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The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (nonsynonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design., French National Research Agency (ANR) European Commission ANR-13-BSV-0007-01, Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2016-04410, Le STUDIUM, Centre Val de Loire, Nouzilly, France - University of Tours

The present study concerned the effect of ageing in laying hens, from 23 to 90 weeks of age, on the regulation of Ca metabolism related to the requirement for eggshell mineralization. Samples were collected from parathyroid gland (PG), liver, jejunum, medullary bone (MB) and kidney for a quantitative study of candidate gene expression. Although parathyroid hormone (PTH) gene expression in the PG did not vary with age, a stronger challenge to Ca homeostasis was suggested in aged hens. Indeed gene expression of Ca transporters , Vitamin D Receptor (VDR) in the jejunum, and that of transient receptor potential channel subfamily V member 5 (TRPV5) in the kidney decreased. This could exacerbate bone resorption and impair bone accretion, as attested by a higher expression of the Carbonic Anhydrase 2 (CA2) gene and a lower expression of collagen type I alpha 1 chain (COL1A1) in the MB. The increased expression of Fibroblast Growth Factor 23 (FGF23) in the MB likely contributed to the decreased plasma levels of 1.25(OH)2D3 and the altered expression of target genes under its regulation. Our data highlights the molecular mechanisms underlying the osteoporotic syndrome previously documented in aged laying hens, thus providing new perspectives for future interventions.

International audience; The Guinea fowl eggshell is a bioceramic material with the remarkable mechanical property of being twice as strong as the chicken eggshell. Both eggshells are composed of 95% calcite and 3.5% organic matrix, which control its structural organization. Chicken eggshell is made of columnar calcite crystals arranged vertically. In the Guinea fowl, the same structure is observed in its inner half, followed by a dramatic change in crystal size and orientation in the outer region. Guinea fowl eggshell is thicker than chicken eggshell. Both structure and shell thickness confer a superior resistance to breakage compared to eggshells of other bird species. To understand the underlying mechanisms controlling the structural organization of this highly resistant material, we used quantitative proteomics to analyze the protein composition of the Guinea fowl eggshell organic matrix at key stages of the biomineralization process. We identified 149 proteins, which were compared to other bird eggshell proteomes and analyzed their potential functions. Among the 149 proteins, 9 are unique to Guinea fowl, some are involved in the control of the calcite precipitation (Lysozyme, Ovocleidin-17-like, Ovocleidin-116 and Ovalbumin), 61 are only found in the zone of microstructure shift and 17 are more abundant in this zone. Significance: The avian eggshell is a critical physical barrier to protect the contents of this autonomous reproductive enclosure from physical and microbial assault. The Guinea fowl (Numida meleagris) eggshell exhibits a unique microstructure (texture), which confers exceptional mechanical properties compared to eggshells of other species. In order to understand the mechanisms that regulate formation of this texture in the Guinea fowl eggshell, we performed comparative quantitative proteomics at key stages of shell mineralization and particularly during the dramatic shift in shell microstructure. We demonstrate that the Guinea fowl eggshell proteome comprises 149 proteins, of which 61 were specifically associated with the change in size and orientation of calcite crystals. Comparative proteomics analysis with eggshell of other bird species leads to new insights into the biomineralization process. Moreover, our data represents a list of organic compounds as potential additives to regulate material design for industrial fabrication of ceramics. This information also provides molecular markers for efficient genomic selection of chicken strains to lay eggs with improved shell mechanical properties for enhanced food safety.

International audience; To meet the high calcium (Ca) demand during eggshell biomineralization (2 g of Ca per egg), laying hens develop specific metabolic regulations to maintain Ca homeostasis. The intake of Ca, its solubilization, and absorption capacity are enhanced at sexual maturity (SM). A better knowledge of the intestinal Ca transporters involved in their variations at this stage could indicate new nutritional strategies to enhance Ca digestive utilization. Transcellular Ca absorption pathway and its major player calbindin-D 28 K (CALB1) mediate a saturable transport, which has been extensively described in this model. Conversely, a contribution by the paracellular pathway involving non-saturable Ca transport through intercellular tight junction has also been suggested. The aim of the present study was to identify candidate genes of these two pathways and their patterns of expression, in immature pullets (12, 15, and 17 wk old) and mature laying hens (23 wk old) in the duodenum, jejunum, and ileum. Using RT-qPCR, this study identifies 3 new candidate genes for transcellular, and 9 for paracellular Ca transport. A total of 5 candidates of the transcellular pathway, transient receptor potential cation channels subfamily C member 1 (TRPC1) and M member 7 (TRPM7); CALB1 and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) and ATPase plasma membrane Ca2+ transporting 2 (ATP2B2) were enhanced with age or after SM in the duodenum, the jejunum or all 3 segments. A total of 4 candidates of the paracellular pathway Claudin 2 (CLDN2) and tight junction proteins 1, 2, and 3 (TJP1, TJP2 and TJP3) increased in the small intestine after SM. Additionally, CALB1, ATP2B2, and CLDN2 were overexpressed in the duodenum or the jejunum or both segments after SM. The enhanced expression of candidate genes of the paracellular Ca pathway after SM, supports that the non-saturable transport could be a mechanism of great importance when high concentrations of soluble Ca are observed in the intestinal content during eggshell formation. Both pathways may work cooperatively in the duodenum and jejunum, the main sites of Ca absorption in laying hens.

International audience; Egg is an encapsulated source of macro and micronutrients that meet all requirements to support embryonic development until hatching. The perfect balance and diversity in its nutrients along with its high digestibility and its affordable price has put the egg in the spotlight as a basic food for humans. However, egg still has to face many years of nutritionist recommendations aiming at restricting egg consumption to limit cardiovascular diseases incidence. Most experimental, clinical, and epidemiologic studies concluded that there was no evidence of a correlation between dietary cholesterol brought by eggs and an increase in plasma total-cholesterol. Egg remains a food product of high nutritional quality for adults including elderly people and children and is extensively consumed worldwide. In parallel, there is compelling evidence that egg also contains many and still-unexplored bioactive compounds, which may be of high interest in preventing/curing diseases. This review will give an overview of (1) the main nutritional characteristics of chicken egg, (2) emerging data related to egg bioactive compounds, and (3) some factors affecting egg composition including a comparison of nutritional value between eggs from various domestic species.

