Identification of Reference Genes for Quantitative Gene Expression Studies in Three Tissues of Japanese Quail

RT-qPCR is the gold standard for candidate gene expression analysis. However, the interpretation of RT-qPCR results depends on the proper use of internal controls, i.e., reference genes. Japanese quail is an agronomic species also used as a laboratory model, but little is known about RT-qPCR reference genes for this species. Thus, we investigated 10 putative reference genes (ACTB, GAPDH, PGK1, RPS7, RPS8, RPL19, RPL32, SDHA, TBP and YWHAZ) in three different female and male quail tissues (liver, brain and pectoral muscle). Gene expression stability was evaluated with three different algorithms: geNorm, NormFinder and BestKeeper. For each tissue, a suitable set of reference genes was defined and validated by a differential analysis of gene expression between females and males (CCNH in brain and RPL19 in pectoral muscle). Collectively, our study led to the identification of suitable reference genes in liver, brain and pectoral muscle for Japanese quail, along with recommendations for the identification of reference gene sets for this species

Etude du rôle du métabolisme de la vitamine A dans les cellules musculaires de poulet

National audienc

Molecular Phenotyping of White Striping and Wooden Breast Myopathies in Chicken

International audienceThe White Striping (WS) and Wooden Breast (WB) defects are two myopathic syndromes whose occurrence has recently increased in modern fast-growing broilers. The impact of these defects on the quality of breast meat is very important, as they greatly affect its visual aspect, nutritional value, and processing yields. The research conducted to date has improved our knowledge of the biological processes involved in their occurrence, but no solution has been identified so far to significantly reduce their incidence without affecting growing performance of broilers. This study aims to follow the evolution of molecular phenotypes in relation to both fast-growing rate and the occurrence of defects in order to identify potential biomarkers for diagnostic purposes, but also to improve our understanding of physiological dysregulation involved in the occurrence of WS and WB. This has been achieved through enzymatic, histological, and transcriptional approaches by considering breast muscles from a slow- and a fast-growing line, affected or not by WS and WB. Fast-growing muscles produced more reactive oxygen species (ROS) than slow-growing ones, independently of WS and WB occurrence. Within fast-growing muscles, despite higher mitochondria density, muscles affected by WS or WB defects did not show higher cytochrome oxidase activity (COX) activity, suggesting altered mitochondrial function. Among the markers related to muscle remodeling and regeneration, immunohistochemical staining of FN1, NCAM, and MYH15 was higher in fast- compared to slow-growing muscles, and their amount also increased linearly with the presence and severity of WS and WB defects, making them potential biomarkers to assess accurately their presence and severity. Thanks to an innovative histological technique based on fluorescence intensity measurement, they can be rapidly quantified to estimate the injuries induced in case of WS and WB. The muscular expression of several other genes correlates also positively to the presence and severity of the defects like TGFB1 and CTGF, both involved in the development of connective tissue, or Twist1, known as an inhibitor of myogenesis. Finally, our results suggested that a balance between TGFB1 and PPARG would be essential for fibrosis or adiposis induction and therefore for determining WS and WB phenotypes

Beta-carotene acts as a regulator of skeletal muscle cells

National audienceThe enzyme beta, beta-carotene-15,15'-monooxygenase (BCMO1) cleaves provitamin A carotenoids into active vitamin A principally in liver and intestine. The BCMO1 gene is expressed at low level in the muscle tissue but little is known about its function. In the chicken muscle, we observed that various BCMO1 expression levels are associated with different carotenoids contents. To investigate the potential role of BCMO1 on skeletal muscle, we assessed the impact of beta-carotene (BC, the prototype substrate of the BCMO1 enzyme) supplementation in vitro on proliferative avian myoblasts. Proliferation was evaluated by BrdU incorporation and by flow cytometry. The BrdU incorporation index was reduced and th e proportion of G0/G1 cells increased following BC supplementation. Cell differentiation was evaluated by immunolabelling of sarcomeric myosin heavy chain (MHC). In this proliferative environment, the proportion of MHC positive cells increased following BC supplementation. The effects of BC were inhibited in the presence of DEAB, an inhibitor of retinaldehyde dehydrogenase, supporting the hypothesis that the BCMO1 enzyme is active in myoblasts and can contribute to retinoic acid production from BC. Our data suggest that provitamin A carotenoids could be used as nutritional regulators of skeletal muscle growt

