Avian Eggshell Research Articles

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.

Molecular and morphological analyses of avian eggshell excavated from a late thirteenth century earth oven

Using ancient DNA (aDNA) extracted from eggshell of the extinct moa (Aves: Dinornithiformes) we determined the species composition and number of eggs found in a late thirteenth century earth oven feature at Wairau Bar (South Island, New Zealand) – one of New Zealand’s most significant archaeological sites. Mitochondrial and nuclear DNA signatures confirmed this oven feature contained fragments of at least 31 moa eggs, representing three moa genera: Emeus; Euryapteryx; Dinornis. We demonstrate through the genetic identification of 127 moa eggshell fragments that thickness is an unreliable character for species assignment. We also present a protocol for assessing the preservation likelihood of DNA in burnt eggshell. This is useful because eggshell fragments found in archaeological contexts have often been thermally modified, and heat significantly increases DNA fragmentation. Eggshell is widely used in radiocarbon dating and stable isotope research, this study showcases how aDNA can also add to our knowledge of eggshell in both archaeological and palaeoecological contexts.

A novel disulfide-rich protein motif from avian eggshell membranes.

Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X4-C-X5-C-X8-C-X6 pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.

Crystallographic contribution to the vital effect in biogenic carbonates Mg/Ca thermometry

ABSTRACTThe processes involved in vital effects, defined as biological processes overriding environmental signals, are not well understood and this hampers the interpretation of environmental parameters such as seawater temperature. Insufficient knowledge is available about changes in physico-chemical parameters, in particular those related to crystallography, associated with biomineral formation and emplacement. This paper assesses the influence of crystallography on Mg2+ concentration and distribution in calcite biominerals of bivalved marine organisms, mussels and rhynchonelliform brachiopods, and considers the implications for Mg/Ca thermometry. In the mussel Mytilus edulis, changes in Mg2+ are not associated with crystallography; but in the brachiopod Terebratulina retusa, increases in Mg2+ concentrations (∼0·5–0·6 wt. ) are associated with the {0001 planes of calcite biominerals. A comparison between mussels and brachiopods with avian eggshells, which form at constant ambient temperature, also reveals that there is at least a common 0·1 wt. variation in magnesium concentration in these calcite biomineral systems unrelated to temperature or crystallography. Results demonstrate that the integration of contextual crystallographic, biological and chemical information may be important to extract accurate environmental information from biominerals.

A 700-year record of mercury in avian eggshells of Guangjin Island, South China Sea

Ancient eggshells over the past 700 years were extracted from an ornithogenic sediment profile on Guangjin Island, South China Sea. Based on SEM and nitrogen isotope analyses, we determined that neither post-depositional processes nor seabirds’ dietary changes had a large influence on eggshell Hg levels. The historical change of Hg in these eggshells was reconstructed. Eggshell Hg was a marker for past Hg deposition in marine environment. The eggshell Hg showed three small peaks at around 1300AD, 1600 AD and 1700–1750AD and rapid increase since 1800 AD. Before 1970 AD the Hg deposition in the Xisha area had global distribution characteristics, with increased Hg emissions due to global anthropogenic activities in industrial times. However, after 1970 AD, a further sharp increase up to present day occurred, implying that the Hg production center had gradually shifted from Europe and America to Asia.

Synthesis of three-dimensional calcium carbonate nanofibrous structure from eggshell using femtosecond laser ablation

BackgroundNatural biomaterials from bone-like minerals derived from avian eggshells have been considered as promising bone substitutes owing to their biodegradability, abundance, and lower price in comparison with synthetic biomaterials. However, cell adhesion to bulk biomaterials is poor and surface modifications are required to improve biomaterial-cell interaction. Three-dimensional (3D) nanostructures are preferred to act as growth support platforms for bone and stem cells. Although there have been several studies on generating nanoparticles from eggshells, no research has been reported on synthesizing 3D nanofibrous structures.ResultsIn this study, we propose a novel technique to synthesize 3D calcium carbonate interwoven nanofibrous platforms from eggshells using high repetition femtosecond laser irradiation. The eggshell waste is value engineered to calcium carbonate nanofibrous layer in a single step under ambient conditions. Our striking results demonstrate that by controlling the laser pulse repetition, nanostructures with different nanofiber density can be achieved. This approach presents an important step towards synthesizing 3D interwoven nanofibrous platforms from natural biomaterials.ConclusionThe synthesized 3D nanofibrous structures can promote biomaterial interfacial properties to improve cell-platform surface interaction and develop new functional biomaterials for a variety of biomedical applications.

