Sex-specific transcriptome of the chicken chorioallantoic membrane.

Dimorphism between male and female embryos has been demonstrated in many animal species, including chicken species. Likewise, extraembryonic membranes such as the chorioallantoic membrane (CAM) are likely to exhibit a sex-specific profile. Analysis of the previously published RNA-seq data of the chicken CAM sampled at two incubation times, revealed 783 differentially expressed genes between the CAM of male and female embryos. The expression of some of these genes is sex-dependant only at one or other stage of development, while 415 genes are sex-dependant at both developmental stages. These genes include well-known sex-determining and sex-differentiation genes (DMRT1, HEGM, etc.), and are mainly located on sex chromosomes. This study provides evidence that gene expression of extra-embryonic membranes is differentially regulated between male and female embryos. As such, a better characterisation of associated mechanisms should facilitate the identification of new sex-specific biomarkers.

RNA-seq analysis of the active chick embryo chorioallantoic membrane reveals genes that encode proteins assigned to ion transport and innate immunity.

The chicken chorioallantoic membrane (CAM) is an extraembryonic membrane that is vital for the embryo. It undergoes profound cell differentiation between 11 and 15 days of embryonic incubation (EID), which corresponds to the acquisition of its physiological functions. To gain insight into the functional genes that accompany these biological changes, RNA sequencing of the CAM at EID11 and EID15 was performed. Results showed that CAM maturation coincides with the overexpression of 4225 genes, including many genes encoding proteins involved in mineral metabolism, innate immunity, homeostasis, angiogenesis, reproduction, and regulation of hypoxia. Of these genes, some exhibit variability in expression depending on the chicken breed (broiler versus layer breeds). Besides the interest of these results for the poultry sector, the identification of new functional gene candidates opens additional research avenues in the field of developmental biology.

Structure, function, and evolution of Gga-AvBD11, the archetype of the structural avian-double-ß-defensin family.

Out of the 14 avian ß-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian ß-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to ß-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed ß-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-ß-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

The Unique Features of Proteins Depicting the Chicken Amniotic Fluid.

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.

Egg serpins: The chicken and/or the egg dilemma.

Twenty-seven serpins belonging to clade A, B, C, D, E, F, G, H and I serpins are currently referenced in chicken genome databases. Phylogenetic analysis of chicken serpins revealed that ovalbumin (Serpinb14) and its paralogs ovalbumin-related protein Y (Serpinb14b) and ovalbumin-related protein X (Serpinb14c) are found in bird species. These clade B serpins are specifically expressed in reproductive tissues and exported in the egg where they constitute major protein components. These data suggest that these three paralogs have probably appeared in birds to face new environments and ensure the extra-uterine development of an embryo in a shell egg. Twelve other serpins have been identified in the newly produced egg, some of them having a specific distribution in the respective egg structures (eggshell, egg white, vitelline membrane and egg yolk). The physiological role of these egg serpins remain largely unexplored, but there is increasing evidence in literature or by homologies with their mammalian counterparts, that some of them participate in cell proliferation, tissue remodeling and/or angiogenesis associated with folliculogenesis and development of extraembryonic structures, eggshell biomineralization, egg defense and nutrition of the embryo. A better knowledge of the phylogenetic evolution of these 15 serpins in other oviparous species, on their egg distribution, on their regulation during embryonic development (activation/degradation/transfer) and on their functional specificity, is needed to better appreciate their role and their bird-specificity. These review shed light on the multiple possibilities that offer the avian egg model to study the role of serpins in reproduction and developmental biology.

Integrative analysis of transcriptomic data related to the liver of laying hens: from physiological basics to newly identified functions.

