An increase in egg incubation temperature was previously shown to enhance the metabolism of mule ducks and increase liver fattening after overfeeding, through a metabolic programming mechanism. Here, we examined whether fasting (F) followed by refeeding (RF) in 11-wk-old mule ducks could become an accelerated model to study the mechanisms of metabolic programming following embryonic thermal manipulation. This study investigated the hepatic response of mule ducks subjected to 23 h of fasting and 1 h of refeeding, in control or thermally programmed animals (with an increase of 1°C, 16 h per day from days 13 to 27 of embryogenesis). Liver weight and energy composition, hepatocyte structure, plasma parameters, and gene expression levels were measured at 1, 2, and 4 h after RF. All these parameters were strongly affected by RF, whereas significant impacts of embryonic programming were measured in cell size (+1 µm on average), lipid composition (+4.2% of saturated fatty acids 4 h after the meal), and relative gene expressions (including HK1, SCD1, ELOVL6, and FASN). In addition to confirming previously identified molecular targets of thermal manipulation, this study revealed new ones, thanks to kinetic sampling after RF. Finally, the detailed description of the impact of the F/RF challenge on the liver structure, composition, and gene expression, but also on plasma parameters allowed us to draw a parallel with these same traits measured during overfeeding. This comparative analysis suggests that this protocol could become a pertinent model to study the mechanisms involved in embryonic liver thermal programming, without overfeeding.

L'épigénétique est communément définie comme l’étude de l'ensemble des mécanismes moléculaires impliqués dans la régulation de l’expression des gènes qui sont réversibles et transmissibles au cours du développement et parfois entre générations, sans altérer la séquence de l'ADN. Plusieurs mécanismes épigénétiques sont maintenant bien connus, comme la méthylation de l'ADN, les variants et modifications post-traductionnelles des histones, ainsi que certains ARN non codants. Grâce au développement technologique du séquençage tout-génome, ces « marques » épigénétiques peuvent être étudiées à l'échelle du génome entier. Il est aujourd’hui clairement établi que l’épigénome, c'est-à-dire l'ensemble des marques épigénétiques d'un tissu, est sensible aux fluctuations de l’environnement, notamment la température ou l’alimentation. Des stratégies de programmation précoce des phénotypes reposant sur ces mécanismes épigénétiques sont ainsi envisagées comme levier pour adapter le phénotype ultérieur des individus à leurs conditions de vie. Par ailleurs, au cours des dernières décennies, la sélection génétique a contribué à l’amélioration considérable des performances des animaux. Bien que la composante génétique puisse être estimée avec précision, une grande partie de la variabilité phénotypique n'est pas directement accessible par les approches actuelles. Dans un contexte de diversification des environnements de production (changement climatique, modes de production plus respectueux du bien-être et de l'environnement), il est nécessaire de comprendre l'impact de l'environnement sur la variabilité phénotypique via les marques épigénétiques, pour optimiser les systèmes d'élevage et mieux prédire le phénotype d'un animal. Comme la sélection génomique il y a quelques années, l'apport de la recherche en épigénétique pourrait contribuer à rendre les systèmes de production avicole plus efficaces et plus durables.

Epigenetics is commonly defined as the set of molecular mechanisms involved in gene expression regulation that are reversible and transmissible during development, and sometimes between generations, without altering the DNA sequence. Several epigenetic marks involved in chromatin regulation are now well known, such as the methylation of DNA cytosines, histone post -translational modifications, and non -coding RNA. Thanks to the technological development of high -throughput DNA sequencing, epigenetic marks can now be investigated at the whole-genome scale. It is also well known that the epigenome (the whole set of epigenetic marks in a tissue) is sensitive to environmental fluctuations, including temperature or nutrition, and can be considered as a mediator between the genes and the environment. Phenotypic programming strategies based on epigenetic mechanisms could thus be used as a lever to adjust the phenotype of individuals with their living conditions. Moreover, over the last decades, genetic selection has contributed to a considerable improvement in animal performances. Although the genetic component can be estimated with some accuracy, a large part of the phenotypic variability, considered to be of environmental origin, is not directly accessible by genetic approaches. In a context of diversification of production environments (climate change, production methods that are more respectful of welfare and the environment...), it is necessary to understand the impact of the environment on phenotypic variation via epigenetic marks, to optimize breeding systems and to predict the phenotype of an animal. Like genomic selection a few years ago, the contribution of epigenetic research could contribute to more efficient and sustainable production systems in poultry farming.

