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Enterococcus faecalis, a member of the natural microbiota of animal and human intestinal tracts, is also present as a natural contaminant in a variety of fermented foods. Over the last decade, E. faecalis has emerged as a major cause of nosocomial infections. We investigated the genetic diversity in 30 clinical and food isolates, including strains V583 and MMH594, in order to determine whether clinical and food isolates could be distinguished. Data were obtained using comparative genomic hybridization and specific PCR with a total of 202 probes of E. faecalis, selected using the available V583 genome sequence and part of the MMH594 pathogenicity island. The cognate genes encoded mainly exported proteins. Hybridization data were analyzed by a two-component mixture model that estimates the probability of any given gene to be either present or absent in the strains. A total of 78 genes were found to be variable, as they were absent in at least one isolate. Most of the variable genes were clustered in regions that, in the published V583 sequence, related to prophages or mobile genetic elements. The variable genes were distributed in three main groups: (i) genes equally distributed between clinical and dairy food isolates, (ii) genes absent from dairy food-related isolates, and (iii) genes present in MMH594 and V583 strains only. Further analysis of the distribution of the last gene group in 70 other isolates confirmed that six of the probed genes were always absent in dairy food-related isolates, whereas they were detected in clinical and/or commensal isolates. Two of them corresponded to prophages that were not detected in the cognate isolates, thus possibly extending the number of genes absent from dairy food isolates. Genes specifically detected in clinical isolates may prove valuable for the development of new risk assessment markers for food safety studies and for identification of new factors that may contribute to host colonization or infection.

Changes in gene activity through epigenetic alterations induced by early environmental challenges during the embryogenesis are known to impact the phenotype, health and disease risk of animals. The epigenome is therefore an essential contributor to phenotypic plasticity, and learning how environmental exposures translate into persisting epigenetic changes may open new doors to improve robustness and resilience of developing animals. Birds are agronomic species of choice to directly manipulate the embryonic environment and study its consequences on the developing animal. We previously showed that the heat tolerance of male commercial chickens was improved by cyclically elevating the egg incubation temperature. This treatment named embryonic thermal manipulation (TM) was associated with changes in gene expression that persisted during the development of chickens and enhanced gene expression response in case of heat challenge at slaughter age, 35 days post-hatch. To further explore the molecular basis of heat acclimation, we took advantage of an inbred line of Japanese quails (Coturnix japonica) to investigate the impact of TM on bird methylome. Among other advantages, quail generation cycle is 3-4 time faster than chicken and the use of an inbred genotype should reduce the phenotype variations associated with genetic variability. A characterization of TM on quail phenotype was performed in interaction with a post-hatch heat stress at the onset of sexual maturity. Notably, we observed an impact of the embryonic treatment on the temperature and the weight of young quails, similarly to what was reported for chicken. Concomitantly we investigated the impact of TM on quail transcriptome by RNA-seq and methylome by whole genome bisulfite sequencing (WGBS) on brain tissues sampled at hatch. Several differentially expressed gene and differential methylation regions were identified. Interestingly, the nature of some transcripts seemed to be affected by the embryonic treatment at both methylation and transcript levels. [ABSTRACT FROM AUTHOR]

Abstract Background Most egg yolk precursors are synthesized by the liver, secreted into the blood and transferred into oocytes, to provide nutrients and bioactive molecules for the avian embryo. Three hundred and sixteen distinct proteins have been identified in egg yolk. These include 37 proteases and antiproteases, which are likely to play a role in the formation of the yolk (vitellogenesis), as regulators of protein metabolism. We used a transcriptomic approach to define the protease and antiprotease genes specifically expressed in the hen liver in relation to vitellogenesis by comparing sexually mature and pre-laying chickens showing different steroid milieu. Results Using a 20 K chicken oligoarray, a total of 582 genes were shown to be over-expressed in the liver of sexually mature hens (1.2 to 67 fold-differences). Eight of the top ten over-expressed genes are known components of the egg yolk or perivitelline membrane. This list of 582 genes contains 12 proteases and 3 antiproteases. We found that “uncharacterized protein LOC419301/similar to porin” (GeneID:419301), an antiprotease and “cathepsin E-A-like/similar to nothepsin” (GeneID:417848), a protease, were the only over-expressed candidates (21-fold and 35-fold difference, respectively) that are present in the egg yolk. Additionally, we showed the 4-fold over-expression of “ovochymase-2/similar to oviductin” (GeneID:769290), a vitelline membrane-specific protease. Conclusions Our approach revealed that three proteases and antiproteases are likely to participate in the formation of the yolk. The role of the other 12 proteases and antiproteases which are over-expressed in our model remains unclear. At least 1/3 of proteases and antiproteases identified in egg yolk and vitelline membrane proteomes are expressed similarly in the liver regardless of the maturity of hens, and have been initially identified as regulators of haemostasis and inflammatory events. The lack of effect of sex steroids on these genes expressed in the liver but the products of which are found in the yolk suggests that these may be passively incorporated into the yolk rather than actively produced for that purpose. These results raise the question of the biological significance of egg yolk proteases and antiproteases, and more generally of all minor proteins that have been identified in egg yolk.

Abstract Background The processing ability of poultry meat is highly related to its ultimate pH, the latter being mainly determined by the amount of glycogen in the muscle at death. The genetic determinism of glycogen and related meat quality traits has been established in the chicken but the molecular mechanisms involved in variations in these traits remain to be fully described. In this study, Chicken Genome Arrays (20 K) were used to compare muscle gene expression profiles of chickens from Fat (F) and Lean (L) lines that exhibited high and low muscle glycogen content, respectively, and of individuals exhibiting extremely high (G+) or low (G-) muscle glycogen content originating from the F2 cross between the Fat and Lean lines. Real-time RT-PCR was subsequently performed to validate the differential expression of genes either selected from the microarray analysis or whose function in regulating glycogen metabolism was well known. Results Among the genes found to be expressed in chicken P. major muscle, 197 and 254 transcripts appeared to be differentially expressed on microarrays for the F vs. L and the G+ vs. G- comparisons, respectively. Some involved particularly in lipid and carbohydrate metabolism were selected for further validation studies by real-time RT-PCR. We confirmed that, as in mammals, the down-regulation of CEBPB and RGS2 coincides with a decrease in peripheral adiposity in the chicken, but these genes are also suggested to affect muscle glycogen turnover through their role in the cAMP-dependent signalling pathway. Several other genes were suggested to have roles in the regulation of glycogen storage in chicken muscle. PDK4 may act as a glycogen sensor in muscle, UGDH may compete for glycogen synthesis by using UDP-glucose for glucoronidation, and PRKAB1, PRKAG2, and PHKD may impact on glycogen turnover in muscle, through AMP-activated signalling pathways. Conclusions This study is the first stage in the understanding of molecular mechanisms underlying variations in poultry meat quality. Large scale analyses are now required to validate the role of the genes identified and ultimately to find molecular markers that can be used for selection or to optimize rearing practices.

