Influence of dietary osteolathyrogens on the ultrastructure of shell and membranes of eggs from laying hens

1. Laying hens were fed osteolathyrogens, either semicarbazide hy‐drochloride at 0.3 g/kg or β‐aminopropionitrile fumarate at 0.6 g/kg, to examine their effects upon the ultrastructure of shell and shell membranes by scanning electron microscopy.

2. Effects of the 2 lathyrogens were similar. Compared with the highly‐branched network of fine fibres in normal membranes, there is widespread lack of separation of fibres in lathyritic specimens and hence, uneven distribution of nucleation sites. Pores are also uneven in size and distribution, which can account for increased permeability.

3. Establishment between shell and membranes is reduced. Within the cone layer both type A and type B mammillary bodies occur. Large interstitial spaces and late fusion of the palisade layer indicate reduced resistance to fracture.     

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.     

The use of the plasma chemistry unit as an aid to the scanning electron microscope study of avian egg‐shell structure

1. In order to study the mammillary layer of the avian egg shell by scanning electron microscopy, it is necessary to separate the outer shell membrane from the calcified shell.

2. Chemical methods of effecting membrane removal are difficult to standardise due to variations in the strength of the membrane‐shell bond.

3. The use of reactive gas plasma provides an alternative, more efficient method for removing membranes without the risk of damage to underlying crystalline structures.     

Bacteriology of washed and unwashed eggs. II. Penetration of Salmonella bacteria through the eggshell

The ability of the eggshell to resist penetration of Salmonella bacteria was studied in unwashed and in hand washed eggs, and in eggs, which had been subjected to industrial, large-scale machine washing under strictly controlled conditions.In one part of the investigation, concerning unwashed and machine washed eggs, a defined amount of a broth culture of Salmonella typhi murium was applied on the shell surface, whereafter the eggs were incubated at 4°C for eight weeks or at 30°C for 12 days. At the end of the storage period, the egg contents were examined for the presence of Salmonella. No Salmonella bacteria were detected in the 120 eggs investigated.In another part of the investigation, dealing with 200 unwashed, 200 hand washed and 200 machine washed eggs, a broth culture of Salmonella was applied on the shell surface of eggs, whose contents had been poured out through an opening. In every second egg of each kind, the shell membranes had been scraped off over an area, corresponding to the placement of the bacterial inoculum. The eggshells were filled with enrichment broth and the occurrence of Salmonella bacteria in this broth was examined after incubation.In the eggs with intact shell membranes, the frequencies of Salmonella positive enrichment broths were for unwashed eggs 0.12, for machine washed eggs 0.10, and for hand washed eggs 0.22. The corresponding frequencies in eggs with removed shell membranes were 0.27 (unwashed), 0.42 (machine washed), and 0.60 (hand washed).No statistical difference occurred between unwashed and machine washed eggs when the shell membranes were intact. When the shell membranes were removed this difference was statistically almost significant. The difference between machine washed and hand washed eggs with intact shell membranes was statistically almost significant, while the same difference was highly significant in eggs with removed shell membranes.The author assumes, from the results of the present and a preceding investigation, that the applied washing procedure in no way harms any essential storage properties of uncracked eggs. Considering the known occurrence of hand washing of eggs in Swedish farms — a treatment with documentedly unfavourable influence — it would be desirable if all eggs could be subjected to machine washing under strictly controlled conditions at the grading stations. Such an arrangement ought to result in an increase in the average bacteriologic-hygienic quality of eggs for market.     

The behaviour of mixed bacterial infections in the shell membranes of the hen's egg

The selection of Gram‐negative bacteria from a heterogenous flora on the shell membranes of the hen's egg occurred during two phases of the infection process. When the infection was confined to the shell membranes, the Gram‐negative bacteria achieved dominance over the Gram‐positive organisms. This dominance was accentuated when the albumen was invaded and it was noted, moreover, that a particular strain of Gram‐negative bacteria predominated whereas several strains co‐existed in the shell membranes.     

Organic matrix morphology and distribution in the palisade layer of eggshells sampled at selected periods during lay

1. 1 cm2 pieces of eggshells from a commercial battery flock were plasma etched to remove the outer shell membranes. 2. They were decalcified using EDTA (200 g/l, pH 6.9 to 7.0) in paraformaldehyde (20 g/l) and 25% gluteraldehyde (20 ml in 0.98 l) in phosphate buffer, then prepared for light and transmission electron microscopy. 3. Light microscopy revealed a differential distribution of matrix material within all 3 regions of the palisade layer at the beginning of lay. 4. Transmission electron microscopy revealed a more even distribution of matrix at the beginning of lay, although morphological differences were observed. At the end of lay all 3 regions showed an increase in % matrix and vesicles/10 cm2 of micrograph compared to the middle and beginning of lay periods. 5. It is hypothesised that matrix vesicles are involved in the regulation of the physiochemical environment within the forming eggshell and that the decline in shell quality associated with the end of lay is related to a concomitant change in matrix quality.     