BACKGROUND: At sexual maturity, the liver of laying hens undergoes many metabolic changes to support vitellogenesis. In published transcriptomic approaches, hundreds of genes were reported to be overexpressed in laying hens and functional gene annotation using gene ontology tools have essentially revealed an enrichment in lipid and protein metabolisms. We reanalyzed some data from a previously published article comparing 38-week old versus 10-week old hens to give a more integrative view of the functions stimulated in the liver at sexual maturity and to move beyond current physiological knowledge. Functions were defined based on information available in Uniprot database and published literature. RESULTS: Of the 516 genes previously shown to be overexpressed in the liver of laying hens, 475 were intracellular (1.23-50.72 fold changes), while only 36 were predicted to be secreted (1.35-66.93 fold changes) and 5 had no related information on their cellular location. Besides lipogenesis and protein metabolism, we demonstrated that the liver of laying hens overexpresses several clock genes (which supports the circadian control of liver metabolic functions) and was likely to be involved in a liver/brain/liver circuit (neurotransmitter transport), in thyroid and steroid hormones metabolisms. Many genes were associated with anatomical structure development, organ homeostasis but also regulation of blood pressure. As expected, several secreted proteins are incorporated in yolky follicles but we also evidenced that some proteins are likely participating in fertilization (ZP1, MFGE8, LINC00954, OVOCH1) and in thyroid hormone maturation (CPQ). We also proposed that secreted proteins (PHOSPHO1, FGF23, BMP7 but also vitamin-binding proteins) may contribute to the development of peripheral organs including the formation of medullar bones to provide labile calcium for eggshell formation. Thirteen genes are uniquely found in chicken/bird but not in human species, which strengthens that some of these genes may be specifically related to avian reproduction. CONCLUSIONS: This study gives additional hypotheses on some molecular actors and mechanisms that are involved in basic physiological function of the liver at sexual maturity of hen. It also revealed some additional functions that accompany reproductive capacities of laying hens, and that are usually underestimated when using classical gene ontology approaches.

The seminal acrosin-inhibitor ClTI1/SPINK2 is a fertility-associated marker in the chicken.

The seminal plasma is a very complex fluid, which surrounds sperm in semen. It contains numerous proteins including proteases and protease inhibitors that regulate proteolytic processes associated with protein activation and degradation. We previously identified a seminal protein, chicken liver trypsin inhibitor 1 (ClTI-1) over expressed in semen of roosters with high fertility, suggesting a role in male fertility. In the present study, we showed that ClTI-1 gene is actually SPINK2. Using normal healthy adult roosters, we showed that SPINK2 amount in seminal plasma was positively correlated with male fertility in chicken lines with highly contrasted genetic backgrounds (broiler and layer lines). Using affinity chromatography combined to mass spectrometry analysis and kinetic assays, we demonstrated for the first time that two chicken acrosin isoforms (acrosin and acrosin-like proteins) are the physiological serine protease targets of SPINK2 inhibitor. SPINK2 transcript was overexpressed all along the male tract, and the protein was present in the lumen as expected for secreted proteins. Altogether, these data emphasize the role of seminal SPINK2 Kazal-type inhibitor as an important actor of fertility in birds through its inhibitory action on acrosin isoforms proteins.

Three-dimensional NMR structure of Hen Egg Gallin (Chicken Ovodefensin) reveals a new variation of the ß-defensin fold.

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 ß-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 ß-sheet and the disulfide bridge array typical of vertebrate ß-defensins. Gallin can therefore be unambiguously classified as a ß-defensin. However, an additional short two-stranded ß-sheet reveals that gallin and presumably the other ovodefensins form a new structural subfamily of ß-defensins. Moreover, gallin and the other ovodefensins calculated by homology modeling exhibit atypical hydrophobic surface properties, compared with the already known vertebrate ß-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.

The Family Secrets of Avian Egg-Specific Ovalbumin and Its Related Proteins Y and X.

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 whose members share a common tertiary structure. Ovalbumin and OVAX are one 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 expression, biochemistry, 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.

Ovalbumin-related protein X is a heparin-binding ov-serpin exhibiting antimicrobial activities.

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 Lys(367)-His(368) 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.

Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens.

Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies.

Passive maternal exposure to environmental microbes selectively modulates the innate defences of chicken egg white by increasing some of its antibacterial activities.

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-1ß, 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-1ß, 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.

Allantoic fluid metabolome reveals specific metabolic signatures in chicken lines different for their muscle glycogen content.