Diversity of the Cuticle Layer of Avian Eggshells

The structure and mineral composition of eggshell cuticles were studied in 5 species of birds. The approximate thickness of the cuticle layer at the top of shell columns was about 1μm in the Red Junglefowl (Gallus gallus), about 130μm in the White Pelican (Pelecanus onocrotalus), about 10μm in the Japanese quail (Coturnix japonica), about 110μm in the Greater Flamingo (Phoenicopterus ruber roseus) and about 45μm in the Humboldt Penguin (Spheniscus humboldti). The matrix of the cuticle layer decalcified with EDTA was composed of vesicles in a variety of sizes in all birds. Major elements in cuticle materials detected by X-ray microanalysis were O, C, Ca and P, and their percentage numbers of atoms decreased in this order. The concentration of P was significantly higher in the cuticles of the quail, flamingo, and penguin than in those of the junglefowl and pelican. Ca mapping on electron-microscopic images showed strong signals in the shell layer and weaker ones in the cuticle layer, whereas P mapping showed that signals were mostly confined in the cuticle layer. X-ray diffraction analyses on the inside of the shell layer showed a profile of calcite crystals of calcium carbonates in all birds. In the cuticle materials, the profile was of calcite in the junglefowl, and a mixture of calcite and vaterite in the pelican. The profiles in cuticle materials of the quail, flamingo and penguin showed no specific signals, indicating that mineral compounds are amorphous in these forms. It was suggested that the diversity of mineral structures in the cuticle layer is caused by the presence of phosphorous, in addition to the structure of the cuticle matrix.

Shell thickness and pore density in relation to shell colouration, female characteristics, and environmental factors in the Collared Flycatcher Ficedula albicollis

Avian eggshell structure may have important consequences for embryonic growth and development, but relatively little is known about the factors responsible for variation in eggshell characteristics of wild birds. In this paper, we explored potential causes of variation in eggshell colour and structure (shell thickness and porosity) in the Collared Flycatcher (Ficedula albicollis). We analysed if eggshell colour is affected by shell structure or pigment level, and whether female traits, laying date, local breeding density, and clutch size affect shell thickness and pore density. We found that eggshell blue-green and UV colours were unrelated to shell thickness, pore density and egg size. Eggs with higher concentration of biliverdin showed lower UV reflection and higher reflection in the blue-green part of the spectrum. We found that females in better nutritional condition, indicated by their higher mass controlled for tarsus length, laid eggs with thicker shells. It is possible that females in better condition have more time available for searching calcium-rich food, and thus could produce eggs with stronger shells. However, female physical characteristics had no significant relationships with shell porosity. In contrast to our expectation, shell thickness and pore density were unrelated to local breeding density and laying date, though very late and repeat clutches were not sampled in our study. However, we found that eggs in larger clutches had lower pore density than eggs in smaller clutches, which may be expected if the rate of water loss and nest humidity are to remain constant in clutches of different egg numbers.

Comparison of micrometer- and scanning electron microscope-based measurements of avian eggshell thickness

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Evaluation of a glossmeter for studying the surface appearance of avian eggs

Gloss is a striking characteristic of avian eggshells that may affect both egg recognition and clutch predation. Previously, gloss has proved difficult to quantify objectively. Newly developed handheld glossmeters seem to offer a solution. We evaluated the use of a handheld glossmeter for a range of different eggshell surfaces, shapes and sizes. In all cases, their measurements were strongly affected by egg size and seem currently suitable only for very large eggs; the size of Rhea sp. eggs or larger. A glossmeter may, however, still prove useful to quantify other structures in comparative ornithology, with flat surfaces such as feathers or nest material.

Antimicrobial Activity of Lipophilic Avian Eggshell Surface Extracts

The avian eggshell cuticle is the waxy outermost layer of the mineralized eggshell in direct contact with the environment. In this study, lipophilic eggshell surface extracts from three domestic species were evaluated for their antimicrobial activity. Chicken and goose extracts demonstrated potent bactericidal activity against both Gram-positive and Gram-negative bacteria, while activity could not be detected for duck eggshell surface extracts. Using the chicken as a model species, evaluation of albumen, fecal material, and uropygial gland extracts eliminated these as a potential source of the observed activity. Results suggest that lipophilic components are incorporated into the egg during its formation and play a role in antimicrobial defense. This study represents the first successful extraction and evaluation of lipophilic antimicrobial components from the avian egg.

