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Effects of organochlorine residues on eggshell thickness, reproduction, and population status of brown pelicans (Pelecanus occidentalis) in South Carolina and Florida, 1969--76.

Shells of brown pelican (Pelecanus occidentalis) eggs collected in South Carolina from 1969 through 1975 and in Florida during 1969, 1970, and 1974 were significantly thinner (P greater than 0.05) than eggshells collected before 1947. Thickness of South Carolina eggshells increased in 1975, and mean thickness of eggshells collected in Florida during 1974 was greater than that of eggshells collected during 1969 and 1970, primarily in Gulf Coast colonies. Residues of 13 organochlorines were found in eggs and tissues of pelicans found dead during 1974 and 1975, although residues in brains of these specimens were not high enough to cause death. Residues of organochlorines, except PCBs, declined through 1975. PCBs increased in eggs from Atlantic Coast colonies. Reproductive success and population status of brown pelicans in South Carolina have improved markedly since authors began their studies in 1969. Good reproductive success was reported in 3 of 5 years from 1973 through 1977.

Animals

Comparative investigations of the morphology and chemical composition of the eggshells of Acanthocephala. I. Macracanthorhynchus hirudinaceus (Archiacanthocephala).

Eggshells of Macracanthorhynchus hirudinaceus (Archiacanthocephala) were investigated for their fine structure as well as their chemical composition. The acanthor larvae are surrounded by four eggshells (E1-4) separated by interstices of low electron density (G1-4). As these envelopes are secreted in different sequences and are reinforced to different degrees, their appearance varies throughout development. The outermost eggshell (E1) of this species has a tripartite appearance; it contains neither chitin nor keratin. Keratin appears in E2 and E3. It was localized electron microscopically using anti-keratin and, for the first time by fluorescence microscopy with the bromobimane reaction. Keratin occurs in two forms: in the second envelope (E2) it consists of twisted struts of filaments, whereas in the innermost sublayer of the third envelope (E3) it shows a conspicuous cross-striation; in the first and second sublayers of E3, neither keratin nor a discernible structure is present. Chitin occurs in the innermost layer (E4). The interstices G1, G3 and G4 seem to contain glycoproteins, whereas interstice G3 seems to contain some type of carbohydrate. After the extraction of proteins including keratin with sodium dodecyl sulfate (SDS) and dithiothreitol (DTE) only layers E1 and E4 remained.

Acanthocephala

Comparative investigations of the morphology and chemical composition of the eggshells of Acanthocephala. III. Eoacanthocephala.

Eggshells of the eoacanthocephalans Neoechinorhynchus rutili (Neoechinorhynchidae) and Paratenuisentis ambiguus (Tenuisentidae) were investigated for their fine structure and their chemical composition. The acanthor larvae of P. ambiguus are surrounded by four eggshells (E1-E4) separated by electron-lucent interstices (G1-G4). This resembles the stratification of the eggshells of archiacanthocephalans and palaeacanthocephalans. However, an additional outer envelope (E0) exists in N. rutili. In mature eggs of this species, the broad interstice between E0 and E1 is densely packed with polysaccharides, but the other interstices also showed an intense Thiéry label. In both species, E2 along with its outer protuberances contains keratin, as does E2 in archiacanthocephalans and palaecanthocephalans, whereas E4 does not contain chitin, which contrasts with findings in the eggs of the two other classes of acanthocephalans. The results are discussed with respect to the classification of the Acanthocephala and to their transmission to the intermediate hosts.

Acanthocephala

Influence of dietary calcium selenium and methylmercury on eggshell thickness in Japanese quail.

A high Ca, semi-purified diet promoted greater egg production with thicker shells in Japanese quail hens. 7 ppm of dietary Se depressed egg weights with no effect on eggshell thickness. The addition of 20 ppm of MHg to either a low or high Ca diet containing Se depressed both egg production and eggshell thickness. Se residues of liver, kidney, and brain in Se-MHg fed quail were significantly higher than in Se treated quail, probably reflecting the ability of Se to bind with Hg. CA, an enzyme required for the formation of the carbonate radical of Ca carbonate in eggshells, was not reduced in the Se-MHg treated Japanese quail hens' blood or oviducts.

