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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↗

Disrupted carbonic anhydrase distribution in the avian shell gland following in ovo exposure to estrogen.

Eggshell thinning among wild birds has been an environmental concern for almost half a century and the underlying mechanisms are still not fully understood. Previously we showed that exposure of quail embryos to ethynylestradiol (EE2) caused disorganization of the tubular glands in the shell gland of adult birds. In this study, we have examined the effect of in ovo exposure to EE2 on carbonic anhydrase (CA) localization, especially in the shell gland, because CA is required for shell formation. In the control birds, CA was localized in the cell membranes of the tubular gland cells of the shell gland, whereas the surface epithelium was always devoid of CA. In ovo treatment with 20ng EE2/g egg resulted in a loss of CA activity in the tubular glands while the surface epithelium showed strong induction of both membrane bound and cytoplasmic CA activity in 49+/-1% of the cells. The dose 2ng EE2/g egg resulted in partial loss of tubular gland CA and strong induction of CA activity in 2.5+/-0.5% of the surface epithelial cells and weaker induction in 22+/-2% of the epithelial cells. In conclusion, this study shows that embryonic exposure to a xenoestrogen disrupts CA distribution in the adult shell gland. We propose that eggshell thinning in avian wildlife could reflect a functional malformation in the shell gland, already induced by xenoestrogen during embryonic development rather than being caused solely by exposure of the adult bird.

Animals↗

Oxygen permeability of the shell and membranes of chicken eggs during development.

Oxygen permeability (KO2) was measured through the shell and shell membranes of chicken eggs throughout incubation. Shell KO2 was constant at 1.90 x 10(-6) cm3 O2 STP . sec-1 . cm-2 . Torr-1. Outer shell membrane KO2 was constant at 1.78 x 10(-6) cm3 O2 STP . sec-1 . cm-2 . Torr-1. Inner membrane KO2 increased from 0.11 x 10(-6) cm3 O2 STP . sec-1 . cm-2. Torr-1 to 1.56 x 10(-6) cm3 O2 STP . sec-1 . cm-2 . Torr-1. Calculations of KO2 from oxygen uptake rates and air cell PO2's were in close agreement with direct measurement of KO2. Resistance to oxygen flux was partitioned at each level. The outer membrane added 6% to the resistance of the shell. The inner membrane initially accounted for 88% of the shell/membrane complex resistance, but fell to 12% of the resistance by the end of incubation. The hypothesis is discussed that the increased permeability of the inner membrane is related to the evaporation of water from the membrane surface. The possibility is rejected that the shell membranes may be potential sites for respiratory adaptation to incubation at altitude.

Adaptation, Physiological↗

Changes in eggshell water vapour conductance during shell formation in the chicken.

Time-related changes in shell porosity and eggshell water vapour conductance were measured in uterine eggs of the domestic fowl (Gallus domesticus) from 10 h in uterus to oviposition. Measurements were carried out in artificially-aborted eggs. It was found that the eggshell water vapour conductance decreased to a minimum level at 15 h in uterus. This coincided with increases in shell thickness and shell porosity as power functions of uterine time. From that time on, water vapour conductance increased as a power function of uterine time to the final value prior to oviposition. Thus, eggshell water vapour conductance is determined by different rates of dynamic changes in shell porosity and shell thickness in the course of shell calcification. The final conductance is reached about 2 h before oviposition.

Animals↗

Involvement of osteopontin in egg shell formation in the laying chicken.

Expression of the osteopontin (OPN) gene in the oviduct of the laying hen was studied. It was detected only in the egg shell gland (ESG), where massive calcification occurs. No OPN gene expression was detected in any other part of the oviduct, such as the magnum and isthmus. The OPN gene was expressed in a circadian fashion during the daily egg cycle only during the period of egg shell calcification. No OPN gene expression was detected in the ESG of a pre-laying hen before the onset of reproduction, or after forced removal of the egg close to its entrance into the ESG. OPN was found to be synthesized by the epithelial cells of the ESG lining the lumen. Upon synthesis, OPN is immediately secreted out of cells and accumulates in the egg shell. These findings demonstrate for the first time temporal and spatial association of OPN with egg shell calcification. OPN, which was found to be part of the organic matrix of the egg shell, may play an important role in egg shell calcification.

Animals↗

The relation between sodium chloride concentration in drinking water and egg-shell damage.