The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents during eggshell biomineralization. Main Body. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization.With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (non-synonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.

International audience; 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 (CaCO3). 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 D3 1.25(OH)2D3 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)2D3 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)2D3 at this stage coincided with increased expression of the 24-hydroxylase gene in the kidney (P < 0.05). In hens fed fine particles of CaCO3, higher plasma levels of 1,25(OH)2D3 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.

The chicken egg is a giant reproductive cell protected by an eggshell, which forms daily in the distal segment of oviduct, in an acellular uterine fluid that contains inorganic and organic precursors of the shell. Its distinctive features, as compared to bone or teeth, are the nature of the mineral and the absence of cell-directed assembly during its fabrication on eggshell membranes. Eggshell formation challenges calcium homeostasis because of the rapidity and intensity of the mineralization. Hens develop numerous physiological adaptations for supplying the shell calcium and carbonate by developing the estrogen-dependent medullary bone, by increasing sixfold intestinal calcium absorption, by stimulating the active metabolite of vitamin D in the kidney, and by developing the uterine capacity to transfer great quantities of Ca2+ and HCO3− ions and to synthesize eggshell matrix proteins. This review described the role of vitamin D and other regulating factors in control of eggshell formation and Ca metabolism in hens.

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].

International audience; In many amniotes, the amniotic fluid is depicted as a dynamic milieu that participates in the protection of the embryo (cushioning, hydration, and immunity). However, in birds, the protein profile of the amniotic fluid remains unexplored, even though its proteomic signature is predicted to differ compared with that of humans. In fact, unlike humans, chicken amniotic fluid does not collect excretory products and its protein composition strikingly changes at mid-development because of the massive inflow of egg white proteins, which are thereafter swallowed by the embryo to support its growth. Using GeLC-MS/MS and shotgun strategies, we identified 91 nonredundant proteins delineating the chicken amniotic fluid proteome at day 11 of development, before egg white transfer. These proteins were essentially associated with the metabolism of nutrients, immune response and developmental processes. Forty-eight proteins were common to both chicken and human amniotic fluids, including serum albumin, apolipoprotein A1 and alpha-fetoprotein. We further investigated the effective role of chicken amniotic fluid in innate defense and revealed that it exhibits significant antibacterial activity at day 11 of development. This antibacterial potential is drastically enhanced after egg white transfer, presumably due to lysozyme, avian beta-defensin 11, vitelline membrane outer layer protein 1, and beta-microseminoprotein-like as the most likely antibacterial candidates. Interestingly, several proteins recovered in the chicken amniotic fluid prior and after egg white transfer are uniquely found in birds (ovalbumin and related proteins X and Y, avian beta-defensin 11) or oviparous species (vitellogenins 1 and 2, riboflavin-binding protein). This study provides an integrative overview of the chicken amniotic fluid proteome and opens stimulating perspectives in deciphering the role of avian egg-specific proteins in embryonic development, including innate immunity. These proteins may constitute valuable biomarkers for poultry production to detect hazardous situations (stress, infection, etc.), that may negatively affect the development of the chicken embryo.

In amniotes, the amniotic fluid is a significant contributor to fetal development and health. While numerous studies have been conducted in mammalian amniotic fluid, the composition of amniotic and other extraembryonic fluids in avian egg along with their physiological functions remain largely unexplored. In such a context, our objective was to characterize the chicken amniotic fluid (AmF) and allantoic fluid (AlF) properties, protein composition, and some associated functions from day 8 to day 16 of incubation. SDS-PAGE combined to mass spectrometry analysis revealed common and specific proteins to each fluid, suggesting distinct properties and functions. Indeed, major AlF proteins are mostly “egg yolk” proteins involved in lipid, vitamin metabolisms, and metal ion transport, while major AmF proteins resemble those of albumen. Drastic changes in the AmF protein profiles were observed during incubation, when the albumen transfers from day 12 onwards, while few changes were detected for the AlF protein profile. The decreases in osmolality (from 231 to 183 mOsm/kg) and pH (from 8.26 to 7.26) observed in the AlF during incubation are associated with water and electrolytes reallocation for the embryo needs. In contrast, AmF pH value remained stable (≈7.5). Active proteolytic enzymes have been identified in the 2 fluids using gelatin zymography, followed by mass spectrometry analysis for protease identification. A total of 12 proteases was detected in the AlF, compared to 5 in the AmF. We have shown that AlF concentrates proteolytic enzymes assumed to participate in digestive processes: aminopeptidase N, dipeptidyl peptidase-4, meprin A, and 72 kDa type IV collagenase preproprotein. The other proteases identified in both fluids also could have a role in morphogenesis (hepatocyte growth factor activator, suppressor of tumorigenicity 14, astacin-like metalloendopeptidase) and hemostasis (prothrombin and coagulation factor X). Altogether, these data suggest that the roles of chicken AlF and AmF are not merely associated with protection of the embryo and regulation of metabolic disposable wastes, but also they could have more sophisticated roles during embryonic development.

Understanding the natural antibacterial defences of egg white and their regulation

International audience; To better appreciate the dynamics of yolk proteins during embryonic development, we analyzed the protein quantitative changes occurring in the yolk plasma at the day of lay and after 12 days of incubation, by comparing unfertilized and fertilized chicken eggs. Of the 127 identified proteins, 69 showed relative abundance differences among conditions. Alpha-fetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, alpha-fetoprotein, similar to thrombin, apolipoprotein B, and apovitellenin-I) showed a major increase in relative abundance, whereas 15 proteins showed a significant decrease in the yolks of fertilized eggs. In unfertilized/table eggs, we observed an accumulation of proteins likely to originate from other egg compartments during incubation. This study provides basic knowledge on the utilization of egg yolk proteins by the embryo and gives some insight into how storage can affect egg quality.