Background Embryonic and fetal development is very susceptible to the availability of nutrients that can interfere with the setting of epigenomes, thus modifying the main metabolic pathways and impacting the health and phenotypes of the future individual. We have previously reported that a 38% reduction of the methyl donor methionine in the diet of 30 female ducks reduced the body weight of their 180 mule ducklings compared to that of 190 ducklings from 30 control females. The maternal methionine-restricted diet also altered plasmatic parameters in 30 of their ducklings when compared to that of 30 ducklings from the control group. Thus, their plasma glucose and triglyceride concentrations were higher while their free fatty acid level and alanine transaminase activity were decreased. Moreover, the hepatic transcript level of 16 genes involved in pathways related to energy metabolism was significantly different between the two groups of ducklings. In the present work, we continued studying the liver of these newly hatched ducklings to explore the impact of the maternal dietary methionine restriction on the hepatic transcript level of 70 genes mostly involved in one-carbon metabolism and epigenetic mechanisms. Results Among the 12 genes ( SHMT1 , GART , ATIC , FTCD , MSRA , CBS , CTH , AHCYL1 , HSBP1 , DNMT3 , HDAC9 and EZH2 ) identified as differentially expressed between the two maternal diet groups ( p -value < 0.05), 3 of them were involved in epigenetic mechanisms. Ten other studied genes ( MTR , GLRX , MTHFR , AHCY , ADK , PRDM2 , EEF1A1 , ESR1 , PLAGL1 , and WNT11 ) tended to be differently expressed (0.05 < p -value < 0.10). Moreover, the maternal dietary methionine restriction altered the number and nature of correlations between expression levels of differential genes for one-carbon metabolism and epigenetic mechanisms, expression levels of differential genes for energy metabolism, and phenotypic traits of ducklings. Conclusion This avian model showed that the maternal dietary methionine restriction impacted both the mRNA abundance of 22 genes involved in one-carbon metabolism or epigenetic mechanisms and the mRNA abundance of 16 genes involved in energy metabolism in the liver of the newly hatched offspring, in line with the previously observed changes in their phenotypic traits.

The foie gras is an emblematic product of French gastronomy composed of waterfowl fatty liver. The organoleptic qualities of this product depend on the liver characteristics such as liver weight (LW) and technological yield (TY) at cooking. One of the main issues for producers is to classify the foie gras with high or low technological quality before cooking them. Thus the study aims at identifying biomarkers of these characteristics with non-invasive biomarkers in duck. 1 H-NMR (nuclear magnetic resonance of the proton) analyses were performed on plasma of male mule ducks at different time points during the overfeeding period to obtain a large range of liver characteristics so as to identify plasmatic biomarkers of foie gras . We used two methods, one based on bucket data from the 1 H-NMR spectra and another one based on the fingerprints of several metabolites. PLS analyses and Linear models were performed to identify biomarkers. We identified 18 biomarkers of liver weight and 15 biomarkers of technological yield. As these two quality parameters were strongly correlated (−0.82), 13 biomarkers were common. The lactate was the most important biomarker, the other were mainly amino acids. Contrary to the amino acids, the lactate increased with the liver weight and decreased with the technological yield. We also identified 5 biomarkers specific to LW (3 carbohydrates: glucuronic acid, mannose, sorbitol and 2 amino acids: glutamic acid and methionine) that were negatively correlated to liver weight. It was of main interest to identify 2 biomarkers specific to the technological yield. Contrary to the isovaleric acid, the valine was negatively correlated to the technological yield.

