Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties.

Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. STATEMENT OF SIGNIFICANCE: Avian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds - it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms.

Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit.

BACKGROUND: 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. CONCLUSION: 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.

Ultrastructure of avian eggshell during resorption following egg fertilization.

For skeletal mineralization, the avian embryo mobilizes calcium from its calcitic eggshell. This occurs through dissolution of specific interior regions of the shell in a process that also weakens the shell to allow hatching. Here, we have examined eggshell ultrastructure during dissolution occurring between laying of a fertilized egg (with incubation) and hatching of the chick (Gallus gallus). We have focused on changes in shell mammillae where the majority of dissolution takes place. Using scanning electron microscopy, we describe differences in matrix-mineral structure and relationships not observed in unfertilized eggs (unresorbed eggshell). We document changes in the calcium reserve body - an essential sub-compartment of mammillae - consistent with it being an early, primary source of calcium essential for embryonic skeletal growth. Dissolution events occurring in the calcium reserve sac and in the base plate of the calcium reserve body, and similar changes in surrounding bulk mammillae structure, all correlate with advancing skeletal embryonic calcification. The changes in mammillae sub-structures can generally be characterized as mineral dissolutions revealing fine surface topographies on remaining mineral surfaces and the exposure of an extensive, intracrystalline (occluded) organic matrix network. We propose that this mineral-occluded network regulates how shell mineral is dissolved by providing dissolution channels facilitating calcium release for the embryonic skeleton.

Avian eggshell structure and osteopontin.

The avian eggshell primarily consists of calcium carbonate mineral (calcite) and matrix proteins. Here we review matrix-mineral relationships in the eggshell at the ultrastructural level using scanning and transmission electron microscopy, and describe the distribution of osteopontin (OPN) as determined by colloidal gold immunolabeling for OPN. A rich protein network integrated within the calcitic structure of the eggshell shows variable, region-specific organization that included layered fibrous planar sheets of matrix, thin filamentous threads, thin film-like surface coatings, vesicular structures and isolated proteins residing on cleaved {104} crystallographic faces of the eggshell calcite. Except for the vesicular structures, these matrix structures all immunolabeled strongly for OPN. Given the potent mineralization- inhibiting function of OPN, we discuss how this protein might regulate eggshell growth rate and inhibit calcification at specific compartmental boundaries to provide eggshell form.

Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro.

We investigated matrix-mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix-mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {104} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {104} calcite faces. OPN inhibited calcite growth in vitro at the {104} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.

Functional whole-colony screening method to identify antimicrobial peptides.

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides. Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aeruginosa and Salmonella typhimurium. These results validated this high throughput AMP identification assay based on filter bound colony array libraries and vital dye inclusion.

Antimicrobial activity of cuticle and outer eggshell protein extracts from three species of domestic birds.

1. The eggshell cuticle is the proteinaceous outermost layer of the eggshell which regulates water exchange and protects against entry of micro-organisms. In this study, we investigated the hypothesis that the cuticle may also reduce microbial contamination by providing a chemical defence. 2. Outer eggshell and cuticle protein was extracted from domestic chicken (Gallus gallus), duck (Anas platyrhynchos) and goose (Anser anser) eggs by HCl and urea treatment, respectively. Antimicrobial activity of the extracts against Gram-positive and Gram-negative bacteria was evaluated. 3. C-type lysozyme, ovotransferrin and ovocalyxin-32 were identified in all extracts by Western blotting. All extracts from all species demonstrated lysozyme enzymatic activity. Immobilised c-type lysozyme retained some enzymatic activity. Protein extracts demonstrated activity against Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis suggesting the action of antimicrobial proteins in addition to lysozyme. 4. The results suggest that the antimicrobial outer eggshell and cuticle proteins present in a number of avian species may be a mechanism which enhances avian reproductive success.

Comparative antibacterial activity of avian egg white protein extracts.

1. Egg white proteins from the eggs of domestic chicken (Gallus gallus), turkey (Meleagris gallopavo), duck (Anas platyrhynchos) and goose (Anser anser) were analysed in order to compare the antimicrobial activity of these products. 2. Albumen from each species was sampled and analysed by SDS-PAGE and Western blotting. Antimicrobial activity and lysozyme activity were measured. 3. Ovotransferrin and ovalbumin were identified in all species while c-type lysozyme was present in chicken, turkey and duck egg white samples, but not in goose. 4. Galliformes appear to possess albumens with greater antimicrobial activity than those of the Anseriformes. This can be attributed to higher concentrations of ovotransferrin and the broad acting c-type lysozyme.