Differences in measurement of membrane thickness in hens' egg shells

The widely varying values given for the membrane thickness in hen's eggs suggest that a critical comparison of the methods of measurement is necessary. The membrane thickness was measured in pieces of isolated membranes, in decalcified sections and in ground sections. The first method gave the lowest values, probably owing to the impossibility of separating the whole of the membranes from the calcified shell. The other two methods showed an average difference in results of only 7.2–10.5 per cent. It was concluded that the actual thickness of dry egg shell membranes can only be measured in sections and that, without correction, such values should not be compared with those taken in isolated membranes.     

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.     

Relationship between the shell membrane‐shell bond and shell deformation in hens’ eggs

1. The influence of the shell membranes on shell strength was studied in 21 Single Comb White Leghorn eggs.

2. Shell deformation and shell thickness were plotted for each egg and the regression line was calculated. The difference between the observed shell deformation and that predicted by the regression line was calculated for each egg and this distance, with changed sign, was called the shell deformation index.

3. The force needed to separate the shell membranes from a 10 mm wide strip of the shell was determined by a tensile testing machine and was termed attachment strength.

4. The correlation between shell deformation index and attachment strength was highly significant (r=0.88, P<0.0005). Thus the shell membranes contribute to shell strength, probably by serving as a reinforcement of the crystalline part of the shell.     

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。     

The distribution and carbohydrate composition of the organic matrix in hen egg shell

After the removal of membranes and cuticle, successive arbitrary layers were dissolved with dilute HC1 from shells laid by a single Thornber “ 404 “ hen. The rate of solution was inversely related to the nitrogen content at a given level but, for a given nitrogen content, was faster from the inside of the shell than from the outside. Matrix is distributed unevenly through the mineralised shell, the concentration of nitrogen and carbohydrates increasing to a maximum about two‐thirds of the way through the shell from the inside. With the exception of hexose, these concentrations then decrease to values as low, or lower than those found on the inside of the shell. The content of chondroitin sulphates in the matrix is fairly constant while that of sialic acid decreases from the inside to the outside. Non‐chondroitin sulphate polysaccharides are more highly concentrated in matrix in the inner and outer strata of the shell than in the region of maximum matrix concentration. Aspects of shell composition and calcification are discussed in relation to these results.     

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.     

A note on the structure and iron‐binding properties of egg‐shell membranes

1. When a solution of ferric ammonium sulphate was added to shell membranes of the domestic fowl, iron infiltrated the mantle (cortex) surrounding the cores of the individual fibres of the membranes.

2. Contraction of warm eggs in ice‐cold colloidal iron caused flooding of pore canals and contamination of the underlying shell membranes with this element.

3. Appreciable contamination of the inner shell membrane with iron persisted for 25 d in infertile eggs stored at 37.5 °C.     

A study of the progressive deposition of shell in the shell gland of the domestic hen

It is known that the organic matrix of the shell is not distributed evenly throughout the calcified portion and an experiment has been described in which incompletely shelled eggs were removed from a group of domestic hens after being in the shell gland for various known periods.

Rate of shell deposition and the timing of the deposition of the various layers in the shell has thus been determined, and it was found that shell deposition was initially slow for the first 3 h in the gland and thereafter more rapid, at a constant rate of 322 mg h^?1, until termination after 17 or 18 h. The narrow layer low in matrix at the top of the mammillary layer of the shell was laid down during the 7th to 9th hour in the gland and did not appear to be associated with any change in shell deposition rate. It was also found that the characteristic pole to pole thickness patterns of shells were present at the earliest measurable stage, that is after about 8 h in the gland.     

