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PubMed2000

Extraction and quantification by ELISA of eggshell organic matrix proteins (ovocleidin-17, ovalbumin, ovotransferrin) in shell from young and old hens.

The eggshell matrix is mainly composed of proteins that are thought to influence shell formation and calcification and, thus, modify the resulting properties of the shell. We investigated the potential of some of these proteins as biomarkers of eggshell quality by developing a competitive indirect ELISA for quantifying ovotransferrin, ovalbumin, and ovocleidin-17 in eggshell extract. Eggshell fragments were demineralized in acetic acid (20%) and freeze-dried. The micro-extraction yield was markedly increased (>50%) when Tween 20 was added to the subsequent extraction and dialysis milieus. Microplates were coated with ovotransferrin and ovalbumin in a 0.1M carbonate-bicarbonate buffer, but ovocleidin-17 was fixed with acetone (-20 C, 20 min). Optimal dilutions of the monoclonal (ovotransferrin) and polyclonal (ovalbumin and ovocleidin-17) antibodies were 1/3,000, 1/25,000 and 1/4,000, respectively. The inhibition curves were optimized by preincubating the antibodies and proteins overnight. The intraassay coefficient (<5%), parallelism of the standards and samples curves, and recovery (101%) were satisfactory for ovotransferrin. Measurements of ovalbumin were less precise because of higher interassay variation and differences between the slopes of standard and sample inhibition curves. Ovocleidin-17 assays showed similar slopes for standard and eggshell extracts. Although the total protein in soluble matrix extracts was not affected by age, the concentrations of these proteins were higher in eggshell extracts from older hens compared with those from young hens: 1.98x for ovotransferrin, 1.86x for ovalbumin, and 1.58x for ovocleidin-17. The quantification of specific eggshell matrix proteins in shell of differing quality is, therefore, a promising tool for analyzing the origin of eggshell faults and may provide useful information for breeding programs.

Aging↗
PubMed2003

Effect of local shell conductance on the vascularisation of the chicken chorioallantoic membrane.

The vascularisation of the chorioallantoic membrane (CAM) of avian embryos is influenced by environmental oxygen partial pressure (P(O(2))) on a global level: incubation at high P(O(2)) reduces the density of pre- and post-capillary vessels of the CAM and decelerates the thinning of the blood-gas barrier, and vice versa. This study investigates the effects of local P(O(2)) on vascular development during the formative period of days ten to fifteen, by making half of the egg hypoxic and the other half hyperoxic. The densities of arterioles, venules and capillaries were reduced under the hypoxic side, compared to untreated eggs, but not significantly changed on the hyperoxic side. Harmonic mean thickness of the tissue barrier and total CAM blood volume were not affected by the treatments. Vascular development of the CAM was therefore only partly influenced by local P(O(2)).

Adaptation, Physiological↗
PubMed1986

Effect of lindane on eggshell characteristics and calcium level in the domestic duck.

Adult laying domestic ducks (Anas platyrhynchos domesticus) were forced fed lindane (gamma-hexachlorocyclohexane) 20 mg/kg body weight daily, thrice/week and twice/week for 8 weeks. Significant shell thinning (18-21%) was observed in ducks with daily and thrice/week lindane feeding. SEM studies of thinned eggshells revealed reduced and tightly clustered mammillae with apparent decrease in intermammillary spaces and signs of inhibition of calcite formation. Such structural changes of thinned shells were associated with a significant reduction in the level of calcium both of the plasma and of shell gland of the ducks. Simultaneously, density of the pore in the shell and pore area/cm2 of the shell in these birds were drastically reduced, presumably because of total absence of 'large' pores in such shells. Such porosity change is suggestive of reduced conductivity of eggshell implying impaired hatching success. Diethyl stilboestrol post-treatment neutralized all the adverse effects of lindane on duck eggshell, indicating induced estrogen deficiency in such birds by lindane.

Animals↗
PubMed1992

Dietary zinc methionine effect on eggshell quality of hens drinking saline water.

In two experiments individually caged 60-wk-old laying hens were exposed to daily temperatures ranging between 18 and 35 C and given various dietary and drinking water treatments. In Experiment 1 these were: 1) basal diet and town water; 2) basal diet and town water supplemented with 2 g NaCl/L; 3) basal diet supplemented with .2 g zinc methionine (Zinpro-200)/kg diet and town water; or 4) basal diet supplemented with .2 g zinc methionine/kg and town water supplemented with 2 g NaCl/L. In Experiment 2, Treatments 1 and 2 were the same as in Experiment 1. Birds on Treatments 3 and 4 received the town water supplemented with 2 g NaCl/L and the basal diet supplemented with either .5 g zinc methionine/kg (Treatment 3) or .28 g ZnSO4H2O/kg to approximate the same dietary zinc concentration in Treatment 3 (Treatment 4). In both experiments, dietary zinc methionine plus 2 g NaCl/L in the drinking water significantly improved shell breaking strength over those birds on the 2 g NaCl/L with no zinc methionine supplementation. This same pattern occurred for shell weight, shell weight per unit of surface area, and percentage of shell defects. There were no improvements in the parameters measured from the supplementation of ZnSO4. The zinc methionine compound apparently was effective in overcoming the negative influence of the added 2 g NaCl/L of town water.