Cuticle and pore plug properties in the table egg.

Food safety of table eggs is vital since many pathogens can contaminate the unfertilized egg, leading to increased risk of foodborne illness for consumers. The eggshell cuticle is the first line of defense to restrict the entry of egg-associated pathogens, such as Salmonella Enteritidis. The thickness and completeness of coverage of the cuticle layer are heritable traits that are strongly associated with egg resistance to bacterial penetration. The present study characterizes the chemical composition of the eggshell cuticle and structure of pore plugs from table eggs. Eggs collected from both brown and white egg laying Lohmann flocks (early, mid, and late lay) were either unwashed or washed. Pore plugs were characterized using scanning electron microscopy (SEM), and elemental composition was determined using energy-dispersive x-ray spectroscopy (EDS). SEM observations confirmed that the plug formed by the cuticle layer within the eggshell pore remains firmly lodged throughout the commercial washing process. The eggshell thickness and cuticle pore length visualized in brown eggs was significantly higher than in white eggs in hens of all ages. EDS analysis revealed that the pore inner surface was enriched in phosphorus and chemically different from the surrounding bulk eggshell mineral. Detailed assessment of the cuticle chemical composition was performed by Fourier transform infrared spectroscopy (FTIR). Washing of eggs removed cuticle from the eggshell surface. There was a trend of lower cuticle coverage with increasing hen age for white eggs. A significant reduction in the amount of proteins and phosphates and polysaccharides was observed in the cuticle of brown unwashed eggs with hen age. In white unwashed eggs, amides and lipids decreased with hen age; by contrast, the amount of sulfate was highest at mid-lay. The results from our research will assist selective breeding programs that target cuticle integrity and pore plug stability to enhance egg resistance to pathogen penetration and improve food safety.

Differentiation of a fibrin gel encapsulated chondrogenic cell line.

Hyaline cartilage has very limited regenerative capacity following damage. Therefore engineered tissue substitutes have been the focus of much research. Our objective was to develop a fibrin-based scaffold as a cell delivery vehicle and template for hyaline cartilage regeneration, and compare its cellular properties against monolayer and pellet culture for chondrogenic cells. The chondrogenic precursor cell line, RCJ 3.1C5.18 (C5.18), was chosen as a test system for evaluating the effect of various culture conditions, including cell encapsulation, on articular chondrogenic cell differentiation. The C5.18 cells in monolayer showed elevated expression of collagen II, an articular chondrogenic marker, but also markers for fibrocartilage differentiation (collagen I and versican) when cultured with chondrogenic medium as compared to basic maintenance medium. Pellets of C5.18 cells cultured in chondrogenic medium were histologically more organized in structure than pellets cultured in control maintenance medium. The chondrogenic medium cultured pellets also secreted an extracellular matrix that was comprised of type II with very little type I collagen, indicating a trend towards a more hyaline-like cartilage. Moreover, when cultured in chondrogenic medium, fibrin-encapsulated C5.18 cells elaborated an extracellular matrix containing type II collagen, as well as aggrecan, which are both components of hyaline cartilage. This indicated a more articular-like chondrogenic differentiation for fibrin encapsulated C5.18 cells. The results of these experiments provide evidence that the C5.18 cell line can be used as a tool to evaluate potential scaffolds for articular cartilage tissue engineering.

The inhibition of cyclic AMP-mediated exocytosis from rat parotid cells by calcium chelation is due to depression of cellular ATP.

The role of second messenger calcium in cAMP-mediated exocytosis from parotid cell aggregates has been assessed following extracellular (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA] and intracellular (quin2) calcium chelation. Only in the former case was inhibition (60-70%) of isoproterenol- and N6,O2-dibutyryl cAMP-stimulated amylase release observed. This inhibition was accompanied by a 70% decrease in cellular ATP levels. Depression of ATP levels with mitochondrial inhibitors was also correlated with inhibition of amylase secretion. Overall, our results suggest that depletion of cellular Ca2+ with EGTA leads to an inhibition of mitochondrial function and that these experimental conditions are therefore inappropriate for the evaluation of the role of cytosolic Ca2+ transients during stimulus-secretion coupling.

