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.

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.

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.

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.

Detection of Frozen-Thawed Duck Fatty Liver by MALDI-TOF Mass Spectrometry: A Chemometrics Study.

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen-thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen-thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing-thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen-thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.