Abstract Background As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses. Results Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid where eggshell formation takes place. Conclusions Our original study provides the first detailed description of the chicken uterus transcriptome during formation of the eggshell. We have discovered a cache of about 600 functional genes and identified a large number of encoded proteins secreted into uterine fluid for fabrication of the eggshell and chemical protection of the egg. Some of these uterine genes could prove useful as biological markers for genetic improvement of phenotypic traits (i.e., egg and eggshell quality).

Abstract Background Difference in adaptability responses to stress has been observed amongst bird species, strains, and individuals. Components of the HPA axis, one of the internal systems involved in homeostasis re-establishment following stress, could play a role in this variability of responses. The aim of the present study was 1) to identify genes involved in the regulation of adrenal activity following ACTH stimulation and 2) to examine adrenal genes differentially expressed in individuals with high and low plasma corticosterone response following ACTH treatment. Results Analysis with 21 K poultry oligo microarrays indicated that ACTH treatment affected the expression of 134 genes. Several transcripts assigned to genes involved in the adrenal ACTH signaling pathway and steroidogenic enzymes were identified as differentially expressed by ACTH treatment. Real-time PCR on 18 selected genes confirmed changes in transcript levels of 11 genes, including MC2R, CREM, Cry, Bmal1, Sqle, Prax1, and StAR. Only 4 genes revealed to be differentially expressed between higher and lower adrenal responders to ACTH treatment. Conclusion The results from the present study reveal putative candidate genes; their role in regulation of adrenal functions and adaptability to stress should be further investigated.

Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT) and polymerase chain reaction (PCR), the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3) and somatic tissues (n = 2), we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round). Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number) but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70%) and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID). However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions) and relevant (biologically validated). In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i) the sample used: brain, ovary or embryos, (ii) the enzymatic properties initially inferred (exponential or linear) and (iii) the preliminary optimization of the protocols. Moreover the use of an in-house developed array, small-sized but well suited to the tissues we worked with, was of real interest for the search of differential expressions.

Abstract Background Analysis of variance is a powerful approach to identify differentially expressed genes in a complex experimental design for microarray and macroarray data. The advantage of the anova model is the possibility to evaluate multiple sources of variation in an experiment. Results AnovArray is a package implementing ANOVA for gene expression data using SAS® statistical software. The originality of the package is 1) to quantify the different sources of variation on all genes together, 2) to provide a quality control of the model, 3) to propose two models for a gene's variance estimation and to perform a correction for multiple comparisons. Conclusion AnovArray is freely available at http://www-mig.jouy.inra.fr/stat/AnovArray and requires only SAS® statistical software.

The chicken chorioallantoic membrane (CAM) is an extraembryonic structure that exhibits many vital functions to support the development of the chicken embryo (gaseous exchange, innate defence, calcium transport from the eggshell to the embryo skeleton, homeostasis). Developing from day 6 of incubation, the CAM progressively differentiates into three functional layers (the chorionic epithelium in contact with the inner eggshell, the highly vascularized mesoderm, and the allantoic epithelium), between 11 and 15 days of incubation. This article describes the RNASeq dataset and the analyses performed on total CAMs collected from male and female embryos after 11 and 15 days of incubation. The datasets are available at the NCBI Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo) using GSE199780 as the accession number. The statistical analysis of the data allowed identifying genes differentially expressed depending on the sex of the embryo at two time points of CAM differentiation. Knowing that the CAM is widely used as a model to study tumour growth, metastasis or wound healing, the resulting analysis highlights the necessity to include this sex variable in experimental assays to avoid any bias of interpretation. Indeed, the functional annotation of genes that are differentially expressed between male and female CAMs revealed an enrichment of activities and functions related to lipid metabolism, bone formation, and morphogenesis suggesting that the response of the CAM to external and experimental stimuli might be different depending on the sex of the embryo., (© 2024 The Author(s).)

The microbial utilization of dietary carbohydrates is closely linked to the pivotal role of the gut microbiome in human health. Inherent to the modulation of complex microbial communities, a prebiotic implies the selective utilization of a specific substrate, relying on the metabolic capacities of targeted microbes. In this study, we investigated the metabolic capacities of 17 commensal bacteria of the human gut microbiome toward dietary carbohydrates with prebiotic potential. First, in vitro experiments allowed the classification of bacterial growth and fermentation profiles in response to various carbon sources, including agave inulin, corn fiber, polydextrose, and citrus pectin. The influence of phylogenetic affiliation appeared to statistically outweigh carbon sources in determining the degree of carbohydrate utilization. Second, we narrowed our focus on six commensal bacteria representative of the Bacteroidetes and Firmicutes phyla to perform an untargeted high-resolution liquid chromatography-mass spectrometry metabolomic analysis: Bacteroides xylanisolvens , Bacteroides thetaiotaomicron , Bacteroides intestinalis , Subdoligranulum variabile , Roseburia intestinalis, and Eubacterium rectale exhibited distinct metabolomic profiles in response to different carbon sources. The relative abundance of bacterial metabolites was significantly influenced by dietary carbohydrates, with these effects being strain-specific and/or carbohydrate-specific. Particularly, the findings indicated an elevation in short-chain fatty acids and other metabolites, including succinate, gamma-aminobutyric acid, and nicotinic acid. These metabolites were associated with putative health benefits. Finally, an RNA-Seq transcriptomic approach provided deeper insights into the underlying mechanisms of carbohydrate metabolization. Restricting our focus on four commensal bacteria, including B. xylanisolvens , B. thetaiotaomicron, S. variabile, and R. intestinalis , carbon sources did significantly modulate the level of bacterial genes related to the enzymatic machinery involved in the metabolization of dietary carbohydrates. This study provides a holistic view of the molecular strategies induced during the dynamic interplay between dietary carbohydrates with prebiotic potential and gut commensal bacteria., Importance: This study explores at a molecular level the interactions between commensal health-relevant bacteria and dietary carbohydrates holding prebiotic potential. We showed that prebiotic breakdown involves the specific activation of gene expression related to carbohydrate metabolism. We also identified metabolites produced by each bacteria that are potentially related to our digestive health. The characterization of the functional activities of health-relevant bacteria toward prebiotic substances can yield a better application of prebiotics in clinical interventions and personalized nutrition. Overall, this study highlights the importance of identifying the impact of prebiotics at a low resolution of the gut microbiota to characterize the activities of targeted bacteria that can play a crucial role in our health., Competing Interests: The authors declare no conflict of interest.