International audience; Background: Ovodefensins are small peptides from eggs, related to avian antimicrobial defensins. Results: The first three-dimensional structure of ovodefensins (gallin) is solved, and its antimicrobial properties are screened. Conclusion: Gallin adopts a -defensin fold, with significant variations. Its antibacterial spectrum was restricted to E. coli. Significance: The first structural features may be related to E. coli specificity and/or other yet unknown functions. Gallin is a 41-residue protein, first identified as a minor component of hen egg white and found to be antimicrobial against Escherichia coli. Gallin may participate in the protection of the embryo during its development in the egg. Its sequence is related to antimicrobial -defensin peptides. In the present study, gallin was chemically synthesized 1) to further investigate its antimicrobial spectrum and 2) to solve its three-dimensional NMR structure and thus gain insight into structure-function relationships, a prerequisite to understanding its mode(s) of action. Antibacterial assays confirmed that gallin was active against Escherichia coli, but no additional antibacterial activity was observed against the other Gram-positive or Gram-negative bacteria tested. The three-dimensional structure of gallin, which is the first ovodefensin structure to have been solved to date, displays a new five-stranded arrangement. The gallin three-dimensional fold contains the three-stranded antiparallel -sheet and the disulfide bridge array typical of vertebrate -defensins. Gallin can therefore be unambiguously classified as a -defensin. However, an additional short two-stranded -sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of -defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate -defensins. These specific structural features of gallin might be related to its restricted activity against E. coli and/or to other yet unknown functions. This work provides initial understanding of a critical sequence-structure-function relationship for the ovodefensin family.

Background: Ovalbumin-related protein X (OVAX) is an uncharacterized ovalbumin-serpin. Results: This egg white-specific serpin lacks protease inhibitory activity, but unlike its ovalbumin homolog, OVAX exhibits antibacterial properties, partly through its heparin-binding site(s). Conclusion: OVAX, a non-inhibitory serpin is a heparin-binding molecule with antibacterial activity. Significance: OVAX participates in egg defense and constitutes a natural agent against Listeria and Salmonella., Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys367-His368 is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.

International audience; The natural protective system of eggs relies on egg yolk immunoglobulins and on antimicrobial proteins/peptides mainly concentrated in the egg white. There is much evidence concerning the specific stimulation of immunoglobulins by antigens but to date, the influence of the hen milieu on the regulation of the egg innate molecular immunity has not been established. To explore the hypothesis of modulation in egg antimicrobial molecules, laying hens were immune-challenged with intravenous injections of Salmonella enterica Enteritidis lipopolysaccharide (LPS) at 24 h intervals. Eggs of the control and LPS groups were collected over a period of 21 days following the first LPS injection and the egg white activities against Staphylococcus aureus and Escherichia coli were assessed. The increase in egg white anti-S. aureus activity reached 20.9% and 23.4% (p

International audience; Egg white is an important contributor to the protection of eggs against bacterial contaminations during the first half of incubation (day zero to 12), prior to the egg white transfer into the amniotic fluid to be orally absorbed by the embryo. This protective system relies on an arsenal of antimicrobial proteins and on intrinsic physicochemical properties that are generally unfavorable for bacterial multiplication and dissemination. Some changes in these parameters can be observed in egg white during egg storage and incubation. The aim of this work was to characterize changes in the antibacterial potential of egg white in embryonated eggs (FE) during the first half of incubation using unfertilized eggs (UF) as controls. Egg white samples were collected at day zero, 4, 8, and 12 and analyzed for pH, protein concentration, and protein profile. Antibacterial properties of egg white proteins were evaluated against Listeria monocytogenes, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis. During incubation, differential variations of egg white pH and protein concentrations were observed between UF and FE. At equal protein concentrations, similar activities against L. monocytogenes and S. uberis were observed for FE and UF egg white proteins. A progressive decline in these activities, however, was observed over incubation time, regardless of the egg group (UF or FE). SDS-PAGE analysis of egg white proteins during incubation revealed discrete changes in the profile of major proteins, whereas the stability of some less abundant antimicrobial proteins seemed more affected. To conclude, the antibacterial activity of egg white proteins progressively decreased during the first half of egg incubation, possibly resulting from the alteration of specific antimicrobial proteins. This apparent decline may be partly counterbalanced in embryonated eggs by the increase in egg white protein concentration. The antibacterial potential of egg white is very effective during early stages of embryonic development but its alteration during incubation suggests that extra-embryonic structures could then progressively ensure protective functions.

The aim of this study was to evaluate the capacity of chickens to adapt to and compensate for early dietary restriction of non-phytate P ( NPP: ) and/or Ca (10 to 21 d) in a later phase (22 to 35 d), and to determine whether compensatory processes depend on the P and Ca concentrations in the finisher diet. Four diets were formulated and fed to broilers from 10 to 21 d in order to generate birds with different mineral status: L1 (0.6% Ca, 0.30% NPP), L2 (0.6% Ca, 0.45% NPP), H1 (1.0% Ca, 0.30% NPP), and H2 (1.0% Ca, 0.45% NPP). On d 22, each group was divided into three groups which received a low (L, 0.48% Ca, 0.24% NPP), moderate (M, 0.70% Ca, 0.35% NPP), or high (H, 0.90% Ca, 0.35% NPP) finisher diet until 35 d, resulting in a total of 12 treatments. Lowering the Ca level enhanced apparent ileal digestibility of P (P AID) at 21 d especially with the high NPP level (Ca × NPP, P < 0.01). The lower bone mineralization observed at 21 d in broilers fed the L1 diet compared to those fed the H2 diet had disappeared by 35 d with long-term stimulation of the P AID with the low NPP level (P

L’œuf de poule est le lieu de développement de l’embryon, mais aussi un aliment de base et une source importante de molécules dotées d’activités biologiques largement utilisées en santé humaine et dans les industries agroalimentaires, cosmétiques et pharmaceutiques. L’identification des protéines de l’œuf initialement réalisée par des techniques classiques de biochimie, a connu un essor considérable grâce aux développements d’outils d’analyse à haut débit, la publication de la séquence génomique de la poule et l’essor de la bioinformatique. Dans cette revue, nous décrirons quel est l’apport de la génomique fonctionnelle et comment l’analyse transcriptomique et protéomique constituent des avancées majeures dans la connaissance des protéines de l’œuf. Ces avancées ont permis en quelques années l’identification et la caractérisation dans les différents compartiments de l’œuf de centaines de nouveaux composés aux fonctions riches et variées. Des études expérimentales supplémentaires seront nécessaires pour explorer les fonctions des composés nouvellement identifiés dans l’œuf. Ces nouvelles molécules constituent potentiellement des composés biologiquement actifs aux propriétés bénéfiques pour l’Homme ou l’animal. Certains de ces composés pourront être utilisés comme marqueurs biologiques dont le polymorphisme d’un nucléotide simple (SNP) permettra de développer une Sélection Assistée par Marqueurs (SAM) pour améliorer les défenses naturelles de l’œuf et réduire le risque de toxi-infections alimentaires humaines.