Nutrient availability in eggs can affect early metabolic orientation in birds. In chickens divergently selected on the Pectoralis major ultimate pH, a proxy for muscle glycogen stores, characterization of the yolk and amniotic fluid revealed a different nutritional environment. The present study aimed to assess indicators of embryo metabolism in pHu lines (pHu+ and pHu-) using allantoic fluids (compartment storing nitrogenous waste products and metabolites), collected at days 10, 14 and 17 of embryogenesis and characterized by 1H-NMR spectroscopy. Analysis of metabolic profiles revealed a significant stage effect, with an enrichment in metabolites at the end of incubation, and an increase in interindividual variability during development. OPLS-DA analysis discriminated the two lines. The allantoic fluid of pHu- was richer in carbohydrates, intermediates of purine metabolism and derivatives of tryptophan-histidine metabolism, while formate, branched-chain amino acids, Krebs cycle intermediates and metabolites from different catabolic pathways were more abundant in pHu+. In conclusion, the characterization of the main nutrient sources for embryos and now allantoic fluids provided an overview of the in ovo nutritional environment of pHu lines. Moreover, this study revealed the establishment, as early as day 10 of embryo development, of specific metabolic signatures in the allantoic fluid of pHu+ and pHu- lines.

Transcriptomic profiling of proteases and antiproteases in the liver of sexually mature hens in relation to vitellogenesis.

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

Concomitant Morphological Modifications of the Avian Eggshell, Eggshell Membranes and the Chorioallantoic Membrane During Embryonic Development.

The chicken eggshell (ES) consists of 95% calcium carbonate and 3.5% organic matter, and represents the first physical barrier to protect the developing embryo, while preventing water loss. During the second half of development, calcium ions from the inner ES are progressively solubilized to support mineralization of the embryonic skeleton. This process is mediated by the chorioallantoic membrane (CAM), which is an extraembryonic structure that adheres to the eggshell membranes (ESM) lining the inner ES. The CAM surrounds the embryo and all egg contents by day 11 of incubation (Embryonic Incubation Day 11, EID11) and is fully differentiated and functionally active by day 15 of incubation (Embryonic Incubation Day 15, EID15). In this study, we explored the simultaneous morphological modifications in the ES, ESM and the CAM at EID11 and EID15 by scanning electron microscopy. We observed that the tips of the mammillary knobs of the ES remain tightly attached to the ESM fibers, while their bases become progressively eroded and then detached from the bulk ES. Concomitantly, the CAM undergoes major structural changes that include the progressive differentiation of villous cells whose villi extend to reach the ESM and the ES. These structural data are discussed with respect to the importance of ES decalcification in providing the calcium necessary for mineralization of embryo's skeleton. In parallel, eggshell decalcification and weakening during incubation is likely to impair the ability of the ES to protect the embryo. It is assumed that the CAM could counteract this apparent weakening as an additional layer of physical, cellular and molecular barriers against environmental pressures, including pathogens, dehydration and shocks. However, such hypothesis needs to be further investigated.

How Egg Storage Duration Prior to Incubation Impairs Egg Quality and Chicken Embryonic Development: Contribution of Imaging Technologies.

Storing fertilised eggs prior to incubation is a frequent practice in commercial hatcheries to coordinate activities and synchronise hatchings. However, the conditions used to store eggs can have major impacts on egg quality and the subsequent viability of chicken embryos. While storage temperatures of 16-18°C are classically used in hatcheries, the duration of storage varies from three to more than 10 days. We explored the effect of storage duration (zero, three or 10 days; D0, D3 and D10, respectively) at 16°C, 80% relative humidity (RH) on egg quality (Broiler, Ross 308), using computed tomography (CT) and classical measurements (egg weight, eggshell strength, egg white pH, Haugh units, yolk index and colour). The results revealed that a storage duration of up to 10 days negatively affected some egg quality traits (yolk index and volume, air chamber volume and egg white pH). Eggs stored for three or 10 days were further incubated for 11, 13 or 15 days (37.8°C, 55% RH). Eggs were analysed by magnetic resonance imaging (MRI) and CT to assess the development of the embryo and internal egg changes occurring during incubation. First, data showed that the fertility and sex ratio of eggs were not affected by storage duration. However, the mortality of viable eggs was increased in the D10 group compared to the D3 group. Results of non-invasive imaging technologies revealed that the storage of eggs for 10 days impaired embryo growth as early as 11 days of incubation (decrease in brain and embryo volumes). Collectively, these data provide new evidence that the duration of egg storage negatively affects embryonic growth. They further corroborate that this parameter is likely to be crucial to synchronising embryonic stages and maybe reducing the hatching window, hence limiting the time spent by newborn chicks in hatchers. In addition, our results highlight that CT and MRI imaging technologies are useful non-invasive tools to evaluate egg quality prior to incubation and the impact of storage (or incubation) practices on developmental growth of the embryo.