Dimorphism between male and female embryos has been demonstrated in many animal species, including chicken species. Likewise, extraembryonic membranes such as the chorioallantoic membrane (CAM) are likely to exhibit a sex-specific profile. Analysis of the previously published RNA-seq data of the chicken CAM sampled at two incubation times, revealed 783 differentially expressed genes between the CAM of male and female embryos. The expression of some of these genes is sex-dependant only at one or other stage of development, while 415 genes are sex-dependant at both developmental stages. These genes include well-known sex-determining and sex-differentiation genes (DMRT1, HEGM, etc.), and are mainly located on sex chromosomes. This study provides evidence that gene expression of extra-embryonic membranes is differentially regulated between male and female embryos. As such, a better characterisation of associated mechanisms should facilitate the identification of new sex-specific biomarkers., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Salmonella is the only bacterium able to enter a host cell by the two known mechanisms: trigger and zipper. The trigger mechanism relies on the injection of bacterial effectors into the host cell through the Salmonella type III secretion system 1. In the zipper mechanism, mediated by the invasins Rck and PagN, the bacterium takes advantage of a cellular receptor for invasion. This study describes the transcriptomic reprogramming of the IEC-6 intestinal epithelial cell line to Salmonella Typhimurium strains that invaded cells by a trigger, a zipper, or both mechanisms. Using S . Typhimurium strains invalidated for one or other entry mechanism, we have shown that IEC-6 cells could support both entries. Comparison of the gene expression profiles of exposed cells showed that irrespective of the mechanism used for entry, the transcriptomic reprogramming of the cell was nearly the same. On the other hand, when gene expression was compared between cells unexposed or exposed to the bacterium, the transcriptomic reprogramming of exposed cells was significantly different. It is particularly interesting to note the modulation of expression of numerous target genes of the aryl hydrocarbon receptor showing that this transcription factor was activated by S . Typhimurium infection. Numerous genes associated with the extracellular matrix were also modified. This was confirmed at the protein level by western-blotting showing a dramatic modification in some extracellular matrix proteins. Analysis of a selected set of modulated genes showed that the expression of the majority of these genes was modulated during the intracellular life of S . Typhimurium.

Background: In the current context of global warming, thermal manipulation of avian embryos has received increasing attention as a strategy to promote heat tolerance in avian species by simply increasing the egg incubation temperature. However, because of their likely epigenetic origin, thermal manipulation effects may last more than one generation with consequences for the poultry industry. In this work, a multigenerational and transgenerational analysis of thermal manipulation during embryogenesis was performed to uncover the long-term effects of such procedure., Results: Thermal manipulation repeated during 4 generations had an effect on hatchability, body weight, and weight of eggs laid in Japanese quails, with some effects increasing in importance over generations. Moreover, the effects on body weight and egg weight could be transmitted transgenerationally, suggesting non-genetic inheritance mechanisms. This hypothesis is reinforced by the observed reversion of the effect on growth after five unexposed generations. Interestingly, a beneficial effect of thermal manipulation on heat tolerance was observed a few days after hatching, but this effect was not transgenerational., Conclusions: Our multigenerational study showed that thermal conditioning of quail embryos has a beneficial effect on post-hatch heat tolerance hampered by transgenerational but reversible defects on growth. Assuming that no genetic variability underlies these changes, this study provides the first demonstration of epigenetic inheritance of traits induced by environmental temperature modification associated with long-term impacts in an avian species., (© 2023. Chinese Association of Animal Science and Veterinary Medicine.)

Enterococcus cecorum is an emerging pathogen responsible for osteomyelitis, spondylitis, and femoral head necrosis causing animal suffering and mortality and requiring antimicrobial use in poultry. Paradoxically, E. cecorum is a common inhabitant of the intestinal microbiota of adult chickens. Despite evidence suggesting the existence of clones with pathogenic potential, the genetic and phenotypic relatedness of disease-associated isolates remains little investigated. Here, we sequenced and analyzed the genomes and characterized the phenotypes of more than 100 isolates, the majority of which were collected over the last 10 years from 16 French broiler farms. Comparative genomics, genome-wide association studies, and the measured susceptibility to serum, biofilm-forming capacity, and adhesion to chicken type II collagen were used to identify features associated with clinical isolates. We found that none of the tested phenotypes could discriminate the origin of the isolates or the phylogenetic group. Instead, we found that most clinical isolates are grouped phylogenetically, and our analyses selected six genes that discriminate 94% of isolates associated with disease from those that are not. Analysis of the resistome and the mobilome revealed that multidrug-resistant clones of E. cecorum cluster into a few clades and that integrative conjugative elements and genomic islands are the main carriers of antimicrobial resistance. This comprehensive genomic analysis shows that disease-associated clones of E. cecorum belong mainly to one phylogenetic clade. IMPORTANCE Enterococcus cecorum is an important pathogen of poultry worldwide. It causes a number of locomotor disorders and septicemia, particularly in fast-growing broilers. Animal suffering, antimicrobial use, and associated economic losses require a better understanding of disease-associated E. cecorum isolates. To address this need, we performed whole-genome sequencing and analysis of a large collection of isolates responsible for outbreaks in France. By providing the first data set on the genetic diversity and resistome of E. cecorum strains circulating in France, we pinpoint an epidemic lineage that is probably also circulating elsewhere that should be targeted preferentially by preventive strategies in order to reduce the burden of E. cecorum -related diseases.

The chicken chorioallantoic membrane (CAM) is an extraembryonic membrane that is vital for the embryo. It undergoes profound cell differentiation between 11 and 15 days of embryonic incubation (EID), which corresponds to the acquisition of its physiological functions. To gain insight into the functional genes that accompany these biological changes, RNA sequencing of the CAM at EID11 and EID15 was performed. Results showed that CAM maturation coincides with the overexpression of 4225 genes, including many genes encoding proteins involved in mineral metabolism, innate immunity, homeostasis, angiogenesis, reproduction, and regulation of hypoxia. Of these genes, some exhibit variability in expression depending on the chicken breed (broiler versus layer breeds). Besides the interest of these results for the poultry sector, the identification of new functional gene candidates opens additional research avenues in the field of developmental biology., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Here, molecular pathways and genes involved in the feed efficiency (FE) and thigh-meat quality of slow-growing Korat chickens were investigated. Individual feed intake values and body weights were collected weekly to the calculate feed conversion ratios (FCR) and residual feed intake. The biochemical composition and meat quality parameters were also measured. On the basis of extreme FCR values at 10 weeks of age, 9 and 12 birds from the high and the low FCR groups, respectively, were selected, and their transcriptomes were investigated using the 8 × 60 K Agilent chicken microarray. A weighted gene co-expression network analysis was performed to determine the correlations between co-expressed gene modules and FE, thigh-meat quality, or both. Groups of birds with different FE values also had different nucleotide, lipid, and protein contents in their thigh muscles. In total, 38 modules of co-expressed genes were identified, and 12 were correlated with FE and some meat quality traits. A functional analysis highlighted several enriched functions, such as biological processes, metabolic processes, nucleotide metabolism, and immune responses. Several molecular factors were involved in the interactions between FE and meat quality, including the assembly competence domain, baculoviral IAP repeat containing 5, cytochrome c oxidase assembly factor 3, and myosin light chain 9 genes.

