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A diabetic-like condition of turkey embryos maintained in shell-less culture.

Serum insulin concentration and pancreatic insulin content were determined for turkey embryos incubated in ovo and in long-term shell-less culture (ex ovo). Insulin was undetectable (less than 10 pg) in serum from 87% of the ex ovo embryos compared with their in ovo counterparts. This was evident at all incubation ages, although insulin was detectable in more of the ex ovo embryos on Day 24. Insulin increased in the embryos incubated in ovo from 122 (Day 15) to levels exceeding 2000 pg/ml at hatching. Total pancreatic insulin content was greater in the cultured embryos on Days 15, 17, and 22 compared with their in ovo counterparts. Serum glucose was significantly greater (P less than 0.05) in the ex ovo embryos at all ages. In response to an infusion of L-arginine, serum insulin increased from 566 to 1256 pg/ml in the in ovo embryos, whereas no change was evident in the ex ovo embryos (233 vs 257 pg/ml). When embryos incubated in ovo were injected with insulin, a significant (P less than 0.05) reduction of serum glucose was observed at 60 min after injection. Serum glucose concentrations remained elevated in the embryos incubated ex ovo despite the insulin injection. Liver glucose 6-phosphatase activity, assessed on Days 15 and 22 of incubation, was found to be significantly (P less than 0.05) lower in the ex ovo embryos. Turkey embryos incubated in shell-less culture exhibited chronic hyperglycemia in concert with extremely low circulating levels of insulin. The pancreatic beta cells of these embryos were not responsive to arginine or elevated glucose. Taken together these findings suggest the occurrence of a diabetic-like condition in the ex ovo embryos. This defect in insulin secretion may, in part, be responsible for some of the developmental abnormalities characteristic of the turkey embryo cultured ex ovo.

Animals↗

Ovotransferrin is a matrix protein of the hen eggshell membranes and basal calcified layer.

The eggshell is an highly ordered structure deposited in the distal oviduct and composed of calcium carbonate and an organic matrix which is believed to influence its fabric. We have identified ovotransferrin as an 80 kDa matrix protein observed at high concentration in the uterine fluid at the initial stage of shell mineralization, by N-terminal sequencing and western blotting using monoclonal and polyclonal antibodies. It is present in extracts from demineralized eggshell and was localized by immunofluorescence in the eggshell membranes and mammillae, which are the sites of calcite nucleation. Northern blotting and RT-PCR demonstrated that ovotransferrin message was expressed in the proximal oviduct (magnum and white isthmus), and at a lower magnitude in the distal oviduct (red isthmus and uterus). Ovotransferrin was revealed by immunofluorescence in the tubular gland cells of the uterus. Calcium carbonate crystals grown in vitro in the presence of purified ovotransferrin showed large modifications of the calcite morphology. These observations and its presence in eggshell and membranes suggest a dual role for ovotransferrin, as a protein influencing nucleation and growth of calcite crystals and as a bacteriostatic filter to reinforce its inhibition of Salmonella growth in egg albumen.

Amino Acid Sequence↗

Presence of calcitonin receptors in shell gland of the guineafowl and changes in binding property during an oviposition cycle.

Radioligand binding assays of membrane fraction of the shell gland endometrium of guineafowl revealed the presence of receptor for calcitonin. The equilibrium dissociation constant (Kd) obtained by Scatchard analyses was 0.50 to 1.25 nM in laying birds and 1.12 to 1.19 nM in nonlaying birds. The maximum binding capacity (Bmax) per milligram of protein was 33.1 to 107.5 femtomoles in laying birds and 101.4 to 114.9 femtomoles in nonlaying birds. Both Kd and Bmax values changed during eggshell formation in laying birds. The results suggest that calcitonin receptor binding may be related to eggshell formation in guineafowl.

Animals↗

Magnesium and phosphorus distribution in the avian eggshell.

Magnesium and phosphorus are major inorganic constituents of the avian eggshell. The Mg/Ca ratio has been used as a palaeothermometer in a range of calcite biominerals. Eggshells provide the opportunity to examine the Mg/Ca ratio of a calcite biomineral produced in a constant temperature environment. Mg distribution is not constant throughout the shell, decreasing from nucleation until after fusion of the mammillary caps and then increasing to termination. This indicates that temperature of deposition is not the only factor controlling the Mg content of this biomineral system. There is a greater increase in magnesium concentration in the outer region of eggshells from older birds. The variation in magnesium concentration does not appear to correlate with organic content. Phosphorus occurs in the outer quarter of the eggshell and rises to termination and is therefore not confined to cuticular vesicles.

Animals↗

Crystal orientation in the shell of the domestic fowl: an electron diffraction study.

