Eggshell Ultrastructure Research Articles

Influence of red light on reproductive performance, eggshell ultrastructure, and eye morphology in Thai-native hens

In total, 120 Thai-native pullets (Gallus domesticus) aged 18 wk were housed in floor pens, located in a conventional open-sided shed under natural daylight (12L:12D) and randomly divided into 3 groups; Groups 1 (DF) and 2 (DR) were reared under natural daylight and supplemented with fluorescent or red light, respectively, whereas group 3 (R) was maintained in light-controlled pens and exposed only to red light. The red light was produced by light-emitting diodes. All treatments were provided with 16 h of light per day (16L:8D) during a 26-wk egg-laying period, and there were 4 replicate pens of 10 hens for each treatment. Photostimulation of these light sources was initiated at 18 wk of age and subsequent effects on reproductive performance were observed during the experimental period. Morphological characteristics of the eyes and eggshell microstructure were examined at the end of the study. Feed and water were provided ad libitum. There were no significant differences in BW, feed intake, egg weight, egg quality, or mortality rate due to the treatment. Pullets in the R and DR treatment groups commenced egg production significantly earlier than those in the DF treatment group. In early-season egg production (0-8 wk), cumulative egg number was significantly (P < 0.05) higher for the R treatment (25.9 eggs/hen) than for the DR (20.9) and DF (19.5) treatment groups. No significant differences in total egg production per hen occurred among the treatment groups. At 2 wk following photostimulation, hens in the R treatment group had significantly (P < 0.05) higher serum estradiol concentrations compared with hens in the other treatment groups. Neither eggshell structure nor eye morphology was affected by the treatments. It was concluded that the spectrum of red light did not affect live performance, egg production, egg quality, eggshell microstructure, or eye morphology of Thai-native hens, except for in accelerating sexual development. The light-emitting diode lighting system would be beneficial for energy savings and the reduction of rearing costs.

Analysis of Elemental Composition of the Eggshell before and after Incubation in the Loggerhead Turtle (Caretta caretta) in Oman

To date, there are limited studies on loggerhead turtle (Caretta caretta) eggshell ultrastructure and its elemental composition. Eggs were collected from turtle nests immediately after oviposition and post hatching. Three eggshell layers were recognized. The outer calcareous layer consists of loose nodular units of different shapes and sizes with loose attachment between the units, resulting in numerous spaces and openings. Each unit consists of CaCO3 crystals in aragonite (99%) and calcite (1%). The middle layer has several strata with numerous openings connecting the calcareous and the inner shell membrane. Crystallites of the middle layer are a mix of amorphous material with aragonite (62%) and calcite (38%). The inner shell membrane has numerous reticular fibers mixed predominantly with halite (NaCl) and small amounts of sylvite. Thermogravimetry analysis of the calcareous showed a low exothermic peak at 425°C, which corresponds to a transitional phase from aragonite to calcite. A high endothermic peak at 814°C corresponds to decomposition of calcite CaCO3 to CaO and CO2. Electron diffraction confirmed the presence of NaCl halite crystal. A significant difference was found in the percentage of elements and crystal configurations in the three layers. This study has value in assessing the emergence success in this endangered species.

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.

PHYLOGENETIC ANALYSIS OF REPRODUCTIVE TRAITS OF MANIRAPTORAN THEROPODS AND ITS IMPLICATIONS FOR EGG PARATAXONOMY

Abstract: A phylogenetic analysis of reproductive and oological (egg) traits of theropod taxa allows determination of the sequence in which these traits evolved in Theropoda. Our results indicate that several avian reproductive traits, such as adults sitting on eggs, asymmetrical eggs, unornamented eggshell surface, and complex eggshell ultrastructure, were already present in non‐avian maniraptorans, and could have evolved in more basal theropods. In addition, non‐avian maniraptorans laid two eggs at a time and orientated their eggs subvertically or subhorizontally in their nests, features not retained by neornithine birds. Based on our cladistic analysis it is also possible to infer the phylogenetic affinity of ootaxa of unknown parentage: Protoceratopsidovum was laid by a maniraptoran more derived than oviraptorids, and Parvoolithus probably belonged to a Cretaceous bird. Finally, our analysis reveals that many of the high‐level categories of egg parataxonomy (morphotypes and basic types) are unnatural groupings (i.e. non‐monophyletic). We recommend that these high‐level categories be abandoned because oofamilies are sufficient to categorize egg taxa.