Molecular phenotyping of white striping and wooden breast defects

International audienc

Characterization of white-striping myopathy in chickens

International audienceIn fast-growing chicken, the white-striping (WS) myopathy is described macroscopically as white striations parallel to the muscle fiber axis with unknown etiology. These myopathies are described in fast-growing (FG) commercial broilers and affect principally the breast Pectoralis Major (PM) muscle. Muscles are generally categorized in three classes: WS0 corresponding to the absence of macroscopic defect, WS1 for muscles moderately affected by WS, and WS2 for muscle severely affected by the defect. The aim of the present study was to evaluate the role of satellite cells and fibro-adipogenic precursors (FAPs) in the development of the WS physiopathology. This was performed by comparing a slow-growing (SG) chicken line, free of the defect, and a FG chicken line whose muscles are frequently affected by the defect. A histological characterization was performed at 42 days of age on the PM muscle of both lines. As expected, SG muscles appeared histologically normal (absence of any injuries) and served as control. Despite 4 FG chickens were classified as WS0, all muscles of this line (n=8) showed injuries like necrosis-regeneration, extension of perimysial and endomysial connective tissue, and infiltration of adipose tissue at various degrees. To assess the macroscopic phenotypic variability we performed quantitative measurements based on immunohistochemistry and/or western blot expression of different markers (i.e. collagen VI and BODIPY 493 for connective tissue and adipose tissue extension, respectively). We observed in the FG line an increase in muscle fiber size, necrotic and regenerative cell proportion, fibrosis and adiposis compared to SG line. These changes were associated with an increase in Pax7/100 fiber ratio and in nuclei percentage in PDGFRA-positive cells (i.e., FAPs). These preliminary results suggest a role of satellite cells and FAPs in the setting up of the WS myopathy. They will be completed by several in vitro and in vivo measurements to assess the proliferation and differentiation abilities of myogenic precursors and FAPs issued of normal and affected muscles, with the objective to improve our knowledge of the WS physiopathology

Ontogeny of hepatic metabolism in two broiler lines divergently selected for the ultimate pH of the Pectoralis major muscle

International audienceAbstract Background Nutrient availability during early stages of development (embryogenesis and the first week post-hatch) can have long-term effects on physiological functions and bird metabolism. The embryo develops in a closed structure and depends entirely on the nutrients and energy available in the egg. The aim of this study was to describe the ontogeny of pathways governing hepatic metabolism that mediates many physiological functions in the pHu + and pHu- chicken lines, which are divergently selected for the ultimate pH of meat, a proxy for muscle glycogen stores, and which differ in the nutrient content and composition of eggs. Results We identified eight clusters of genes showing a common pattern of expression between embryonic day 12 (E12) and day 8 (D8) post-hatch. These clusters were not representative of a specific metabolic pathway or function. On E12 and E14, the majority of genes differentially expressed between the pHu + and pHu- lines were overexpressed in the pHu + line. Conversely, the majority of genes differentially expressed from E18 were overexpressed in the pHu- line. During the metabolic shift at E18, there was a decrease in the expression of genes linked to several metabolic functions (e.g. protein synthesis, autophagy and mitochondrial activity). At hatching (D0), there were two distinct groups of pHu + chicks based on hierarchical clustering; these groups also differed in liver weight and serum parameters (e.g. triglyceride content and creatine kinase activity). At D0 and D8, there was a sex effect for several metabolic pathways. Metabolism appeared to be more active and oriented towards protein synthesis ( RPS6 ) and fatty acid β-oxidation ( ACAA2 , ACOX1 ) in males than in females. In comparison, the genes overexpressed in females were related to carbohydrate metabolism ( SLC2A1, SLC2A12 , FoxO1 , PHKA2 , PHKB , PRKAB2 and GYS2 ). Conclusions Our study provides the first detailed description of the evolution of different hepatic metabolic pathways during the early development of embryos and post-hatching chicks. We found a metabolic orientation for the pHu + line towards proteolysis, glycogen degradation, ATP synthesis and autophagy, likely in response to a higher energy requirement compared with pHu- embryos. The metabolic orientations specific to the pHu + and pHu- lines are established very early, probably in relation with their different genetic background and available nutrients

Embryonic thermal manipulation impacts the postnatal transcriptome response of heat-challenged Japanese quails

International audienceBackground The thermal-manipulation (TM) during egg incubation is a cyclic exposure to hot or cold temperatures during embryogenesis that is associated to long-lasting effects on growth performance, physiology, metabolism and temperature tolerance in birds. An increase of the incubation temperature of Japanese quail eggs affected the embryonic and post-hatch survival, growth, surface temperatures and blood characteristics potentially related to thermoregulation capacities. To gain new insights in the molecular basis of TM in quails, we investigated by RNA-seq the hypothalamus transcriptome of 35 days-old male and female quails that were treated by TM or not (C, control) during embryogenesis and that were exposed (HC) or not (RT) to a 36 °C heat challenge for 7 h before sampling.Results For males, 76, 27, 47 and 0 genes were differentially expressed in the CHC vs. CRT, CRT vs. TMRT, TMHC vs. TMRT and CHC vs. TMHC comparisons, respectively. For females, 17, 0, 342 and 1 genes were differentially expressed within the same respective comparisons. Inter-individual variability of gene expression response was observed particularly when comparing RT and HC female animals. The differential expression of several genes was corroborated by RT-qPCR analysis. Gene Ontology functional analysis of the differentially expressed genes showed a prevalent enrichment of terms related to cellular responses to stimuli and gene expression regulation in both sexes. Gene Ontology terms related to the membrane transport, the endoplasmic reticulum and mitochondrial functions as well as DNA metabolism and repair were also identified in specific comparisons and sexes.Conclusions TM had little to no effect on the regulation of gene expression in the hypothalamus of 35 days-old Japanese quails. However, the consequences of TM on gene expression were revealed by the HC, with sex-specific and common functions altered. The effects of the HC on gene expression were most prominent in TM females with a ~ 20-fold increase of the number of differentially expressed genes, suggesting that TM may enhance the gene response during challenging conditions in female quail hypothalamus. TM may also promote new cellular strategies in females to help coping to the adverse conditions as illustrated by the identification of differentially expressed genes related to the mitochondrial and heat-response functions