Calmodulin regulation of the ATP-dependent calcium uptake by inverted vesicles prepared from rabbit synaptosomal plasma membranes.

Calmodulin has been shown to activate the ATP-dependent Ca2+ uptake in inside-out vesicles which have been prepared from rabbit synaptosomal plasma membranes by the methodology of Gill et al. (Gill, D.L., Grollman, E.F. and Kohn, L.D. (1981) J. Biol. Chem. 256, 184-192). Following extensive washings of these membranes with EGTA/EDTA solutions, the Ca2+ uptake activity demonstrated an affinity for calmodulin of 30 nM and an affinity for Ca2+ of 2 microM. The activity was completely inhibited by the anticalmodulin compound R24571 (Ki congruent to 8 microM). The molecular weight of the ATPase molecule, revealed by a combination of the [125I]calmodulin overlay technique and [32P]phosphoenzyme electrophoresis, was 145 000. The overlay technique also revealed that the mechanism of activation is via a direct binding of calmodulin to the pump molecule.

Regulation of calcium accumulation and efflux from platelet vesicles. Possible role for cyclic-AMP-dependent phosphorylation and calmodulin.

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

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.

Distribution of calcium/calmodulin-dependent kinase 2 in the brain of Apteronotus leptorhynchus.

Antibodies directed against the mammalian alpha and beta subunits of calcium/calmodulin-dependent kinase 2 (CaMK2) and brain dissection were used for immunoblot analysis of these proteins in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the CaMK2alpha antibody labeled a single band of the expected molecular mass (approximately 50 kDa) for this enzyme in rat cortex and electric fish brain. CaMK2alpha was enriched in fish forebrain and hypothalamus and also strongly expressed in midbrain sensory areas. Western blots revealed that CaMK2beta antibodies labeled bands in an appropriate molecular mass range (approximately 58-64 kDa) for this enzyme in mammalian cortex and electric fish brain. However, a higher molecular mass band (approximately 80 kDa) was also labeled; because all these bands were eliminated by preadsorbtion with the CaMK2-derived peptide antigen, they may all represent CaMK2beta-like isoforms. We mapped the brain distribution of CaMK2 isoforms with emphasis on the electrosensory system. CaMK2alpha was present at high density in dorsal forebrain, hypothalamic nuclei, torus semicircularis, and tectum. It was also enriched in discrete fiber tracts in forebrain, diencephalon, and rhombencephalon. CaMK2beta-like isoforms were enriched in ventral forebrain, hypothalamic nuclei, torus semicircularis and the reticular formation. Unlike CaMK2alpha, CaMK2beta -like isoforms were predominantly present in cell bodies and rarely found in fiber tracts or neuropil. In the electrosensory lateral line lobe, CaMK2alpha was restricted to specific feedback fibers, i.e., tractus stratum fibrosum and its terminal field in the ventral molecular layer. In contrast, CaMK2beta-like isoforms were enriched in somata and dendrites of pyramidal cells and granular interneurons.

Inositol 1,4,5-trisphosphate receptor localization in the brain of a weakly electric fish (Apteronotus leptorhynchus) with emphasis on the electrosensory system.

Inositol 1,4,5-trisphosphate is a widespread intracellular second messenger that mobilizes intracellular Ca2+ stores. The inositol 1,4,5-trisphosphate receptor involved is associated with the endoplasmic reticulum in neurons. In mammalian brain, inositol 1,4,5-trisphosphate receptor-containing neurons are found in many diverse regions, with cerebellar Purkinje cells containing the highest density of these receptors. We used immunohistochemical methods to identify the distribution of inositol 1,4,5-trisphosphate receptor-containing neurons in the brain of the weakly electric fish and Western blotting to confirm that a protein similar to the inositol 1,4,5-trisphosphate receptor of mammalian brain was recognized in the fish brain. In the telencephelon, the dorsal forebrain regions had low amounts of inositol 1,4,5-trisphosphate receptor. In the diencephalon, only the nucleus tuberis posterior was moderately immunoreactive. In the mesencephalon, only the optic tectum contained cells with intense immunoreactivity, similar to our findings for the ryanodine receptor (G.K.H. Zupanc, J.A. Airey, L. Maler, J. Sutko, and M.H. Ellisman, 1992, J. Comp. Neurol. 325:135-151), which also mobilizes intracellular calcium. In the rhombencephalon, a subset of the pyramidal cells of the electrosensory lateral line lobe contained inositol 1,4,5-trisphosphate receptor. These cells have been shown to contain ryanodine receptor (Zupanc et al., 1992). However, unlike the ryanodine receptor, the distribution of inositol 1,4,5-trisphosphate receptor in these cells is constrained to the soma and proximal dendrites. This compartmentalization may indicate the limit of the range of second-messenger action. Other regions containing immunoreactive cells were the nucleus praeminentialis dorsalis (multipolar and boundary cells), nucleus medialis and crista cerebellaris, and the cerebellum, whose Purkinje cells were the most intensely labeled. The functional implications of inositol 1,4,5-trisphosphate receptor localization in the electrosensory lateral line lobe are discussed.