Eggshell decalcification and skeletal mineralization during chicken embryonic development: defining candidate genes in the chorioallantoic membrane.

During chicken embryonic development, skeleton calcification mainly relies on the eggshell, whose minerals are progressively solubilized and transported to the embryo via the chorioallantoic membrane (CAM). However, the molecular components involved in this process remain undefined. We assessed eggshell demineralization and calcification of the embryo skeleton after 12 and 16 d of incubation, and analyzed the expression of several candidate genes in the CAM: carbonic anhydrases that are likely involved in secretion of protons for eggshell dissolution (CA2, CA4, CA9), ions transporters and regulators (CALB1, SLC4A1, ATP6V1B2, SGK1, SCGN, PKD2) and vitamin-D binding protein (GC). Our results confirmed that eggshell weight, thickness, and strength decreased during incubation, with a concomitant increase in calcification of embryonic skeletal system. In the CAM, the expression of CA2 increased during incubation while CA4 and CA9 were expressed at similar levels at both stages. SCL4A1 and SCGN were expressed, but not differentially, between the two stages, while the expression of ATP6V1B2 and PKD2 genes decreased. The expression of SGK1 and TRPV6 increased over time, although the expression of the latter gene was barely detectable. In parallel, we analyzed the expression of these candidate genes in the yolk sac (YS), which mediates the transfer of yolk minerals to the embryo during the first half of incubation. In YS, CA2 expression increases during incubation, similar to the CAM, while the expression of the other candidate genes decreases. Moreover, CALB1 and GC genes were found to be expressed during incubation in the YS, in contrast to the CAM where no expression of either was detected. This study demonstrates that the regulation of genes involved in the mobilization of egg minerals during embryonic development is different between the YS and CAM extraembryonic structures. Identification of the full suite of molecular components involved in the transfer of eggshell calcium to the embryo via the CAM should help to better understand the role of this structure in bone mineralization.

Antimicrobial Proteins and Peptides in Avian Eggshell: Structural Diversity and Potential Roles in Biomineralization.

The calcitic avian eggshell provides physical protection for the embryo during its development, but also regulates water and gaseous exchange, and is a calcium source for bone mineralization. The calcified eggshell has been extensively investigated in the chicken. It is characterized by an inventory of more than 900 matrix proteins. In addition to proteins involved in shell mineralization and regulation of its microstructure, the shell also contains numerous antimicrobial proteins and peptides (AMPPs) including lectin-like proteins, Bacterial Permeability Increasing/Lipopolysaccharide Binding Protein/PLUNC family proteins, defensins, antiproteases, and chelators, which contribute to the innate immune protection of the egg. In parallel, some of these proteins are thought to be crucial determinants of the eggshell texture and its resulting mechanical properties. During the progressive solubilization of the inner mineralized eggshell during embryonic development (to provide calcium to the embryo), some antimicrobials may be released simultaneously to reinforce egg defense and protect the egg from contamination by external pathogens, through a weakened eggshell. This review provides a comprehensive overview of the diversity of avian eggshell AMPPs, their three-dimensional structures and their mechanism of antimicrobial activity. The published chicken eggshell proteome databases are integrated for a comprehensive inventory of its AMPPs. Their biochemical features, potential dual function as antimicrobials and as regulators of eggshell biomineralization, and their phylogenetic evolution will be described and discussed with regard to their three-dimensional structural characteristics. Finally, the repertoire of chicken eggshell AMPPs are compared to orthologs identified in other avian and non-avian eggshells. This approach sheds light on the similarities and differences exhibited by AMPPs, depending on bird species, and leads to a better understanding of their sequential or dual role in biomineralization and innate immunity.