The ultrastructure of sea turtle eggshell does not contribute to interspecies variation in fungal invasion of the egg

The eggshells of green ( Chelonia mydas (L., 1758)), loggerhead (Caretta caretta (L., 1758)), hawksbill ( Eretmochelys imbricata (L., 1766)), and flatback ( Natator depressus (Garman, 1880)) sea turtles nesting in eastern Australia were examined by scanning electron microscopy to determine if the ultrastructure was contributing to interspecific variation in fungal invasion of eggs. The eggshells of all species investigated were of similar structure (outer inorganic layer of aragonite crystals and an inner organic fibrillar layer) and of similar thickness. Well-defined pores that would allow direct entry of fungal hyphae or spores were not present in any species. It was concluded that the eggshell ultrastructure does not allow direct access by fungal hyphae or spores and does not contribute to interspecific variation in the vulnerability of loggerhead sea turtle eggs to fungal infection.

Testing phylogenetic implications of eggshell characters in side-necked turtles (Testudines: Pleurodira)

Amniote egg and eggshell morphology is a rich source of characters to link aspects of reproductive biology with systematics. Extensive work concerning both anatomy and phylogenetic assignability has been done on fossil bird and dinosaur eggs, but little is known for extant sauropsids. The utility of eggshell characters for phylogenetic analyses is tested and discussed for extant side-necked turtles (Pleurodira), and the diversity of egg ultrastructure is examined in several species. Egg gross morphology and eggshell ultrastructure of 12 species of extant side-necked turtles was documented using scanning electron microscopy. Thirteen eggshell characters were scored and mapped on a composite phylogeny and ancestral character states were reconstructed. Many of the characters do not show a phylogenetic signal according to a test comparing the number of steps on the chosen phylogeny with that on randomly generated trees. The presence of conservative, clade-supporting features could be demonstrated, and the following clades are supported by several characters: the Elseya– Emydura entity, short-necked Australasian chelids, is backed by two characters, and two additional characters could potentially support this group. Three characters support the monophyly of South American chelids, whereas two characters argue for the exclusion of Hydromedusa, a long-necked form resembling Australian chelids rather than South American forms, from this clade.

Avian eggshell mineralization: biochemical and functional characterization of matrix proteins

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

Heterodera glycines: eggshell ultrastructure and histochemical localization of chitinous components

The eggshell in most nematodes consists of an outer vitelline layer, a middle chitinous and an inner lipid layer. Earlier work with eggs of Heterodera glycines suggests the presence of two chitinous layers but the vitelline layer was not observed. From our observation the outer chitin layer described in past literature is actually a vitelline layer. Histochemical analysis has demonstrated that chitin is absent from the outer envelope. Electron microscope observations of the eggshell show a waxy appearance and osmium staining consistent with that of the proteinaceous vitelline layer found in other nematodes. Lectin localization also shows that the eggshell continues to develop past fertilization with the delivery and integration of eggshell precursors. Contrary to previous reports, we propose that the ultrastructure of the eggshell H. glycines follows the common three-layer structure observed in other nematodes. Index Descriptions and Abbreviations: BSA, bovine serum albumin; egg; eggshell; electron microscopy; fine structure; hatching; Heterodera glycines; histochemical; nematode; parasite; PBS, phosphate buffered saline; RT, room temperature; SEM, scanning electron microscopy; SDS, sodium dodecyl sulfate; soybean cyst nematode; TEM, transmission electron microscopy; WGA, wheat germ agglutinin; WGA-CG, wheat germ agglutinin-colloidal gold; WGA-HRP, wheat germ agglutinin-horseradish peroxidase; vitelline

Correlation between shell colour and ultrastructure in pheasant eggs

1. The histochemistry and ultrastructure of pheasant eggs were compared on the basis of blue or brown shell colour. 2. Differences in lectin histochemistry of the outer shell membrane calcification surface indicate a biochemical disruption of the calcification sites in blue eggs. 3. Significant differences were observed in all aspects of eggshell ultrastructure with blue eggs having thinner shells with structural defects. 4. Poor hatchability of blue eggs may reflect high rates of weight loss associated with a defective eggshell.