1. A significant linear increase in egg-shell defects from 60-week-old laying hens, and corresponding significant linear decreases in various egg-shell-quality measurements, were observed in response to increasing concentrations of sodium chloride in the drinking water, to the maximum concentration of 600 mg/l used in the present study. 2. The incidence of damaged egg shells was increased 3-fold by including NaCl in the drinking water at a concentration of 600 mg/l. 3. Shell defects declined when birds were placed on normal water for 5 weeks but were still 1.4- to 2.1-fold greater than control values. 4. After an induced rest from lay on normal water, shell defects were still 1.3- to 3.2-fold greater in birds which had previously received the NaCl in the drinking water. 5. The increased incidence of shell damage was not related to decreased food intake or increased egg weight or production.

Animals↗

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.

Air Microbiology↗

Responses in egg shell quality to sodium chloride supplementation of the diet and/or drinking water.

1. Supplementing the drinking water of 50-week-old laying hens with sodium chloride (NaCl) concentrations between 0.5 and 2 g/l for 7 weeks significantly increased the incidence of egg shell defects and significantly decreased egg shell quality. Dietary NaCl concentrations between 0 and 2 g/kg had little effect on this response. 2. At similar total NaCl intakes egg shell defects were much greater when the NaCl was obtained from the drinking water rather than from the diet. 3. Hens producing eggs with defective shells as a result of receiving saline drinking water failed to recover the ability to lay eggs with good shells after 8 weeks on normal water. 4. The increased incidence of shell damage was not related to decreased food intake or increased egg weight or production.

Animal Feed↗

Prevalence of Salmonella enteritidis in poultry shell eggs in Arkansas.

The objective of this study was to determine whether poultry shell eggs are a major reservoir of Salmonella enteritidis in Arkansas. One hundred dozen commercially purchased shell eggs were cultured for the presence of Salmonella sp. After each dozen eggs was examined, the contents of the 12 eggs were separated from their shells. The contents and the shells were separately pooled and cultured. One dozen of the 100 dozen egg shells cultured were found to be externally contaminated with S heidelberg, while none of the contents of the 100 dozen eggs were found to contain Salmonella organisms. The reevaluation of previously obtained telephone follow-up data on 204 patients with Salmonella infections from 1992-1993 revealed that 30 had consumed raw eggs before their salmonellosis but only one patient was infected with S enteritidis. These data suggest that poultry shell eggs are not a major cause of human illness due to S enteritidis in Arkansas.

Animals↗

Effects of PCBs, DDT, and mercury compounds upon egg production, hatchability and shell quality in chickens and Japanese quail.

Dietary polychlorinated biphenyls (PCBs), DDT and related compounds, in well controlled experiments, produced no detrimental effects upon egg shell quality in Single Comb White Leghorm chickens or in Japanese quail. PCBs caused some decrease in egg production and a drastic reduction in hatchability in chickens, but not in Japanese quail. Inorganic mercury as HgSO4 or HgCl2, at dietary levels up to 200 p.p.m. of Hg, had only small effects, if any, upon egg production, hatchability, shell quality, morbidity and mortality. However, methyl mercury chloride at levels which provided 10 or 20 mg. of Hg per kg. of diet caused severe effects upon egg weight, egg production, fertility, hatchability, egg shell strength, morbidity and mortality. The results of these experiments demonstrate that the decrease in egg shell quality which has occurred in eggs of White Leghorn hens over the past three decades is not due to contamination of commercial feeds with DDT or its derivatives, or with PCBs. The extent to which environmental contamination with methyl mercury is responsible for decreased egg shell strength in commercial laying hens, and possible synergistic relationships between methyl mercury, DDT, DDE and PCBs in reducing egg production, hatchability and shell strength, remain to be determined.

Adipose Tissue↗

Physiological profile of caged layers during one production year, molt, and postmolt: egg production, egg shell quality, liver, femur, and blood parameters.

A longitudinal study of a flock of Single Comb White Leghorn pullets was initiated at 19 weeks of age (preproduction) and continued through a production year, a forced molt, and for 4 months of postmolt production. A representative sample of hens was obtained at 12-week intervals during the first year and at subsequent selected times. Liver lipid, femur weight, femur volume, femur density, egg weight, shell weight, percent shell, milligrams shell/square centimeter of shell surface area, serum calcium, serum phosphorus, and serum alkaline phosphatase were determined. Percent hen-day production peaked at 90% and then declined by .6 to .7% each week during the first production year. After molting, percent hen-day egg production peaked at 80% and declined .9% per week over the subsequent 20 weeks. Egg weight increased continually during the first production year. Shell weight was greatest immediately postmolt; thereafter it declined. Shell thickness was greatest at 31 weeks of age and declined throughout the first year. After molting, the shell thickness of 83-week-old hens was similar to values of hens about 37 weeks of age. Serum calcium and phosphorus of laying hens were influenced by age, feed intake and environmental temperature. The lowest values occurred during hot weather. Liver lipid was lowest in nonlaying hens (17 to 20%) and was approximately 42% of dry weight in laying hens. Femur density was greater in laying than nonlaying hens.

