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.     

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.     

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.     

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.     

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.     

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.     

New approach of testing the effect of heat stress on eggshell quality: mechanical and material properties of eggshell and membrane

1. The effect of high temperature on eggshell quality was investigated by measuring the mechanical and material properties of shell and membranes. 2. Heat exposure resulted in a decrease in zootechnical performance and eggshell thickness, increase in egg breakage, and unchanged egg shape index. 3. The static stiffness (Kstat), dynamic stiffness (Kdyn) and modulus of elasticity of the eggshell were not significantly affected by high temperature. Membrane prolongation increased significantly while membrane attachment strength and breakage strength tended to decrease and increase, respectively. The relationships between these variables were changed by high temperature. 4. Neither Kstat nor Kdyn could give a reasonable explanation for the changed eggshell quality induced by heat stress. The decreased eggshell thickness and changed properties of shell membrane may be responsible, at least partially, for the decreased shell quality of eggs from heat-stressed hens.     

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.     

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。     

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。     

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.     

The effect of dietary calcium source, concentration and particle size on calcium retention, eggshell quality and overall calcium requirement in laying hens

1. Four different sources of calcium in isonitrogenous and isoenergeric diets were fed to laying hens for 2 weeks when they were 56 and 57 weeks old. The calcium source blends were as follows: 29% fine limestone (LF)+71% large limestone (LG), 32% LF+68% eggshell, 32% LF+68% oyster shell, 50% LF+50% LG. The contents of these blends of calcium in the diets were as follows: 103.3, 93.3, 93.3 and 93.3 g/kg, respectively. 2. The coefficients of calcium retention were significantly higher in 50:50 LF:LG (0.578) and 32:68 LF:eggshell (0.576). The midnight feeding significantly improved the coefficient of calcium retention in all mixtures except 50:50 LF:LG. 3. In the mixtures 29:71 LF:LG and 32:68 LF:oyster shell, there were significantly greater eggshell quality, eggshell weight, eggshell thickness and eggshell strength. Midnight feeding had no significant effect on eggshell quality. 4. In the ration with oyster shells, 96.5% of the retained calcium was deposited in the eggshell, but in rations 32:68 LF:eggshell and 50:50 LF:LG the utilisation was only 73.9 and 78.6%, respectively. 5. To ensure good quality eggshells in the last third of production, the recommendation for calcium is 4.1 g/kg (900 g/kg dry matter, feed intake 110 g/d). As a source of calcium in this stage of production, a feed mixture containing two-thirds large particles should be used (limestone grit or oyster shell).     

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.     

Influence of strain and flock age on geometrical and mechanical attributes of eggs produced from Japanese quail birds

We investigated how the geometrical and mechanical properties of eggshell of Japanese quail are affected by strain and flock age. Two strains of quail (white and gray) were used in the current experiment. The results showed that there was no significant difference for all geometric measurements due to strain effect. Eggs produced from the older birds showed significantly higher (P < 0.01) values compared with younger age for all studied traits. Eggs produced from quails at 22 weeks had a significantly (P < 0.01) darker yolk color than that of the younger age. Superiority in shell thickness, shell weight, and breaking force was detected in eggs of gray quails compared with white quails. On the other hand, the eggs from white quails had significantly higher values for static stiffness and Young's modulus as compared with those of gray counterparts. A significant decrease (P < 0.01) was found for fracture toughness and Young's modulus in eggs of aged birds. A significant negative relationship was found between the breaking force and both static stiffness and Young's modulus. A significant positive relationship was observed between breaking force and both shell thickness and shell percentage. The phenotypic correlation between eggshell breaking force and toughness was relatively high.     

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.     

Measuring the eggshell strength of 6 different genetic strains of laying hens: techniques and comparisons

1. Eggshell quality was compared in 6 different strains of laying hens. Three strains were commercial; the three others were experimental. 2. Four different variables describing the strength of eggshells were investigated. Three of them were the classical ones eggshell thickness, shell stiffness measured during quasi-static compression and breaking force. Dynamic stiffness, introduced by Coucke (Ph.D. Thesis, KU Leuven, 1998), was the 4th. The fact that this measurement is dynamic could be helpful in genetic selection for eggshell breakage, because forces applied to the egg in practice are dynamic, rather than static. 3. Hisex White hens produce eggs with the strongest eggshell, in terms of all 4 eggshell variables. However, their shell quality in terms of breaking force did not remain constant over the laying period, unlike 4 other strains. 4. All strains showed a decline in quasi-static stiffness over time. 5. The eggshell thickness of three strains showed a decline over time. 6. Dynamic stiffness remained constant or improved in all strain. 7. All variables describing the mechanical eggshell strength gave different information.     

Eggshell penetration of hen's eggs by Salmonella enterica serovar Enteritidis upon various storage conditions

1. The survival and penetration of Salmonella enterica serovar Enteritidis (SE) inoculated on the eggshell was examined upon storage for up to 20 d at real-life conditions (15 to 25 degrees C and 45 to 75% relative humidity (RH)). 2. Penetration was assessed by emptying the egg contents and filling the eggs with a selective medium that allowed visualising Salmonella growth on the inside of the shell and membrane complex. 3. The study of survival on the eggshells was based on viable counts and showed that numbers of surviving organisms decreased over time. Survival was inversely related to storage temperature and RH. Although the average counts decreased over time, a limited proportion of shells carried high numbers of SE at all storage conditions. 4. Penetration spots were observed earlier using an increased storage temperature due to increased growth rates of SE on the agar. After 20 d of storage a similar percentage (c. 44.7%) of eggshells became penetrated, irrespective of the storage conditions tested in this study. 5. The higher the Salmonella shell contamination at the end of storage, the higher the probability that the eggshell was penetrated. Salmonella shell counts exceeding 4 log cfu yielded more than a 90% probability of eggshell penetration occurring.     

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.     

Quantitative expression of candidate genes affecting eggshell color

There are three pigments that affect the color of an eggshell: protoporphyrin, biliverdin and biliverdin‐zinc chelate. Protoporphyrin is the main pigment in brown and light‐brown eggshells, whereas very little protoporphyrin is found in white eggshells. Eggshell protoporphyrin is derived from the heme formation in birds. Coproporphyrinogen III oxidase (CPOX) and ferrochelatase (FECH) represent rate‐limiting enzymes for the heme‐biosynthetic pathway. Breast cancer resistance protein (BCRP), feline leukemia virus receptor (FLVCR), and heme‐responsive gene‐1 (HRG1) serve as primary transporters for both protoporphyrinogen and heme. Finally, four organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. Here we measured gene expression levels in key tissues of egg‐producing hens. We analyzed three different types of hens that generated distinct eggshell colors: white, pink or brown. Our data revealed three ways in which eggshell color was genetically influenced. First, high‐level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High‐level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, as high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. As such, we have identified seven genes in which expression levels in different tissues were associated with eggshell color.