L’œuf est une source très digestible de protéines et de lipides qui contient par ailleurs une part importante des vitamines et minéraux nécessaires à l’Homme. La composition en acides aminés, équilibrée par rapport aux besoins de l’Homme, fait de l’œuf l’une des meilleures sources protéiques et les lipides de l’œuf fournissent des quantités importantes d’acides gras insaturés et de phospholipides. Les constituants majeurs de l’œuf sont relativement stables; ils dépendent essentiellement de la proportion de blanc et de jaune, elle-même largement influencée par l’âge de la poule. En revanche, la composition de l’œuf en acides gras, vitamines, minéraux et pigments peut fortement varier. La dépendance de ces éléments vis-à-vis de l’alimentation de la poule offre ainsi des opportunités d’enrichissement de l’œuf en constituants d’intérêt pour la santé humaine. Des études nutritionnelles ont par exemple démontré chez l’Homme l’impact positif de la consommation d’œufs enrichis en acides gras polyinsaturés; de même, la lutéine et la zéaxanthine de l’œuf sont particulièrement bien absorbés par l’organisme humain et biodisponibles pour la rétine. Le concept d’augmentation du risque cardio-vasculaire lié à la forte teneur de l’œuf en cholestérol est quant à lui clairement contredit par de nombreuses études épidémiologiques. Une limite à la consommation d’œuf réside finalement dans son pouvoir allergène, en particulier chez le jeune enfant.

La qualité de l’œuf se révèle fortement influencée par les modifications physiologiques liées à l’âge de la poule, à une mue ou par les conditions environnementales (température, lumière et système d’élevage). Le poids de l’œuf augmente avec l’âge des poules, du fait notamment d’un accroissement de la part de jaune. L’âge de la poule réduit la qualité de la coquille ainsi que les propriétés fonctionnelles des œufs. La mue restaure, pour un cycle plus court, les performances des poules et la qualité des œufs, dégradée en fin de premier cycle. L’augmentation de la température ambiante en élevage entraîne, dès 30°C, des perturbations de l’équilibre acido-basique et une chute de la consommation d’aliment. Celles-ci réduisent la production d’œufs, leur poids et leur qualité de coquille. Les programmes lumineux mis en place avant et après l’entrée en ponte, conditionnent la courbe de ponte. L’application de nycthémères supérieurs à 24h ou de programmes fractionnés symétriques augmente le poids d’œuf et l’épaisseur de coquille mais les directives européennes interdisent leur usage. La qualité sensorielle, nutritionnelle ou technologique de l’œuf est faiblement affectée et de manière inconstante par le système de production, volière ou cage. Certaines cages aménagées ont initialement engendré une détérioration de l’intégrité des œufs mais des modifications de leur conception, de la disposition des aménagements et de la taille du groupe aboutissent progressivement à l’obtention de résultats comparables à ceux obtenus en cages conventionnelles.

La maîtrise des rejets de phosphore (P) dans les déjections animales passe par une meilleure connaissance des besoins des animaux et de l’utilisation de l’apport alimentaire de P. Les méthodes d’étude sont un peu différentes chez les animaux monogastriques (porcs et volailles) ou les ruminants. En règle générale, la détermination des besoins repose sur une démarche factorielle (estimation des dépenses d’entretien et de production) ; toutefois, une méthode plus globale peut être appliquée chez les monogastriques (optimisation de la minéralisation osseuse). L’intégration des connaissances a permis de préciser les systèmes, l’expression des besoins (et des apports) passant de P total à P digestible apparent (porcs) ou absorbé réel (ruminants). L’évaluation de l’efficacité digestive de l’apport alimentaire de P nécessite, chez le monogastrique, la prise en compte de l’utilisation du phosphore phytique. Cette dernière peut être très différente selon l’activité phytasique propre des constituants du régime, l’utilisation de phytases microbiennes et l’éventuelle dénaturation des phytases lors de la granulation des aliments. Pour ces raisons, deux valeurs de P digestible apparent (activité phytasique intacte ou dénaturée) figurent dans les tables INRA-AFZ ; de plus des équations sont proposées pour tenir compte de l’incorporation de phytase microbienne. Pour les ruminants, la compilation des données de la littérature a permis d’établir un coefficient d’absorption réelle de P pour les grandes catégories d’aliments (phosphates concentrés et fourrages). Ces démarches, en permettant une meilleure adéquation des apports aux besoins doivent se traduire par une réduction substantielle des rejets de phosphore dans les effluents d’élevage.

International audience; The ovalbumin gene family in Gallus gallus is composed of three homologous genes located within a 46 kb locus on chromosome 2: ovalbumin, ovalbumin-related protein Y (OVAY) and ovalbumin-related protein X (OVAX) genes. The expression of these genes in hen oviduct is under estrogen control but their relative hormonal responsiveness and subsequent protein concentration in egg, is distinctive. Interestingly, all three proteins lack the classical signal peptide for secretion. Ovalbumin, OVAX and OVAY belong to the serine protease inhibitor (serpin) family of which members share a common tertiary structure. Ovalbumin and OVAX are ones of the few members of this family that do not express any protease inhibition activity whereas OVAY has been predicted to be inhibitory, by comparison with the consensus sequence for inhibitory serpins. In contrast to ovalbumin and OVAY, OVAX interacts with heparin, a negatively charged glycosaminoglycan, via a positively charged domain exposed at the surface of the molecule. Ovalbumin is the major egg white protein and might be a source of amino-acids for the developing embryo. The physiological function of OVAY is not known but recent data have revealed a possible role of this protein in early embryonic development. Considering the antibacterial activities of OVAX, this protein might play a role in egg defense. This review sheds light on the expressional, biochemical and structural specificities of these three highly similar paralogs. It gives new clues in favor of diverging functions, which are likely to have arisen by duplication events from a common ancestral gene.