Animals

P,p-DDT and p,p'-DDE effects on egg production, eggshell thickness, and reproduction of Japanese quail.

p,p' -DDT and p,p' -DDE were investigated for effects on egg production and eggshell thickness in Japanese quail. p,p' -DDT was examined for effects on hatchability and fertility. DDE was tested at 0, 2, 10, 40, and 200 ppm in the diet. No evidence suggested that DDE affected number of eggs laid, egg weight, or eggshell thickness at any level of DDE tested. DDT was tested at 1,2.5, 10 and 40 ppm in the diet. In one experiment, quail fed DDT at 40 ppm and caged in male-female pairs broke more eggs than quail caged similarly but fed lower amounts of DDT or than quail fed an equal amount of DDT but caged alone. DDT did not detectably reduce eggshell thickness, number of eggs laid, fertility, or hatchability. However, paired quail laid fewer eggs than did single quail in two experiments and laid eggs with thinner shells in one experiment.

Animals

Identification of a putative eggshell precursor protein of the female Schistosoma japonicum.

In adult worms of Schistosoma japonicum, a prominent radiolabelled female-specific protein (34 kDa) was demonstrated on fluorography of SDS gels with the pulse incorporation of 14C-tyrosine in vitro, though it was difficult to detect major female-specific proteins by direct staining methods. This female-specific protein was demonstrated to localize exclusively in the vitelline cells by indirect immunofluorescence using the rabbit anti-34 kDa female protein antiserum. It was shown that 14C-tyrosine was selectively incorporated into the vitelline cells by the pulse labelled autoradiographs. Two days after the exposure of worms to radio-tyrosine, the shells of eggs in the uterus were demonstrated to have become radioactive, indicating that 14C-tyrosine-labelled protein was used as a material for the eggshell. In the fluorograph of proteins extracted from newly laid eggs in vitro, the prominent band was not found at the 34 kDa region, but a lot of radioactivity appeared at higher than 100 kDa. The results suggested that a 34 kDa female protein was a precursor of the eggshell and became a much larger protein molecule as a result of cross-linking during eggshell hardening.

Animals

Incorporation of radiolabelled amino acids by adult Schistosoma japonicum: further characterization of a putative eggshell precursor protein.

The synthesis patterns of female-specific proteins of Schistosoma japonicum were further investigated with particular reference to the 34 kDa putative eggshell precursor protein. Adult male and female worms of S. japonicum were metabolically labelled with 14C-tyrosine, 14C-glycine and 35S-methionine in vitro. The rates of amino acid incorporation for female worms were significantly higher than for males in all radiolabelling experiments. Labelled proteins were resolved by two-dimensional gel electrophoresis and visualized by fluorography. By using 14C-tyrosine and 14C-glycine, the 34 kDa female protein band resolved into three major spots with pI 6.0, 5.8 and 5.6. On the other hand, labelling studies using 35S-methionine failed to reveal synthesis of any corresponding spots at Mr 34 kDa. These results, together with the observations that eggshell hydrolysates are very rich in glycine but poor in methionine, suggested that the 34 kDa putative eggshell precursor protein of S. japonicum consists of at least three isoelectric forms. In addition, we have demonstrated several other female-specific polypeptides synthesized by this worm.

Amino Acids

Pores in avian eggshells: gas conductance, gas exchange and embryonic growth rate.

The number of pores (N) in eggshells of birds were counted in 161 species ranging in egg mass (W) from 0.9 to 500 g. In addition the water vapor conductance of the shell (G) mg X (d X Torr)-1, the water loss in the nest (M) mg X d-1, the incubation duration (I) d, and the shell thickness or pore length (L) micron are listed for each species when available. Allometric equations for all variables are given when regressed on egg mass. When log G is regressed on log N the slope is essentially 1.0 indicating that the G X N-1 for average pores in bird eggshells is 1.5 micrograms H2O X (d X Torr)-1 regardless of egg mass or incubation duration. According to Fick's law of diffusion such pores have a cross-sectional area to pore length ratio of 0.67 micron 2 X micron-1. Further analysis show that N, G, M, and the rate of oxygen consumption at the pre-internal pipping stage, are all directly proportional to the absolute mean growth rate of embryos (defined as 0.67 [W/I], g X d-1). Thus, single pores of typical eggshells not only have a similar conductance G X N-1, but also the metabolic rate and rate of water loss are matched to the pore conductance so that O2, CO2 fluxes per pore at the pre-internal pipping stage and water vapor flux per pore are similar among species, namely 68, 49 and 50 microliters X d-1, respectively. The partial pressure differences across the shell at the same stage are 42, 40 and 27 Torr, respectively. Ecological as well as taxonomical variation may alter some of the relationships predicted for the 'typical' egg in order to conserve a typical overall diffusive water loss.