Phylogenetic and taxonomical relationships of the eight species in the melanogaster subgroup of the genus Drosophila (Sophophora) based on the electrophoretic mobility of the major chorion proteins and the eggshell ultrastructure

The eight species of the melanogaster species subgroup of the genus Drosophila were compared to each other with respect to: (a) the electrophoretic mobility of the major chorion proteins under SDS–PAGE; (b) the overall eggshell ultrastructure. The phylogenetic tree obtained using the first criterion showed that the eight species are divided into three clusters that contain the species of the melanogaster complex (D. melanogaster, D. sechellia, D. mauritiana and D. simulans), the two species of the yakuba complex (D. yakuba and D. teissieri) and the erecta complex species (D. erecta and D. orena). This clustering is in agreement to the generally accepted phylogeny of the subgroup based on exclusive criteria. The eggshell ultrastructure of the species at the main body of the egg revealed that the phylogenetically closely related species exhibit similar eggshell structure. We conclude that the criteria applied in this study could be eventually used in groups with unknown phylogeny to trace the phylogenetic relationships of the species.

Phylogenetic and taxonomical relationships of the eight species in the melanogaster subgroup of the genus Drosophila ( Sophophora ) based on the electrophoretic mobility of the major chorion proteins and the eggshell ultrastructure

The eight species of the melanogaster species subgroup of the genus Drosophila were compared to each other with respect to: (a) the electrophoretic mobility of the major chorion proteins under SDS–PAGE; (b) the overall eggshell ultrastructure. The phylogenetic tree obtained using the first criterion showed that the eight species are divided into three clusters that contain the species of the melanogaster complex (D. melanogaster, D. sechellia, D. mauritiana and D. simulans), the two species of the yakuba complex (D. yakuba and D. teissieri) and the erecta complex species (D. erecta and D. orena). This clustering is in agreement to the generally accepted phylogeny of the subgroup based on exclusive criteria. The eggshell ultrastructure of the species at the main body of the egg revealed that the phylogenetically closely related species exhibit similar eggshell structure. We conclude that the criteria applied in this study could be eventually used in groups with unknown phylogeny to trace the phylogenetic relationships of the species.

Organic matrix composition and ultrastructure of eggshell: A comparative study

1. The avian eggshell is a biomineralised composite ceramic consisting of calcium carbonate embedded in an organic matrix. Matrix components are supposed to be involved in the control of mineralisation, crystallographic texture and biomechanical properties of eggshell. 2. The structure and eggshell matrix composition of various domesticated bird species were compared to gain insight into the universality of the eggshell mineralisation process. 3. The SDS-PAGE profiles of soluble eggshell matrix were specific within groups of birds (a: laying hen, breeder hen, quail, pheasant and possibly turkey; b: guinea fowl; c: duck and goose) but some of the protein bands were common to all groups. 4. Analogies between species were confirmed by Western blotting using hen protein antibodies. Ovocleidin-17 (OC-17) and ovalbumin were revealed in all species (except quail for OC-17). Lysozyme was present only in hen eggshell. Another egg white protein: ovotransferrin showed a positive signal in hens, turkey and quail. Osteopontin was observed in laying and breeder hens and quail. 5. Different proteoglycans were localised to discrete regions within the eggshell. Dermatan sulphate was observed within the matrix of the calcified shell of all species except quail which contained chondroitin-6- sulfate. Keratan sulphate was observed in mammillary bodies of breeder and laying hen, quail, pheasant and turkey while chondroitin sulphate was also present in guinea fowl and duck. 6. The general structural organisation of the different avian eggshells was similar but specific differences were observed in the ultrastructure of the mammillary layer. Species of the same taxonomic family could be grouped according to their structural analogies: breeder hen, turkey and pheasant resembled that of the domestic fowl. Guinea fowl was unique. Goose and duck were quite similar with large and confluent mammillary bodies. 7. Some matrix components are therefore common to eggshells of various species but more information is needed to relate differences in matrix composition between taxonomic groups with differences in ultrastructure.

EGGSHELL ULTRASTRUCTURE IN ARGULUS JAPONICUS (BRANCHIURA)

ABSTRACT Eggshell ultrastructure is described in a branchiuran, Argulus japonicus. Growing oocytes of various sizes are found on the outer surface of the ovary. They are protruded outward, covered by a thin basal lamina of the ovarian epithelium which connects them on the ovarian surface. Many microvilli develop on the surface of oocytes from the late previtellogenic stage. These microvilli elongate to raise the basal lamina from the oocyte surfaces. In the early vitellogenic oocytes, large and small electron-dense granules appear and increase in number in the outer and inner regions, respectively, of the perivitelline space between the basal lamina and the oocyte surfaces. In the late vitellogenic oocytes, other large electron-lucent granules appear and increase in number in the innermost region of the perivitelline space. Mature eggs in the oviduct have a two-layered eggshell: the outer layer composed of large electron-dense granules and the inner layer of small electron-dense granules and large electron-lucent granules. Some unique features on the origin and formation of the eggshell in A. japonicus are discussed.