Remerciements : GenoToul, Plateforme Bioinformatique de Toulouse Midi-Pyrénées; International audience; 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 4) and 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. Significance The avian eggshell is a natural protective barrier of the egg as long as it remains intact. It prevents microbial penetration to ensure a harmonious development of the embryo and table eggs free of pathogens for consumers. The eggshell is a calcitic biomineral structure initiated on organic nucleation sites which are evenly disposed on eggshell membranes. Its structural organisation results from an interaction between matrix proteins and calcium carbonate, in particular calcite crystal during the growth phase. The initial steps of the calcification process during which calcitic mineral phase is selected and the process of crystal orientation selection by competition for space are crucial in the determination of the whole eggshell columnar ultrastructure and its resulting exceptional mechanical properties. Recent studies have also described the physical characteristics of calcium carbonate during the initial sequence of calcium carbonate biomineralisation in many species and have highlighted the key role of amorphous calcium carbonate. In order to determine precisely the importance of particular proteins relative to these key events at the onset of mineralisation, this project used quantitative proteomics of matrix proteins in forming eggshell, assuming that a protein more abundant at a particular stage of the process has an higher probability to be involved in its control. This information was coupled with bioinformatic analysis predicting the function of proteins in the stabilisation of disordered forms of calcium carbonate and their influence on crystal polymorph and morphology. Eggshell calcification is a relevant model for studying calcium carbonate biomineralisation on a membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressures and temperatures and might provide means of reinforcing its solidity.

1. The hygienic properties of eggs produced in two designs of furnished cage were investigated and compared to two standard cage designs. 2. At 28, 37, 47 and 58 weeks of age, the proportion of dirty eggs was higher in one of the furnished cage designs while, in the other, it was similar to standard cages. 3. At 27, 33 and 60 weeks, the bacterial load on the eggshell (total aerobic bacteria and enterococci) was higher in furnished cage designs. A seasonal effect was observed with lower counts at 60 weeks (winter) than at 27 weeks (summer). 4. More dirty eggs and a higher bacterial load were observed in eggs laid outside the nests, which suggests egg hygiene in furnished cages could be similar to standard cages if the equipment in furnished cages was improved to enhance nest laying.

Le phosphore en excès est un élément néfaste pour l’environnement et coûteux pour l’alimentation des volailles. Une méta-analyse a été conduite sur les données de 62 publications représentant 1271 observations chez les poulets de chair, dindes et canards afin de visualiser les réponses des performances de volaille aux apports de phosphore (P) alimentaire. Pour le GMQ et l’IC des poulets de chair, les concentrations alimentaires optimales obtenues varient de 6 à 7 g de P total par kg d’aliment, pour un rapport Ca/P compris entre 1,4 et 1,6. Des valeurs supérieures sont observées chez les dindes. La rétention du P par l’animal diminue avec la teneur en P de l’aliment mais augmente avec l’ajout de phytases microbiennes. Cette supplémentation qui libère une partie du phosphore phytique, autorise une réduction des apports de phosphore minéral et par conséquent réduit le rejet de phosphore. Les apports alimentaires de P et de phytases microbiennes renforcent la minéralisation. La minéralisation osseuse est optimale pour des niveaux d’ingestion moyens de 1 à 21 j de 250 à 300 mg de P par jour. La présente méta-analyse a été conduite en utilisant la mesure exacte du P total de l’aliment, contrairement à la pratique, justifiée, d’utiliser le niveau de P disponible estimé. La prochaine étape sera d’analyser les résultats en fonction du P disponible après avoir fiabilisé son estimation en prenant en compte les effets matières premières, phytase et autres (vitamine D, rapport Ca/P…). Enfin, ce travail a souligné que si de nombreuses données sont disponibles chez le jeune poulet de chair, elles sont rares chez les dindes et les canards notamment pendant les périodes de finition où des marges de progrès réelles sont envisageables.

The effect of moult on eggshell mechanical properties, on composition and concentrations of organic matrix components and on eggshell microstructure was investigated. The observed changes were studied to understand the role of organic matrix and eggshell microstructure in eggshell strength. Moult was induced by zinc oxide (20 g zinc/kg diet) in 53 ISA Brown laying hens at 78 weeks of age. No difference was observed for egg or eggshell weights after moult. In contrast, moult improved the shell breaking strength (28.09 vs 33.71 N). After moult, there was a decrease in the average size of calcite crystals composing the eggshell and in their heterogeneity, whereas crystal orientation remained basically the same. After moulting, the total protein concentration in eggshell increased slightly. The comparisons of SDS-PAGE profiles of the organic matrix constituents extracted before and after moulting showed changes in staining intensity of certain bands. After moult, bands associated with main proteins specific to eggshell formation (OC-116 and OC-17) showed higher staining intensity, while the intensity of the egg white proteins (ovotransferrin, ovalbumin and lysozyme) decreased. ELISA confirmed the decrease in ovotransferrin after moult. Its concentration was inversely correlated with breaking strength before moult. These observations suggest that changes in eggshell crystal size could be due to changes in organic matrix composition. These changes may provide a mechanism for the improvement in shell solidity after moulting.

The eggshell of the hen is a highly ordered and mineralised structure, which is sequentially deposited within an acellular milieu – the uterine fluid secreted by the distal oviduct. Spherulitic crystal growth of calcite is initiated on organic aggregates on surface of the eggshell membranes, followed by competition between radial crystallites for space to form a compact columnar biomineral. The exceptional mechanical properties associated with the well-defined eggshell ultrastructure and texture arise from the control of crystal morphology and growth by the organic matrix, and, amongst them, proteins specific to the uterus and eggshell (ovocleidins and ovocalyxins). The changes in uterine fluid constituents with stages of egg calcification, their effects on morphology of calcite grown in vitro, and the relationship between eggshell texture and mechanical properties point to this control of eggshell fabric. To cite this article: Y. Nys et al., C. R. Palevol 3 (2004) .

Thirty-six pigs, weaned at 28 days of age at an average body weight of 9.3 kg were used in a 19-day experiment to assess the bioavailability of Zn. A Zn–methionine complex with a molar methionine to Zn ratio of 2:1 was compared to Zn sulphate using a basal diet based on maize and soya bean meal supplemented with or without microbial phytase. Six experimental diets were formulated: the basal diet containing 32 mg Zn/kg, the basal diet supplemented with 20 mg Zn/kg from either sulphate (ZnSO 4 ·7H 2 O) or the organic source (ZnOrg), and the same three previous diets supplemented with 1200 units (U) of microbial phytase per kilogram. The replacement of ZnSO 4 by the organic source did not modify Zn retention, the concentration of Zn in bone and plasma or plasma alkaline phosphatase (AP) activity. The addition of microbial phytase considerably improved Zn bioavailability. Considering plasma AP activity, plasma Zn concentration, bone Zn concentration and Zn retention, the effect of adding 1200 U phytase/kg in the basal diet largely exceeded the effect of adding 20 mg Zn/kg. When phytase was added to the basal diet, these indicators of Zn status increased by 198, 269, 111 and 189%, respectively. Dietary phytase and Zn also modified the utilisation of Ca, P, Mg, Fe and Cu by piglets.

