Ultrastructural analysis of the eggshell: contribution of the individual calcified layers and the cuticle to hatchability and egg viability in broiler breeders

1. Scanning electron microscopy was used to assess the relationship between the layers of the eggshell and egg viability. The relative thickness (absolute and %) of the mammillary, palisade, vertical crystal and cuticle layers relative to the total eggshell were measured over a 30-week laying period in 2 broiler breeder lines having different hatchability rates. 2. A significant difference in hatchability of 5.5% between the breeder lines was observed. However, there was no significant difference in either egg viability or fertility between lines. 3. The relative thickness of the individual calcified layers and the cuticle did not differ between lines. Therefore, variations in the thickness of the individual eggshell layers are unlikely to explain the 5.5% difference in hatchability rate between lines. 4. The thickness of the calcified region did not alter as the broiler hen aged. However, the percent contribution of each calcified layer significantly differed over the egg production period. 5. The cuticle was significantly thinner at the beginning and at the end of the laying period compared to 38 weeks of age. 6. No significant correlation was observed between egg viability and the relative thicknesses of the mamillary, palisade or cuticle layers. In contrast, there was a positive relationship between egg viability and the thickness of the vertical crystal layer. 7. The percent contribution of the mammillary and palisade layers was similar in the thinnest and thickest eggshells, suggesting conservation of the proportions of these layers independent of eggshell thickness.     

Relationship between shell structure and movement of Salmonella enteritidis across the eggshell wall.

1. Ultrastructural analyses of the eggshells of one strain of commercial layers indicated that the cuticular layer is rarely present as an even covering over the shell surface and the paired shell membranes are invariably pitted with holes larger than bacterial dimensions. 2. The above conditions pertain irrespective of the age of the laying bird. 3. Bacterial penetration of the eggshell is independent of pore numbers. 4. In the absence of a functional cuticle and with the shell membranes removed, bacteria are checked in their movement by structural modifications in the mammillary layer.     

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.     

An egg ist ein ei,es un huevo,est un oeuf

1. Many of the mechanical tests devised to measure shell quality are inadequate because they fail to recognise the complex interaction between the organic and inorganic aspects of the eggshell. 2. Twelve structural modifications have been observed at the level of the mammillary layer and their presence correlated with a variety of environmental stress events. Occurring as they do in the basal layers of the shell, these morphological variants influence its mechanical properties. 3. The organic matrix proteins which complex with the calcium carbonate derive from a variety of sites within the oviduct and vary in their location within the fully formed shell. In vitro mineralisation reveals the significance of these proteins in the crystal growth mechanism. 4. The isolation and identification of the protein moiety in well-structured eggshells is an essential prerequisite to understanding the abnormalities in crystal growth observed in the shells of older birds challenged by disease and other undesirable 'on farm' events. 5. The eggshell is the daily indicator of the bird's harmony with its environment and as such provides a readily accessible and non invasive measure of welfare. The integration of these data with those derived from behavioural and biochemical testing should provide industry with a reliable numerical welfare index.     

高温条件下蛋壳结构变化的研究

蛋壳是鸡蛋保持新鲜、防止变质的重要组成部分。将高温条件下鸡蛋与新鲜蛋的蛋壳进行比较,结果发现,高温条件下蛋壳外表面的护膜局部脱落;乳锥层的纤维变细,乳锥减少;蛋壳结构也发生了变化,柱状层中孔的密度和直径均有所增大。     

Morphological and histochemical observations of the organic components of ostrich eggshell

The organic component of the avian eggshell can be divided into 3 portions, the shell membranes, the matrix and the cuticle. These have been well characterised in the chicken but little has been published with regard to the ostrich (Struthio camelus). A number of recent studies have indicated that the cause of intra-shell embryonic deaths in the ostrich is similar to intra-shell embryonic deaths that occur in the chicken. These deaths in the chicken are associated with the loss of or damage to the waxy cuticle and other organic components of the eggshell, which is reported to be absent in the ostrich eggshell. In this study, preliminary morphological and histochemical analyses, at the level of the light and electron microscope, have characterised the various organic components of the ostrich eggshell. The results of the histochemical and electron microscopical analyses suggest that there may only be 1 shell membrane in this species, which could play a major role in the limitation of bacterial penetration to the embryonic chamber The shell membrane has a distinct elemental profile as determined by EDS analysis. The matrix is shown to decrease in mesh size from the mammillary layer to the vertical crystal layer. The closer packing of the mesh may indicate the presence of a morphologically discernible termination signal to calcification or the remnants of an evolutionary calcified cuticle. The matrix of the pores may also form a defensive barrier against bacterial invasion, which could be damaged as a result of dipping the eggs before incubation.     

