Utilisation of bacterial (Rubrivivax gelatinosus) biomass for egg yolk pigmentation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulation of plasma levels and percentages of incorporation of ω-3 PUFAs in egg yolk under the influence of supplementation sources rich in omega 3 to diet of laying

Hundred forty-four Shaver White laying hens were used over a 4 week experimental period to investigate the effect of 3% of soybean oil, corn oil (MIL), canola oil, flaxseed oil (LIN), salmon oil (SAL) or tuna and sardine oil (SR/AT) added to the diets, upon the fatty acid egg yolk composition, blood plasma levels and incorporation time of each fatty acid into the egg yolk. Hens were allocated into 72 cages and the experimental design was a 6 x 6 randomized factorial model. Hens fed 3% of different oils, responded with increased polyunsaturated fatty acids omega 3 (ω-3 PUFAs), except for corn oil. The addition of flaxseed, soybean or corn oil into the diet increased the PUFAs levels into the egg yolk and in the blood plasma. Adding tuna and sardine oil into the diet increased the concentration of yolk saturated fatty acids. The levels of ω-3 PUFAs were increased in the tuna and sardine oil treatment, while the flaxseed oil increased the plasma fatty acids. The deposition of 349.28 mg/yolk of a-linolenic fatty acids (ALA) was higher in the group fed LIN, while the higher equal to 157.13 mg DHA/yolk was observed in group SR/AT. In the plasma, deposition increased from 0.33% (MIL) for 6.29% ALA (LIN), while that of DHA increase of 0.47% (MIL) for 4.24% (SAL) and 4.48% (SR/AT) and of 0.98% (MIL) for 6.14% (SR/AT) and 8.44% (LIN) of ω-3 PUFAs. The percentage of EPA into the yolk and plasma was higher for the hens fed 3% tuna and sardine oil diet, as well as the levels of yolk DHA. The concentration of DHA into the plasma was higher for the salmon and tuna/sardine oil treatments. The PUFAs yolk decreased during the first eight days of experiment, while the ω-3 PUFAs increased during the same period. The concentration of ALA increased until ten days of experiment, while the percentage of EPA and DHA increased up to the eighth experimental day

(Table 1) Enantiomer fractions of egg yolk and plasma samples from Svalbard glaucous gulls (Larus hyperboreus)

Glaucous gulls (Larus hyperboreus) and their eggs from Svalbard (Norwegian Arctic) have been used as biomonitors of contaminants in the marine environment. In this study, the enantiomer fractions (EFs) of chiral chlordanes and atropisomeric polychlorinated biphenyl (PCB) congeners were determined in the blood plasma of adult male and female glaucous gulls from three breeding colonies in Svalbard. Plasma EFs were similar in magnitude and direction to EFs previously reported in glaucous gulls from other arctic food webs, suggesting overall similarities in the biochemical processes influencing the EFs of bioaccumulated organochlorine (OC) contaminants within the food webs at those locations. Additionally, EFs in yolk of eggs collected concurrently from within the same nesting colonies varied with location, laying date, and OC concentrations, and may be influenced by changes in the local feeding ecology between those colonies. No differences were found between the EFs for any analyte in female gulls compared to those found in egg yolk, indicating that processes involved in the maternal transfer of chlordanes and PCBs to eggs do not modulate the stereochemical ratio between enantiomers. Therefore, the use of eggs as a valuable and noninvasive means of OC biomonitoring may also extend to enantiomer compositions in glaucous gulls, and perhaps also in other seabird species from arctic regions.

Use of hemoglobin as alternative to peroxidases in cholesterol amperometric biosensors

Sophisticated molecular architectures can be produced with the layer-by-layer (LbL) method, which may combine distinct materials on the same film. In this study, we take advantage of this capability to produce cholesterol amperometric biosensors from LbL films containing hemoglobin (Hb) and cholesterol oxidase in addition to the polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(ethylene imine) (PEI). Following an optimization procedure, we found that an LbL film deposited onto ITO substrates, with the architecture ITO(PEI/Hb)5(PEI/COx)10, yielded a sensitivity of 93.4 μA μmol L-1 cm-2 for cholesterol incorporated into phospholipid liposomes, comparable to state-of-the-art biosensors. Hb acted as efficient electron mediator and did not suffer interference from phospholipids. Significantly, cholesterol could also be detected in real samples from chicken egg yolk, with no effects from potential interferents, including phospholipids. Taken together these results demonstrate the possible fabrication of low cost, easy-to-use cholesterol amperometric biosensors, whose sensitivity can be enhanced by further optimizing the molecular architectures of the LbL films. © 2012 Elsevier B.V.

Refinement of a cell line based artificial diet for rearing the parasitoid wasp, Trichogramma pretiosum

An artificial diet incorporating insect cells originally developed for Trichogramma australicum Girault (Hymenoptera: Tricho-grammatidae) was successfully used to rear Trichogramm pretiosum Riley (Hymenoptera: Trichogrammatidae). To refine the diet, individual components were removed. Chicken egg yolk and the insect cells were identified as the most important components for T. pretiosum development. Their removal resulted in few pupae and no adults. Removal of Grace's insect medium, a common component of artificial diets, was found to markedly improve the development of T pretiosum, producing 60% larva to pupa transition and 19% pupa to adult transition. There was no significant difference in T pretiosum development on diets in which milk powder, malt powder or infant formula were interchanged, despite differences in nutrient composition. The use of yeast extract resulted in significantly higher survival to the adult stage when compared with yeast hydrolysate enzymatic and a combination of yeast extract and yeast hydrolysate enzymatic. Comparison of four antimicrobial agents showed the antibacterial agent Gentamycin and the antifungal agent Nystatin had the least detrimental effect on T pretiosum development. The use of insect cell line diets has the potential to simplify artificial diet production and significantly reduce T pretiosum production costs in Australia compared to diets using insect hemolymph or the use of natural or factitious hosts. (c) 2005 Elsevier Inc. All rights reserved.

Characterization of sea urchin yolk glycoproteins

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, S. purpuratus, a polyclonal antibody to a 90-kDa polymannose glycoprotein was prepared. lmmunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa egg yolk glycoprotein during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified and peptide mapping revealed that they were cleavage products derived from the major yolk glycoprotein. The antibody identified a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: L. pictus, A. punctulata, and D. excentricus. However, eggs from other echinoderm classes and from chicken, frog, fruit fly, and nematode did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked, polymannose carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160-170 kDa) from each of the three sea urchin species was purified and analyzed, revealing striking similarities in pI and in amino acid and monosaccharide composition. Peptide mapping showed that the 160-kDa glycoprotein from the four echinoids are structurally homologous. The major yolk glycoprotein appeared to be proteolyzed by a thiol protease, which could be activated in yolk particles prepared from unfertilized eggs by low pH. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout embryonic development, and that externalization of the glycoproteins was not detectable. The yolk glycoprotein precursor began to be synthesized in premetamorphosis larvae, and continued in adult males and females. Both the yolk glycoproteins and the yolk platelets disappeared during larval development. This disappearance has special significance because there were no yolk proteins in the direct developing sea urchin, H. erthryogramma, which bypasses larval development and metamorphoses directly into an adult. ^

Purification and properties of riboflavin-binding protein from the egg white of the duck (Anas platyrhynckos).

Riboflavin-binding protein was purified from the egg white of domestic duck and some of its properties were investigated. The protein was homogeneous by the criteria of gel filtration on Sephadex G-100 and electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, had molecular weight of 36 000 ± 1000 and, unlike the chicken egg white protein (Mr 32 000 ± 2000), was devoid of covalently-bound carbohydrate. It was similar to the chicken riboflavin-binding protein in its behavior on ion-exchange celluloses and affinity to interact with the flavin and its coenzymes, but differed significantly in amino acid composition in that it completely lacked proline and contained less of methionine and arginine. The protein partially cross-reacted with the specific antiserum to chicken riboflavin-binding protein with a spur during immunodiffusion analysis.

Oestrogen induction of riboflavin-binding protein in immature chicks. Nature of the secretory protein.

The riboflavin-binding protein isolated from sera of oestrogen-treated male chicks as well as that synthesized and secreted in vitro by the chicken liver have the same molecular size as that of the egg-yolk protein. Functionally the serum and yolk proteins are similar. This is in contrast with the hormone-induced synthesis, secretion and deposition of phosvitin and lipovitellin in the ovary.

Oestrogen-induced synthesis of thiamin-binding protein in immature chicks. Kinetics of induction, hormonal specificity and modulation

A specific radioimmunoassay procedure was developed to monitor the plasma concentrations of thiamin-binding protein, a minor yolk constituent of the chicken egg. By using this sensitive assay, the kinetics of oestrogen-induced elaboration of this specific protein in immature chicks was investigated. After a single injection of the steroid hormone, with an initial lag period of 4–5h the thiamin-binding protein rapidly accumulated in the plasma, attaining peak concentrations around 75h and declining thereafter. A 4-fold amplification of the response was noticed during the secondary stimulation, and this increased to 9-fold during the tertiary stimulation with the steroid hormone. The magnitude of the response was dependent on the hormone dose, and the initial latent period and the duration of the ascending phase of induction were unchanged for the hormonal doses tested during both the primary and secondary stimulations. The circulatory half-life of the protein was 6h as calculated from the measurement of the rate of disappearance of the exogenously administered 125I-labelled protein. Simultaneous administration of progesterone, dihydrotestosterone or corticosterone did not alter the pattern of induction. On the other hand, hyperthyroidism markedly decreased the oestrogenic response, whereas propylthiouracil-induced hypothyroidism had the opposite effect. The anti-oestrogen E- and Z-clomiphene citrates, administered 30min before oestrogen, effectively blocked the hormonal induction. α-Amanitin and cycloheximide administered along with or shortly after the sex steroid severely curtailed the protein elaboration. A comparison of the kinetics of induction of thiamin- and riboflavin-binding proteins by oestrogen revealed that, beneath an apparent similarity, a clear-cut difference exists between the two vitamin-binding proteins, particularly with regard to hormonal dose-dependent sensitivity of induction and the half-life in circulation. The steroid-mediated elaboration of the two yolk proteins thus appears to be not strictly co-ordinated, despite several common regulatory features underlying their induction.

Structural studies of the hen's egg lipovitellins. NMR and fluorescence investigations

The pH dependent reversible association-dissociation reaction of α- and β-lipovitellins from egg yolk has been studied by 1H NMR and fluorescence probe methods. Increased mobility of the choline methyl groups has been demonstrated on dissociation. The lipid methylene resonance of β-lipovitellin shows clear doublet character suggesting that the fatty acid chains exist in distinct environments. The high field component increases with temperature but is suppressed on treatment with pronase, suggesting a significant role for proteins in maintaining the differences in lipid environments. 1-Anilino-8-naphthalene sulfonate has been shown to bind less effectively to the monomeric lipovitellins. This is in agreement with earlier results suggesting that dissociation may be accompanied by increased hydration and conformational changes.