Animals

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

CO2 and avian eggshell formation at high altitude.

We tested the hypothesis that altitude-induced hypocapnia in hens reduces eggshell conductance to water vapor (GH2O). Seven laying hens (Gallus domesticus) native to 1200 m were chronically exposed to high altitude (3800 m), and then to high altitude with sufficient inspired CO2 to relieve hypocapnia (3800 m + CO2). Egg GH2O was measured gravimetrically, shell thickness was measured with a micrometer, and aggregate pore area was calculated from measured values using Fick's law. Comparing results at 1200 m (n = 118) and 3800 m (n = 102), GH2O was reduced from 13.9 +/- 0.2 to 12.6 +/- 0.2 mg/(d.Torr)(mean +/- SE), shell thickness was reduced from 0.297 +/- 0.003 mm to 0.287 +/- 0.003 mm, and calculated aggregate pore area per egg was reduced from 1.97 +/- 0.03 mm2 to 1.72 +/- 0.03 mm2. When hypocapnia was relieved at 3800 m + CO2 (n = 82), GH2O was reduced even further to 11.1 +/- 0.2 mg/(d.Torr), shell thickness increased to 0.305 +/- 0.003 mm, and aggregate pore area was reduced to 1.61 +/- 0.03 mm2. Based on these results we reject our hypothesis. We conclude that hypocapnia is responsible for thin eggshells at altitude. Other physiological stimuli must cause the reductions in eggshell GH2O and pore area.

Altitude

Predicted structure of a major Schistosoma mansoni eggshell protein.

The complete sequence for a major Schistosome mansoni eggshell protein gene has been determined from a genomic DNA fragment. The use of an open reading frame encoding a glycine-rich polypeptide was confirmed by in vitro translation of schistosome mRNA in the presence of [3H]glycine and comparison with the amino acid composition of purified, schistosome eggshells. Apart from the extraordinary abundance of glycine and tyrosine which are evenly distributed throughout the polypeptide chain, the most striking features of the deduced amino acid sequence are the presence of five well-conserved tandem repeats of 16-18 residues in the N-terminal region and the asymmetrical distribution of charged residues. Acidic residues (Asp) are confined to the N-terminal region, while basic residues (Lys, His), with the exception of a single histidine, are found in the C-terminal region. A model structure composed of short anti-parallel beta-strands is proposed, in which glycines and residues with small side chains lie within the strands and tyrosines and cysteines are arranged at the bends, where they would be available for cross-linking. Four such strands form one of the tandem repeats which are predicted in turn to form a stack of five closely packed beta-sheets, each of three strands and linked by the more variable fourth strand. The C-terminal region may form a similar but less compact structure. The ordered structure demonstrated by birefringence studies of the schistosome eggshell [Kusel, J. (1970) Parasitology 60, 79-88] could be formed by packing of the polypeptides such that the N-terminal domain contributes counter ions or cross-links to the C-terminal domain of adjacent molecules.

Amino Acid Sequence

The gene family encoding eggshell proteins of Schistosoma japonicum.

The four closely related genes encoding eggshell proteins in the human parasite Schistosoma japonicum are described. A cDNA and a genomic DNA library were constructed and members of the eggshell protein gene family isolated. The four genes in this family do not contain introns, and differ in organization and nucleotide sequence from the related set of genes in Schistosoma mansoni and Schistosoma haematobium. The coding sequences of two of the S. japonicum genes and their flanking regions were determined. Transcription start sites for these genes were shown by primer extension analysis to occur 47 and 50 nucleotides in front of the start codon. A female-specific component in nuclear extracts binds to a DNA fragment containing conserved sequences upstream of the transcription start sites. The deduced protein sequences of 207 and 212 amino acids are composed of 50% glycine with continuous glycine regions as long as 11 residues. In vitro translations of male and female RNAs revealed female-specific translation products, the sizes of which were consistent with the eggshell proteins.