Identification of the major proteins of the organic matrix of emu (Dromaius novaehollandiae) and rhea (Rhea americana) eggshell calcified layer

1. The eggshell is a composite consisting of 95% calcite and an organic matrix. 2. While many proteins of the chicken eggshell matrix have already been identified, little is known about the matrix of other birds. 3. Isolation of the emu and rhea eggshell matrix and analysis of its major constituents showed that the predominant components were C-type lectin-like proteins related to those of ostrich, chicken and goose. 4. Serum albumin, vitelline membrane outer layer protein I (VMO-I) and the turpentine-induced acute phase serum protein 18-B were identified as minor components of the emu shell matrix. Both eggshell matrices also contained a novel proline- and alanine-rich protein. 5. Like ostrich, and unlike chicken and goose, both emu and rhea eggshell matrix contained two different C-type lectin-like proteins as major components, indicating that the occurrence of two proteins of this family may be widespread among ratites.     

Features of eggshell formation in guinea fowl: kinetics of shell deposition, uterine protein secretion and uterine histology

1. Rate of calcium carbonate deposition, duration of eggshell formation, organic composition of the uterine fluid, morphology of the egg shells and histochemistry of the uterus were studied in guinea fowl to analyse the origin of such thick, strong egg shells. 2. The egg shell was linearly deposited from 6.4 h to 21.8 h after the oviposition of the previous egg. The rate of egg shell deposition was similar to that in laying hens. However, the duration of linear shell deposition was increased by 2.1 h relative to that in hens. This explained the increased egg shell weight observed in the guinea fowl. 3. Intervals between oviposition of intra-clutch eggs were 24 h throughout the laying period. Ovulation occurred just after oviposition of the previous egg in the guinea fowl, as previously observed in hens but the duration of egg white protein deposition, of plumping and of initiation of shell mineralisation were all 1.5 h shorter than in domestic hen. 4. Uterine fluid can only be collected during the growth and terminal phase of shell formation. The electrophoretic profiles of the uterine fluid differed between phases and were somewhat different from those previously observed in the hen. Ovalbumin and ovocleidin-17 were both present in the uterine fluid and also in egg shell extract. Ovocleidin-17 was predominant during the growth phase. 5. The histology of the uterus differed slightly in guinea fowl compared to hens. Ovocleidin and ovalbumin are both secreted by the tubular glands. 6. Examination of radial ultrathin sections of eggshell showed, above the mammillary layer, intricate interlacing of adjacent exospherite in guinea fowl in contrast to the continuous columnar microstructure in hens. 7. The kinetics of egg shell deposition largely explains the increased egg shell weight of guinea fowl. The organic matrix proteins may be associated with the contrast between the structural organisation of the guinea fowl egg shell and that of the hen egg shell.     

Effect of production system and flock age on egg quality and total bacterial load in commercial laying hens

The shell of the egg is essential in providing shape to the egg and ensuring safe packaging of the internal contents; however, shell defects have been shown to increase the risk of microbial contamination of eggs. Eggs were collected from commercial cage and free-range flocks at the ages of 25, 35, 45, 55, 65, and 75 wk. From each collection per flock, 30 eggs were processed for the eggshell and egg internal quality determination, 30 eggs for cuticle estimation, 30 eggs for mammillary layer ultrastructural features scoring, and 60 eggs for egg microbial enumeration. Translucency score and shell reflectivity were significantly higher in free-range eggs and increased with flock age in both production systems. Egg weight, shell weight, percentage shell, shell thickness, albumen height, Haugh unit, and yolk color were higher for cage eggs. The amount of cuticle was higher in cage eggs and fluctuated with flock age in both production systems. For the mammillary layer ultrastructural variables, a significant effect of production system and flock age was observed for early fusion, Type A bodies, and Type B bodies, whereas aragonite, depression, erosion, and hole were rarely observed. Variability of mammillary cap size, the incidence of poor mammillary cap quality, incidence of late fusion, alignment, Type A bodies, Type B bodies, and cubic cone formation were greater in the free-range versus cage system and increased with flock age in both production systems. The incidence of confluence and early fusion were greater in cage eggs and decreased with age in both production systems. Significantly lower total microbial load was observed for cage compared with free-range eggs, but the overall bacterial load recorded in this study was low. It can be concluded that cage eggs were better in overall quality when directly compared with free-range eggs.     