Ultrastructural Studies of Chicken Embryo Chorioallantoic Membrane during Incubation

Structural changes taking place in the chorioallantoic membrane (CAM) of embryonated hen's eggs during incubation were followed up by histological, histochemical and electron microscopic methods. The allantoic sac surrounds the amnionic sac and also the yolk sac by the 7th to 11th day, respectively, and forms together with the chorion the CAM. The mesenchymal layer placing between the chorion of ectodermal and the allantoic layer of entodermal origin develops from the somatic and splanchnic-pleure of the parietal mesoderm. The CAM is composed of three different layers, i.e. chorionic epithelium, mesenchyme and allantoic epithelium. The chorion comprises 2 layers of cubical epithelial cells. In between the 2 layers capillaries and directly under the shell membrane vascular sinuses can be found. Blood circulating in the sinuses is separated from the air in the pores of the shell membrane by the adacent epithelial cell-projections of 0,2 μm thickness, the basal membrane (0.1 μm) and the sinus endothelial layer of 0.2 μm thickness. The mesenchymal cells are of star-like form. The allantioc epithelium is built of one layer of fusiform cells with oval-shaped or rod-like nuclei.     

The hen's egg: Evidence on the mechanism relating shell strength to loading rate

1. The decrease in strength of an egg shell which is known to accompany an increase in the period of time over which a load is applied might come about by either of two mechanisms: a decrease in the strength of the material constituting the strong, outer layer of the mineral shell, or a decrease in the thickness of this layer brought about by deepening of the crevices that are normally present between adjacent crystal columns in the weak, inner layer of the shell.

2. Experiments designed to discriminate between these mechanisms are described: the results indicate the second mechanism.

3. This mechanism offers explanations for various other shell phenomena, including delayed fracture under a static, cyclical or recurrent load.

4. The main implication for the poultry industry is that any external insult to a shell is likely to weaken it by doing damage that is local, internal, irreparable and cumulative, even though it may be invisible from the outside.     

Changes in structure of the limiting membrane and in oxygen permeability of the chicken egg integument during incubation

1. Oxygen permeabilities (K_O2 ) of the shell and shell membranes of fertile and infertile chicken eggs were measured at 37.5 °C and a relative humidity of 0.60 throughout 14 d incubation, with turning. The K_O2 of the shell and membranes of infertile eggs was around 1.0 × 10^?7 cm³ O₂STP sec^?1 cm^?2 Torr^?1 (1 Torr = 133.322 Pa) throughout incubation. With fertile eggs, from which there was a linear loss of water during incubation, the K_O2 of the shell and shell membranes was about 1.0 × 10^?7 cm³ O₂STP sec^?1 cm^?2 Torr^?1 for the first four days of incubation. Thereafter the majority of shells and membranes had a K_o2 of about 1.0 × 10^?6 cm³ O₂STP sec^?1 cm^?2 Torr^?1.

2. A diminution of the Na⁺ and K⁺ content of the shell membranes of fertile eggs was not associated with changes in the dimensions of the glyco‐protein mantle on the cores of the individual fibres of the membranes. There was, however, a progressive deterioration in the limiting membrane of fertile but not of infertile eggs.

3. It was concluded that changes in the O₂ resistance of the integument of fertile eggs were not a product of change in either of the shell membranes but of damage caused to the limiting membrane by the chorioallantois.     

不同蛋鸡品种鸡蛋品质的比较分析

针对当前市场对高品质鸡蛋需求增加的现状，本实验比较了在相同饲养管理条件下的当地几个主要蛋鸡品种的鸡蛋品质，结果表明：不同蛋鸡品种在鸡蛋品质的许多方面存在差异，综合考虑柴鸡、绿壳蛋鸡在生产优质鸡蛋方面具有一定的遗传优势。     

The outer membrane of the avian egg shell as a reticular structure

The shell membranes of fowl, duck, quail and turkey eggs were examined by electron microscopy. A proportion of the outer membrane fibres from all sources were interconnected by lamellae about 90 A thick. The lamellae, which are probably protein, endow the outer membrane with a secondary reticulum reinforcing the reticulum formed by the fibres themselves.     

Studies on the isthmus region of the somestic fowl.

The isthmus extends from the aglandular zone, which delimits it from the magnum, to the tubular shell gland, which to the naked eye is marked by a distinct colour change from off-white to brown. 2. The surface epithelium comprises three cell types, ciliated, non-ciliated and mitochondrial, of which only the non-ciliated cells contribute towards the carbohydrate moiety of the shell membranes. 3. The gland cells are distinctive, containing granules of variable electron density, variations also occurring within individual granules. 4. Although two types of gland cell have been observed, they may merely represent different phases of development. 5. In the type 1 cell, the rough endoplasmic reticulum and Golgi complex are typical of the normal protein secreting cell; in the type 2 cell the RER is sparse, dilated and filled with intracisternal granules while the Golgi complex is likewise distended.