Background: 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.

Purpose: A protective effect of anti-Müllerian hormone (AMH) on follicle atresia was recently demonstrated using long-term treatments, but this effect has never been supported by mechanistic studies. This work aimed to gain an insight into the mechanism of action of AMH on follicle atresia and on how this could account for the increased follicle pool observed in women with polycystic ovary syndrome (PCOS)., Methods: In vivo and in vitro experiments were performed to study the effects of AMH on follicle atresia and on the proliferation and apoptosis of granulosa cells (GCs). RNA-sequencing was carried out to identify new AMH target genes in GCs. The expression of some of these genes in GCs from control and PCOS women was compared using microfluidic real time quantitative RT-PCR., Results: A short-term AMH treatment prevented follicle atresia in prepubertal mice. Consistent with this result, AMH inhibited apoptosis and promoted proliferation of different models of GCs. Moreover, integrative biology analyses of 965 AMH target genes identified in 1 of these GC models, confirmed that AMH had initiated a gene expression program favoring cell survival and proliferation. Finally, on 43 genes selected among the most up- and down-regulated AMH targets, 8 were up-regulated in GCs isolated from PCOS women, of which 5 are involved in cell survival., Main Conclusions: Our results provide for the first time cellular and molecular evidence that AMH protects follicles from atresia by controlling GC survival and suggest that AMH could participate in the increased follicle pool of PCOS patients., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sustainability of poultry farming relies on the development of more efficient and autonomous production systems in terms of feed supply. This implies a better integration of adaptive traits in breeding programs, including digestive efficiency, to favor the use of a wider variety of feedstuffs. The objective of the study was to better characterize the kinetics of development of the digestive tract in broilers, in relationship with digestive efficiency by measuring various digestive parameters as well as serum color. Absolute and relative growth of gastrointestinal tract organs were compared between 2 divergent chicken lines selected on digestive efficiency (AMEn) during 7 wk of development. We show that as early as 7 d of age, these 2 lines differs for several organs developments and that these differences remain visible later on. In addition, the allometry of the gizzard and intestine segments is different between the 2 lines, with efficient birds putting more effort in the upper part of the digestive tract during postnatal development and less-efficient birds putting more effort in the lower part of the gastrointestinal tract. Interestingly, we also showed that differences in serum pigmentation, which is a good biomarker for digestive capacity, could be a convenient diagnostic tool to discriminate between chickens with high or low digestive efficiency at early stages of development. In conclusion, this study showed that selection of chickens for AMEn had large impacts in gastrointestinal development including at early stages and is a valuable resource for further studies on the genetic and physiological control of the response of the animal to feed variations., (Copyright © 2020. Published by Elsevier Inc.)

This study provides an integrative description of candidate gene expression across tissues involved in calcium (Ca) metabolism during the egg laying cycle, using the well-defined model of Ca supply as fine or coarse particles of calcium carbonate (CaCO 3 ). Plasma and tissue samples were collected from hens at the peak of laying at 0 to 1, 9 to 10, and 18 to 19 h postovulation (PO). After mRNA preparation from the parathyroid gland, medullary bone, liver, kidney, duodenum, and jejunum, gene expressions were quantified using RT-qPCR. The highest levels of parathyroid hormone (PTH) mRNA in the parathyroid gland (P < 0.05), and of the active form of vitamin D 3 1.25(OH) 2 D 3 in the plasma (P < 0.01) were observed at 18 to 19 h PO. During this active phase of eggshell formation, bone resorption was attested to high levels of plasma inorganic phosphorus (iP) and the receptor activation of nuclear factor-κB expression in the bone (P < 0.001 and P < 0.05, respectively). At this stage, 5 genes of the transcellular and the paracellular Ca absorption pathways in the intestine (P < 0.05) and the Ca channel transient receptor potential cation channel subfamily V member 5 (P < 0.05), involved in its reabsorption in the kidney, were overexpressed. At 0 to 1 h PO during the subsequent daylight period, 2 candidates of the transcellular and the paracellular Ca pathways (P < 0.05) remained at high levels in the intestine, while calbindin D 28K expression was the highest in the kidney (P < 0.05). As PTH mRNA and 1.25(OH) 2 D 3 were low, bone accretion was likely active at this stage. The phosphaturic hormone fibroblast growth factor 23 (FGF23) was overexpressed at 18 to 19 h PO (P < 0.05) in the bone when plasma iP was high, which suggested a role in the subsequent reduction of P reabsorption in the kidney, as attested to the decreased expression of P cotransporters, leading to iP clearance from the plasma at 0 to 1 h PO (P < 0.05). The low levels of 1.25(OH) 2 D 3 at this stage coincided with increased expression of the 24-hydroxylase gene in the kidney (P < 0.05). In hens fed fine particles of CaCO 3 , higher plasma levels of 1,25(OH) 2 D 3 and higher expression of several genes involved in bone turnover reflected a stronger challenge to Ca homeostasis. Altogether, these data support the hypothesis that FGF23 could drive vitamin D metabolism in the laying hen, as previously documented in other species and explain the tight link between P and Ca metabolisms., (Copyright © 2019. Published by Elsevier Inc.)