Properties, Genetics and Innate Immune Function of the Cuticle in Egg-Laying Species.

Cleidoic eggs possess very efficient and orchestrated systems to protect the embryo from external microbes until hatch. The cuticle is a proteinaceous layer on the shell surface in many bird and some reptile species. An intact cuticle forms a pore plug to occlude respiratory pores and is an effective physical and chemical barrier against microbial penetration. The interior of the egg is assumed to be normally sterile, while the outer eggshell cuticle hosts microbes. The diversity of the eggshell microbiome is derived from both maternal microbiota and those of the nesting environment. The surface characteristics of the egg, outer moisture layer and the presence of antimicrobial molecules composing the cuticle dictate constituents of the microbial communities on the eggshell surface. The avian cuticle affects eggshell wettability, water vapor conductance and regulates ultraviolet reflectance in various ground-nesting species; moreover, its composition, thickness and degree of coverage are dependent on species, hen age, and physiological stressors. Studies in domestic avian species have demonstrated that changes in the cuticle affect the food safety of eggs with respect to the risk of contamination by bacterial pathogens such as Salmonella and Escherichia coli. Moreover, preventing contamination of internal egg components is crucial to optimize hatching success in bird species. In chickens there is moderate heritability (38%) of cuticle deposition with a potential for genetic improvement. However, much less is known about other bird or reptile cuticles. This review synthesizes current knowledge of eggshell cuticle and provides insight into its evolution in the clade reptilia. The origin, composition and regulation of the eggshell microbiome and the potential function of the cuticle as the first barrier of egg defense are discussed in detail. We evaluate how changes in the cuticle affect the food safety of table eggs and vertical transmission of pathogens in the production chain with respect to the risk of contamination. Thus, this review provides insight into the physiological and microbiological characteristics of eggshell cuticle in relation to its protective function (innate immunity) in egg-laying birds and reptiles.

Storage Temperature or Thermal Treatments During Long Egg Storage Duration Influences Hatching Performance and Chick Quality.

This study was designed to improve the hatching performance, chick robustness and poultry health in the event of long-term egg storage and suboptimal age of the reproductive flock. A total of 9,600 eggs from one young breeder flock (28 weeks of age, batch B) and 9,600 eggs from an older breeder flock (59 weeks of age, batch E) were used (ROSS 308). Each batch was separated into three sub-groups and stored for 14 days. The first sub-group of eggs (Cool, group C) was stored at 11.6°C. The second sub-group of eggs (Warm, group W) was stored at 18.3°C with two pre-incubation on days 6 and 10 of the storage period. The final sub-group of eggs (Control, group Ct) was stored at 18.3°C throughout the storage period. Eggs were similarly incubated and hatched birds were raised on the same experimental farm. In both batches, embryonic development was significantly more advanced in W eggs than in C and Ct eggs ( p < 0.01). In both batches, C and W treatments decreased early embryonic mortality by more than 10% compared with Ct, decreased the proportion of late-hatched chicks and improved the percentage of first grade chicks: in batch E, 42% of Ct eggs were first grade chicks vs. 57% in group W and 59% in group C. Benefits were even higher in batch B, where only 60% of Ct eggs gave first grade chicks vs. 83% in others groups. The hatching rate was thus higher in groups C and W regardless of flock age: for batch B eggs, 85% hatched in W and 84% in C vs. 62% in Ct, while for batch E eggs, 59% hatched in W and 61% in C vs. 45% in Ct. Day-old Ct chicks from batch E were heavier than W and C ones, and heavier than W chicks from batch B ( p < 0.05). Long-term parameters on farm were not significantly different between groups. Thermal treatments during the storage of eggs from both young and old breeder flocks counterbalance the negative effects of prolonged egg storage on hatching rate, without altering chicken performance during rearing.