The eggshell of the domestic fowl has been studied by transmission electron microscopy and diffraction. Thin sections of shell were prepared by chemical and ion-beam thinning techniques. Each calcite column of the palisade layer consisted of crystallites of diameter 20 to 30 micrometer with some tendency for crystallite alignment within a single column. Evidence indicates that there was no significant preferred orientation in the palisade layer as a whole. Only in the surface layer was any preferred orientation detected, and here (1014) planes tended to lie parallel to the surface. The results are compared with previously published data, and calcite nucleation and growth are discussed.

Animals↗

Extraction of glycosaminoglycans from chicken eggshell.

The objectives of this study were to analyze glycosaminoglycan (GAG) and mineral composition in the chicken eggshell. Eggshells were decalcified with acetic acid, and GAG was extracted from the decalcified shell by digestion with papain. The eggshell contained an average of 0.024% of its dry weight as uronic acid, a carbohydrate moiety of GAG. The eggshell GAG consisted of approximately 48% hyaluronic acid and and 52% galactosaminoglycan. In the latter, chondroitin sulfate-dermatan sulfate copolymers were the major galactosaminoglycans with dermatan sulfate disaccharide as a relatively minor component. The inorganic material recovered after decalcification accounted for approximately 140% of dry weight of the eggshell and contained 24.11% calcium, 0.04% phosphorous, and 0.23% magnesium, with an undetectable amount of nitrogen.

Acetates↗

Biochemical analysis of a lithopedion.

Lithopedions are very rare; the medical literature mentions 330 cases. In the present case, a calcified lithopedion was found in the abdominal cavity of a 69-year-old woman who had died of unrelated causes. In general, the different organs of the lithopedion were easily distinguishable macroscopically. Microscopic examination detected reasonably well preserved structure only in skeletal muscle. Dry weight studies showed a definite dehydration (mummification) had taken place. Chemical analysis revealed a shell consisting mostly of inorganic constituents. Quantitative inorganic analysis of tissues was done and results compared to normal tissues. These results showed significant decrease of K and Cl, significant increase in Mg, P, Na and enormous increase in Ca. These data are compatible with the histologically observed calcification of the tissues. Under proper conditions, presumably dormant enzymes were reactivated in brain, liver, and muscle, although the total activity was low. Discernible LDH isoenzymes were found in liver and muscle. Discernible CK isoenzymes were found in brain and muscle.

Aged↗

Evaluation of the potential beneficial effects of ostrich eggshell combined with eggshell membranes in healing of cranial defects in rabbits.

Potential beneficial effects of outer and inner ostrich eggshell membranes alone and in combination with the shell powder on bone regeneration in cranial defects of 18 rabbits were investigated using radiological, clinical and histological methods. Three 6 mm wide, half-thickness defects were made in the calvarial bones of each rabbit. One defect remained empty as a control; prepared eggshell membranes were extended across the other two defects; and prepared eggshell powder was inserted inside the membrane in one case. Either alone or in combination with eggshell powder, the outer and inner ostrich eggshell membranes produced little adjunctive effect under the conditions and duration of this experiment.

Animals↗

Differential regulation of calbindin-D28K mRNA in the intestine and eggshell gland of the laying hen.

The effect of shell calcification and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on calbindin-D28K (previously known as vitamin D-dependent calcium-binding protein) and calbindin mRNA was investigated in the intestine and eggshell gland (ESG) of juvenile female chicks, laying hens and non-laying female birds with active gonads. Increasing amounts of 1,25-(OH)2D3 were fed to laying hens and juvenile birds treated with oestradiol to develop the ESG. The intestinal concentration of calbindin was increased 30-fold by 1,25-(OH)2D3 in chicks treated with oestradiol and fed a vitamin D-deficient diet. In these same animals, 1,25-(OH)2D3 had no effect on the formation of calbindin mRNA or calbindin in the ESG even though fully viable 1,25-(OH)2D3 receptors are present in this tissue. In laying birds fed adequate amounts of vitamin D3, intestinal, but not ESG, calbindin was increased by the addition of 1,25-(OH)2D3 to the diet. At the onset of egg production the concentrations of calbindin and calbindin mRNA were increased in the intestine and ESG. This increase occurred within the period of calcification of the first egg, through a process unaffected by vitamin D. Calcification of the first egg increased the concentration of calbindin in the ESG by eight- to tenfold, although the concentration of calbindin mRNA was increased by only two- to threefold. These results suggest that the induction of calbindin synthesis by 1,25-(OH)2D3 or by the egg calcification process is associated with an increase in the concentration of calbindin mRNA in the ESG and intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Raman investigation of calcium carbonate bone substitutes and related biomaterials.