Hen nutrition is crucial for optimising egg quality. Hen's weight at point of lay, energy and protein densities of the diets, amino acid and fatty acids contents but also the mode of feed delivery throughout the day affect egg weight and also, but to a lesser extent, the proportion of yolk and albumen. Egg profile in fatty acid and in trace elements (iodine, selenium, manganese) and fat-soluble vitamins (E, D, A and K) reflects the dietary supply of these nutrients. The yolk colour also depends on the dietary carotenoids even if effectiveness of colouring varies according to the source of the xanthophyll. Finally, this chapter describes the effects of dietary calcium and other minerals and vitamins on shell strength.

This chapter provides an overview on the structure, composition and formation of bird eggs with emphasis on chicken, Gallus gallus . The first part describes the global structure of the egg and its compartments (yolk, white/albumen, eggshell). The second part reviews their chemical composition in terms of lipids, proteins, carbohydrates, minerals, vitamins and pigments. The last part explains the key steps of egg formation in the liver, ovary and oviduct, providing a broadly based view on the physiology, regulation and kinetics of synthesis or secretion of egg components. It also describes the development, anatomy and role of the genital tract in this process.

The development of molecular biology in the late 1980s, the recent publication of the chicken genome sequence and the development of new bioinformatics tools were major scientific advances leading to the characterisation of several egg components previously unidentified. Using recent data on the characterisation of egg proteins, the chapter describes the most recent developments in egg biochemistry (proteomics), and molecular biology (transcriptomics). The newly identified molecules will stimulate research in understanding avian biology and identifying biologically active compounds of potential interest to the pharmaceutical, cosmetic and food industries.

Clusterin is a widely expressed secretory glycoprotein which is found in mammals as a disulfide-bonded alpha/beta heterodimer generated by cleavage of the single-chain precursor. In contrast, clusterin occurs in the chicken mainly as an intracellular single-chain form and is not observed in serum. The present report identifies chicken clusterin as a component of the eggshell. This extracellular clusterin originates in the uterine fluid, where it is a disulfide-bonded heterodimer derived from the precursor polypeptide by proteolytic cleavage at the same site as in mammals. Clusterin message expression in the oviduct was measured by real time RT-PCR, and levels were found to be highest in magnum and uterus. Western blotting using protein extracts of oviduct tissues indicated major clusterin production in the magnum, while immunostaining of the oviduct identified clusterin in the tubular glands of the uterus and the magnum. In addition, clusterin was detected in egg white by Western blotting. In the decalcified eggshell, immunofluorescence and colloidal-gold immunocytochemistry revealed that clusterin was predominantly localized in the palisade and mammillary layers, but also in the mantle and core of the inner and outer shell membranes. It has been suggested recently that clusterin acts as an extracellular chaperone. Thus clusterin could function in the uterine fluid to prevent the premature aggregation and precipitation of eggshell matrix components before and during their assembly into the rigid protein scaffold necessary for ordered mineralization.

Le zinc est un oligo-élément essentiel qui intervient dans la plupart des fonctions biologiques de l’animal. La perte d’appétit, la diarrhée, le retard de croissance ou la parakératose sont les principaux symptômes d’une carence en zinc qui peuvent apparaître chez le porc recevant une alimentation non supplémentée en zinc. La méconnaissance des besoins en zinc du porc et des facteurs de variation de sa disponibilité conduit à l’incorporer de manière excessive dans les aliments. Cette pratique génère des rejets importants et augmente le risque de pollution des sols cultivés. L’ajustement des apports de zinc aux besoins de l’animal et l’amélioration de sa disponibilité sont des solutions qui permettent d’en réduire les apports. Une revue des données de la littérature nous permet d’estimer les besoins en zinc du porc en fonction du poids vif. L’utilisation d’additifs, notamment de phytase microbienne, permet d’améliorer la disponibilité du zinc. A partir des résultats de la littérature, nous estimons que l’activité de 1000 unités de phytase microbienne par kg d’aliment est équivalente à une addition de 24 et 19 mg/kg de zinc sous forme de sulfate chez le porcelet de respectivement 15 et 25 kg. Ces estimations sont à valider expérimentalement. D’autres voies d’amélioration de la disponibilité du zinc doivent également être explorées.

La coquille de l’œuf est une structure minérale parfaitement définie. Elle est déposée chaque jour en moins de 24 h dans un milieu acellulaire, le fluide utérin, sécrété par la partie distale de l’oviducte. La poule exporte une quantité considérable de calcium, et adapte à de nombreux niveaux son métabolisme calcique. La coquille résulte d’une croissance cristalline radiale de la calcite initiée sur des amas organiques présents en surface des membranes coquillières. Cette croissance se poursuit pour former une couche compacte dont les cristaux présentent progressivement une direction privilégiée du fait d’une compétition spatiale entre sites adjacents. Les propriétés mécaniques exceptionnelles de la coquille résultent de la quantité et de l’organisation cristallographique de ce biomatériau, elles-mêmes contrôlées par la matrice organique. Celle-ci est composée de nombreuses protéines spécifiques, uniquement synthétisées par l’utérus de la poule. La spécificité du profil électrophorétique du fluide utérin à un stade de formation de la coquille, le contrôle du polymorphe (calcite) et de la morphologie des cristaux in vitro par les protéines de ce milieu plaident en faveur de ce contrôle de la fabrication de la coquille par sa matrice protéique. Cette hypothèse est renforcée par les observations in vivo d’une association entre texture ou solidité de la coquille avec les teneurs en protéines de la matrice organiques ou avec la présence de polymorphisme simple nucléotidique de gènes codant ces protéines., The eggshell of the hen is a highly ordered and mineralized structure, which is sequentially deposited within an acellular milieu - the uterine fluid secreted by the distal oviduct. Each day, the hen exports a huge amount of calcium to form the eggshell and undergoes tremendous changes in its calcium metabolism. The eggshell results from spherulitic crystal growth of calcite initiated on organic aggregates on the surface of eggshell membranes, followed by competition between radial crystallites for space, resulting in a compact columnar biomineral. The exceptional mechanical properties associated with the well-defined eggshell ultrastructure and its texture arise from the control of crystal morphology and growth by the organic matrix, amongst them proteins specific to the uterus and eggshell (ovocleidins and ovocalyxins). The uterine fluid constituents varies at different stages of egg calcification, and alter the morphology of calcite crystals grown in vitro. Moreover, there is a relationship between levels of matrix proteins or their single nucleotide polymorphisms upon eggshell texture or mechanical properties. All these features point to the control of eggshell fabric by the organic matrix.

National audience; The egg is a highly digestible source of proteins and lipids, which contains a large range of vitamins and minerals needed by humans. The amino acid content, perfectly balanced for human needs, classifies eggs amongst the highest nutritional value of protein sources. Egg lipids provide large amounts of unsaturated fatty acids and phospholipids. The major egg components show limited variability; it essentially depends on egg white/yolk ratio, which is largely controlled by the hen's age. On the contrary, large differences can be observed for egg content in fatty acids, vitamins, minerals and pigments. The influence of the hen's diet on egg composition for these elements enables the enrichment of eggs for human health benefit. For example, nutritional studies have demonstrated in humans the positive impact of consumption of unsaturated fatty acid enriched eggs; in addition, lutein and zeaxanthin are very efficiently absorbed by humans and highly available for the retina when provided by eggs. In addition, the negative image of the egg due to its high cholesterol content tends to be attenuated because the assumption of increased cardiovascular risk is not supported by the majority of epidemiological studies. One constrain to egg consumption is associated with allergenic proteins, and care should be taken especially for young children.; L’œuf est une source très digestible de protéines et de lipides qui contient par ailleurs une part importante des vitamines et minéraux nécessaires à l’Homme. La composition en acides aminés, équilibrée par rapport aux besoins de l’Homme, fait de l’œuf l’une des meilleures sources protéiques et les lipides de l’œuf fournissent des quantités importantes d’acides gras insaturés et de phospholipides. Les constituants majeurs de l’œuf sont relativement stables ; ils dépendent essentiellement de la proportion de blanc et de jaune, elle-même largement influencée par l’âge de la poule. En revanche, la composition de l’œuf en acides gras, vitamines, minéraux et pigments peut fortement varier. La dépendance de ces éléments vis-à-vis de l’alimentation de la poule offre ainsi des opportunités d’enrichissement de l’œuf en constituants d’intérêt pour la santé humaine. Des études nutritionnelles ont par exemple démontré chez l’Homme l’impact positif de la consommation d’œufs enrichis en acides gras polyinsaturés ; de même, la lutéine et la zéaxanthine de l’œuf sont particulièrement bien absorbés par l’organisme humain et biodisponibles pour la rétine. Le concept d’augmentation du risque cardio-vasculaire lié à la forte teneur de l’œuf en cholestérol est quant à lui clairement contredit par de nombreuses études épidémiologiques. Une limite à la consommation d’œuf réside finalement dans son pouvoir allergène, en particulier chez le jeune enfant.

Fractionation of the soluble chicken eggshell matrix by chromatographic methods yielded 13 endogenous proteolytic fragments of the eggshell-specific proteoglycan core protein ovocleidin-116. The N-terminal amino acid sequences of these fragments in general confirmed the recently cDNA-deduced sequence of ovocleidin-116, with one exception. One fragment yielded a completely new sequence and was instrumental in detecting a frame shift error in the nucleotide sequence. The correction yielded a new sequence which was 38 amino acids shorter than before and contained a 57-amino acid long novel C-terminal sequence. 1 The predicted sequence of ovocleidin-116 contained two consensus N-glycosylation sites, only one of which (Asn62) was found to be fully modified. A disulfide bond was identified between Cys31 and 42 implying that Cys329 and 421 form a second disulfide bond. Finally, the yield of fragments indicated that ovocleidin-116 is a major component of the chicken eggshell matrix.

Thirty-two pigs, weaned at 28 days of age with an average body weight of 9 kg, were used in an experiment devoted to the assessment of the bioavailability of an organic Zn source (ZnOrg) compared to Zn sulfate. Pigs were fed a basal diet containing 28 mg·kg -1 of Zn supplemented with ZnSO4 or ZnOrg to provide 0, 10, 20 and 30 mg·kg -1 of supplemental Zn. In order to reduce the amount and the variability of their Zn stores, pigs were fed the basal diet for a 7-day adjustment period preceding a 19-day experimental period. Growth performance and bone bending moment were not affected by the level nor the source of zinc. On the contrary, plasma zinc concentration and alkaline phosphatase activity (P < 0.001), bone zinc concentration (P < 0.001), liver and empty body zinc concentrations (P < 0.01) as well as the amount of Zn retained estimated both by the balance tech- nique (BT) (P < 0.001) and by the comparative slaughter technique (CST) (P < 0.01) increased lin- early in response to supplemental Zn. However, the two Zn sources exhibited a similar bioavailability. Twenty-seven percent of Zn intake was retained when measured by means of the balance technique, regardless of the dietary level or zinc source. Zn retention was 54% lower when measured by means of the CST as compared to the BT. pig / zinc / bioavailability / source / indicator

The eggshell of the hen is a highly ordered, mineralised structure deposited within an acellular milieu – the uterine fluid secreted by the distal oviduct. Spherulitic crystal growth is initiated b...

Cet article analyse l’intérêt et le rôle des oligo-éléments comme facteurs favorisant la croissance pondérale ou osseuse et la défense immunitaire du poulet de chair. L’objectif est d’analyser les conséquences de leur supplémentation à des doses correspondant au besoin de croissance ou à des apports très supérieurs, une pratique actuellement remise en question par la législation afin de limiter le rejet des oligo-éléments dans l’environnement. Des excès de Zn, Fe ou Mn n’ont pas d’effet positif sur la croissance du poulet de chair. Une concentration élevée de Cu (100 à 250 mg/kg) dans l’aliment tend à améliorer légèrement la croissance, mais est à l’origine d’une accumulation de Cu néfaste pour l’environnement dans les zones de production intensive. Zn, Se, Cu et Fe sont des éléments essentiels pour le bon fonctionnement du système immunitaire chez l’oiseau. Les infections et la coccidiose affectent leur métabolisme ce qui peut nécessiter une correction par voie alimentaire. Les ultra-oligo-éléments (Al, Se, Bo, Cd, Cr, F, I, Si, V…) peuvent jouer un rôle important chez des poussins soumis à divers stress nutritionnels, ou se révéler toxiques lorsqu’ils sont utilisés en excès., Trace elements as related to growth and health in chickens This review summarizes the interest and the role of trace element factors favouring body weight or bone growth and the immune response in poultry. The aim is to analyse consequences of supplementation in diets at levels corresponding or higher than the growth requirement for optimising performance of broilers; especially as the use of high dietary levels of trace elements is currently questioned to protect the environment. Cu shows a slight growth promoting effect on chick growth when added at high concentrations, but Zn, Fe or Mn do not. However, such high dietary level can be deleterious for the environment in areas of intensive production. Zn, Se, Cu and Fe are essential elements for the immune system but there is no clear evidence that high dietary levels stimulate the immune response Infection and coccidiosis affect trace mineral metabolism and, therefore, may need nutritional adjustment. Ultra-trace element (Al, Se, Bo, Cd, Cr, F, I, Si, V.) can be of nutritional importance for chicks submitted to some nutritional stress or, when in excess, can be toxic.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.

Two experiments were conducted to study the effects of microbial phytase at two dietary levels of phytate and those of vegetal phytase on Zn and Mn availability in broiler chicks (21 days of age). In each experiment, 160 chicks were fed on 10 experimental diets. In experiment 1a, the basal diet (37 mg/kg Zn and 29 mg/kg Mn, corn-soybean meal (CSM)), was supplemented with two levels of Zn (14 and 35 mg/kg) and Mn (16 and 40 mg/kg) in the presence or absence of microbial phytase (800 U/kg) and (experiment 1b) with or without heated bran in order to increase phytate level without increasing vegetal phytase activity. In experiment 2a, the basal CSM diet (34 and 31 mg/kg Zn and Mn, respectively), was supplemented with two levels of Zn (10 and 30 mg/kg) and Mn (12 and 35 mg/kg), in presence or absence of microbial phytase (1200 U/kg). At both Zn and Mn supplementation, heated or non heated wheat was substituted for corn to introduced 480 U/kg vegetal phytase (experiment 2b). No differences were noted in weight gain, feed intake and tibia ash with dietary Zn and Mn level or with presence of phytase. Feed conversion ratio (FCR) decreased, however, with dietary Zn and Mn in one of the trial (2a). Zn concentration in the tibia and plasma as well as tibia Mn increased with dietary mineral levels (22–45 mg Zn/kg; 29–75 mg Mn/kg). Adding microbial phytase improved Zn level in tibia when dietary Zn was low (experiment 1a; 37 mg/kg: experiment 2a; 34 and 44 mg/kg). At higher dietary Zn, microbial phytase had no effect even when dietary phytate was increased to 3.1 g/kg. Vegetal phytase increased plasma and tibia Zn concentrations at the lower Zn dietary supplementation (10 mg/kg) but had no effect on Mn availability. This investigation demonstrates that addition of microbial (800 UP equivalent to 14 mg/kg in a CSM diet) and vegetal (to a lower extend) phytases increase Zn availability in broilers.

Background Egg defence against bacterial contamination relies on immunoglobulins (IgY) concentrated in the yolk and antimicrobial peptides/proteins predominantly localized in the egg white (EW). Hens contaminated with pathogenic microorganisms export specific IgYs to the egg (adaptative immunity). No evidence of such regulation has been reported for the antimicrobial peptides/proteins (innate immunity) which are preventively secreted by the hen oviduct and are active against a large range of microbes. We investigated whether the egg innate defences can be stimulated by the environmental microbial contamination by comparing the antimicrobial activity of EW of hens raised in three extreme breeding conditions: Germ-free (GF), Specific Pathogen Free (SPF) and Conventional (C) hens. Results The difference in the immunological status of GF, SPF and C hens was confirmed by the high stimulation of IL-1β, IL-8 and TLR4 genes in the intestine of C and SPF groups. EW from C and SPF groups demonstrated higher inhibitory effect against Staphylococcus aureus (13 to 18%) and against Streptococcus uberis (31 to 35%) as compared to GF but showed similar activity against Salmonella Enteritidis, Salmonella Gallinarum, Escherichia coli and Listeria monocytogenes. To further investigate these results, we explored putative changes amongst the three main mechanisms of egg antimicrobial defence: the sequestration of bacterial nutrients, the inactivation of exogenous proteases and the direct lytic action on microorganisms. Lysozyme activity, chymotrypsin-, trypsin- and papain-inhibiting potential of EW and the expression of numerous antimicrobial genes were not stimulated suggesting that these are not responsible for the change in anti-S. aureus and anti-S. uberis activity. Moreover, whereas the expression levels of IL-1β, IL-8 and TLR4 genes were modified by the breeding conditions in the intestine of C and SPF groups they were not modified in the magnum where egg white is formed. Conclusions Altogether, these data revealed that the degree of environmental microbial exposure of the hen moderately stimulated the egg innate defence, by reinforcing some specific antimicrobial activities to protect the embryo and to insure hygienic quality of table eggs.

1. Extra and intramineral eggshell matrix proteins were solubilised before and after demineralisation by sequential extractions using guanidine hydrochloride and EDTA. 2. The intramineral electrophoretic profile of SDS‐PAGE showed the presence of 80, 66, 43, 36 and 15 kDa bands with a predominance of a 17 kDa band. In the extramineral part, the major protein was the 15 kDa band. 3. The introduction of intramineral extract to a metastable solution of calcium carbonate delayed the rate of crystal growth. The delay in the rate of precipitation was elicited by a single fraction (MW 50–80 kDa), isolated by gel filtration chromatography, of eggshell extracts. Extramineral extracts had no effect. 4. Addition in vitro of intramineral eggshell extracts modified the morphology of calcite; the crystals aggregated and showed irregular surfaces. 5. These observations suggest that constituents of the eggshell matrix are involved in the control of calcite growth and crytallographic structure of the hen's eggshell.

Background In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3 -) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. Results A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3 - movement) and duodenum (high rates of Ca2+/HCO3 - trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1). We propose that the HCO3 - is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3 - is secreted through the HCO3 -/Cl- exchanger SLC26A9. HCO3 - synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3 -/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. Conclusions Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3 - and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.