Stimulation of rat parotid secretion by cAMP analogues that synergistically activate the type II isoenzyme of the cAMP-dependent protein kinase.

Beta-adrenergic-stimulated parotid secretion is believed to be mediated by activation of the cAMP-dependent protein kinase (PK-A). However, the relative roles of the type I and II PK-A isoenzymes are still unclear. Combinations of site-selective, lipophilic cAMP analogues that synergistically activate each PK-A were used to investigate this problem. The selectivity of synergistic activation with these combinations was verified with the partially purified parotid PK-A isoenzymes, using kemptide as a substrate. Synergism in activation of PK-AII was only seen with 8-thiomethyl cAMP (8-TM) and N-6-benzoyl cyclic AMP (N6B), while PK-AI was only synergistically activated by 8-(6-aminohexyl) amino cyclic AMP (AHA) and N6B. Additive activation of each isoenzyme was observed for the combination of 8-TM and AHA. Rates of amylase secretion from dispersed parotid acini in response to secretagogues were determined with a coupled enzyme assay for amylase activity, which was adapted for use in a microplate reader. Cells were stimulated to secrete during 30 min with different doses (0.1-1.0 mM) and combinations of the cyclic nucleotide analogues. Alone, N6B was most effective in stimulating amylase secretion. The basal amylase secretory rate was stimulated by these secretagogues (0.44 mM) to the following extent: 53-fold (N6B), 8-fold (8-AHA), 2-fold (8-TM). In combination at a series of concentrations, only 8-TM + N6B produced synergistic stimulation of secretion, while AHA + N6B and 8-TM + AHA did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Extraction and quantification by ELISA of eggshell organic matrix proteins (ovocleidin-17, ovalbumin, ovotransferrin) in shell from young and old hens.

The eggshell matrix is mainly composed of proteins that are thought to influence shell formation and calcification and, thus, modify the resulting properties of the shell. We investigated the potential of some of these proteins as biomarkers of eggshell quality by developing a competitive indirect ELISA for quantifying ovotransferrin, ovalbumin, and ovocleidin-17 in eggshell extract. Eggshell fragments were demineralized in acetic acid (20%) and freeze-dried. The micro-extraction yield was markedly increased (>50%) when Tween 20 was added to the subsequent extraction and dialysis milieus. Microplates were coated with ovotransferrin and ovalbumin in a 0.1M carbonate-bicarbonate buffer, but ovocleidin-17 was fixed with acetone (-20 C, 20 min). Optimal dilutions of the monoclonal (ovotransferrin) and polyclonal (ovalbumin and ovocleidin-17) antibodies were 1/3,000, 1/25,000 and 1/4,000, respectively. The inhibition curves were optimized by preincubating the antibodies and proteins overnight. The intraassay coefficient (<5%), parallelism of the standards and samples curves, and recovery (101%) were satisfactory for ovotransferrin. Measurements of ovalbumin were less precise because of higher interassay variation and differences between the slopes of standard and sample inhibition curves. Ovocleidin-17 assays showed similar slopes for standard and eggshell extracts. Although the total protein in soluble matrix extracts was not affected by age, the concentrations of these proteins were higher in eggshell extracts from older hens compared with those from young hens: 1.98x for ovotransferrin, 1.86x for ovalbumin, and 1.58x for ovocleidin-17. The quantification of specific eggshell matrix proteins in shell of differing quality is, therefore, a promising tool for analyzing the origin of eggshell faults and may provide useful information for breeding programs.