a better understanding of the mechanisms that regulate bone development in fast-growing chickens. In this study, we highlighted the importance of cell adhesion and extracellular matrix related genes in triggering FHS. Furthermore, we have shown new insights on the involvement of lipidemia and immune response/inflammation with FHS in broilers. Understanding the changes in the GP transcriptome might support breeding strategies to address poultry robustness and to obtain more resilient broilers.