Amino Acid Sequence

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

Relationship between thinning of eggshells and reduction in number of eggs produced after administration of some saluretic drugs in domestic fowls.

1. The effect of some diuretic drugs (acetazolamide, hydrochlorothiazide, chlorthalidone, furosemide, ethacrynic acid and amiloride) on eggshell formation and egg production in domestic fowls were investigated. All of the tested compounds significantly inhibited eggshell formation, furosemide being the most potent drug. 2. The number of laid eggs was also reduced by several of the diuretics. There was a correlation between reduction of shell thickness and number of eggs (r = 0.77; P less than 0.01). 3. If the tested diuretics were subdivided into two groups according to their different modes of action, where furosemide, ethacrynic acid and amiloride represent sodium transport inhibitors, and acetazolamide, hydrochlorothiazide and chlorthalidone (contributory action of the two latter) are inhibitors of carbanhydrase, the highest correlation coefficient (r = 0.96) between reduction of shell thickness and egg production was found for sodium transport inhibitors. The corresponding correlation coefficient for carbanhydrase inhibitors (r = 0.44) was not significant. 4. The probable mechanisms of action of the diuretics on eggshell formation and ovulation are discussed.

Animals

Optimal dietary level of 1 alpha,25-dihydroxycholecalciferol for eggshell quality in laying hens.

The optimal dietary level of 1 alpha,25-dihydroxycholecalciferol [1,25-(OH)2D3] for eggshell quality was established. White Leghorn hens, 59 wk of age, were fed one of eight diets that contained the same basal ingredients, including 3.1% calcium, but different levels (microgram/kg) or forms of calciferol supplements: no calciferol supplement of any form (56 hens); 27.5 (control) or 55.0 micrograms of cholecalciferol (56 hens each); 3, 5, or 7 micrograms of 1,25-(OH)2D3 (28 hens each); 5 micrograms of 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] with 28 hens; 5 micrograms each of 1,25-(OH)2D3 and 24,25-(OH)2D3 (28 hens). All groups were fed the control diet prior to the 21-wk treatment. The group fed 5 micrograms 1,25-(OH)2D3/kg diet ranked first in specific gravity (SG), e.g., 1.081 versus 1.077 for the control group at Week 21 (P less than .05). The group fed 7 micrograms 1,25-(OH)2D3/kg consumed 30% less feed and laid 20% fewer eggs than the control, but shell quality was not affected. The groups receiving no calciferol supplement or receiving only 24,25-(OH)2D3 laid eggs with significantly lower SG than the control after 2 wk of treatment (1.072 or less versus 1.082 at Week 2). The rest of the treatment groups mentioned were comparable to the control in eggshell quality and egg production. Groups fed the combination of 1,25-(OH)2D3 and 24,25-(OH)2D3 per kilogram of feed, or 1,25-(OH)2D3 alone at 5 micrograms/kg, had significantly higher tibial weights relative to the control group. All groups receiving the diets without cholecalciferol supplementation had markedly reduced hatchability. It was concluded that the optimal dietary level of 1,25-(OH)2D3 for improving eggshell quality without affecting egg production was approximately 5 micrograms/kg and the toxic level was 7 micrograms/kg.

Animals

Effect of cimetidine on eggshell quality and plasma 25-hydroxycholecalciferol in laying hens.