Improving the digestive efficiency of broiler chickens (Gallus gallus) could reduce organic waste, increase the use of alternative feed not used for human consumption and reduce the impact of feed in production costs. By selecting chicken lines divergently for their digestive efficiency, we showed previously that digestive efficiency is under genetic control and that the two resulting divergent lines, D+ (high digestive efficiency or "digestibility +") and D- (low digestive efficiency or "digestibility -"), also differ for the abundance of specific bacteria in their caeca. Here we perform a more extensive census of the bacteria present in the digestive microbiota of 60 chickens selected for their low apparent metabolizable energy corrected for nitrogen balance (AMEn-) or high (AMEn+) digestive efficiency in a [D+ x D-] F8 progeny of 200 individuals. We sequenced the 16S rRNA genes of the ileal, jejunal and caecal microbiotas, and compared the compositions and predicted functions of microbiotas from the different intestinal segments for 20 AMEn+ and 19 AMEn- birds. The intestinal segment of origin was the main factor structuring the samples. The caecal microbiota was the most impacted by the differences in digestive efficiency, with 41 bacterial species with abundances differing between highly and poorly efficient birds. Furthermore, we predicted that the caecal microbiota of efficient birds might be enriched in genes contributing to the degradation of short chain fatty acids (SCFA) from non-starch polysaccharides. These results confirm the impact of the genetic selection led on digestibility on the caecal microbiota taxonomic composition. They open the way toward the identification of specific, causal genes of the host controlling variations in the abundances of bacterial taxons., Competing Interests: The authors have declared that no competing interests exist.

In mammalian ovaries, the theca layers of growing follicles are critical for maintaining their structural integrity and supporting androgen synthesis. Through combining the postnatal monitoring of ovaries by abdominal magnetic resonance imaging, endocrine profiling, hormonal analysis of the follicular fluid of growing follicles, and transcriptomic analysis of follicular theca cells, we provide evidence that the exposure of ovine fetuses to testosterone excess activates postnatal follicular growth and strongly affects the functions of follicular theca in adulthood. Prenatal exposure to testosterone impaired androgen synthesis in the small antral follicles of adults and affected the expression in their theca cells of a wide array of genes encoding extracellular matrix components, their membrane receptors, and signaling pathways. Most expression changes were uncorrelated with the concentrations of gonadotropins, steroids, and anti-Müllerian hormone in the recent hormonal environment of theca cells, suggesting that these changes rather result from the long-term developmental effects of testosterone on theca cell precursors in fetal ovaries. Disruptions of the extracellular matrix structure and signaling in the follicular theca and ovarian cortex can explain the acceleration of follicle growth through altering the stiffness of ovarian tissue. We propose that these mechanisms participate in the etiology of the polycystic ovarian syndrome, a major reproductive pathology in woman.

In vertebrates, the embryonic environment is known to affect the development and the health of individuals. In broiler chickens, the thermal-manipulation (TM) of eggs during the incubation period was shown to improve heat tolerance at slaughter age (35 days of age) in association with several modifications at the molecular, metabolic and physiological levels. However, little is known about the Japanese quail (Coturnix japonica), a closely related avian species widely used as a laboratory animal model and farmed for its meat and eggs. Here we developed and characterized a TM procedure (39.5°C and 65% relative humidity, 12 h/d, from days 0 to 13 of incubation) in quails by analyzing its short and long-term effects on zootechnical, physiological and metabolic parameters. Heat-tolerance was tested by a heat challenge (36°C for 7h) at 35 days of age. TM significantly reduced the hatching rate of the animals and increased mortality during the first four weeks of life. At hatching, TM animals were heavier than controls, but lighter at 25 days of age for both sexes. Thirty-five days after hatching, TM decreased the surface temperature of the shank in females, suggesting a modulation of the blood flow to maintain the internal temperature. TM also increased blood partial pressure and oxygen saturation percentage at 35 days of age in females, suggesting a long-term modulation of the respiration physiology. Quails physiologically responded to the heat challenge, with a modification of several hematologic and metabolic parameters, including an increase in plasma corticosterone concentration. Several physiological parameters such as beak surface temperature and blood sodium concentration revealed that TM birds responded differently to the heat challenge compared to controls. Altogether, this first comprehensive characterization of TM in Japanese quail showed durable effects that may affect the response of TM quails to heat., Competing Interests: The authors have declared that no competing interests exist.

Changes in gene activity through epigenetic alterations induced by early environmental challenges during embryogenesis are known to impact the phenotype, health, and disease risk of animals. Learning how environmental cues translate into persisting epigenetic memory may open new doors to improve robustness and resilience of developing animals. It has previously been shown that the heat tolerance of male broiler chickens was improved by cyclically elevating egg incubation temperature. The embryonic thermal manipulation enhanced gene expression response in muscle ( P. major ) when animals were heat challenged at slaughter age, 35 days post-hatch. However, the molecular mechanisms underlying this phenomenon remain unknown. Here, we investigated the genome-wide distribution, in hypothalamus and muscle tissues, of two histone post-translational modifications, H3K4me3 and H3K27me3, known to contribute to environmental memory in eukaryotes. We found 785 H3K4me3 and 148 H3K27me3 differential peaks in the hypothalamus, encompassing genes involved in neurodevelopmental, metabolic, and gene regulation functions. Interestingly, few differences were identified in the muscle tissue for which differential gene expression was previously described. These results demonstrate that the response to embryonic thermal manipulation (TM) in chicken is mediated, at least in part, by epigenetic changes in the hypothalamus that may contribute to the later-life thermal acclimation., (Copyright © 2019 David, Vitorino Carvalho, Gimonnet, Brionne, Hennequet-Antier, Piégu, Crochet, Couroussé, Bordeau, Bigot, Collin and Coustham.)

The broiler industry is facing an increasing prevalence of breast myopathies, such as white striping (WS) and wooden breast (WB), and the precise aetiology of these occurrences remains poorly understood. To progress our understanding of the structural changes and molecular pathways involved in these myopathies, a transcriptomic analysis was performed using an 8 × 60 K Agilent chicken microarray and histological study. The study used pectoralis major muscles from three groups: slow-growing animals (n = 8), fast-growing animals visually free from defects (n = 8), or severely affected by both WS and WB (n = 8). In addition, a weighted correlation network analysis was performed to investigate the relationship between modules of co-expressed genes and histological traits. Functional analysis suggested that selection for fast growing and breast meat yield has progressively led to conditions favouring metabolic shifts towards alternative catabolic pathways to produce energy, leading to an adaptive response to oxidative stress and the first signs of inflammatory, regeneration and fibrosis processes. All these processes are intensified in muscles affected by severe myopathies, in which new mechanisms related to cellular defences and remodelling seem also activated. Furthermore, our study opens new perspectives for myopathy diagnosis by highlighting fine histological phenotypes and genes whose expression was strongly correlated with defects.

