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.     

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.     

蛋壳胶护膜研究进展

蛋产品安全一直是人们重点关注的问题,也是研究的热点。随着福利养殖的推广,鸡蛋被微生物污染的机率增大。除了加强饲养管理之外,强化鸡蛋自身保护屏障从而抵抗细菌入侵也十分重要。胶护膜是蛋壳最外层覆盖的无色透明膜,鸡蛋产出前1.5~2.0 h于子宫部形成,是蛋抵御微生物污染的重要屏障,研究表明胶护膜较厚的蛋不易被细菌入侵。鸡蛋蛋壳胶护膜主要由糖蛋白（90%）、多糖（4%）、脂质（3%）和以羟基磷灰石晶体形式存在的无机磷（3%）组成。胶护膜中含有溶菌酶C、卵转铁蛋白、ovocleidin-32等多种抗菌蛋白,可有效地阻碍细菌跨壳污染。此外,胶护膜能终止蛋壳矿化、帮助蛋产出前在子宫中翻转、调节水气交换以延长鸡蛋的保存时间。本文对胶护膜的结构、生物学功能、胶护膜品质评价方法及胶护膜质量的影响因素等方面进行了阐述,并对尚待解决的问题展开了探讨。     

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.     

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.     

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.     

Quality assessment of chicken eggshell cuticle by infrared spectroscopy and staining techniques: a comparative study

1. The cuticle is a very thin organic layer that coats the eggshell surface and plugs the eggshell pores preventing bacterial penetration. It also reduces eggshell permeability which is important to maintain internal quality of the egg. Thus, the eggshell cuticle quality is crucial to ensure the food safety and quality of eggs.

2. A new methodology to assess eggshell cuticle quality, based on attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), is compared with a more traditional method based on cuticle staining techniques.

3. Both techniques are useful to determine the amount of cuticle; however, the ATR-FTIR technique is independent of egg colour, more sensitive and provides complete information about the cuticle and its composition. Additionally, it provides information about eggshell permeability.

4. The methodology for cuticle quality assessment described in this work can be very useful for genetic selection programmes aimed to improve the safety and quality of eggs.     

Genetic parameters for eggshell traits in ostriches

1. A study was conducted on ～14000 ostrich eggs to estimate genetic parameters for eggshell traits that could benefit the hatchability of ostrich eggs. Traits measured included the number of pores on the eggshell, the average diameter of these pores, the total area of pores on the eggshell, permeability (pore area/shell thickness) and eggshell thickness.

2. Heritability estimates ranged from 0·16 for total pore area to 0·41 for the natural logarithm of pore count. The heritability estimates for water loss on 21 and 35?d (WL21 and WL35) of incubation were high at 0·23 and 0·24, respectively.

3. On a genetic level, pore count was negatively correlated with average pore diameter (?0·73) and shell thickness (?0·28), whereas it was positively correlated with total pore area (0·58), WL21 (0·24) and WL35 (0·34). The direct and maternal genetic correlations of pore count with total pore area (0·58) and permeability (0·59) were high and significant. Permeability was positively correlated to WL21 and WL35, both on the direct and maternal genetic levels.

4. The estimated genetic parameters indicate that it should be possible to select for the various eggshell traits in ostrich eggs, or for permeability and water loss. However, as a trait with an intermediate optimum, direct selection for permeability and other eggshell traits would not be straightforward, and the possible application of these results to improve hatchability of ostrich eggs in the future needs consideration.     