Foie gras is a traditional dish in France that contains 50 to 60% of lipids. The high-fat content of the liver improves the organoleptic qualities of foie gras and reduces its technological yield at cooking (TY). As the valorization of the liver as foie gras products is strongly influenced by the TY, classifying the foie gras in their potential technological quality before cooking them is the main challenge for producers. Therefore, the current study aimed to identify hepatic biomarkers of foie gras qualities like liver weight (LW) and TY. A group of 120 male mule ducks was reared and overfed for 6–12 days, and their livers were sampled and analyzed by proton nuclear magnetic resonance ( 1 H-NMR). Eighteen biomarkers of foie gras qualities were identified, nine for LW and TY, five specific to LW, and four specific to TY. All biomarkers were strongly negatively correlated to the liver weights and positively correlated to the technological yield, except for the lactate and the threonine, and also for the creatine that was negatively correlated to foie gras technological quality. As a result, in heavy livers, the liver metabolism was oriented through a reduction of carbohydrate and amino acid metabolisms, and the plasma membrane could be damaged, which may explain the low technological yield of these livers. The detected biomarkers have been strongly discussed with the metabolism of the liver in nonalcoholic steatohepatitis.

Embryonic thermal programming has been shown to improve foie gras production in overfed mule ducks. However, the mechanisms at the origin of this programming have not yet been characterized. In this study, we investigated the effect of embryonic thermal manipulation (+1°C, 16 h/24 h from embryonic (E) day 13 to E27) on the hepatic expression of genes involved in lipid and carbohydrate metabolisms, stress, cell proliferation and thyroid hormone pathways at the end of thermal manipulation and before and after overfeeding (OF) in mule ducks. Gene expression analyses were performed by classic or high throughput real-time qPCR. First, we confirmed well-known results with strong impact of OF on the expression of genes involved in lipid and carbohydrates metabolisms. Then we observed an impact of OF on the hepatic expression of genes involved in the thyroid pathway, stress and cell proliferation. Only a small number of genes showed modulation of expression related to thermal programming at the time of OF, and only one was also impacted at the end of the thermal manipulation. For the first time, we explored the molecular mechanisms of embryonic thermal programming from the end of heat treatment to the programmed adult phenotype with optimized liver metabolism.

Background : The production of foie gras involves different metabolic pathways in the liver of overfed ducks such as lipid synthesis and carbohydrates catabolism, but the establishment of these pathways has not yet been described with precision during embryogenesis. The early environment can have short- and long-term impacts on the physiology of many animal species and can be used to influence physiological responses that is called programming. This study proposes to describe the basal hepatic metabolism at the level of mRNA in mule duck embryos in order to reveal potential interesting programming windows in the context of foie gras production. To this end, a kinetic study was designed to determine the level of expression of selected genes involved in steatosis-related liver functions throughout embryogenesis. The livers of 20 mule duck embryos were collected every four days from the 12 th day of embryogenesis (E12) until 4 days after hatching (D4), and gene expression analysis was performed. The expression levels of 50 mRNAs were quantified for these 7 sampling points and classified into 4 major cellular pathways. Results : Interestingly, most mRNAs involved in lipid metabolism are overexpressed after hatching (FASN, SCD1, ACOX1), whereas genes implicated in carbohydrate metabolism (HK1, GAPDH, GLUT1) and development (HGF, IGF, FGFR2) are predominantly overexpressed from E12 to E20. Finally, regarding cellular stress, gene expression appears quite stable throughout development, contrasting with strong expression after hatching (CYP2E1, HSBP1, HSP90AA1). Conclusion : For the first time we described the kinetics of hepatic ontogenesis at mRNA level in mule ducks and highlighted different expression patterns depending on the cellular pathway. These results could be particularly useful in the design of embryonic programming for the production of foie gras.

Like many other species, the duck genome has been sequenced thanks to the technological breakthrough provided by the emergence of Next Generation Sequencing (NGS). The resulting de novo assemblies are however made of thousands of scattered scaffolds. To achieve chromosome-scale contiguity, long-range intermediate genome maps remain indispensable. Radiation Hybrid (RH) maps have been used to assist the generation of chromosome-scale genome assemblies by taking advantage of the high density SNP chips that provide a large number of markers that can be efficiently genotyped on the panel. In the absence of such a resource in duck, we sequenced 100 hybrid clones of a duck RH panel enabling direct genotyping of the assembly scaffolds on the panel. The rationale is to use scaffolds as markers and to genotype the scaffolds by sequencing the clones: the presence/absence of a scaffold in a particular sequenced hybrid is attested by the presence/absence of reads mapping specifically to this scaffold. The detection of scaffolds exhibiting a chromosomal breakage resulting from the irradiation process revealed itself to be a critical issue of this genotyping by sequencing process. This process resulted in the construction of RH vectors for 2,027 scaffolds, representing a total of about 1 Gb of sequences (95% of the current Duck genome assembly). The subsequent linkage analysis enabled the construction of RH maps and therefore to organize, i.e. order and orient, the scaffolds into pseudomolecules associated to the corresponding duck chromosomes. We describe here the whole mapping process, from sequence-based genotyping to the construction of comparative maps, as well as few examples of intra-chromosomal rearrangements that have been identified by the comparison with the chicken, turkey and zebra finch genomes and subsequently confirmed by FISH. We describe a method to order and orient sequence scaffolds into super-scaffolds spanning entire chromosomes. The method, which requires a pre-existing RH panel and sequence scaffolds from an NGS assembly, relies on a shallow sequencing of the RH clones. This approach was applied to the duck genome and produced chromosome-scale scaffolds for 29 out of the 41 duck chromosomes.

Animal studies have shown that very early life events may have programing effects on adult metabolism and health. In this study, we aim, for the first, time to elucidate the effects of embryonic thermal manipulation (TM) on the performance of overfed mule ducks, in particular for the production of foie gras (fatty liver). We designed three embryonic TMs with different protocols for increasing the incubation temperature during the second part of embryogenesis, to determine whether hepatic metabolism could be "programed" to improve its fattening response to overfeeding at the age of three months. Initial results confirm that an increase in the incubation temperature leads to faster development (observed for all treated groups compared to the control group), and a decrease in the body surface temperature at birth. Thereafter, in a very innovative way, we showed that the three TM conditions specifically increased liver weights, as well as liver lipid content after overfeeding compared to the non-TM control group. These results demonstrate that embryonic TM effectively "programs" the metabolic response to the challenge of force-feeding, resulting in increased hepatic steatosis. Finally, our goal of improving foie gras production has been achieved with three different embryonic thermal stimuli, demonstrating the high reproducibility of the method. However, this repeatability was also perceptible in the adverse effects observed on two groups treated with exactly the same cumulative temperature rise leading to a reduction in hatchability (75 and 76% vs. 82% in control), in addition to an increase in the melting rate after cooking. These results suggest that embryonic thermal programing could be an innovative and inexpensive technique for improving foie gras production, although the specific protocol (duration, level or period of temperature increase), remains to be elucidated in order to avoid adverse effects.

In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokines mediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immune defense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledge about the function of avian leptin and TNF is limited and they are absent or lowly expressed in adipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped TNF and its syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC) region. This mapping position suggests that avian TNF has a role in regulating immune response. To test its possible interaction with leptin within the immune system and beyond, we compared the transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis of GenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in the brain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected and LPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistry staining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals, in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be unique to mammals.