The main objective of ViSEAGO package is to carry out a data mining of biological functions and establish links between genes involved in the study. We developed ViSEAGO in R to facilitate functional Gene Ontology (GO) analysis of complex experimental design with multiple comparisons of interest. It allows to study large-scale datasets together and visualize GO profiles to capture biological knowledge. The acronym stands for three major concepts of the analysis: Vi sualization, S emantic similarity and E nrichment A nalysis of G ene O ntology. It provides access to the last current GO annotations, which are retrieved from one of NCBI EntrezGene, Ensembl or Uniprot databases for several species. Using available R packages and novel developments, ViSEAGO extends classical functional GO analysis to focus on functional coherence by aggregating closely related biological themes while studying multiple datasets at once. It provides both a synthetic and detailed view using interactive functionalities respecting the GO graph structure and ensuring functional coherence supplied by semantic similarity. ViSEAGO has been successfully applied on several datasets from different species with a variety of biological questions. Results can be easily shared between bioinformaticians and biologists, enhancing reporting capabilities while maintaining reproducibility. ViSEAGO is publicly available on https://bioconductor.org/packages/ViSEAGO ., Competing Interests: Competing interestsThe authors declare that they have no competing interests.

Background: The sustainability of poultry farming relies on the development of more efficient and autonomous production systems in terms of feed supply. This implies a better integration of adaptive traits in breeding programs, including digestive efficiency, in order to favor the use of a wider variety of feedstuffs. The aim of the project was to improve the understanding of genes involved in digestive functions by characterizing the transcriptome of different sections of the digestive tract: the junction between the proventriculus and the gizzard, the gizzard, the gastroduodenal junction, and the jejunum., Results: Total RNA from the four tissues were sequenced on a HiSeq2500 for six 23-day-old chickens from a second generation (F2) cross between two lines that were divergent for their digestive efficiency (D+/D-). Bioinformatics and biostatistics analyses of the RNA-seq data showed a total of 11,040 differentially expressed transcripts between the four tissues. In total, seven clusters of genes with markedly different expression profiles were identified. Functional analysis on gene groups was performed using "Gene Ontology" and semantic similarity. It showed a significant enrichment of body immune defenses in the jejunum, and an enrichment of transcriptional activity in the gizzard. Moreover, an interesting enrichment for neurohormonal control of muscle contraction was found for the two gizzard's junctions., Conclusion: This analysis allows us to draw the first molecular portrait of the different sections of the digestive tract, which will serve as a basis for future studies on the genetic and physiological control of the response of the animal to feed variations.

The increasing cost of conventional feedstuffs has bolstered interest in genetic selection for digestive efficiency (DE), a component of feed efficiency, assessed by apparent metabolisable energy corrected to zero nitrogen retention (AMEn). However, its measurement is time-consuming and constraining, and its relationship with metabolic efficiency poorly understood. To simplify selection for this trait, we searched for indirect metabolic biomarkers through an analysis of the serum metabolome using nuclear magnetic resonance ( 1 H NMR). A partial least squares (PLS) model including six amino acids and two derivatives from butyrate predicted 59% of AMEn variability. Moreover, to increase our knowledge of the molecular mechanisms controlling DE, we investigated 1 H NMR metabolomes of ileal, caecal, and serum contents by fitting canonical sparse PLS. This analysis revealed strong associations between metabolites and DE. Models based on the ileal, caecal, and serum metabolome respectively explained 77%, 78%, and 74% of the variability of AMEn and its constitutive components (utilisation of starch, lipids, and nitrogen). In our conditions, the metabolites presenting the strongest associations with AMEn were proline in the serum, fumarate in the ileum and glucose in caeca. This study shows that serum metabolomics offers new opportunities to predict chicken DE.

Background: The understanding of the biological determinism of meat ultimate pH, which is strongly related to muscle glycogen content, is a key point for the control of muscle integrity and meat quality in poultry. In the present study, we took advantage of a unique model of two broiler lines divergently selected for the ultimate pH of the pectoralis major muscle (PM-pHu) in order to decipher the genetic control of this trait. Two complementary approaches were used: detection of selection signatures generated during the first five generations and genome-wide association study for PM-pHu and Sartorius muscle pHu (SART-pHu) at the sixth generation of selection., Results: Sixty-three genomic regions showed significant signatures of positive selection. Out of the 10 most significant regions (detected by HapFLK or FLK method with a p-value below 1e-6), 4 were detected as soon as the first generation (G1) and were recovered at each of the four following ones (G2-G5). Another four corresponded to a later onset of selection as they were detected only at G5. In total, 33 SNPs, located in 24 QTL regions, were significantly associated with PM-pHu. For SART-pHu, we detected 18 SNPs located in 10 different regions. These results confirmed a polygenic determinism for these traits and highlighted two major QTL: one for PM-pHu on GGA1 (with a Bayes Factor (BF) of 300) and one for SART-pHu on GGA4 (with a BF of 257). Although selection signatures were enriched in QTL for PM-pHu, several QTL with strong effect haven't yet responded to selection, suggesting that the divergence between lines might be further increased., Conclusions: A few regions of major interest with significant selection signatures and/or strong association with PM-pHu or SART-pHu were evidenced for the first time in chicken. Their gene content suggests several candidates associated with diseases of glycogen storage in humans. The impact of these candidate genes on meat quality and muscle integrity should be further investigated in chicken.

Background: White striping (WS) is an emerging muscular defect occurring on breast and thigh muscles of broiler chickens. It is characterized by the presence of white striations parallel to the muscle fibers and has significant consequences for meat quality. The etiology of WS remains poorly understood, even if previous studies demonstrated that the defect prevalence is related to broiler growth and muscle development. Moreover, recent studies showed moderate to high heritability values of WS, which emphasized the role of genetics in the expression of the muscle defect. The aim of this study was to identify the first quantitative trait loci (QTLs) for WS as well as breast muscle yield (BMY) and meat quality traits using a genome-wide association study (GWAS). We took advantage of two divergent lines of chickens selected for meat quality through Pectoralis major ultimate pH (pHu) and which exhibit the muscular defect. An expression QTL (eQTL) detection was further performed for some candidate genes, either suggested by GWAS analysis or based on their biological function., Results: Forty-two single nucleotide polymorphisms (SNPs) associated with WS and other meat quality traits were identified. They defined 18 QTL regions located on 13 chromosomes. These results supported a polygenic inheritance of the studied traits and highlighted a few pleiotropic regions. A set of 16 positional and/or functional candidate genes was designed for further eQTL detection. A total of 132 SNPs were associated with molecular phenotypes and defined 21 eQTL regions located on 16 chromosomes. Interestingly, several co-localizations between QTL and eQTL regions were observed which could suggest causative genes and gene networks involved in the variability of meat quality traits and BMY., Conclusions: The QTL mapping carried out in the current study for WS did not support the existence of a major gene, but rather suggested a polygenic inheritance of the defect and of other studied meat quality traits. We identified several candidate genes involved in muscle metabolism and structure and in muscular dystrophies. The eQTL analyses showed that they were part of molecular networks associated with WS and meat quality phenotypes and suggested a few putative causative genes.