Bacterial shell contamination in the egg collection chains of different housing systems for laying hens

The bacterial eggshell contamination of eating eggs in different commercial housing systems; two conventional cages, one organic aviary system and one barn production, were compared. The total counts of aerobic bacteria and the total counts of Gram-negative bacteria on the shell were used to detect key points where contamination occurred and to study the progress of contamination in the egg collection and transportation chains. The key points in the chain were those where eggs accumulated on a short conveyor belt, initial shell contamination in the alternative housing systems and extra nest-boxes placed on the ground. The high bacterial load of floor eggs (>6.3 log CFU total aerobic flora/eggshell) explains why they cannot be used for eating. On average higher initial shell contamination with total counts of aerobic bacteria was found for eggs from the alternative housing systems compared to the conventional systems; respectively 5.46 compared to 5.08 log CFU/eggshell. However, initial contamination with total counts of Gram-negative bacteria on the shells was less in the alternative systems: 3.31 compared to 3.85 log CFU/shell. Initial bacterial shell contamination tended to correlate positively with the concentration of bacteria in the air of the poultry houses. Storing shell eggs, whether temporarily refrigerated or not, for 9 d or more, resulted in a decrease in bacterial eggshell contamination for both bacterial variables.     

Soluble protein constituents of the domestic fowl's eggshell.

1. The protein components of the domestic fowl's eggshell are believed to influence appreciably the mechanical properties of the shell and/or its biomineralisation. The purpose of this study was to compare the protein species composing the eggshell matrix in different parts of the shell structure, by SDS-PAGE and chromatography, utilising eggshell cleaned by different methodologies. 2. Protein species were identified whose absence was associated with the removal of the mammillary knobs. In particular, a prominent 81 kDa protein, as well as 38 and 54 kDa calcium-binding proteins, were concentrated within the mammillary layer, as was a 129 kDa insoluble protein. By contrast, soluble proteins of 54, 33, 22, and 14 kDa were enriched in the palisade layer. 3. Our results demonstrate that the mineralised layers of the fowl's eggshell possess a complex array of distinct proteins. The different proteins which have been detected in the mammillary and palisade layers may be related to the distinct crystallisation patterns of calcium carbonate in these zones of the eggshell.     

不同品种鸡蛋品质的比较研究

随机抽取40周龄尼克粉鸡、仿草鸡、苏禽黄鸡、青壳蛋鸡的蛋各100个,进行蛋品质测定。结果表明不同品种鸡蛋的蛋品质存在着差异,除蛋形指数差异不显著外,4种鸡蛋的蛋壳颜色和蛋黄颜色相互间存在极显著差异(P<0.01);尼克粉鸡蛋、苏禽黄鸡蛋与仿草鸡蛋及青壳蛋鸡蛋的蛋重相互间存在极显著差异;尼克粉鸡蛋、青壳蛋鸡蛋与仿草鸡蛋、苏禽黄鸡蛋的蛋黄比率存在极显著差异;尼克粉鸡蛋与仿草鸡蛋、苏禽黄鸡蛋、青壳蛋鸡蛋的蛋壳厚度存在极显著差异;4种鸡蛋的哈氏单位相互间表现出显著(P<0.05)或极显著的差异;蛋壳强度只在尼克粉鸡与仿草鸡之间有显著差异。     

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.     

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.     

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。     

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.     

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.     

Effects of some egg characteristics on the mass loss and hatchability of ostrich (Struthio camelus) eggs

1. This study was conducted to examine some egg characteristics and determine the effects of eggshell thickness and eggshell porosity on water loss and hatchability of eggs in ostriches. 2. Shell thickness did not correlate significantly with hatchability. However, eggs of low shell thickness lost more mass (13.03%) than those with intermediate (11.22%) and high (10.36%) shell thickness. Mass loss during incubation was higher in hatched (11.98%) than unhatched eggs (11.09%). Shell thickness was negatively correlated to egg mass loss (r = -0.65). 3. The pore density was correlated with hatchability. Hatchability was 50% lower in eggs with low pore densities (40.93%) than with high densities (80.94%). Pore density was positively correlated with egg mass loss (r = 0.63). Incubation mass losses of hatched and unhatched eggs were not significantly different. 4. Mean eggshell water vapour conductance (G) value and shell conductance constant (k) were 87.77 +/- 4.21 mg H2O/d/Torr and 2.44 respectively (n = 15). 5. Because of eggshell functional properties and resulting low egg mass loss hatchability is low when ostrich eggs are artificially incubated. The mass of eggs used in the experiment was relatively high and their eggshell water vapour conductance was low. As a result, egg incubation mass loss was lower than it should be. It is concluded that incubator humidity should be low (25%) to allow enough mass loss during incubation from the eggs.     

Influence of the microstructure on the shell strength of eggs laid by hens of different ages

1. The eggshell is a bioceramic material constructed of columnar calcite crystals preferentially oriented with their c-axis perpendicular to the shell surface. 2. The influence of microstructure (crystal size, shape and crystallographic orientation of crystal grains) on the mechanical properties of eggshells (shell strength) was investigated using eggs from hens of different ages. 3. There was a strong correlation between crystallographic texture and the strength of the eggshell in the case of eggs laid by young hens. The strength of eggshells increased as the preferential orientation of the crystals constituting the eggshell decreased. 4. By comparing two age populations, the effect of hen age on eggshell properties was evaluated. In general, eggshells from aged hens had a lower breaking strength (less than half that of those laid by young hens) and showed a greater variability in their structural properties such as thickness, grain morphology and crystallographic texture. 5. Texture analysis revealed that shells from eggs laid by aged hens have two preferred crystal orientations, after (001) and (104), compared with mainly one, after (001), in eggs laid by young hens. 6. These observed changes in eggshell properties could be due to changes in the organic matrix of the eggshell associated with ageing of the hens.     

Eggshell characteristics and penetration by Salmonella enterica serovar Enteritidis through the production period of a layer flock

1. Egg weight, shell thickness, number of pores, cuticle deposition and ability of Salmonella enterica serovar Enteritidis (SE) to penetrate the shell were determined for eggs from one layer flock through the entire production period. 2. Penetration was assessed by filling the eggs with a selective medium that allowed visualising Salmonella growth on the inside of the shell and membrane complex. After inoculation of each shell with on average 2.59 log cfu, the eggs were stored for up to 20 d at 20 degrees C and 60% relative humidity (RH). 3. On average 38.7% of the eggshells became penetrated. Mostly penetration occurred on d 3. Although it affected all shell characteristics studied, hen age did not significantly influence eggshell penetration. 4. No correlations were observed between any of the shell characteristics studied and the ability of SE to penetrate the shell. The growth of SE on the shell is of major importance because shell contamination at 20 d of storage and SE penetration were highly correlated.     

Effect of lighted incubation on embryonic growth and hatchability performance of two strains of layer breeder eggs

1. Eggs from two layer-type breeder flocks (ISA-W vs Leghorn) between 30 and 45 weeks of age were used in 4 trials to study the effects of lighted incubation on embryonic growth from 5 to 18 d of age and hatchability per cent (HP). The physical dimensions, eggshell characteristics and conductance (EC) of eggs of the two strains were compared. 2. Eggs were set in an incubator on trays either in the dark-control or under two tubes of 20-watt white fluorescent light during the first 18 d of incubation. The light intensity ranged from 1230 to 1790 lux at the surface of the eggs. Eggs were transferred to dark hatching compartments at d 19 of incubation. 3. The genetic make-up of birds influenced the physical dimensions and eggshell characteristics of eggs. ISA-W eggs had higher weight, surface area (ESA), volume (EV), width, EC, shell volume and HP, and lower ESA:EV ratio, per cent shell, shell density and dead embryos than those of Leghorn eggs. 4. Lighted incubation increased daily embryonic growth (mg/d) and HP by 3.9 and 5.9%, respectively, when compared with the dark-control incubation. 5. The physical dimensions and eggshell characteristics of eggs influenced the effects of lighted incubation on embryonic growth and HP. Lighted incubation increased embryonic growth and HP of ISA-W eggs. The increase in embryonic growth and HP of the Leghorn eggs was not significant. 6. The genetic make-up of birds influenced the physical dimensions and eggshell characteristics of their eggs and these differences in the characteristics of hatching eggs influenced embryonic growth and HP when incubated under light.