The interaction between biomaterials used in surgical procedures and the host bone is not yet perfectly understood. It appears that these problems may have been encountered because of insufficient characterisation of the basic component used in the synthesis of such biomaterials. Calcium carbonate (CaCO3) is interesting for bone filling or regeneration procedures because of its resorbability. The aim of this work is to compare different CaCO3 biomaterials and their basic source with the help of microRaman spectroscopy. Bionacre and Biocoral are analysed. The main bands of carbonate internal modes are observed around 1084-86 and 704-12 cm-1. In the lattice modes region, for both Biocoral and synthetic aragonite, two bands at 206 and 155 cm-1 are observed. The eggshell, oyster shell and synthetic calcite samples exhibit bands at 281 and 155 cm-1. Three bands are present at 280, 206 and 155 cm-1 on the Bionacre sample. The 206 and the 280 cm-1 bands are due to aragonite and calcite forms respectively. Therefore it appears to be a mixture of aragonite and calcite whereas Biocoral is pure aragonite. Additional Raman investigations should be of great interest in evaluating the structural modifications and their influence on the biological behaviour of these biomaterials. Lity index, the percentage of prematurity and of low birth weight are the indices of national health status.

Animals↗

Effects of reducing dietary protein, methionine, choline, folic acid, and vitamin B12 during the late stages of the egg production cycle on performance and eggshell quality.

A series of four experiments was conducted to determine whether-shell quality during the late stages of egg production can be improved by using diets that are effective in reducing egg size. The experiments involved dietary manipulation of protein, methionine, choline, folic acid, and vitamin B12. In experiment 1, reducing dietary protein in combination of reducing the dietary methionine and choline or this diet without supplemental folic acid and vitamin B12 resulted in reduced egg weight and improved shell quality. However, egg production also was drastically reduced. In experiment 2, reducing the dietary level of methionine, without adding supplemental choline, folic acid, and vitamin B12 reduced egg size and improved shell quality, but egg production was reduced as well. In this experiment reducing the dietary methionine without supplemental folic acid and vitamin B12 reduced egg size and improved shell quality with no adverse effect on egg production. In experiment 3, reducing the dietary level of methionine and choline or reducing the dietary level of choline, folic acid, and vitamin B12 reduced egg size and improved shell quality without adverse effects on egg production. On the other hand, reducing dietary methionine, folic acid, vitamin B12, and supplemental choline reduced egg weight and improved shell quality but lowered egg production. In experiment 4, reducing dietary methionine together with reducing choline and vitamin B12 reduced egg size and improved shell quality with no adverse effect on egg production. The results of this series of experiments generally indicate that certain manipulations of the combination of methionine, choline, folic acid, and vitamin B12 have the potential to reduce egg weight and improve shell quality without affecting egg production during the latter stages of the egg production cycle.

Animal Feed↗

Abnormal characteristics of the blood from chick embryos maintained in "shell-less" culture.

Chick embryos were maintained in shell-less culture up to a total age of 15 days. The composition of their blood was analyzed together with the blood from coontrol embryos of comparable degree of differentiation. The blood from cultured embryos had lower hematocrit values; their serum contained a reduced concentration of proteins, phospholipids and total calcium and an increased concentration of inorganic phosphorus. In view of the reduced concentration of proteins and phospholipids, the concomitant hypocalcemia must represent, at least in part, a reduction in the binding capacity of the serum and not only a decrease in the ionic fraction of serum calcium.

Animals↗

Effects of varying chamber construction and embryo pre-incubation age on survival and growth of chick embryos in shell-less culture.

Shell-less culture involves culturing chick embryos with associated yolk and albumen outside of the eggshell and shell membranes. The technique allows direct access to and continuous observation of cultured embryos almost to the time of hatching. The plastic wrap/culture tripod technique described in this paper allows normal embryonic growth and differentiation from 48 hours (in ovo pre-incubation age) through at least 10 days of total incubation. As the duration of in ovo pre-incubation is decreased below 36 hours, there is a concomitant increase in the percentage of grossly abnormal embryos associated with decreased survival and retarded growth and differentiation. Survival of embryos pre-incubated for 72 hours through 13 and 18 days of total incubation is greater than 80% and 40%, respectively. The wrap/tripod technique allows substantially better survival and more normal development of cultured embryos than does the petri dish technique of Auerbach et al. (1974). Embryonic growth in 7.8-cm-diameter chambers is significantly greater than in either larger (10.6 cm) or smaller (5.2 cm) diameter chambers. Some gas exchange through the culture chamber walls appears necessary for optimal embryonic survival and growth. Suspending egg contents in either Safeway or Handi-Wrap plastic wrap (both of intermediate permeability) in 7.8-cm tripods resulted in superior growth and/or survival compared to suspension in Silastic sheeting (high permeability), Saran Wrap (low permeability), glass dishes (nonpermeable), or glass dishes lined with an inner layer of Safeway wrap(nonpermeable).

Animals↗

Role of type X collagen on experimental mineralization of eggshell membranes.