Ultrastructural characteristics of ostrich eggshell: outer shell membrane and the calcified layers

The ultrastructure of the eggshell of the domestic hen has been well researched and structural studies of other avian species, such as the ostrich, often base their interpretation of egg shell structure on that of the chicken. In the ostrich, lowered hatchability and hatching trauma may be due to shell ultrastructural abnormalities. In the present study the ultrastructure of the calcified portion, and the outer shell membrane (OSM), of domesticated ostrich eggshells was investigated using standard electron microscopic techniques. Transmission and scanning electron microscopy studies demonstrated intimate contact between cup-shaped structures present on the OSM and the mammillary layer of the calcified portion of the shell. The initial calcium carbonate growth of the calcified shell was of a dendritic nature with nucleation sites on the surface of the cup's contents. The dendritic growth gave way to a more randomly-orientated, smaller crystallite growth structure, which changed in form as it neared the vertical crystal layer (VCL). The VCL is described as being both amorphous and 'crumbly' depending on the plane of fracture. These observations suggest that firstly, initial calcification is contained within the cups and is then directed outwards to form the shell and that secondly, the VCL may contain an evolutionary, calcified cuticular layer. These observations serve as a baseline for studies investigating the effect of shell structure and strength on hatchling trauma and the influence of maternal diet.     

Effect of beta‐aminoproprionitrile on eggshell formation

1. Eggshells are bioceramic‐biopolymer composites made by a cell‐mediated deposition of an extracellular matrix which drives the organisation of the inorganic phase. Ultrastructurally, eggshells are composed of shell membranes, mammillary knobs, palisade, and cuticle. Shell membranes are two nets of type X collagen‐containing fibrils. On to these membranes, the mammillary knobs, that is, the crystal nucleation sites, are deposited. Type X collagen is highly cross‐linked and insoluble.

2. In order to evaluate the role of type X collagen cross‐linking on eggshell formation, hens were injected with different doses of β‐aminoproprionitrile, which specifically interferes with cross‐link formation.

3. Changes in egg size and shape were observed. Scanning electron micrographs analysis of these eggs demonstrated marked changes in crystal growth and shell membrane structure and arrangement. A dot‐blot analysis, using a monoclonal antibody against chicken type X collagen, shows a dose‐dependent increase in shell membrane collagen extractability.

4. It is concluded that the formation of β‐aminoproprionitrile‐sensitive cross‐links among the type X collagen molecules of the shell membranes play an essential role in normal eggshell formation.     

Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers

Eggs exhibiting eggshell apex abnormalities (EAA) were evaluated for changes in shell characteristics such as strength, thickness, and ultrastructure. Mycoplasma synoviae (MS) infection was confirmed by serological assay along with isolation of MS from the trachea and oviduct. Changes in eggshell quality were shown to be statistically significant (p < 0.01). We also identified ultrastructural changes in the mammillary knob layer by Scanning Electron Microscopy. While eggs may seem to be structurally sound, ultrastructural evaluation showed that affected eggs do not regain their former quality. In our knowledge, this is the first report describing the occurrence of EAA in Korea.     

Structure and relationship of the mammillary gore to membrane fibres and initial calcification of the avian Egg Shell 1

1. The interior of the mammillary knob is shown to be composed of: (1) outer shell membrane fibres that penetrate the basal portion of the knob and become associated with (2) The mammillary core, and (3) the bulk calcification of the main knob.

2. The mammillary knob consists of radial crystals which are arranged in a hemisphere on the outer shell membrane fibres, and are composed of a central zone which is a homogeneous solid, crystalline in nature, and an outer heterogeneous vesiculated zone.

3. The walls of the mammillary knob are continuous with the base and sides of the mammillary core radial crystals; the dorso‐central zone of the core crystals are exposed to a cavity that communicates with the upper zones of the mammillary knob and palisade layer via the central channel. The dorsal surface of the core cavity is formed by a layer of unorganised crystals of the mammillary knob proper.

4. Initial calcification of the knob begins at the base and sides of the mammillary core and expands over the organic core as the walls of the knob thicken during subsequent calcification. Incomplete fusion of the central expansions from peripheral knob areas results in the central channel.     

鸡蛋壳内部组成、构造及其质量的基因调控技术

蛋壳质量问题一直困扰着蛋鸡养殖业的发展.本文结合蛋壳形成过程,针对蛋壳组成、构造特点,分析蛋壳钙和基质蛋白沉积量对蛋壳质量的影响,结合传统蛋壳质量改善措施及现存问题,探讨钙结合蛋白-D28k和基质蛋白外源基因注射蛋鸡调控蛋壳质量的应用可能性.随着研究的深入,外源基因表达调控蛋鸡输卵管和子宫内相关产物表达量,将为改善蛋壳质量提供新的技术手段.但蛋壳内部组成、构造和蛋鸡体内相关腺体分泌物对蛋壳形成机制影响等基础研究尚不完善,是制约外源基因调控技术在蛋壳质量改善研究领域应用的主要因素.     

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.     