Background: In quail, two feather colour phenotypes i.e. fawn-2/beige and yellow are associated with the ASIP locus. The aim of our study was to characterize the structural modifications within this locus that explain the yellow mutation (large deletion) and the fawn-2/beige mutation (assumed to be caused by a different structural modification). Results: For the yellow phenotype, we identified a complex mutation that involves a 141,162-bp long deletion. For the fawn-2/beige phenotype, we identified a 71-kb tandem duplication that comprises one unchanged copy of ASIP and one copy present in the ITCH-ASIP fusion gene, which leads to a transcript coding for a normal ASIP protein. Although this agrees with previous reports that reported an increased level of ASIP transcripts in the skin of mutant animals, we show that in the skin from fawn-2/beige embryos, this level is higher than expected with a simple duplication of the ASIP gene. Thus, we hypothesize that the 5 region of the ITCH-ASIP fusion gene leads to a higher transcription level than the 5 region of the ASIP gene. Conclusions: We were able to conclude that the fawn-2 and beige phenotypes are caused by the same allele at the ASIP locus. Both of the associated mutations fawn-2/beige and yellow lead to the formation of a fusion gene, which encodes a transcript for the ASIP protein. In both cases, transcription of ASIP depends on the promoter of a different gene, which includes alternative up-regulating sequences. However, we cannot exclude the possibility that the loss of the 5 region of the ASIP gene itself has additional impacts, especially for the fawn-2/beige mutation. In addition, in several other species including mammals, the existence of other dominant gain-of-function structural modifications that are localized upstream of the ASIP coding sequences has been reported, which supports our hypothesis that repressors in the 5 region of ASIP are absent in the fawn-2/beige mutant.

Les animaux d'élevage sont confrontés à de multiples contraintes environnementales auxquelles ils doivent s'adapter. De plus en plus d'études s'intéressent à l'impact de l'environnement précoce sur les phénotypes des animaux et leurs capacités à s'adapter aux différents challenges rencontrés ultérieurement. Dans cette revue, nous nous intéresserons aux connaissances actuelles sur l'influence de l'environnement précoce sur la construction et la variabilité des phénotypes et de l'adaptation des animaux d'élevage à leur milieu, en prenant des exemples dans différentes espèces. Nous nous focaliserons sur les rôles de trois contributeurs importants de la construction des phénotypes : les mécanismes épigénétiques, le microbiote et les processus comportementaux. Les mécanismes épigénétiques, qui modulent l'expression du génome sous l'effet de perturbations environnementales intervenues pendant le développement, peuvent induire une variété de phénotypes dont les caractéristiques peuvent perdurer jusqu'à l'âge adulte, voire se transmettre à la génération suivante. La constitution du microbiote digestif est elle aussi très dépendante de l'environnement précoce, et joue un rôle important dans l’expression des phénotypes, notamment dans le domaine de la santé. Enfin, l'expérience précoce influence considérablement le comportement de l'animal, en particulier ses capacités d'apprentissage, qui peuvent lui conférer une meilleure adaptation à des situations nouvelles ultérieures. Les études menées dans ces domaines permettent de définir de nouveaux leviers d'action pour tenter d'optimiser les capacités d’adaptation de nos animaux, notamment par les voies de l'alimentation des animaux jeunes ou de leurs parents voire de leurs grands-parents, de l'acclimatation pendant le développement, ou plus généralement par des modifications de l'environnement, en particulier de l'environnement social.

Farm animals face multiple environmental constraints to which they must adapt. More and more studies are investigating the impact of the early environment on animal phenotypes and their ability to adapt to the various challenges encountered later. Here we review current knowledge on the influence of the early environment on the construction and variability of phenotypes and adaptation of farm animals to their environment, taking examples from different species. We focus on the roles of three major contributors to the construction of phenotypes: epigenetic mechanisms, microbiota, and behavioural processes. Epigenetic mechanisms, which modulate genome expression under the influence of environmental disturbances during development, can induce a variety of phenotypes whose characteristics can persist into adulthood, or even be transmitted to the next generation. The constitution of the digestive microbiota is also highly dependent on the early environment, and plays an important role in the expression of phenotypes, especially in the field of animal health. Finally, early experiences greatly influence an animal's behaviour, especially its learning abilities, which can give it a better chance of adapting to new situations later on. The studies carried out in these fields make it possible to define new levers for action to try to optimise the adaptability of our animals, in particular through the feeding of young animals or their parents or even grandparents, acclimatisation during development, or more generally through changes in the environment, in particular the social environment.