Animal Feed↗
PubMed1995

Purification and immunochemistry of a soluble matrix protein of the chicken eggshell (ovocleidin 17).

The protein components of biomineralized structures (matrix proteins) are believed to modulate crystal nucleation and growth, and thereby influence the shape and strength of the final structure. The chicken eggshell contains a complex array of distinct matrix proteins. The most abundant of these was purified to homogeneity by a combination of anionic exchange and hydroxyapatite chromatographies. Antibodies to this protein were raised in rabbit, and utilized for Western blotting and immunohistochemistry. These studies indicated that the 17 kDa antigen (ovocleidin 17, OC-17) is found in the shell gland mucosa, and that only the tubular gland cells were positive. Immunohistochemistry with decalcified shell indicated that OC-17 is uniformly distributed throughout the shell matrix, but concentrated in the mammillary bodies. Our results indicate that this protein is secreted during shell formation and becomes incorporated into this structure. It may therefore play a role in the crystallization process and influence the properties of the resulting eggshell.

Amino Acid Sequence↗
PubMed2000

Monoclonal antibodies to mineralized matrix molecules of the avian eggshell.

The extracellular matrix of the mineralizing eggshell contains molecules hypothesized to be regulators of biomineralization. To study eggshell matrix molecules, a bank of monoclonal antibodies was generated that bound demineralized eggshell matrix or localized to oviduct epithelium. Immunofluorescence staining revealed several staining patterns for antibodies that recognized secretory cells: staining for a majority of columnar lining cells, staining for a minor sub-set of columnar lining cells, intensified staining within epithelial crypts, and staining of the entire tubular gland. Western blotting with the antibody Epi2 on eggshell matrix showed binding to molecules with the apparent molecular weight of eggshell matrix dermatan sulfate proteoglycan (eggshell DSPG). Immunoblots of cyanogen bromide-cleaved eggshell DSPG revealed broad band of reactivity that shifted to 25 kDa after chondroitinase digestion; indicating that the Epi2 binding site is located on a fragment which contains dermatan sulfate side chains. Immunogold labeling showed that Epi2 binds to secretory vesicles within the non-ciliated cells of the columnar epithelium, while the antibodies Tg1 and Tg2 bind to secretory vesicles of tubular gland cells. Immunogold labeling of demineralized shell matrix showed binding of Epi2, Tg1, and Tg2 to the matrix of the palisade layer, and showed little reactivity to other regions of the shell matrix. Quantification of the immunogold particles within the eggshell matrix revealed that antibodies Epi2 and Tg1 bind all calcified regions equally while antibody Tg2 has a greater affinity for the baseplate region of the calcium reserve assembly.

Animals↗
PubMed1978

Calcium-45 uptake by shell gland, oviduct, plasma and eggshell of DDT-dosed ducks and chickens.

Experiments were conducted with ducks and chickens to determine the effects of acute doses of DDT and DDE on calcium-45 balance and calcium-45 distribution among plasma, femur, tibia, oviduct, shell gland, and eggshell. Species differences in distribution of calcium-45 were evident between control ducks and control chickens. Ducks contained more radiocalcium in plasma, shell glands, tibias, and femurs than chickens and eliminated less radiocalcium in their droppings than chickens. The percentage of radiocalcium deposited in eggshells by control birds of either species was about equal. Ducks and chickens treated with DDT or DDE contained more radiocalcium in their shell glands than their respective controls. Treatment with DDT or DDE caused eggshell thinning in ducks but not in chickens; however, treatment with DDT or DDE did not alter significantly the percentage of radiocalcium that appeared in eggshells of either species.

Animals↗
PubMed1997

Preservation of hatchery waste by lactic acid fermentation. 1. Laboratory scale fermentation.

In Experiment 1, two types of hatchery wastes, including cockerel chicks (CC), and shell waste (SW) blended with CC (60:40 CC:SW), were ground and mixed with a by-product carbohydrate (5, 10, and 15% wt/wt) with or without a bacterial culture and fermented for a period of 21 d. Cockerel chicks fermented with 15% carbohydrate and the culture had a pleasant smell and little H2S production. Elevated H2S concentrations were recorded for CC:SW samples at all carbohydrate levels when the culture was not added. No NH3 was detected from any treatments during fermentation. The addition of culture to the CC and CC:SW by-products resulted in pH values lower (P < 0.05) than those without culture on Day 21, and the 15% carbohydrate treatment significantly reduced pH beyond the 5% carbohydrate. Final proximate composition of CC and CC:SW samples with culture were not significantly different from those without culture added. In Experiment 2, carbohydrate was added at 10.0, 13.3, 16.7, and 20.0% wt/wt to CC and CC:SW in the presence of the bacterial culture. Shell waste alone was fermented with 15, 20, and 25% carbohydrate and the culture. Moisture level in this experiment was adjusted to approximately 70% for all treatments. The lowest pH for the CC and CC:SW treatments was observed at the 16.7% carbohydrate level. Shell waste pH was better maintained at the 20 and 25% carbohydrate levels. After fermentation for 21 d CC, CC:SW and SW treatments from Experiments 1 and 2 contained negligible Escherichia coli, and no Salmonella were detected.

Analysis of Variance↗
PubMed2002

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.

Animals↗
PubMed1991

Collagens of the chicken eggshell membranes.

An immunohistochemical analysis of the eggshell membranes shows the occurrence of type X collagen while type I collagen was not detected by using an appropriate monoclonal antibody with untreated shell membranes. A positive immuno-reaction for type I collagen was obtained after digestion of the shell membranes with pepsin. These observations indicate the possibility that type I collagen epitope was masked by type X collagen and that type X collagen may serve as an inhibitory boundary for biomineralization.

Acetates↗
PubMed2005

Sodium and anion transport across the avian uterine (shell gland) epithelium.

The uterine (shell gland) epithelium from the domestic chicken was mounted in Ussing chambers, bathed in symmetric avian saline solution on both apical and basolateral aspects and voltage clamped at 0 mV. The epithelium exhibited a basal short circuit current (I(sc)) that was partially inhibited by the epithelial Na(+) channel (ENaC) blockers, amiloride and benzamil (IC(50) values of 0.8 and 0.12 micromol l(-1), respectively). Inhibition of basal Na(+) absorption by 10 micromol l(-1) amiloride was confirmed by measurements of transepithelial Na(+) and Cl(-) fluxes, where inhibition of the apical-to-basolateral and net Na(+) flux occurred, but no significant effects on Cl(-) fluxes were detected. The amiloride-insensitive portion of the basal I(sc) was both Cl(-) and HCO(3)(-) dependent and was inhibited by the Cl(-) channel blocker, diphenyl-2-carboxylate (DPC; 100 micromol l(-1)). Stimulation with 8-(4-chlorophenylthio)-cyclic 3'-5', adenosine monophosphate (8-cpt cAMP) produced a sustained increase in I(sc) that was dependent on both Cl(-) and HCO(3)(-). The magnitude of the amiloride-sensitive I(sc) was approximately twofold greater in birds where shell formation was complete, but oviposition had not yet occurred. In addition, the amiloride-sensitive I(sc) was greater in hens over the age of 55 weeks and in molting birds. The anion-dependent component of the basal I(sc) was reduced in older birds, and electrogenic HCO(3)(-) transport was nearly absent in molting birds. These results demonstrated that electrogenic Na(+) transport in avian shell gland was similar to the mammalian uterine epithelium and increased with age and during molting. Electrogenic Cl(-) and HCO(3)(-) transport were coupled under basal and cAMP stimulated conditions and basal anion transport decreased with age and during molting.

Aging↗
PubMed1995

Daily energy allotments and performance of broiler breeders. 2. Females.

Arbor Acres broiler breeder hens, 21 to 61 wk of age, were given the daily ME level recommended by the primary breeder for their age and level of production (100% AA) or were given 94 or 88% of that amount. The reduction in daily energy allotment had no effect on age at 50% or age at peak production, but there was a highly significant linear decrease in the eggs per hen from 165 +/- 2.8 in hens given 100% AA to 149 +/- 3.2 and 141 +/- 3.8 in hens given 94 and 88% AA. The percentage of nonlaying hens between 44 and 60 wk of age was substantially increased as daily energy allotment was reduced. The percentage settable eggs was significantly lower in the 100% AA group, but no differences in fertility or hatchability were seen. Chicks per hen fell from 123 +/- 2.4 to 116 +/- 2.4 to 102 +/- 2.6 as energy allotment was progressively reduced. Dietary energy allotment had no effect on egg weight, shell weight, shell characteristics, or proportion of albumen and yolk. Chick weight was unaffected but chicks' liver weight increased with increasing dam energy intake. Percentage lipid in hens' carcasses increased with increasing energy intake and percentage protein and percentage ash decreased.

Animals↗
PubMed1996

Ultrastructure and characteristics of eggshells of the olive ridley turtle (Lepidochelys olivacea) from Gahirmatha, India.

The structure of a chelonian eggshell is of prime importance for the developing embryo. It acts as a protective covering as well as mediator in the exchange of heat and water. The fresh eggshell of the olive ridley (Lepidochelys olivacea) turtle displays a variety of structural forms in their aragonite framework. Its netted substrate, loose texture and poorly organised crystallites favour the easy exchange of air and water during its development. The shell consists mostly of calcium in carbonate form and K, Mg, Fe, Ni, Pb, Zn, Co, Cr, Cu, Mn and Cd in traces. Ba and Sr in traces are also a feature of the thermal analysis graph. Phosphorous was not detected in the shell and its absence eliminates an earlier suggestion of it being a key factor in the development of aragonite crystals in the inorganic structure of marine turtle eggshells. Its combustion characteristics, which have not been previously reported, are recorded.

Animals↗
PubMed1984

Development of preferred orientation in the eggshell of the domestic fowl.

Preferred orientation in the shell of the domestic fowl is shown by x-ray diffractometry to develop gradually throughout the shell, beginning immediately after the start of shell deposition and reaching a maximum at the exterior surface. Only 2 out of 20 shells examined exhibited a single preferred orientation: This was one in which the pole of the (001) plane lies parallel to the shell surface normal. The remaining shells had two preferred orientations present simultaneously, one in which the (001) pole is parallel and the other in which the (104) pole is parallel to the surface normal. Previous work has resulted in conclusions that are in conflict with these; they are discussed in relation to the present work.

Animals↗
PubMed1984

Comparison of the effect of DDE on the Ca metabolism of the eggshell gland and its subcellular fractions of the duck and the domestic fowl.

In a strain of ducks sensitive to the eggshell-thinning effect of p-p'-DDE, administration of 40 mg kg of the compound in the food for 45 days reduced the eggshell index (EI) by 13% and the content of calcium in the fluid of the shell gland forming an eggshell by 36%, and raised the calcium content of the shell gland mucosa by 19%, compared with the control values. DDE inhibited the translocation (secretion) of calcium between the gland mucosa and the uterine cavity. The ATP-dependent binding of Ca2+ to the subcellular fractions of the gland mucosa was reduced in DDE-treated ducks. The Ca2+ binding to a microsomal subfraction (FI) rich in fragments of the plasma membrane was reduced by 16%, whereas that to a subfraction FIII which bound Ca2+ at a very high rate was reduced by 36%. The latter may contain calcium-secreting granules of the gland. In the mitochondrial fraction the Ca2+ binding was reduced by 35%. In the domestic fowl DDE did not lower EI or interfered with the translocation of calcium between the shell gland mucosa and uterine cavity. DDE administration increased the Ca2+ binding to FI by 26%; the binding to other subfractions was not changed significantly. DDE may interfere with the stimulus-secretion mechanism of the eggshell gland in ducks through its effect on Ca2+ binding.

Adenosine Triphosphate↗
PubMed1992

Research note: influence of ahemeral light:dark cycles on egg traits in brown egg pullets.

Brown egg pullets (DeKalb Sex-Sal) were subjected to an ahemeral lighting program to determine their response in terms of egg traits. All birds were reared on a conventional lighting program of 10 h light (L):14 h dark (D) to 16 wk. At 16 wk, all birds received a schedule of 11L:13D followed by a weekly 1-h increase in photoperiod to 14L:10D at 19 wk. Control treatment (CON) birds were continued on this schedule. Ahemeral treatment (AHM) birds were given a 28-h schedule 14L:14D at 23 wk that was continued to 28 wk, then returned to a 24-h cycle of 14L:10D. Shell weight responded quickly to the ahemeral treatment and showed a significant (P less than .05) increase during the 2nd 28-h cycle. Shell thickness and total egg weight showed significant increases on the 3rd cycle; albumen weight showed an increase on Cycle 4, and yolk weight increased significantly only on the 8th cycle. When AHM treatment birds were returned to a conventional cycle (14L:10D) at 28 wk, the total weight remained significantly higher (P less than .05) as late as Cycle 6.

Animals↗
PubMed1986

Calcium regulation in the embryonic chick. II. Ultrastructure of the parathyroid glands in shell-less and in ovo embryos.

The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell-less culture (after removal of the eggshell). Shell-less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell-less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12-day shell-less embryos. However, the parathyroids of the 15-day shell-less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of the two groups is indistinguishable, both appearing to be more active than the 15-day shell-less group. Thus, protein synthetic activity of the parathyroid glands, as detected by ultrastructural alterations of the chief cells, normally appears to be initiated during the latter part of embryogenesis (by approximately 15 days incubation) and its onset can be stimulated at least 3 days prematurely by hypocalcemia.

Animals↗