Relationship between eggshell strength and keratan sulfate of eggshell membranes

Eggshell strength is an important factor in an effort to minimize eggshell breakage, which is a significant problem in the egg production industry. In the current study, we isolated and quantified the specific glycosaminoglycans (GAGs) from the calcified eggshell and shell membranes, which are related to eggshell strength. Our data suggest that GAGs exist in calcified eggshell may influence morphology of shell but do not affect on increase of shell amount while GAGs of shell membranes are maybe highly associated with shell strength with an increase of shell weight. Shell strength showed a strong correlation with the content of GAGs (r=0.942, p<0.0005) and a weak relationship with uronic acid content (r=0.564, p=0.056) in shell membranes. Monosaccharides in shell membranes were determined by Bio-LC analysis for the identification of any specific GAGs related with shell strength. It indicates that the galactose content as a component of keratan sulfate (KS) has a significant correlation with eggshell strength (r=0.985, p<0.0005). These results suggest that eggshell strength is proportional to the KS content of eggshell membranes with an increase of eggshell weight.     

Structure of the shell and tertiary membranes of eggs of softshell turtles (Trionyx spiniferus)

Eggs of the turtle Trionyx spiniferus are rigid, calcareous spheres averaging 2.5 cm in diameter. The eggshell is morphologically very similar to avian eggshells. The outer crystalline layer is composed of roughly columnar aggregates, or shell units, of calcium carbonate in the aragonite form. Each shell unit tapers to a somewhat conical tip at its base. Interior to the crystalline layer are two tertiary egg membranes: the outer shell membrane and the inner shell membrane. The outer shell membrane is firmly attached to the inner surface of the shell, and the two membranes are in contact except at the air cell, where the inner shell membrane separates from the outer shell membrane. Both membranes are multi-layered, with the inner shell membrane exhibiting a more fibrous structure than the outer shell membrane. Numerous pores are found in the eggshell, and these generally occur at the intersection of four or more shell units.     

Influence of Temperature on Bacterial Infection of the Hen''s Egg

Temperature of incubation had a marked effect on infection of eggs in which the air cells had been inoculated with a washed suspension of Serratia marcescens. There was no evidence of bacterial multiplication or spoilage in eggs held at 10 C for 42 days. Multiplication occurred in the shell membranes of eggs held at 30 or at 37 C when the yolk made contact with these membranes, and continued in the contents of the egg, at which time the first signs of spoilage appeared. In a few eggs, very large populations were present in the shell membranes and in the albumen. In eggs inoculated with Pseudomonas fluorescens and held at 10 C, bacterial multiplication occurred in the shell membranes in the first 7 days of incubation. These populations did not appear to change in size in the 7- to 14-day period of incubation. Renewed multiplication and concomitant spoilage of the contents was observed in many of the eggs thereafter.     

Influence of Temperature on Bacterial Infection of the Hen's Egg

Temperature of incubation had a marked effect on infection of eggs in which the air cells had been inoculated with a washed suspension of Serratia marcescens. There was no evidence of bacterial multiplication or spoilage in eggs held at 10 C for 42 days. Multiplication occurred in the shell membranes of eggs held at 30 or at 37 C when the yolk made contact with these membranes, and continued in the contents of the egg, at which time the first signs of spoilage appeared. In a few eggs, very large populations were present in the shell membranes and in the albumen. In eggs inoculated with Pseudomonas fluorescens and held at 10 C, bacterial multiplication occurred in the shell membranes in the first 7 days of incubation. These populations did not appear to change in size in the 7- to 14-day period of incubation. Renewed multiplication and concomitant spoilage of the contents was observed in many of the eggs thereafter.     

Levels of calcium and soluble collagen in turkey egg shell membranes

1. This experiment examined the effect of weeks in egg production and type of housing confinement of turkey hens on calcium and soluble collagen levels in egg shell membranes; and discussion was given to their apparent relationship to gas exchange in turkey eggs. 2. The high level of acid-soluble collagen in inner and outer egg shell membranes of aging caged hens compared with the same aged floor-penned hens may have a relationship with the low hatchability generally recognized in caged hens. 3. The levels of calcium found in the outer shell membrane are low and appeared to decrease with the age of the hen. 4. There were no differences over time in levels of total collagen and neutral salt-soluble collagen (newly formed collagen) found in egg shell membranes of turkey hens confined in cages or floor pens. 5. It is suggested that the acid-soluble collagen levels found in inner shell membranes may have a relationship in limiting respiratory gas exchange during latter incubation time, and thus limit embryo survival.     