中草药复合添加剂对高龄蛋鸡生产性能、蛋品质及蛋壳超微结构的影响

研究旨在探讨中草药复合添加剂对高龄蛋鸡生产性能、蛋品质及蛋壳超微结构的影响.选取900只440日龄海兰褐蛋鸡,分为对照组、试验组,每组各5个重复,每个重复90只鸡.对照组饲喂基础饲粮,试验组在基础饲粮中添加0.2％中草药复合添加剂,预饲期7d,试验期56 d.结果 显示:与对照组相比,试验组死淘率与破(软)蛋率分别降低...     

Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens

The effect of moult on eggshell mechanical properties, on composition and concentrations of organic matrix components and on eggshell microstructure was investigated. The observed changes were studied to understand the role of organic matrix and eggshell microstructure in eggshell strength. Moult was induced by zinc oxide (20 g zinc/kg diet) in 53 ISA Brown laying hens at 78 weeks of age. No difference was observed for egg or eggshell weights after moult. In contrast, moult improved the shell breaking strength (28.09 vs 33.71 N). After moult, there was a decrease in the average size of calcite crystals composing the eggshell and in their heterogeneity, whereas crystal orientation remained basically the same. After moulting, the total protein concentration in eggshell increased slightly. The comparisons of SDS-PAGE profiles of the organic matrix constituents extracted before and after moulting showed changes in staining intensity of certain bands. After moult, bands associated with main proteins specific to eggshell formation (OC-116 and OC-17) showed higher staining intensity, while the intensity of the egg white proteins (ovotransferrin, ovalbumin and lysozyme) decreased. ELISA confirmed the decrease in ovotransferrin after moult. Its concentration was inversely correlated with breaking strength before moult. These observations suggest that changes in eggshell crystal size could be due to changes in organic matrix composition. These changes may provide a mechanism for the improvement in shell solidity after moulting.     

不同周龄母鸡鸡蛋及不同蛋壳质地鸡蛋的蛋品质比较

本文比较了同一品种不同周龄(26、40周龄和64周龄)母鸡所产蛋的蛋壳品质和蛋品质,包括蛋重、蛋壳相对重、蛋形指数、蛋壳强度、蛋壳厚度、蛋黄颜色、蛋黄指数、蛋黄比率、哈氏单位、蛋清pH值。之后,还比较了该鸡种不同蛋壳质地鸡蛋(正常蛋和砂壳蛋)之间上述指标的差异。结果表明:在不同周龄母鸡所产蛋之间,蛋重、蛋黄指数、蛋黄比率、哈氏单位等均存在显著差异(p<0.05),而蛋壳厚度之间差异不显著(p>0.05)。此外,蛋重、蛋形指数、蛋黄颜色、蛋黄比率随着母鸡周龄的增加呈上升趋势,而蛋壳强度、蛋壳相对重、哈氏单位、蛋黄指数、蛋清pH值呈下降趋势。最后,蛋壳质地不同的鸡蛋(正常蛋与砂壳蛋)之间,蛋重、蛋壳相对重、蛋壳强度、蛋清pH值之间存在极显著差异(p<0.01),而其他参数之间差异不显著(p>0.05)。     

鸡蛋壳的超微结构研究

本研究采用高分辨率的扫描电镜观察海赛克斯鸡蛋壳的超微结构,详细分析了其结构模式。蛋壳从内到外包括壳膜、锥体层、柱状层、表面晶体层、覆盖层五部分。同时研究结果表明,鸡蛋壳强度的大小可能与壳膜层壳膜的厚度,壳膜纤维的粗细,锥体层乳头间的空隙大小以及覆盖层上裂隙的深浅和数量有关。     

Physical and mechanical properties of eggshell as affected by chicken breed and flock age

ABSTRACT

1. A study was conducted to evaluate the relationship among physical and mechanical properties of the eggshell, as affected by breed and hen’s age.

2. Data on eggshell quality (external characteristics and derived measurements) were obtained from 322 laying hens, from three breeds (Fayoumi, Dandarawi and Hy-Line Brown) during the laying cycle, starting from 38 wks of age for four experimental periods (38, 46, 54 and 62 wks).

3. Eggs obtained from the Fayoumi breed exhibited the highest shell thickness and breaking force. There was a linear improvement in eggshell quality attributes associated with hen’s age up to 54 wks, thereafter a deterioration was found for all breeds.

4. Generally, eggs laid by native breeds (Fayoumi and Dandarawi) had better mechanical properties compared to those produced by the commercial strain (Hy-Line Brown). In addition, the interaction between breed and hen’s age was not significant for any physical property or mechanical attribute. There was a highly significant (P ≤ 0.01) positive correlation between the breaking force and either eggshell toughness or shell thickness, and regression analyses suggested that eggshell toughness was the best predictor for breaking force, followed by shell thickness.