SeMet alleviates LPS-induced eggshell gland necroptosis mediated inflammation by regulating the Keap1/Nrf2/HO-1 pathway.

Exposure to lipopolysaccharide (LPS) can lead to inflammation in a variety of tissues and organs. Selenium (Se) plays a crucial role in mitigating inflammatory damage. Compared with inorganic selenium, organic selenium, such as selenomethionine (SeMet), has the advantages of a higher absorption rate and lower toxicity in animals. This study examined the protective effects of SeMet on eggshell gland tissue damage caused by LPS. Hy-Line Brown laying hens were chosen as the experimental animals and were randomly assigned to four groups: control group (C), lipopolysaccharide group (LPS), SeMet group (Se), and SeMet + lipopolysaccharide group (Se + LPS). H&E staining and transmission electron microscope were performed to observe the pathological changes of eggshell glands, oxidative stress related indicators were measured using relevant kits, qRT‒PCR and western blotting were used to evaluate the mRNA and protein levels of the Nrf2 pathway, necroptosis, and inflammation related indicators. The results showed that LPS treatment increased the content of malondialdehyde (MDA), decreased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), and decreased the content of glutathione (GSH). LPS increased the levels of Keap1, RIPK1, RIPK3, MLKL, TNF-α, COX-2, and NF-κB, while decreasing the levels of HO-1, NQO1, Nrf2, and Caspase-8. However, SeMet treatment effectively reversed the changes of the above indicators, indicating that SeMet alleviates eggshell gland cell necroptosis-mediated inflammation induced by LPS via regulating the Keap1/Nrf2/HO-1 pathway. This study elucidated the mechanism by which SeMet alleviates LPS-induced eggshell gland tissue damage in Hy-Line Brown laying hens and provided a new direction for expanding the application of SeMet in the feeding and production of laying hens.

Effect of dietary supplementation of zinc proteinate on performance, egg quality, blood biochemical parameters, and egg zinc content in White Leghorn layers.

A study was conducted to investigate the effects of zinc proteinate (Zn-P) on laying performance, egg quality, antioxidant indices, and egg zinc content in laying hens from 38 to 49 weeks of age. A total of 150 White Leghorn layers were randomly assigned to five treatments, each with six replicates with five birds per replication. Dietary treatments included a corn-soybean meal-based basal diet with no zinc addition and basal diet supplemented with Zn-P at 40, 80, 120, or 160 mg/kg of feed for 12 weeks. The analyzed zinc concentrations of the five diets were 29.5, 70.8, 110.2, 147.5, and 187.5 mg Zn/kg, respectively. Dietary Zn-P supplementation had no effect on feed intake and egg production. However, raising the zinc level improved egg weight (P < 0.01) and egg mass (P < 0.05) and lowered the feed conversion ratio (P < 0.05) during the later (46-49 weeks) period. The Zn-P supplementation also significantly (P < 0.05) increased Haugh units, egg shell strength, and shell thickness and had no influence on other egg quality parameters. Increasing zinc levels in the diet resulted in increase in egg zinc contents and serum zinc level. The serum triglyceride and LDL-cholesterol levels significantly decreased (P < 0.05) in Zn-P-supplemented groups. Supplementation of Zn-P significantly (P < 0.05) increased serum Cu-Zn-SOD activity and reduced MDA concentration. It could be concluded that dietary supplementation of higher levels of Zn-P, more than 80 mg/kg diet, significantly improved the egg zinc content, some egg quality traits, antioxidant activity, and serum zinc levels.

Superiority of coarse eggshell as a calcium source over limestone, cockle shell, oyster shell, and fine eggshell in old laying hens.

Chicken eggshell (ES) waste is a rich source of calcium carbonate (CaCO3); however, the potential of ES as dietary calcium (Ca) in old laying hens has not been explored. This study compared the effects of feeding limestone, cockle shell, oyster shell, fine ES, and coarse ES as the sole Ca source on production performance, egg quality, blood biochemical constituents, and tibia characteristics in old laying hens. A total of 450 ISA-Brown laying hens at 73 wk of age with similar egg production rate (EPR) were randomly assigned to 5 treatment groups (90 hens/group, 9 hens/replicate) for 7 wk. Dietary treatment groups comprised a corn-soybean meal based diet containing different Ca sources: (i) limestone (LS; < 2 mm and 2-4 mm mixed in the ratio of 3:7) as control, (ii) cockle shell (CS; 1-4 mm), (iii) oyster shell (OS; 3-16 mm), (iv) ES fine particles (ESF; < 1 mm), and (v) ES coarse particles (ESC; 3-5 mm). Results indicated that dietary inclusion of coarse ES particles significantly increased average egg weight (P < 0.001) and daily egg mass (P < 0.05), and decreased feed conversion ratio (P < 0.001) as compared with the other treatments. However, no significant differences in EPR, feed intake, cracked egg proportion, and mortality were observed among the dietary treatments (P > 0.05). Notably, the use of ESF led to a lower proportion of cracked eggs than ESC (P < 0.05). ESC fed hens produced the heaviest eggs whereas CS fed hens produced the lightest (P < 0.001); the particle size of ES also affected the egg weight (P < 0.05). The eggs from OS and ESC fed hens showed a greater albumen height in comparison to eggs from CS group (P < 0.05); but no significant difference was observed among the LS, OS, ESF, and ESC groups (P > 0.05). The yolk color was darker in the eggs of group ESF as compared with other dietary groups (P < 0.01). However, no significant effects on Haugh units and shell properties were observed among the treatments (P > 0.05). The blood biochemistry results were not affected by the dietary Ca (P > 0.05) except for lower levels of high-density lipoprotein percentage (HDL %) in OS and ESC fed hens (P < 0.05). The tibia characteristics including weight, length, width, and breaking strength did not differ among the dietary groups (P > 0.05). However, the ESC and OS fed hens showed higher tibia bone mineral density (BMD) than the other groups (P < 0.001). In conclusion, coarse ES as a sole Ca source had beneficial effects on the production performance, egg quality, and tibia BMD in old laying hens.

Comparison the Zn-Threonine, Zn-Methionine, and Zn Oxide on Performance, Egg Quality, Zn Bioavailability, and Zn Content in Egg and Excreta of Laying Hens.

The experiment was conducted to investigate the effect of supplementary zinc-threonine (Zn-Thr), zinc-methionine (Zn-Met), and zinc oxide (ZnO) on the laying performance, egg quality, Zn content in egg and excreta. One hundred and sixty hens (Hy-Line W36) were randomly divided into 10 treatments with 4 replications of 4 hens each. During the first 4 weeks, groups were fed a corn-soy basal diet without extra zinc (30.3 mg Zn/kg diet) to depletion storage zinc from the body and it was used as the control diet. During the ensuing 10 weeks from 36 to 45 weeks of age, 3 levels of 30, 60, and 90 mg Zn/kg were added to the diet by ZnO, Zn-Met, and Zn-Thr sources. Results showed that there were no significant differences between the experimental treatments in terms of egg weight, feed intake, eggshell weight, eggshell percentage, and albumen weight. In total experimental period, the supplementation of Zn-Met and Zn-Thr decreased feed conversion ratio and increased egg mass and egg production compared with ZnO 30 and 60 mg Zn/kg and control treatments (P < 0.05). The results showed that treatment effect on Zn content in egg and excreta was significant (P < 0.05). It is concluded that diets supplied with lower levels of organic Zn can maintain egg production performance, improve egg quality, enrich eggs, increase bioavailability, and reduce the negative effects of age on egg quality characteristics in laying hens compared with their oxide form.

Dietary cadmium chloride impairs shell biomineralization by disrupting the metabolism of the eggshell gland in laying hens.

In this study, we identified cadmium (Cd) as a potential endocrine disruptor that impairs laying performance, egg quality, and eggshell deposition and induces oxidative stress and inflammation in the eggshell glands of laying hens. A total of 480 38-wk-old laying hens were randomly assigned into 5 groups that were fed a basal diet (control) or a basal diet supplemented with Cd (provided as CdCl2·2.5 H2O) at 7.5, 15, 30, and 60 mg Cd per kg feed for 9 wk. The results showed that, when compared with the control group, a low dose of dietary Cd (7.5 mg/kg) had positive effects on egg quality by improving albumen height, Haugh unit, yolk color, and shell thickness at the third or ninth week. However, with the increase in the dose and duration of Cd exposure, the laying performance, egg quality, and activities of eggshell gland antioxidant enzymes (catalase [CAT], glutathione peroxide [GSH-Px]), and ATPase (Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase) deteriorated, and the activity of total nitric oxide synthase (T-NOS) and the level of malondialdehyde (MDA) increased significantly (P < 0.05). The histopathology and real-time quantitative PCR results showed that Cd induced endometrial epithelial cell proliferation accompanied by upregulation of the mRNA levels of progesterone receptor (PgR) and epidermal growth factor receptor (EGFR), downregulation of the mRNA levels of estrogen receptor α (ERα) and interleukin 6 (IL6), and inflammation of the eggshell gland accompanied by significantly increased expression of complement C3 and pro-inflammatory cytokine tumor necrosis factor α (TNFα) (P < 0.05). In addition, the ultrastructure of the eggshell showed that dietary supplementation with 7.5 mg/kg Cd increased the palisade layer and total thickness of the shell, but with the increase in dietary Cd supplementation (30 and 60 mg/kg) the thickness of the palisade layer and mammillary layer decreased significantly (P < 0.05), and the outer surface of the eggshell became rougher. Correspondingly, the expression of calbindin 1 (CALB1), ovocalyxin-32 (OCX-32), ovocalyxin-36 (OCX-36), osteopontin (SPP1), and ovocledidin-17 (OC-17) decreased significantly (P < 0.05) with increasing dietary Cd supplementation. Conclusively, the present study demonstrates that dietary supplementation with Cd negatively affects laying performance, egg quality, and eggshell deposition by disturbing the metabolism of eggshell glands in laying hens but has a positive effect on egg quality at low doses.

Effects of Dietary Different Levels of Nano, Organic and Inorganic Zinc Sources on Performance, Eggshell Quality, Bone Mechanical Parameters and Mineral Contents of the Tibia, Liver, Serum and Excreta in Laying Hens.

This study was conducted to determine the effect of dietary zinc (Zn) sources and their levels on the performance, egg quality, tissue mineral concentrations and bone mechanical traits of laying hens. Two hundred seventy, 44-week-old, Super Nick white laying hens were randomly distributed to 15 experimental groups in a 3 (sources of Zn) × 5 (levels of Zn) factorial arrangement, using groups of 18 birds with 6 replicates. The 15 experimental diets consisted of three sources of Zn (zinc-oxide as an inorganic form, zinc-proteinate as an organic form and nano zinc-oxide powder as a nano form) and five different levels of Zn (20, 40, 60, 80 and 100 mg Zn/kg diet). The experiment lasted 12 weeks. Dietary Zn sources, Zn levels and their interactions had no significant effect on the performance parameters between the treatment groups, nor a significant effect on eggshell weight and eggshell breaking strength. Eggshell thickness and eggshell Ca and P contents were significantly affected by the different dietary Zn sources. Dietary Zn sources, Zn levels and their interactions had no significant effect on tibia Ca and P contents, and also had no significant effect on tibia Zn content. Liver Zn content was significantly affected by the dietary Zn sources. Serum Zn content was not affected by the dietary treatments. Dietary Zn sources, Zn levels and their interactions had no significant effect on tibia weight, tibia stress and tibia breaking strength, as tibia mechanical parameters. Excreta Zn content decreased linearly when hens were fed nano Zn compared to organic and inorganic Zn, and excreta Zn content also linearly increased with increasing dietary Zn levels. In conclusion, 20 mg/kg of supplemental Zn is optimal for sustaining performance, good eggshell quality and bone status, while also reducing Zn excretion and soil pollution, with organic (Zn-proteinate) and nano (ZnO) Zn as the preferred forms in laying hen diets.

Effects of Dietary Cadmium and Boron Supplementation on Performance, Eggshell Quality and Mineral Concentrations of Bone in Laying Hens.

This study was conducted to determine the effects of supplementation of different levels of cadmium and boron on performance, eggshell quality, and mineral concentrations of bone in layer diets. In this trial, a total of 144 layer chickens, 21 weeks old, were randomly divided into 12 experimental groups. In each experimental group, there were four replicates, and in each of the replicates, there were three hens. Experimental diets consisted of all possible combinations of four levels of added cadmium (0, 5, 15, and 45 mg/kg) and three levels of added boron (0, 60, and 120 mg/kg) to the basal diet. Added cadmium (15 or 45 mg/kg) had a significant adverse effect on performance parameters (P < 0.01). Eggshell thickness increased with the addition of 5 mg/kg level of cadmium to the diet (P < 0.01). Tibia cadmium content increased with the addition of cadmium and boron in the diet (P < 0.01). However, tibia boron content decreased with the supplementation of cadmium (P < 0.01). These results indicate that the addition of boron to the diet did not prevent adverse effect of cadmium on performance and eggshell quality, or accumulation of cadmium in bone.

Interactive effects of vitamin D3 and strontium on performance, nutrient retention and bone mineral composition in laying hens.

BACKGROUND: Strontium is currently prescribed for patients with osteoporosis to increase bone density and reduce bone fractures but its relevance in animal nutrition is obscure. In order to investigate the effect of supplemental strontium and vitamin D3 on performance, egg quality and skeletal integrity in poultry a total of 108 laying hens, 99 weeks of age, were fed three levels of strontium (0, 500, 1000 mg kg(-1) ) and two levels of vitamin D3 (2500, 5,000 iu kg(-1)) over a 12-week period. RESULTS: There was an improvement (P < 0.05) in egg production and feed conversion efficiency with strontium at 500 mg kg(-1) and a significant increase in egg weight in those hens fed additional vitamin D3 . Supplemental strontium increased phosphorus, sodium and strontium retention in birds fed 2500 iu D3 kg(-1) but reduced phosphorus, sodium and strontium retention in birds fed 5000 iu D3 kg(-1), resulting in an interaction (P < 0.01) between strontium and vitamin D3 . Addition of 5000 iu D3 kg(-1) increased egg weight (P < 0.05); predominantly by increased albumen content (P < 0.05), whereas strontium supplementation reduced egg weight (P < 0.001). Similarly, 5000 iu kg(-1) D3 increased apparent metabolizable energy (P < 0.05); in contrast, strontium supplementation reduced (P < 0.05) apparent metabolizable energy. CONCLUSION: The addition of 500 mg kg(-1) strontium significantly improved egg production and feed efficiency; however, further investigation needs to be undertaken to refine the optimum level of strontium required to maximize hen performance. The interrelationship between strontium and vitamin D3 requires further exploratory study.

Integrating de novo transcriptome assembly and cloning to obtain chicken Ovocleidin-17 full-length cDNA.

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

Effects of the levels of methionine, linoleic Acid, and added fat in the diet on productive performance and egg quality of brown laying hens in the late phase of production.

Two trials were conducted to study the effect of reducing the methionine (MET, 0.36 vs. 0.31%), linoleic acid (LIN, 1.60 vs. 1.12%), and supplemental fat (SFAT, 3.0 vs. 1.1%) content of isoenergetic diets on the productive performance and egg quality of brown laying hens late in the production cycle. The 8 treatments were arranged factorially (2 x 2 x 2), with 6 replicates of 20 hens per treatment (Hy-Line, from 59 to 70 wk of age in trial 1, and Lohmann, from 56 to 75 wk of age in trial 2). Except for SFAT content, dietary treatment had little effect on laying hen performance and egg quality. The only effect of a reduction in MET content on hen performance was the decrease in the percentage of large and extra large eggs (79.8 vs. 85.9%; P < 0.05) from 60 to 67 wk of age in trial 2. A decrease in the level of SFAT reduced egg production (79.3 vs. 77.0%; P < 0.05), egg weight (66.3 vs. 64.9 g; P < 0.001), egg mass (52.5 vs. 49.8 g/d; P < 0.001), feed conversion ratio (2.26 vs. 2.36 kg of feed/kg of eggs; P < 0.001), and percentage of extra large eggs (13.1 vs. 8.2%; P < 0.05) in trial 1, but no significant differences were detected in trial 2. Reducing the LIN content of the diet from 1.60 to 1.12% did not affect any trait in either of the 2 trials. We conclude that reducing the level of SFAT from 3.0 to 1.1% might decrease productivity and the percentage of extra large eggs. However, a reduction in the MET level from 0.36 to 0.31% and in LIN from 1.60 to 1.12% did not affect any trait in hens late in the production cycle. Eggshell quality was not affected by any of the dietary treatments.

Transgenic birds for the production of recombinant proteins.

Transgenic birds were expected to be an excellent transgenic bioreactor for the production of recombinant pharmaceutical proteins. However, the only successful transgenic bioreactors have been based on mammals. We have developed two key techniques for obtaining transgenic birds. For bird embryo culture, we identified that the low rate of hatchability of cultured embryos is caused by limited oxygen and calcium availability. In quail embryo culture using a chicken eggshell as a culture vessel, hatchability increased to 80% by the supplement of calcium lactate in addition to oxygen aeration. A fully artificial vessel for quail embryo culture using a gas-permeable Teflon membrane was also designed. Although the hatchability was lower than that of cultures using a surrogate eggshell, we succeeded in hatching of bird embryos using a fully artificial vessel. For transgene introduction, a replication-defective pantropic retroviral vector based on Moloney murine leukemia virus (MoMLV) pseudotyped with vesicular stomatitis virus G protein (VSV-G) was injected to laid embryos at the blastodermal stage, and the embryos were hatched in vitro to generate G0 birds. The viral vector sequence was detected in the tissues of all G0 birds. The germ-line transmission efficiency was more than 80%. Plural copies of the transgene were inserted into the genome of G1 transgenic progeny.

Effect of hens fed dietary flaxseed with and without a fatty liver supplement on hepatic, plasma and production characteristics relevant to fatty liver haemorrhagic syndrome in laying hens.

1. Two long-term experiments were conducted with Single Comb White Leghorn (SCWL) hens (line UCD-003) predisposed to fatty liver haemorrhagic syndrome (FLHS). The first investigated the effect of adding a fatty liver supplement to the diet of laying hens prior to the onset of lay, and continuing either until peak production or throughout 39 weeks into lay. The second experiment, lasting 9 months into lay, investigated the effect of adding a fatty liver supplement, with or without 100 g/kg dietary ground flaxseed, to the diet. Body weight, feed intake, plasma triglycerides (in experiment 2) and egg production were measured throughout the experiment. Liver weight, liver fat content, liver malondialdehyde (MDA) content and liver haemorrhage score and fatty acid content of liver fat (in experiment 2) were measured at the end of each experiment. 2. In experiment 1, hens given diets containing the fatty liver supplement had higher egg production and eggshell strength, but there was no difference in liver parameters including MDA content or haemorrhage score compared with controls. 3. At the end of experiment 2, hens on 100 g/kg flaxseed diets had lower body weight, liver weight, liver dry matter and fat content, and plasma triglyceride concentrations than hens given the control diets. 4. Liver haemorrhage score was positively correlated with liver weight, but not with liver fat content, plasma triglyceride concentration or liver MDA content. This suggests that reducing the liver lipid content or feeding fatty liver supplements may not be as effective in controlling FLHS as controlling the size of the liver.

Production performance, serum/yolk cholesterol and immune competence of white leghorn layers as influenced by dietary supplementation with probiotic.

An experiment was conducted to measure the influence of a dietary probiotic on the production performance, the concentrations of cholesterol in the serum and yolk and immune competence in White Leghorn layers from 25 to 72 weeks of age. One hundred and twenty commercial White Leghorn layers, aged 24 weeks, with an average of 62% hen-day egg production, were equally and randomly distributed into three groups, with eight replicates of 5 birds in each. The birds were reared in individual laying cages. They were placed on one of three dietary treatments: basal, or basal with probiotic supplementation at a rate of 100 or 200 mg/kg feed. The addition of probiotic significantly increased the egg production, shell weight, shell thickness and serum calcium, and reduced the concentrations of cholesterol in the serum and yolk. However, no differences in these traits was observed between the groups receiving 100 or 200 mg probiotic. Feed conversion, egg weight, serum phosphorus and serum alkaline phosphatase activity were not influenced by supplementation with probiotic. Antibody production in response to the inoculation of sheep red blood cells and the cutaneous basophilic hypersensitivity (CBH) responses to inoculation with phytohaemagglutinin did not differ significantly among the dietary groups at either 24 or 40 weeks of age. The antibody titre was significantly higher in the groups supplemented with probiotic at 64 weeks of age. The addition of 100 mg/kg of probiotic in the diet significantly increased the CBH response at 64 weeks of age.

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.

Re-evaluation of calcium and phosphorus requirements in aged laying hens.

1. A series of 5 trials was conducted with 401- to 650-d-old Lohmann, Yafa (local breed with brown eggshells) and Yarkon (local breed with white eggshells) hens fed for 56 to 84 d with diets containing 25 to 50 g/kg calcium (Ca) and 4.5 to 7.6 g/kg phosphorus (P). 2. Increasing dietary Ca from 24-25 to 36-40 g/kg improved egg production, shell weight (SW) and shell thickness (ST), and decreased mortality. 3. Increasing dietary Ca to 48 to 50 g/kg did not affect egg production but increased SW and/or ST. In one out of the 4 trials, this effect was not significant, most likely because of the high shell quality of the eggs from the Yafa hens used for this trial. 4. Dietary P content of 4.5 g/kg (1.0 g/kg added inorganic P) appears to be sufficient for maintaining egg production and shell quality in aged laying hen given 36 to 40 g/kg Ca. 5. Increasing dietary Ca above 40 g/kg may require a higher dietary P content.

Biosorption of actinides from dilute waste actinide solution by egg-shell membrane.

Removal of radioactive elements from the effluent and waste aqueous solutions is an important problem. In previous laboratory batch experiments, hen egg-shell membrane (ESM) was stable as an insoluble protein and was very capable of binding heavy metal ions from aqueous solution. Batch laboratory pH profile, time dependency, and capacity experiments were performed to determine the binding of uranium (U) and thorium (Th) to ESM. Batch pH profile experiments indicated that the optimum pH for binding these actinides was approx 6.0 (U) or 3.0 (Th). The adsorption isotherms were developed at pH 5.0 (U) or 3.0 (Th) at 25 degrees C, and the adsorption equilibrium data fitted both Langmuir and Freundlich models. The maximum uptakes by the Langmuir model were about 240 mg U/g and 60 mg Th/g dry weight ESM. In addition, their adsorption capacities increased as salt concentration increased. ESM could also accumulate uranium from dilute aqueous solution by adjusting to the optimum pH. These results showed that ESM was effective for removing actinides from solution and would be useful in filtration technology to remove actinides from aqueous solution.

Calcium metabolism in olive ridley turtle eggs during embryonic development.

Analyses of calcium, magnesium, sulphur, potassium and phosphorus content of the eggshell, yolk-albumen and embryos of olive ridley turtle, Lepidochelys olivacea, have been carried out at various stages of embryonic development. Calcium is the major inorganic constituent in the egg (shell and yolk-albumen) and embryos. Other elements are present either in trace or in minute trace amounts. The egg contents (yolk and albumen) provide only 40% of the embryonic calcium requirement of the hatchling. The remaining 60% is provided by the eggshell. The eggshell also undergoes a similar reduction in its calcium content from laying to hatching. Elements other than calcium present in the yolk-albumen are sufficient for normal embryonic development. The movement of calcium from the eggshell to the embryo starts at about the 40th day of development at 29.5 degrees C. Birds, turtles and crocodiles use their eggshell as the secondary source of embryonic calcium requirement. This dependence on the eggshell varies in different groups which is highest in birds and lowest in crocodiles.

Timing of the calcium intake and effect of calcium deficiency on behaviour and egg laying in captive great tits, Parus major.

The calcium demand of egg-laying birds is much higher than in other vertebrates during reproduction. We showed elsewhere that a low level of calcium availability can greatly affect the eggshell quality and reproduction of free-living passerines. However, there are few data on calcium demand and calcium intake in relation to egg laying and behaviour and egg-laying performance under conditions of calcium shortage in nondomesticated birds. We examined these aspects in an experiment with captive great tits, Parus major, on a diet deficient in calcium, with or without snail shells as an additional calcium source. More than 90% of the calcium intake for egg production took place during the egg-laying period. Females ingested about 1.7 times as much calcium as they deposited in eggshells. Removing the snail shells after the first egg resulted in eggshell defects and interruptions of laying after 1-3 d. Females without snail shells doubled their searching effort and started to burrow in the soil and to eat sand, small stones, and their own eggs. Most calcium was consumed in the evening, probably to supplement the calcium available from the medullary bone with an additional calcium source in the gut during eggshell formation. The results demonstrated that eggshell formation requires accurate timing of the calcium intake and that obtaining sufficient calcium is time-consuming, even in calcium-rich environments. These factors pertaining to calcium intake greatly affect the ability of birds to collect sufficient calcium for eggshell formation in calcium-poor areas.

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.

Calcium homeostasis in in ovo and ex ovo turkey embryos.

Calcium homeostasis of in ovo (normal) and ex ovo (shell-less) turkey embryos was investigated at 15, 18, and 21 days of incubation. Hypocalcemia and an elevation in circulating 1,25(OH)2D3 in ex ovo embryos were observed by 15 days. Calcium and phosphorus concentrations in femora and tibiae in ex ovo embryos were significantly lower compared to their normal counterparts. These results suggest that shell calcium mobilization is required prior to 15 days of incubation for maintaining serum calcium and supporting bone mineralization. Furthermore, the elevation of 1,25(OH)2D3 is indicative of a functional calcium homeostatic mechanism responding to the absence of the primary calcium source (eggshell) during the second half of turkey embryonic development.