Alkaline Phosphatase↗

Shell quality: potential for improvement by dietary means and relationship with egg size.

Three experiments were conducted by sampling a total of 15,705 eggs from Leghorn hens fed diets varying in methionine, Na, and Ca content. In Experiment 1, birds of four different ages were housed in a common environment and fed diets containing between .233 and .383% methionine. In Experiment 2, birds of five different ages were housed in a common environment and fed diets containing .15, .30, and .45% Na and .25, .45, and .65% nonphytate phosphorus (NPP). In Experiment 3, eggs were sampled from 42 to 62 wk of age in 4-wk intervals from hens fed diets containing from 3 to 9% Ca in increments of 1.5%. Feeding lower levels of methionine (.233%) produced significantly lower egg weights and greater shell strength but at the expense of a decline in egg production at early ages. Reducing the NPP level from .65 to .25% produced lower egg weights and greater shell strength. A corresponding drop in production was not observed except at the .45%-Na level. No significant effects of Na on shell strength were observed. Higher Ca levels produced greater shell strength and had variable effects on egg weight. Increasing the Ca level beyond 6% resulted in a significant decline in production. Regression analyses indicated that within a population, the relationship between egg weight and shell strength is positive. In some instances, the relationship was curvilinear, where the positive association between egg weight and shell strength decreased with increasing egg weights.

Animals↗

Effect of sodium aluminosilicate, oyster shell, and their combinations on acid-base balance and eggshell quality.

Three experiments were conducted to determine the effect of sodium aluminosilicate (SAS), oyster shell (OS), and their combinations on production performance, eggshell quality, and acid-base balance. Experiments 1 and 2 were conducted during summer and Experiment 3 in winter. In Experiment 1, the effect of two levels of SAS (0 and .75%) and two levels of OS (0 and 50% substitution for pulverized limestone) was studied. In Experiment 2, the effect of SAS (.75%) with or without Na adjustment was investigated. When Na was adjusted, various sources of chloride were used to maintain an adequate level of this mineral. Calcium and available P were maintained at a constant 3.5 and .4%, respectively in Experiments 1 and 2. In Experiment 3, the levels of SAS and OS were similar to those of Experiment 1, but dietary Ca was either 2.8 or 3.5%. Egg production performance was not influenced by dietary treatments in Experiments 1 and 2 (P greater than .05). Egg production, but not egg mass, was reduced due to SAS in Experiment 3 (P less than .05). Dropping moisture increased when SAS was used in the diets with or without Na correction. Shell quality increased (P less than .05) due to SAS in the summer (Experiments 1 and 2) but not in the winter (Experiment 3). The shell quality response due to SAS was independent of Na correction or the source of dietary chloride. The OS increased shell quality in both summer and winter (P less than .05). Combinations of SAS and OS did not have an additive effect on shell quality (P greater than .05). Blood acid-base balance, plasma Ca and P, bone ash, bone Ca, and Ca retention were not influenced by dietary treatments. The results suggest that elevated environmental temperatures may be required in order for SAS to show its optimum effect on shell quality.

Acid-Base Equilibrium↗

Variations in external and internal microbial populations in shell eggs during extended storage.

The current project was conducted to determine the microbial quality of commercially processed shell eggs during extended storage. Unwashed eggs were collected at the accumulator before entering the processing line. Washed eggs were retrieved after placement in flats. All eggs were stored on pulp flats at 4 degrees C for 10 weeks. Twelve eggs from each treatment were rinsed on the day of collection and during each week of storage. After rinsing, eggs were sanitized in ethanol, and contents were aseptically collected. Total aerobes, yeasts and molds, Enterobacteriaceae, and pseudomonads were enumerated from shell rinses and pooled egg contents. During storage, no differences were found between unwashed and washed eggs for Enterobacteriaceae and pseudomonads in either shell rinses or contents. No differences were found between treatments for population levels of total aerobes or yeasts and molds in the egg contents throughout the storage period. Significant differences between treatments were found at each week of storage for external shell contamination by total aerobes. The highest unwashed egg contamination occurred at week 8 of storage and the lowest was at weeks 0 and 1 of storage. The highest shell contamination with aerobic bacteria on the washed eggs was found at week 0 of storage and the lowest was at week 7. Yeast and mold contamination determined by shell rinses was also significantly different between treatments at each week of storage. Commercially washed eggs were significantly less contaminated than were unwashed eggs for the populations monitored.