Formation of the prismatic layer in the freshwater bivalve Hyriopsis cumingii: the feedback of crystal growth on organic matrix

The squeezing hypothesis and the organic frameworks preformation hypothesis propose two different mechanisms to explain the interaction between organic frameworks and crystals during biomineralization of the prismatic layer of the mollusk shell. In this study, we began to study Hyriopsis cumingii shell formation and discover that this species seemed to follow the squeezing hypothesis. During the formation of the aragonite prismatic layer in the freshwater bivalve H. cumingii, we found that crystal growth was involved in controlling initiation of formation of the interprismatic organic membranes. First, newly formed crystals were embedded in the periostracum. Next, the interprismatic organic membranes of the prismatic layer were produced via squeezing between neighboring crystals. The organic matrix secreted by the mantle continuously self‐assembled into the interprismatic organic membranes as the crystals grew. In the mature stage, the interprismatic organic membranes were shaped by crystal growth. These findings provide evidence to support the squeezing hypothesis and add to the existing knowledge about interactions that occur at the organic–inorganic interfaces during mollusk shell biomineralization.     

The Properties and Classification of the Predominant Bacteria in Rotten Eggs

A collection of 226 strains of bacteria was assembled from eggs which had rotted on the premises of the producer and from others which had been allowed to rot in the laboratory at 10, 20 or 30°. A majority of the eggs had a mixed infection. All but 8 of the isolates were Gram negative rods. The predominant types were Alcaligenes faecalis, Aeromonas liquefaciens, Proteus vulgaris, Cloaca spp., Citrobacter sp. and Pseudomonas fluorescens. A nonpigmented pseudomonad could not be identified with any of the species included in Pseudomonas.     

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.     

Short Notes

Buntings on a Yorkshire farm, by D. Summers-Smith

The sex ratio of migrant Ruffs, by M. E. Greenhalgh

Cloaca pecking in the Dunnock, by R. F. Sanderson     

Cameral membranes in prolecanitid and goniatitid ammonoids from the Permian Arcturus Formation, Nevada, USA

We describe cameral membranes in prolecanitid and goniatitid ammonoids from the Lower Permian Arcturus Formation, Nevada, USA. The membranes are preserved as phosphatic sheets and were originally composed of organic material such as conchiolin. Because the phragmocones are filled with micritic calcite, the cameral membranes can be exposed by etching with weak acetic acid. The membranes are associated with the siphuncle and also coat the septal faces and chamber walls. The siphuncular membranes are much more extensive in the prolecanitids than in the goniatites. These membranes appear in the prolecanitids at the beginning of the third whorl, corresponding to a shell diameter of 3-4 mm, and become more complex through ontogeny. Additional membranes, called transverse membranes, appear in some of the septal saddles on the ventrolateral side. The siphuncular membranes in prolecanitids are very similar to those in the Ceratitina plus Mesozoic Ammonoidea, suggesting that such membranes are widely distributed in this group. However, the origin and function of these membranes are unclear. We argue that the siphuncular membranes were sequentially secreted by the rear mantle during forward movement of the body and were not produced by desiccation of cameral liquid after the formation of the chambers. The most compelling arguments for this interpretation are the abrupt appearance of these membranes at a shell diameter of approximately 3-4 mm in prolecanitids, ceratites, and ammonitids, coincident with the end of the neanic stage, and the uniform increase in complexity of the membranes through ontogeny. The shape of the siphuncular membranes in prolecanitids suggests the presence of an invagination on the dorsal side of the siphuncle during part of the chamber formation cycle. Cameral membranes may have served a variety of functions including stabilizing the cameral liquid to reduce rocking motion during swimming, anchoring the siphuncle to the chamber wall, and facilitating cameral liquid removal, permitting a faster rate of growth.     

A scanning and transmission electron microscopic study of the membranes of chicken egg.