Type X collagen is a transient and developmentally regulated collagen that has been postulated to be involved in controlling the later stages of endochondral bone formation. However, the role of this collagen in these events is not yet known. In order to understand the function of type X collagen, if any, in the process of biomineralization, the properties of type X collagen in eggshell membranes were further investigated. Specifically, calvaria-derived osteogenic cells were tested for their ability to mineralize eggshell membranes in vitro. Immunohistochemistry with specific monoclonal antibodies was used to correlate the presence or absence of type X collagen or its propeptide domains with the ability of shell membranes to be mineralized. The extent of mineralization was assessed by Von Kossa staining, scanning electron microscopy and energy-dispersive spectroscopy. The results indicate that the non-helical domains of type X collagen must be removed to facilitate the cell-mediated mineralization of eggshell membranes. In this tissue, intact type X collagen does not appear to stimulate or support cell-mediated mineralization. We postulate that the non-helical domains of type X collagen function in vivo to inhibit mineralization and thereby establish boundaries which are protected from mineral deposition.

Amino Acid Sequence↗

The influence of line and laying period on the relationship between different eggshell and membrane strength parameters.

We investigated the correlations between different eggshell and membrane strength parameters and their changes over time. Eggshell and membrane characteristics were measured for 2 different lines of laying hens, Hisex Brown and Bovans Brown, from wk 33 up to wk 78. Several parameters were investigated: 2 parameters concerning total eggshell strength (static and dynamic stiffness), 1 structural parameter (i.e., eggshell thickness), 2 parameters describing the material characteristics of the shell [dynamic Young's modulus (EM) and nitrogen content], and 3 parameters to measure the membrane characteristics (attachment force breaking strength and nitrogen content). This study shows that the correlations among parameters change during the laying period and among lines. The EM of the eggshell increased during the laying period for the Hisex line. No difference was observed in case of the Bovans line. In general, a negative correlation was obtained between eggshell thickness and the EM. The correlation between static and dynamic stiffness increased during lay. Finally, the attachment force and breaking strength of the membranes declined during the laying period.

Animals↗

Oviducal structure in four species of gekkonid lizard differing in parity mode and eggshell structure.

Oviducal structure was analysed in vitellogenic females from four species of gekkonid lizard exhibiting variation in parity mode and eggshell structure: Hemidactylus turcicus (oviparous) which produces a hard, calcareous eggshell; Saltuarius wyberba (oviparous) which produces a soft, parchment-like eggshell; and Hoplodactylus maculatus and Hoplodactylus duvaucelii (both viviparous). Oviducts were analysed by light, scanning electron and transmission electron microscopy. The uterus exhibited differences among species that were directly attributable to parity mode. H. turcicus and S. wyberba (oviparous) had numerous uterine shell glands; H. maculatus and H. duvaucelii (viviparous) had very few. The uterus also exhibited differences between the two oviparous species (H. turcicus and S. wyberba) which may be related to the type of eggshell produced. Variations were noted in the staining properties of the uterine glandular and epithelial cells. The structure of the infundibulum, uterine tube, isthmus and vagina also differed among species, but differences could not be directly related to parity mode or eggshell structure. Instead, the differences may be related to how prepared the oviduct is for ovulation in individuals analysed from the different species. This study confirms, in the Gekkonidae, aspects of oviducal structure that have been associated with parity mode in other squamate taxa.

Animals↗

The avian eggshell extracellular matrix as a model for biomineralization.

The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest that the dermatan sulfate proteoglycans may be important in modulating crystal morphology in the hen eggshell and correlate with mineralization-modulating biomolecules from other calcified tissue, which are generally anionic.

Animals↗

Effect of breed and age of hens on the yolk to albumen ratio in two different genetic stocks.

A commercial egg-type chicken (ISA Brown; CEC) and a dual-purpose pure breed of the Korean Native Chicken (KNC) were compared for yolk to albumen (Y:A) ratio and their major egg characteristics at seven different ages. The eggs from KNC were significantly (P < 0.01) lighter in egg, albumen, and shell weight than those from CEC. However, the yolk weight and Y:A ratio of the eggs from KNC were significantly (P < 0.01) heavier or larger than the ones of CEC. The effect of age on the yolk, shell weight, and shell thickness was highly significant (P < 0.01), whereas significant differences were not found for Y:A ratio and other major egg components. The breed-by-age-of-hen interactions were significant for shell weight (P < 0.03) and shell thickness (P < 0.01). The yolk, albumen, and shell weight were positively (P < 0.001) associated with egg weight in both breeds. The association of Y:A ratio with egg weight in both breeds also were highly significant (P < 0.001) but negative. The results of the present study indicated that the different effects on the Y:A ratio were due to breed rather than age. Results suggest that the eggs from KNC are more desirable for use in products made of yolk than for consumption as table eggs.

Aging↗