Animals↗

Inactivation of Salmonella enterica serovar Enteritidis on shell eggs by ozone and UV radiation.

The presence of Salmonella enterica serovar Enteritidis in shell eggs has serious public health implications. Several treatments have been developed to control Salmonella on eggs with mixed results. Currently, there is a need for time-saving, economical, and effective egg sanitization treatments. In this study, shell eggs externally contaminated with Salmonella (8.0 x 10(5) to 4.0 x 10(6) CFU/g of eggshell) were treated with gaseous ozone (O3) at 0 to 15 lb/in2 gauge for 0 to 20 min. In other experiments, contaminated shell eggs were exposed to UV radiation at 100 to 2,500 microW/cm2 for 0 to 5 min. Treatment combination included exposing contaminated eggs to UV (1,500 to 2,500 microW/cm2) for 1 min, followed by ozone at 5 lb/in2 gauge for 1 min. Eggs that were (i) noncontaminated and untreated, (ii) contaminated and untreated, and (iii) contaminated and treated with air were used as controls. Results indicated that treating shell eggs with ozone or UV, separately or in combination, significantly (P < 0.05) reduced Salmonella on shell eggs. For example, contaminated eggs treated with ozone at 4 to 8 degrees C and 15 lb/in2 gauge for 10 min or with UV (1,500 to 2,500 microW/cm2) at 22 to 25 degrees C for 5 min produced 5.9- or 4.3-log microbial reductions or more, respectively, when compared with contaminated untreated controls. Combinations including UV followed by ozone treatment resulted in synergistic inactivation of Salmonella by 4.6 log units or more in about 2 min of total treatment time. Salmonella was effectively inactivated on shell eggs in a short time and at low temperature with the use of a combination of UV radiation and ozone.

Animals↗

Shell and core in monkey and human nucleus accumbens identified with antibodies to calbindin-D28k.

The neurochemical division of the rodent nucleus accumbens into shell and core is now a widely accepted concept. However, such divisions in the primate nucleus accumbens have yet to be fully clarified and described. In the present study, the forebrains of three primates--marmoset, rhesus monkey, and human--and a Wistar rat, were immunoreacted with antibodies directed against calbindin-D28k. The patterns of immunoreactivity in the primates' ventral striatum were mapped and compared to that of rat. Calbindin staining was uneven in all species and there was no evidence of a bicompartmental organization, i.e., striosome/patch and matrix, in central parts of the nucleus. Nucleus accumbens in primates, as in rat, could be divided immunohistochemically into a crescent-shaped outer shell--medially, ventrally and laterally--and an inner core. In general, medial parts of the shell stained less intensely for calbindin than did lateral parts. However, interspecific variation in the intensity of the immunoreactive staining and the mediolateral extent of the shell was obvious. The core, which immunostained unevenly, was consistently more intensely immunoreactive than either medial or lateral shell in all species except the marmoset. These results suggest that the neurochemical subdivisions of shell and core established for nucleus accumbens of rodents are also present in primates. However, further work is needed to establish whether these territories are homologous and, if so, the full extent of that homology.

Animals↗

Calcium carbonate modifications in the mineralized shell of the freshwater snail Biomphalaria glabrata.

The mineralized shell (consisting of calcium carbonate) of the tropical freshwater snail Biomphalaria glabrata was investigated with high resolution synchrotron X-ray powder diffractometry and X-ray absorption spectroscopy (EXAFS). Parts from different locations of the snail shell were taken from animals of different age grown under various keeping conditions. Additionally, eggs with ages of 60, 72, 120, and 140 hours were examined. Traces of aragonite were found as first crystalline phase in 120 h old eggs, however, Ca K-edge EXAFS indicated the presence of aragonitic structures already in the X-ray amorphous sample of 72 h age. The main component of the shell of adult animals was aragonite in all cases, but in some cases minor amounts of vaterite (below 1.5%) are formed. The content of vaterite is generally low in the oldest part of the shell (the center) and increases towards the mineralizing zone (the shell margin). In juvenile snails, almost no vaterite was detectable in any part of the shell.

Aging↗

Asprich: A novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina rigida.

Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk-shell formation. To date about 16 proteins from mollusk-shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shell-forming cells of a bivalve, Atrina rigida, using probes for short Asp-containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid-rich proteins, which we named "Asprich"; a subfamily of the unusually acidic shell-matrix proteins. Polyclonal antibodies raised against a synthetic peptide of the conserved acidic1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous layer. Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We can identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium-binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg-binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well fulfill basic functions in shell formation.

Amino Acid Sequence↗