Impact of time since collection on avian eggshell color: a comparison of museum and fresh egg specimens

Studies of avian eggshell coloration have been a long-standing research focus in behavioral evolutionary ecology. Museum collections have provided a widely used resource because they allow efficient sampling across broad temporal, geographical, and taxonomic ranges, even for species that are rare and for which sampling in the wild is ethically or practically unwarranted. We used reflectance spectrophotometry across the avian visual spectrum to compare eggshell color of specimens of the song thrush (Turdus philomelos) in two museums (Natural History Museum, UK and Auckland Museum, New Zealand) with each other and with eggshells collected fresh in New Zealand. These data enabled us to test the effects of source and storage in different museums, as well as time since collection, across four metrics of eggshell coloration: blue-green and ultraviolet chroma, overall brightness, and the spectral coefficient of variation. Variation within an egg, within a clutch, and among clutches, was similar between the two museum datasets but different from those of fresh eggs. We found significant differences in all four metrics between the collections, and that fresh eggshells reflected stronger in the blue-green wavelength than in museum eggs. This difference is most likely due to different preservation techniques and storage histories. Furthermore, an effect of time since collection was only apparent in the blue-green chroma and was higher in more recent museum eggshell samples. Our results support the use of historic museum samples in intraspecific studies of shell coloration providing that efforts are made to compare specimens, which were collected during similar periods.

Variability in Avian Eggshell Colour: A Comparative Study of Museum Eggshells

BackgroundThe exceptional diversity of coloration found in avian eggshells has long fascinated biologists and inspired a broad range of adaptive hypotheses to explain its evolution. Three main impediments to understanding the variability of eggshell appearance are: (1) the reliable quantification of the variation in eggshell colours; (2) its perception by birds themselves, and (3) its relation to avian phylogeny. Here we use an extensive museum collection to address these problems directly, and to test how diversity in eggshell coloration is distributed among different phylogenetic levels of the class Aves.Methodology and ResultsSpectrophotometric data on eggshell coloration were collected from a taxonomically representative sample of 251 bird species to determine the change in reflectance across different wavelengths and the taxonomic level where the variation resides. As many hypotheses for the evolution of eggshell coloration assume that egg colours provide a communication signal for an avian receiver, we also modelled reflectance spectra of shell coloration for the avian visual system. We found that a majority of species have eggs with similar background colour (long wavelengths) but that striking differences are just as likely to occur between congeners as between members of different families. The region of greatest variability in eggshell colour among closely related species coincided with the medium-wavelength sensitive region around 500 nm.ConclusionsThe majority of bird species share similar background eggshell colours, while the greatest variability among species aligns with differences along a red-brown to blue axis that most likely corresponds with variation in the presence and concentration of two tetrapyrrole pigments responsible for eggshell coloration. Additionally, our results confirm previous findings of temporal changes in museum collections, and this will be of particular concern for studies testing intraspecific hypotheses relating temporal patterns to adaptation of eggshell colour. We suggest that future studies investigating the phylogenetic association between the composition and concentration of eggshell pigments, and between the evolutionary drivers and functional impacts of eggshell colour variability will be most rewarding.

Influence of Ovalbumin on CaCO3Precipitation duringin VitroBiomineralization

As a major constituent of egg white matrix, ovalbumin has long been perceived to be implicated in the formation of avian eggshells, in particular, the mammillary layer. However, very little is known about the detailed mechanism by which this protein mediates shell calcification. By the combined studies of AFM, SEM, and TEM, we have investigated the influence of ovalbumin on CaCO(3) precipitation under in vitro mineralization conditions. We observed that the influence was multifold. This protein modified the morphology of calcite crystals through a distinct anisotropic process with respect to the four crystal step edges. AFM characterization revealed that the modification was initiated at the obtuse-obtuse step corner and propagated predominantly along the obtuse steps. Furthermore, the protein favored the existence of unstable phases such as amorphous calcium carbonate and crystalline vaterite. In contrast, lysozyme, another protein also present in the system, played a very different role in modifying calcite morphology. The mechanistic understanding gained from this study is clearly also of practical significance in developing advanced inorganic CaCO(3) materials with the aid of morphological manipulation of crystalline structures via different protein mediation.

Fossil avian eggshell preserves ancient DNA

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.

Can museum egg specimens be used for proteomic analyses?

BackgroundMass spectrometry and proteomic analyses have become powerful tools for the analysis of proteins and peptides. Investigation of proteins contained in the various layers of the avian eggshell has focused entirely on domesticated species. It has been widely assumed that this existing research can inform the study of wild bird species despite the fact that the vast majority of the diversity in avian species (~95%) exists outside the Orders to which domestic and poultry species belong. Museum collections offer a potentially valuable source of material for studying composition of wild avian eggshell matrix proteins. We used museum and fresh eggshells of common quails Coturnix coturnix to compare the protein composition of their organic matrices. Four eggs of domestic chickens were analysed simultaneously as a control for comparison to the fresh and museum quail eggs. The determination of the proteins was carried out using enzymatic cleavage followed by high-performance mass spectrometry.ResultsWe found that some of the expected key eggshell proteins (3 out of 11) were not present in the samples of museum quail egg. These proteins were either entirely absent from the museum eggs or the technique was unable to detect them. There was no pattern in the absent proteins in the sense of protein function or where they are located within the eggshell.ConclusionWe conclude it is likely that such studies on museum specimens using a proteomic approach will be limited in coverage of proteins and may, therefore, be misleading.

The role of blunt egg pole characteristics for recognition of eggs in the song thrush (Turdus philomelos)

Summary Colour and speckling of avian eggshells play important roles in egg recognition behaviour. However, only a few studies have paid attention to whether specific parts of eggs may be more important than others in rejection decisions by hosts. We hypothesised that the blunt pole in song thrush (Turdus philomelos) eggs possesses important cues for recognition of own eggs because this is the most visible part of the egg in the nest. We examined host response towards conspecific eggs painted blue either at the blunt or sharp poles, and used unpainted conspecific eggs as a control. We found, as predicted, that the experimental eggs painted blue at the blunt poles were rejected significantly more often than the experimental eggs unpainted at this pole. Rejection rates of experimental eggs were higher with increasing differences in number of spots between the two poles of own eggs. Our results confirmed that the essential cues used for egg discrimination in song thrushes are found at the blunt egg pole.

Detecting pigments from colourful eggshells of extinct birds

The known chemical basis of diverse avian eggshell coloration is generated by the same two classes of tetrapyrrole pigments in most living birds. We aimed to extend the evolutionary scope of these patterns by detecting pigments from extinct birds’ eggs. In our samples biliverdin was successfully extracted from subfossil shell fragments of the blue-green egg-laying upland moa Megalapteryx didinus, while protoporphyrin was extracted from the beige eggs of two other extinct moa species. Our data on pigment detection from eggshells of other extant paleognath birds, together with published information on other modern lineages, confirm tetrapyrroles as ubiquitous and conserved pigments contributing to diverse eggshell colours throughout avian evolution.

Photodynamic antimicrobial activity of avian eggshell pigments

Pigmentation in avian eggshells appears to be associated with shell strength, temperature regulation, and camouflage. The pigments found in eggshells are mainly porphyrins, which have been utilized therapeutically as photosensitizers. Here, we examined the photoinactivation of gram-positive (Staphylococcus aureus, Bacillus cereus) and gram-negative bacteria (Escherichia coli, Salmonella enteritidis) by hen eggshells and their pigments. The results indicated that eggshells have a light-dependent antimicrobial activity against gram-positive, but not gram-negative, bacteria. Our results indicate the possibility that the natural pigments used therapeutically have evolved in nature as a defence system.

Determination of insoluble avian eggshell matrix proteins

The organic components of bones and other mineralized tissues have a high impact on the organization and deposition of calcium, and consequently influence the mechanical properties of those tissues. The extractable proteins of avian eggshells have been studied extensively and many of them have been identified; insoluble (non-extractable) proteins have been sparsely studied, however. In the work discussed in this paper we studied EDTA-insoluble proteins by gradual decalcification of eggshell with EDTA. The insoluble proteinaceous films were chemically treated with cyanogen bromide and the mixtures of large fragments obtained were gradually precipitated with salt. The separated fractions were digested with trypsin and analyzed by HPLC-MS-MS (ion trap mass spectrometer). Analysis of the entire eggshell matrix (without precipitation steps) only enabled 6 proteins to be determined (ovocalyxins 32 and 36, ovocleidin 17 and 116, clusterin, and ovalbumin). Pretreatment of the individual eggshell layers and gradual precipitation with salt markedly increased the number of proteins identified - 28 proteins were determined. We identified for the first time collagens I (two chains) and III in the eggshell matrix, and Kunitz-like protease inhibitor as a major shell matrix protein. Besides the above mentioned proteins we can also mention EDIL3, fibronectin, sulfhydryl oxidase, tubulin alpha 1, lysozyme, Dickkopf-related protein 3, keratins, and ovotransferrin. The relative abundances of proteins in all eggshell layers were determined using the exponentially modified protein abundance index (emPAI). In the cuticle layer seven proteins were identified, whereas 16 proteins were described in the palisade layer and 23 in the mammillary layer.