Questions regarding the structure of the inner and outer shell membranes of the chicken egg were addressed in this study by correlating observations from light microscopy and scanning and transmission electron microscopy. The egg membrane had a limiting membrane, which measured .9 to .15 microns in thickness and appeared to be a continuous and an impervious layer, but the shell membrane did not. Under the SEM, each membrane was seen to be made up of several fibre layers. In the tear preparations viewed under the SEM two layers were observed in the egg membranes and three to five layers in the shell membrane, with an apparent plane of cleavage between each layer. Each fibre was made up of a central core and an outer mantle layers. The central core was perforated by channels which measured .08 to 1.11 microns in diameter and ran longitudinally along the length of the fibre. Between the mantle layer and the fibre core was a gap or cleft measuring between .03 to .07 microns. The diameter of the fibres of the inner layer of the egg membrane ranged between .08 to .64 microns, whereas those of the outer layer of the same membrane ranged from .05 to 1.11 microns. Fibres in the shell membrane ranged from .11 to 4.14 microns diameter.     

The fine structure of the outer surface of the incubated eggshells of the Helmeted guinea fowl (Numidia meleagris)

The surface of the eggshells of the Helmeted guinea fowl (Numidia meleagris) was polished during incubation by the parent. Examination with the light microscope showed that the cuticle had been removed from the ridges on the outer surface of the shell and that the plugs in the outer orifice of the pore canals had acquired extraneous materials including grease. Studies with a scanning electron microscope revealed that the spheres that made up the pore plugs retained their identity even though they were stained. It was concluded that ridges on the shell surface protected the pore plugs from damage by attrition and that the plugs acted as filters thereby preventing nest debris from occluding the pore canals or contaminating the shell membranes.     

Non-invasive measurement of oxygen partial pressure, lateral diffusion and chorioallantoic blood flow under the avian eggshell.

We measured P(O2) under the shell of avian eggs indirectly, by sealing 0.05 mL glass tubes to the shell, sealing them with mercury and using an oxygen microelectrode to measure the contained gas that equilibrates with the gas in the shell membranes. This technique requires a smaller area of contact with the shell and a shorter equilibration period than established techniques, and allows measurements at several locations simultaneously and over a long period of time without endangering the embryo. P(O2) under the shell of chicken eggs decreased to 14.3 kPa on the day before hatching (day 19). P(O2) was unstable during late development and differences up to 3.1 kPa occurred transiently on opposite sides of the equator. By waxing the shell around sampling tubes, we estimated Krogh's coefficient for lateral oxygen diffusion in the shell membranes at 1.1 mmol cm(-1) d(-1) kPa(-1), a value about a third of a previous estimate. Sampling of gas under sufficiently large regions of waxed shell allowed indirect measurements of chorioallantoic venous P(O2), without affecting embryonic respiration. Venous P(O2) was 3.8 kPa on day 19. Assuming 14.3 kPa represents arterialized blood leaving the chorioallantois, it became possible to calculate the effective chorioallantoic blood flow rate, which was 3.5 mL min(-1) on day 19.     

Non-invasive measurement of oxygen partial pressure, lateral diffusion and chorioallantoic blood flow under the avian eggshell

We measured P(O2) under the shell of avian eggs indirectly, by sealing 0.05 mL glass tubes to the shell, sealing them with mercury and using an oxygen microelectrode to measure the contained gas that equilibrates with the gas in the shell membranes. This technique requires a smaller area of contact with the shell and a shorter equilibration period than established techniques, and allows measurements at several locations simultaneously and over a long period of time without endangering the embryo. P(O2) under the shell of chicken eggs decreased to 14.3 kPa on the day before hatching (day 19). P(O2) was unstable during late development and differences up to 3.1 kPa occurred transiently on opposite sides of the equator. By waxing the shell around sampling tubes, we estimated Krogh's coefficient for lateral oxygen diffusion in the shell membranes at 1.1 mmol cm(-1) d(-1) kPa(-1), a value about a third of a previous estimate. Sampling of gas under sufficiently large regions of waxed shell allowed indirect measurements of chorioallantoic venous P(O2), without affecting embryonic respiration. Venous P(O2) was 3.8 kPa on day 19. Assuming 14.3 kPa represents arterialized blood leaving the chorioallantois, it became possible to calculate the effective chorioallantoic blood flow rate, which was 3.5 mL min(-1) on day 19.     

Novel reactor for aqueous-organic two-phase biocatalysis: A packed bed hollow fiber membrane bioreactor

Summary Hollow fiber membranes were potted in a tubular shell with a particulate, microporous, enzyme bearing support packed in the shell space. A bicontinuous system was thus formed with the reactants, supplied through the shell and the fiber lumen, forming an interface at the surface of the particles. Acid production rates, without any reactor optimization, up to four times greater than with membrane reactors were obtained during the lipase catalyzed hydrolysis of ethyl laurate and olive oil.     

Influence of counterions on the interaction of pyridinium salts with model membranes

The interaction of pyridinium salts (PS) with red blood cells and planar lipid membranes was studied. The aim of the work was to find whether certain cationic surfactant counterion influence its possible biological activity. The counterions studied were Cl-, Br-, I-, ClO4-, BF4- and NO3-. The model membranes used were erythrocyte and planar lipid membranes (BLM). At high concentration the salts caused 100% erythrocyte hemolysis (C100) or broke BLMs (CC). Both parameters describe mechanical properties of model membranes. It was found that the efficiency of the surfactant to destabilize model membranes depended to some degree on its counterion. In both, erythrocyte and BLM experiments, the highest efficiency was observed for Br-, the lowest for NO3-. The influence of all other anions on surfactant efficiency changed between these two extremities; that of chloride and perchlorate ions was similar. Some differences were found in the case of BF4- ion. Its influence on hemolytic possibilities of PS was significant while BLM destruction required relatively high concentration of this anion. Apparently, the influence of various anions on the destructive action of PS on the model membrane used may be attributed to different mobilities and radii of hydrated ions and hence, to different possibilities of particular anions to modify the surface potential of model membranes. This can lead to a differentiated interaction of PS with modified bilayers. Moreover, the effect of anions on the water structure must be taken into account. It is important whether the anions can be classified as water ordering kosmotropes that hold the first hydration shell tightly or water disordering chaotropes that hold water molecules in that shell loosely.     

Alligators provide evidence for the evolution of an archosaurian mode of oviparity.

The female reproductive tract of birds is different from that of other oviparous amniotes in that the eggshell membranes and calcareous layer are formed in separate regions of the uterus; the isthmus and shell gland, respectively. Phylogenetically, birds are included among the archosaurs, along with crocodilians and dinosaurs. Many dinosaurs were oviparous, producing hard-shelled eggs, yet the reproductive system of dinosaurs has proven difficult to investigate, due to poor preservation of soft anatomy. In this study, we examined functional morphology and eggshell formation in a reptilian archosaur, the American alligator, and demonstrated that the crocodilian reproductive tract has separate uterine regions for formation of the eggshell membranes and calcareous layer. These uterine regions are ultrastructurally comparable to the isthmus and shell gland of birds, and may be homologous. This similarity of reproductive functional morphology between crocodilians and birds may implicate the evolution of an archosaurian mode of oviparity that may shed light on dinosaur reproduction.     

Acid glycosidases in the isthmus of the hen oviduct and egg shell membranes

Specific activities of seven acid glycosidases: beta-hexosaminidase, alpha- and beta-galactosidase, alpha- and beta-mannosidase, alpha-glucosidase and alpha-fucosidase were determined in various parts of the domestic hen oviduct (infundibulum, isthmus, shell gland and vagina). The activity of most enzymes was the highest in the isthmus. Multiple forms of all acid glycosidases from the isthmus were separated by strong anion exchange chromatography at pH 6.0. The isoelectric points of the isthmus forms of beta-hexosaminidase, beta-galactosidase and alpha- and beta-mannosidase were determined by chromatofocusing. For the first time the high beta-galactosidase activity was found in hen egg shell membranes.     

Physical structure of shell membranes

The results obtained in the present work bring out clearly that the dried shell membrane is an open lattice-like network containing micropores of the order of 1 micron in mean equivalent diameter and whose surface density is roughly 20 millions per sq. cm. The gas transfer through this network is largely by viscous flow and accordingly does not obey Graham's law of diffusion. The data make clear that dried shell membranes are extremely permeable to all the gases examined and that wet membranes are completely impermeable. This fact leads one to suspect that during embryonic development, the rate of influx of gases is controlled in some measure by the moisture content of the shell membrane. The permeability technique developed here should be of value in the study of other membranes and especially natural membranes of biological importance.     

Partial biochemical and immunochemical characterization of avian eggshell extracellular matrices.

There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.