Background: Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue., Results: Fixed and unfixed chromatin were prepared from chicken muscle tissues ( Pectoralis major ). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study., Conclusions: Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.

The processing ability and sensory quality of chicken breast meat are highly related to its ultimate pH (pHu), which is mainly determined by the amount of glycogen in the muscle at death. To unravel the molecular mechanisms underlying glycogen and meat pHu variations and to identify predictive biomarkers of these traits, a transcriptome profiling analysis was performed using an Agilent custom chicken 8 × 60 K microarray. The breast muscle gene expression patterns were studied in two chicken lines experimentally selected for high (pHu+) and low (pHu-) pHu values of the breast meat. Across the 1,436 differentially expressed (DE) genes found between the two lines, many were involved in biological processes related to muscle development and remodelling and carbohydrate and energy metabolism. The functional analysis showed an intensive use of carbohydrate metabolism to produce energy in the pHu- line, while alternative catabolic pathways were solicited in the muscle of the pHu+ broilers, compromising their muscle development and integrity. After a validation step on a population of 278 broilers using microfluidic RT-qPCR, 20 genes were identified by partial least squares regression as good predictors of the pHu, opening new perspectives of screening broilers likely to present meat quality defects.

Background: Meat type chickens have limited capacities to cope with high environmental temperatures, this sometimes leading to mortality on farms and subsequent economic losses. A strategy to alleviate this problem is to enhance adaptive capacities to face heat exposure using thermal manipulation (TM) during embryogenesis. This strategy was shown to improve thermotolerance during their life span. The aim of this study was to determine the effects of TM (39.5 °C, 12 h/24 vs 37.8 °C from d7 to d16 of embryogenesis) and of a subsequent heat challenge (32 °C for 5 h) applied on d34 on gene expression in the Pectoralis major muscle (PM). A chicken gene expression microarray (8 × 60 K) was used to compare muscle gene expression profiles of Control (C characterized by relatively high body temperatures, Tb) and TM chickens (characterized by a relatively low Tb) reared at 21 °C and at 32 °C (CHC and TMHC, respectively) in a dye-swap design with four comparisons and 8 broilers per treatment. Real-time quantitative PCR (RT-qPCR) was subsequently performed to validate differential expression in each comparison. Gene ontology, clustering and network building strategies were then used to identify pathways affected by TM and heat challenge., Results: Among the genes differentially expressed (DE) in the PM (1.5 % of total probes), 28 were found to be differentially expressed between C and TM, 128 between CHC and C, and 759 between TMHC and TM. No DE gene was found between TMHC and CHC broilers. The majority of DE genes analyzed by RT-qPCR were validated. In the TM/C comparison, DE genes were involved in energy metabolism and mitochondrial function, cell proliferation, vascularization and muscle growth; when comparing heat-exposed chickens to their own controls, TM broilers developed more specific pathways than C, especially involving genes related to metabolism, stress response, vascularization, anti-apoptotic and epigenetic processes., Conclusions: This study improved the understanding of the long-term effects of TM on PM muscle. TM broilers displaying low Tb may have lower metabolic intensity in the muscle, resulting in decreased metabolic heat production, whereas modifications in vascularization may enhance heat loss. These specific changes could in part explain the better adaptation of TM broilers to heat.

Variations in muscle glycogen storage are highly correlated with variations in meat ultimate pH (pHu), a key factor for poultry meat quality. A total of two chicken lines were divergently selected on breast pHu to understand the biological basis for variations in meat quality (i.e., the pHu- and the pHu+ lines that are characterized by a 17% difference in muscle glycogen content). The effects of this selection on bird metabolism were investigated by quantifying muscle metabolites by high-resolution NMR ((1)H and (31)P) and serum metabolites by (1)H NMR. A total of 20 and 26 discriminating metabolites between the two lines were identified by orthogonal partial least-squares discriminant analysis (OPLS-DA) in the serum and muscle, respectively. There was over-representation of carbohydrate metabolites in the serum and muscle of the pHu- line, consistent with its high level of muscle glycogen. However, the pHu+ line was characterized by markers of oxidative stress and muscle catabolism, probably because of its low level of energy substrates. After OPLS-DA multiblock analysis, a metabolic set of 15 high-confidence biomarkers was identified that could be used to predict the quality of poultry meat after validation on an independent population.

Background: The avian eggshell membranes surround the egg white and provide a structural foundation for calcification of the eggshell which is essential for avian reproduction; moreover, it is also a natural biomaterial with many potential industrial and biomedical applications. Due to the insoluble and stable nature of the eggshell membrane fibres, their formation and protein constituents remain poorly characterized. The purpose of this study was to identify genes encoding eggshell membrane proteins, particularly those responsible for its structural features, by analyzing the transcriptome of the white isthmus segment of the oviduct, which is the specialized region responsible for the fabrication of the membrane fibres., Results: The Del-Mar 14 K chicken microarray was used to investigate up-regulated expression of transcripts in the white isthmus (WI) compared with the adjacent magnum (Ma) and uterine (Ut) segments of the hen oviduct. Analysis revealed 135 clones hybridizing to over-expressed transcripts (WI/Ma + WI/Ut), and corresponding to 107 NCBI annotated non-redundant Gallus gallus gene IDs. This combined analysis revealed that the structural proteins highly over-expressed in the white isthmus include collagen X (COL10A1), fibrillin-1 (FBN1) and cysteine rich eggshell membrane protein (CREMP). These results validate previous proteomics studies which have identified collagen X (α-1) and CREMP in soluble eggshell extracts. Genes encoding collagen-processing enzymes such as lysyl oxidase homologs 1, 2 and 3 (LOXL1, LOXL2 and LOXL3), prolyl 4 hydroxylase subunit α-2 and beta polypeptide (P4HA2 and P4HB) as well as peptidyl-prolyl cis-trans isomerase C (PPIC) were also over-expressed. Additionally, genes encoding proteins known to regulate disulfide cross-linking, including sulfhydryl oxidase (QSOX1) and thioredoxin (TXN), were identified which suggests that coordinated up-regulation of genes in the white isthmus is associated with eggshell membrane fibre formation., Conclusions: The present study has identified genes associated with the processing of collagen, other structural proteins, and disulfide-mediated cross-linking during eggshell membrane formation in the white isthmus. Identification of these genes will provide new insight into eggshell membrane structure and mechanisms of formation that will assist in the development of selection strategies to improve eggshell quality and food safety of the table egg.

Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units and 4) development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages. Variations in abundance according to these calcification events were observed for 175 proteins. A putative function related to the mineralisation process was predicted by bioinformatics for 77 of them and was further characterised. We confirmed the important role of lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116 for shell calcification process, characterised major calcium binding proteins (EDIL3, ALB, MFGE8, NUCB2), and described novel proteoglycans core proteins (GPC4, HAPLN3). We suggest that OVAL and OC-17 play a role in the stabilisation of ACC. Finally, we report proteins involved in the regulation of proteins driving the mineralisation. They correspond to numerous molecular chaperones including CLU, PPIB and OCX21, protease and protease inhibitors including OVM and CST3, and regulators of phosphorylation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.

In cattle, early embryonic failure plays a major role in the limitation of reproductive performance and is influenced by genetic effects. Suboptimal oocyte quality, including an inadequate store of maternal factors, is suspected to contribute to this phenomenon. In the present study, 13 Montbeliarde cows were phenotyped on oocyte quality, based on their ability to produce viable embryos after in vitro maturation, fertilisation and culture for 7 days. This discriminated two groups of animals, exhibiting developmental rates below 18.8% or above 40.9% (relative to cleaved embryos). Using microarrays, transcriptomic profiles were compared between oocytes collected in vivo from these two groups of animals. The difference in oocyte development potential was associated with changes in transcripts from 60 genes in immature oocytes and 135 genes in mature oocytes (following Bonferroni 5% correction). Of these, 16 and 32 genes were located in previously identified fertility quantitative trait loci. A subset of differential genes was investigated on distinct samples by reverse transcription-quantitative polymerase chain reaction. For SLC25A16, PPP1R14C, ROBO1, AMDHD1 and MEAF6 transcripts, differential expression was confirmed between high and low oocyte potential animals. Further sequencing and searches for polymorphisms will pave the way for implementing their use in genomic selection.

Gallus gallus eggshell is a bioceramic composed of 95% calcium carbonate in calcitic form and 3.5% extracellular organic matrix. The calcification process occurs in the uterine fluid where biomineralization follows a temporal sequence corresponding to the initiation, growth and termination stages of crystal growth. Eggshell texture and its ultrastructure are regulated by organic matrix proteins, which control mineralization process and influence the eggshell biomechanical properties. We performed proteomic qualitative analyses and identified 308 uterine fluid proteins. Quantitative analysis showed differential abundances at the three stages of shell biomineralization for 64 of them. Cluster analysis revealed a first group of proteins related to mineralization and mainly present at the onset of calcification including OVOT, OVAL, OC-17, and two novel calcium binding proteins (EDIL3, MFGE8). A second group of proteins mainly present at the initiation and termination of shell formation was potentially involved in the regulation of the activity of the uterine fluid proteins (e.g. molecular chaperones, folding proteins, proteases and protease inhibitors). OCX21, a protein highly concentrated in the fluid and the shell, belongs to this group. A third group equally represented at all stages of shell mineralization corresponded to antibacterial proteins that could protect the forming egg against microbial invasion., Biological Significance: The calcitic avian eggshell protects the developing embryo and, moreover, ensures that the nutritious table egg remains free of pathogens. The eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes) followed by an interaction between the growing mineral crystals and the shell organic matrix. This interaction leads to a highly ordered shell microstructure and texture which contribute to its exceptional mechanical properties. Shell mineralization occurs in three distinct phases of calcification (initiation, growth and termination), which are associated with distinct populations of matrix proteins that are secreted into the acellular uterine fluid as modulators of the process. The recent development of high-throughput methods has led to the identification of many proteins in the shell, but little is known concerning their role in shell formation. In order to determine precisely the importance of particular proteins relative to eggshell mineralization, this project used qualitative and quantitative proteomics of the uterine fluid constituents, coupled with bioinformatic analysis, to predict the functional role of proteins secreted at each of the three main stages of shell calcification. Besides its relevance to food production and to hen reproduction, eggshell calcification is furthermore a relevant model for studying calcium carbonate biomineralization on a two-dimensional membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressure and temperature., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chicken eggshell is the protective barrier of the egg. It is a biomineral composed of 95% calcium carbonate on calcitic form and 3.5% organic matrix proteins. Mineralization process occurs in uterus into the uterine fluid. This acellular fluid contains ions and organic matrix proteins precursors which are interacting with the mineral phase and control crystal growth, eggshell structure and mechanical properties. We performed a proteomic approach and identified 308 uterine fluid proteins. Gene Ontology terms enrichments were determined to investigate their potential functions. Mass spectrometry analyses were also combined to label free quantitative analysis to determine the relative abundance of 96 proteins at initiation, rapid growth phase and termination of shell calcification. Sixty four showed differential abundance according to the mineralization stage. Their potential functions have been annotated. The complete proteomic, bioinformatic and functional analyses are reported in Marie et al., J. Proteomics (2015) [1].

In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Fast-growing chickens have a limited ability to tolerate high temperatures. Thermal manipulation during embryogenesis (TM) has previously been shown to lower chicken body temperature (Tb) at hatching and to improve thermotolerance until market age, possibly resulting from changes in metabolic regulation. The aim of this study was to evaluate the long-term effects of TM (12 h/d, 39.5°C, 65% RH from d 7 to 16 of embryogenesis vs. 37.8°C, 56% RH continuously) and of a subsequent heat challenge (32°C for 5 h at 34 d) on the mRNA expression of metabolic genes and cell signaling in the Pectoralis major muscle and the liver. Gene expression was analyzed by RT-qPCR in 8 chickens per treatment, characterized by low Tb in the TM groups and high Tb in the control groups. Data were analyzed using the general linear model of SAS considering TM and heat challenge within TM as main effects. TM had significant long-term effects on thyroid hormone metabolism by decreasing the muscle mRNA expression of deiodinase DIO3. Under standard rearing conditions, the expression of several genes involved in the regulation of energy metabolism, such as transcription factor PGC-1α, was affected by TM in the muscle, whereas for other genes regulating mitochondrial function and muscle growth, TM seemed to mitigate the decrease induced by the heat challenge. TM increased DIO2 mRNA expression in the liver (only at 21°C) and reduced the citrate synthase activity involved in the Krebs cycle. The phosphorylation level of p38 Mitogen-activated-protein kinase regulating the cell stress response was higher in the muscle of TM groups compared to controls. In conclusion, markers of energy utilization and growth were either changed by TM in the Pectoralis major muscle and the liver by thermal manipulation during incubation as a possible long-term adaptation limiting energy metabolism, or mitigated during heat challenge.