Relationship of Eggshell Ultrastructure and Shell Strength to the Soundness of Shell Eggs

Factors affecting the soundness of shell eggs are of primary concern to egg processors due to substantial financial losses from cracked and leaker eggs. Ultrastructural analyses were used to examine the palisade layer width and mammillary knob layer thickness of sound, cracked, and leaker eggshells. Subjective observations were also made. There was no significant difference (P > 0.05) in the width of the palisade layer or the mammillary knob layer among sound, cracked, and leaker eggshells. The eggshell strengths of sound, cracked, and leaker eggs were evaluated using puncture force and shell thickness measurements. Sound eggshells were found to have a significantly (P < 0.05) higher mean puncture force (35.3 N) than cracked (30.4 N) and leaker (28.4 N) eggshells. The mean puncture force values for cracked and leaker eggshells were not significantly different from each other (P > 0.05). Regression and correlation analyses indicated a significant correlation (P < 0.01; r = 0.61) between palisade layer width and puncture force. The mean shell thickness values for cracked (0.36 mm) and leaker (0.35 mm) eggs were significantly lower (P < 0.05) than those of sound eggs (0.38 mm), although cracked and leaker eggshell thickness did not differ significantly (P > 0.05). The correlation coefficient between puncture force and shell thickness was significant (P < 0.01; r = 0.56), indicating a possible relationship.

Ultrastructure and characteristics of eggshells of the olive ridley turtle (Lepidochelys olivacea) from Gahirmatha, India.

The structure of a chelonian eggshell is of prime importance for the developing embryo. It acts as a protective covering as well as mediator in the exchange of heat and water. The fresh eggshell of the olive ridley (Lepidochelys olivacea) turtle displays a variety of structural forms in their aragonite framework. Its netted substrate, loose texture and poorly organised crystallites favour the easy exchange of air and water during its development. The shell consists mostly of calcium in carbonate form and K, Mg, Fe, Ni, Pb, Zn, Co, Cr, Cu, Mn and Cd in traces. Ba and Sr in traces are also a feature of the thermal analysis graph. Phosphorous was not detected in the shell and its absence eliminates an earlier suggestion of it being a key factor in the development of aragonite crystals in the inorganic structure of marine turtle eggshells. Its combustion characteristics, which have not been previously reported, are recorded.

Reflections on the origin of the amniote egg in the light of reproductive strategies and shell structure

On the basis of evolutionary pattern of reproductive strategies (r‐ and K‐selected animals) and the ultrastructure of modern amniote eggshells, a new model of the origin of the amniote egg is presented. In contrast to the well‐known idea of Romer (1957) that the egg came first while adult reptiles in the Carboniferous remained in water, it is suggested that the early evolution of the amniote egg (including the gradual formation of membranes) happened within the aquatic realm. Increasing enlargement of eggs and yolk is interpreted as an adaptation of reproduction strategies to lakes with poor nutrient contents. The first accumulation of Ca‐ions in the outer membrane, paralleled by many modern noncalcified lepidosaurian eggs, was a process of detoxification, according to new ideas in biomineralization. The function of the shell, to protect the embryo against microbial activity and to prevent water loss, which is necessary for the preamniote egg to become a fully terrestrial egg, was the terminal step in thi...

Eggshell ultrastructure in Onychogomphus forcipatus unguiculatus (Vander Linden) (Odonata: Gomphidae)

The eggshell ultrastructure of the exophytic riverine dragonfly Onychogomphus forcipatus unguiculatus (Odonata: Gomphidae) is presented using transmission electron microscopy. A homogeneous vitelline envelope is surrounded by a thick, many-layered endochorion and a relatively thick, homogeneous exochorion. The micropylar projection is covered by a jelly-layer. The structure of the shell is intermediate between known endophytic and exophytic eggshells in the order. A possible evolution of jelly-covered exophytic eggs from endophytic riverine ancestors is discussed.

Ultrastructure and taxonomy of ostrich eggshells from upper Palaeolithic sites of India

Ultrastructure and taxonomy of ostrich eggshells from upper Palaeolithic sites of India