Experiments were conducted to investigate the effect of feeding cimetidine (CIMET), ranging from 0 to 750 mg/kg, on vitamin D3 metabolism and eggshell calcification in laying hens fed two levels of vitamin D3 (500 and 2,000 ICU/kg). Final BW and feed intake were not significantly affected by either CIMET or vitamin D3 level. Feeding 500 and 750 mg of CIMET significantly decreased total egg production in hens fed either level of vitamin D3, but no differences were observed at lower CIMET levels. Tibia ash decreased significantly in hens fed 150 to 750 mg of CIMET, regardless of the vitamin D3 level. Plasma Ca and inorganic P concentrations were decreased in hens fed high CIMET levels (500 and 750 mg/kg) at Week 2, but no differences were observed at Week 4. Feeding CIMET (500 and 750 mg/kg) significantly decreased plasma 25-hydroxycholecalciferol (25-OHD3) levels at Week 2 in hens fed both vitamin D3 diets but not at Week 4. Eggshell breaking force, shell thickness, and percentage shell weight were decreased significantly by CIMET in all experiments; however, in one experiment, shell quality recovered by Week 8. These results suggest that the CIMET-induced reduction in bone mineralization, eggshell quality, and plasma 25-OHD3 levels could be due to interference of CIMET with vitamin D3 metabolism in vitamin D3-replete laying hens. Shell quality decreased in CIMET-treated hens fed the higher vitamin D diet even though 250-HD3 plasma levels were three times higher than in hens fed the lower vitamin D diet, suggesting that CIMET affected shell quality through some mechanism other than inhibition of 250-HD3 synthesis.

Animal Feed

Influence of vitamin D3, 1 alpha-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 on eggshell quality, tibia strength, and various production parameters in commercial laying hens.

Four hundred 53-wk-old Hyline W36 laying hens were randomly allocated to 10 treatments. The effects of feeding two vitamin D3 metabolites, 1 alpha-hydroxyvitamin D3 [1 alpha-(OH) D3] and 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3], each at five dietary levels (0, .75, 1.50, 3.00, and 4.50 micrograms/kg of feed) were determined on eggshell quality and tibia strength in commercial laying hens (Experiment 1). In Experiment 2, 1,440 Hyline W36 65-wk-old laying hens were used to determine the effects of four levels of vitamin D3 (0, 500, 1,000, and 1,500 ICU vitamin D3/kg) and three levels of dietary 1,25-(OH)2 D3 (0, .5, and 1.0 microgram/kg of feed) on eggshell quality, tibia strength, and egg production. In Experiment 1, neither 1,25-(OH)2 D3 nor 1 alpha-(OH) D3 affected eggshell quality or production criteria. Tibia weight was increased by adding either 1,25-(OH)2 D3 or 1 alpha-(OH) D3. In Experiment 2, 1,25-(OH)2 D3 increased percentage of shell, shell weight, and egg breaking strength when 0 ICU D3/kg was fed but had no effect at higher levels of vitamin D3. Egg production, feed consumption, and egg weight were also increased with supplemental 1,25-(OH)2 D3 when 0 ICU D3/kg was fed. Tibia weight and tibia breaking strength were also increased by adding 1,25-(OH)2 D3 to the diet. The commercial laying hen metabolizes sufficient 1,25-(OH)2 D3 from dietary vitamin D3 to maintain shell quality but not enough to maintain tibia strength.

Animals

Effects of selection for growth and selection diet on eggshell quality and embryonic development in Japanese quail.

Decreased hatchability in Japanese quail following selection for growth and relative improvements in hatchability following selection during goitrogen treatment were investigated. An unselected quail line (Line C) and lines selected for high 4-wk body weight while being fed diets containing either 20% CP and .2% thiouracil (TU) (Line T), or 28% CP and no TU (Lines P and H-CD) were used. Egg weight loss between 0 and 4, and 0 and 14 days of incubation, hatchability, and developmental stage of embryonic mortalities were determined in Experiment 1. Length of the incubation period, and percentages of body water and dry body weight were determined for 10- and 14-day embryos and 18-day-old chicks in Experiment 2. Eggshell quality was determined in Experiment 3. Hatchability was lower in selected than in unselected quail due to increased early and late embryonic mortality. Egg weight loss during incubation was greater in Line P than in Line C eggs. Weight loss, measured across lines, was lower in eggs that hatched than in those in which embryos died early. Percentage body water was higher in Line C than in Lines P and T at 14 and 18 days of incubation; however, percentage body water in Line T was lower than Line P at 14 days. Percentage of dry body weight was greater in Lines P and T than in Line C at 14 and 18 days. Eggshell thickness was greater in Line H-CD when compared with Line C. Increases in eggshell permeability occurring during selection for growth were associated with increased embryonic mortality and decreased hatchability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals