iTRAQ-Based Quantitative Proteomic Analysis of Duck Eggshell During Biomineralization.

The avian egg is a valuable model for the calcitic biomineralization process as it is the fastest calcification process occurring in nature and is a clear example of biomineralization. In this study, iTRAQ MS/MS is used to detect and study for the first time: 1) the overall duck eggshell proteome; 2) regional differences in the proteome between the inner and outer portions of the duck eggshell. The new reference protein datasets allow us to identify 179 more eggshell proteins than solely using the current release of Ensembl duck annotations. In total, 484 proteins are identified in the entire duck eggshell proteome. Twenty-eight novel proteins of unknown function that are involved in eggshell formation are also identified. Among the identified eggshell proteins, 54 proteins show differential abundances between the inner, partially mineralized eggshell (obtained 16 h after ovulation) compared to the overall complete eggshell (normally expulsed eggshell). At least 64 of the abundant matrix proteins are common to eggshell of 4 different domesticated bird species (chicken, duck, quail, turkey) and zebra finch. This study provides a new resource for avian eggshell proteomics, and augments the inventory of eggshell matrix proteins that will lead to a deeper understanding of calcitic biomineralization.

Genome-wide association study revealed a promising region and candidate genes for eggshell quality in an F2 resource population.

BACKGROUND: Eggshell is subject to quality loss with aging process of laying hens, and damaged eggshells result in economic losses of eggs. However, the genetic architecture underlying the dynamic eggshell quality remains elusive. Here, we measured eggshell quality traits, including eggshell weight (ESW), eggshell thickness (EST) and eggshell strength (ESS) at 11 time points from onset of laying to 72 weeks of age and conducted comprehensive genome-wide association studies (GWAS) in 1534 F2 hens derived from reciprocal crosses between White Leghorn (WL) and Dongxiang chickens (DX). RESULTS: ESWs at all ages exhibited moderate SNP-based heritability estimates (0.30 ~ 0.46), while the estimates for EST (0.21 ~ 0.31) and ESS (0.20 ~ 0.27) were relatively low. Eleven independent univariate genome-wide screens for each trait totally identified 1059, 1026 and 1356 significant associations with ESW, EST and ESS, respectively. Most significant loci were in a region spanning from 57.3 to 71.4 Mb of chromosome 1 (GGA1), which together account for 8.4 ~ 16.5% of the phenotypic variance for ESW from 32 to 72 weeks of age, 4.1 ~ 6.9% and 2.95 ~ 16.1% for EST and ESS from 40 to 72 weeks of age. According to linkage disequilibrium (LD) and conditional analysis, the significant SNPs in this region were in extremely strong linkage disequilibrium status. Ultimately, two missense SNPs in GGA1 and one in GGA4 were considered as promising loci on three independent genes including ITPR2, PIK3C2G, and NCAPG. The homozygotes of advantageously effective alleles on PIK3C2G and ITPR2 possessed the best eggshell quality and could partly counteract the negative effect of aging process. NCAPG had certain effect on eggshell quality for young hens. CONCLUSIONS: Identification of the promising region as well as potential candidate genes will greatly advance our understanding of the genetic basis underlying dynamic eggshell quality and has the practical significance in breeding program for the improvement of eggshell quality, especially at the later part of laying cycle.

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.

Uterine fluid proteins and egg quality characteristics for 2 commercial and 2 heritage laying hen lines in response to manipulation of dietary calcium and vitamin D3.

The aim of this study was to evaluate the quality of eggs from 2 selected commercial strains of laying hens and 2 unselected lines of chickens fed diets with different combinations of Ca and vitamin D and relate it to the profile of uterine proteins and ultrastructure of the shell. A group of 4 chickens was housed in each of 24 cages. The group consisted of one representative from each of the following breeds: Lohmann LSL- Lite, Lohmann Classic-Brown, Fayoumi, and Light Sussex. Six dietary combinations of Ca and vitamin D(3) (3.35%, 2,500 IU; 4.10%, 2,500 IU (control); 4.85%, 2,500 IU; 3.35%, 200 IU; 4.10%, 200 IU; and 4.85%, 200 IU) were randomly assigned to 4 replicate cages for 2 treatment periods (26-29 and 56-59 wk of age). Data were analyzed as a split-plot design with cage as the main plot and hen as the subplot. Egg quality traits were different (P < 0.0001) between commercial and heritage breeds. Lohmann Brown had stronger shells with higher specific gravity compared with other breeds. Both commercial and heritage birds responded to a drop in vitamin D3 level by marked reduction in shell thickness. The SDS-PAGE profiles of uterine fluid samples revealed a decrease (P < 0.05) in 200-, 150-, 116-, and ≤6.5-kDa proteins, whereas proteins with molecular weight (MW) of 80, 55, 52, 45, 42, and 28 kDa increased with bird age. A 36- and 52-kDa protein band was most intense for Fayoumi compared with other breeds. Ultrastructural characteristics showed flattened and deeply etched mammillary caps for Lohmann Brown and the presence of type A and type B bodies between mammillary cones in eggshells from Fayoumi and Lohmann Lite. The negative correlation between ultrastructural characteristics, which decrease with bird age, and the 116-kDa uterine protein band could provide insight into reduced eggshell quality as hens age.

Effects of propolis on eggshell microbial activity, hatchability, and chick performance in Japanese quail (Coturnix coturnix japonica) eggs.

Propolis is a sticky resin produced by worker honeybees from substances collected from plants, and it has strong antibacterial and antifungal properties. The purpose of this study was to establish the effects of propolis on egg weight loss, hatchability, chick performance, and to control microbial activity naturally occurring on eggshells. A total of 750 fresh eggs was randomly divided into 5 groups. Eggs from the first group were sprayed with ethyl alcohol (70%, A), the second group was sprayed with benzalkonium chloride (B), and the third, fourth, and fifth groups were sprayed with propolis at 3 doses: 5, 10, and 15%. Eggs sprayed with propolis had lower egg weight loss than eggs from groups A and B (P < 0.001). Bacterial activity was reduced significantly in all propolis groups. There were no significant differences between treatments for hatchability, embryonic mortality, BW gain, and relative growth. Results of the present study indicated that propolis could be an alternative hatching egg disinfectant versus a chemical disinfectant, without adverse effects on hatchability and performance of quail chicks.

Residual yolk energetics and postnatal shell growth in Smooth Softshell Turtles, Apalone mutica.

We examined functions of residual yolk (RY) in hatchling Smooth Softshell Turtles (Apalone mutica). Removal of RY did not affect survival, shell growth, or resting metabolic rates of turtles for 40 d after hatching. Our estimates of metabolic rate suggest that RY can fuel maintenance and activity metabolism for approximately 25 days. A. mutica absorb more than 1g of water in the first 2 weeks of life, which appears to be the basis of post-hatch shell expansion rather than yolk-provisioned growth. Post-hatch growth may be limited by the magnitude of RY remaining at hatching, but RY protein and lipid proportions do not differ from those of freshly-laid eggs. In addition, A. mutica did not use RY to fuel nest emergence. Our results suggest that RY does not fulfill several hypothetical functions in A. mutica, including postnatal growth, catabolic fuel for nest emergence, and long-term nutritional sustenance for maintenance, activity, or hibernation. Instead, A. mutica appear to absorb most yolk prior to hatching, and are left with a minimum of RY. Variation in RY mass with incubation regime in other species suggests that mothers may overprovision their eggs to ensure successful development across a diversity of possible incubation conditions.

Development of a method to control the water evaporation of hatching eggs during incubation.

Three experiments were conducted to develop methods to control the amount of water loss and to evaluate the metabolic effects of water condition in the White Leghorn breeder eggs during incubation. One hundred twenty, 54, and 90 Julia strain White Leghorn breeder eggs were incubated at 37.8 degrees C, 60% RH in experiments 1, 2, and 3. In experiment 1, eggs were drilled with various bore diameters of 0, 0.5, 1, 2, 3, 4, and 5 mm on the blunt end of the eggshell. In experiment 2, 4 x 4 mm(2) windows were cut into the eggs or the eggs were drilled with 5 holes of bore diameter 2 mm on the blunt end of eggshell. In experiment 3, eggs were drilled with 1, 3, 5, and 7 holes of diameter 2 mm on the blunt end of eggshell. Eggs were treated on d 3 of each experiment and the amount of water loss was recorded on d 19 of incubation. Embryo growth was evaluated in experiments 2 and 3. In addition, the livers of embryos were collected in the 0-, 1-, 3-, and 5-hole treatment groups after weighing eggs to determine 3-hydroxy acyl coenzyme A dehydrogenase activity. In experiment 1, although higher water loss was observed in all windowed eggs than in control, there were no differences in amount of water loss among all bore diameters. Accordingly, that was not successful to control amount of water loss. In experiment 2, higher water loss was observed in drilled eggs at the same levels in windowed eggs as in control. Drilling holes was a more useful treatment to control amount of water loss on incubated eggs than windowing. In experiment 3, amount of water loss increased linearly with increasing number of holes on the blunt end of eggshell. Hepatic 3-hydroxy acyl coenzyme A dehydrogenase activity increased with increasing the number of drilled holes.

Reproductive and developmental effects of transovarian exposure to o,p'-DDT in Japanese quails.

Avian species have the possible risk of embryonic exposure to persistent, lipophilic environmental contaminants, such as dichlorodiphenyltrichloroethane (DDT), by transfer of chemicals accumulated in mother birds to eggs. To model developmental and reproductive disorders of wild birds living in contaminated areas, we exposed Japanese quails in ovo to o,p'-DDT prior to incubation. A positive estrogenic substance diethylstilbestrol (DES; 1 and 10 ng/g of egg) and o,p'-DDT (1-100 microg/g of egg) were injected into the yolk before incubation. Treatment with o,p'-DDT (10 or 100 microg/g) but not with DES significantly reduced the hatchability of eggs. After sexual maturation, o,p'-DDT affected eggshell formation in female quails but had little influence on laying; high doses of o,p'-DDT significantly reduced eggshell strength, shell weight, and shell thickness, and several females treated with 100 microg o,p'-DDT/g laid eggs lacking shells. Diethylstilbestrol decreased egg production itself but had little effect on the eggshell. Both o,p'-DDT and DES caused dose-dependent shortening of the left oviduct and abnormal development of the right oviduct in females, while testis asymmetry was observed in males treated with a high dose of DES. In the uterus of the oviduct, the mRNAs for calcium-regulating factors osteopontin and calbindin D28K were reduced by both treatments, particularly that with o,p'-DDT. The results indicated that transovarian exposure to o,p'-DDT could bring about population declines in avian species through loss of fecundity caused by depression of hatchability and dysfunction of the reproductive tract.

Organochlorine pesticides, PCBs, dioxins, and metals in postterm peregrine falcon (Falco peregrinus) eggs from the Mid-Atlantic states, 1993-1999.

Peregrine falcons were extirpated from the eastern United States by 1964 due to the effects of dichloro-diphenyl-trichlorethane (DDT) (Peakall and Kiff 1988). As a result of restoration efforts, peregrines have largely recovered in the region but remain a barometer of environmental contamination. In the course of monitoring nests, biologists in the mid-Atlantic states collected peregrine falcon eggs that failed to hatch. In the period 1993-1999, 93 eggs were collected from 66 nests in 31 locations in New Jersey, Pennsylvania, Delaware, Maryland, and Virginia. We analyzed eggs for organochlorine pesticides, PCBs, and metals, and calculated toxic equivalencies (TEQs) for dioxins and furans. Organochlorine contaminants were detected in eggs from all parts of the region. Although nest success in all parts of the region was good, the PCB TEQ in the Atlantic-New Jersey region was significantly related to nest success, and the regionwide PCB TEQ was nearly significant for nest success across the five-state area. dichloro-diphenyl-dichloroethylene (DDE), DDT, and total PCBs were negatively correlated with eggshell thickness, although eggshell thinning (10.4%) was not at a level associated with deleterious population effects. The five states represented in this study are productive for peregrine falcons and have contributed to the recovery of this species. However, the results suggest that Atlantic coastal peregrines might be subject to contaminant burdens that have the potential to decrease nest success and productivity.

Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro.

We investigated matrix-mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix-mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {104} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {104} calcite faces. OPN inhibited calcite growth in vitro at the {104} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.

Do egg-laying crocodilian (Alligator mississippiensis) archosaurs form medullary bone?

It is beyond question that Mesozoic dinosaurs, like Aves and Crocodylia, are archosaurs. However, within the archosaurian clade, the origin and distribution of some major features are less clear, particularly with respect to reproductive physiology. Medullary bone, a highly mineralized, bony reproductive tissue present in the endosteal cavities of all extant egg-laying birds thus far examined, has recently been reported in Tyrannosaurus rex. Its presence or absence in extant crocodilians, therefore, may shed light on the timing of its evolutionary appearance. If medullary bone is present in all three taxa, it arose before the three lineages diverged. However, if medullary bone arose after this divergence, it may be present in both extinct dinosaurs and birds, or in birds only. If present in extinct dinosaurs and birds, but not crocodilians, it would indicate that it arose in the common ancestor of this clade, thus adding support to the closer phylogenetic relationship of dinosaurs and birds relative to crocodilians. Thus, the question of whether the crocodilian Alligator mississippiensis forms medullary bone during the production of eggs has important evolutionary significance. Our examination of long bones from several alligators (two alligators with eggs in the oviducts, one that had produced eggs in the past but was not currently in reproductive phase, an immature female and an adult male) shows no differences on the endosteal surfaces of the long bones, and no evidence of medullary bone, supporting the hypothesis that medullary bone first evolved in the dinosaur-bird line, after the divergence of crocodilians from this lineage.

Effect of dietary supplementation of organic or inorganic zinc on carbonic anhydrase activity in eggshell formation and quality of aged laying hens.

This study evaluated the effects of different dietary levels and sources of zinc (Zn) on performance and carbonic anhydrase (CA) activity in eggshell formation and quality in aged laying hens. A total of 504 Hy-line Grey layers aged 59 wk were fed a basal diet (Zn, 28.4 mg/kg) for 4 wks, then randomly allocated to 7 groups that were fed a basal diet or a basal diet supplemented with inorganic (ZnSO4·H2O) or organic (amino acid metals, 9.58%) Zn at 35, 70, or 140 mg Zn per kg of feed for 6 weeks. Each group had 6 replicates of 12 hens. Results showed that egg weight decreased linearly with the supplemental level of organic Zn (P < 0.05). Dietary Zn supplementation had linear and quadratic effects on the CA activity in plasma (P < 0.05), and it was higher in the organic Zn-added groups at wks 2 and 4 (P < 0.05). Dietary Zn supplementation had a quadratic effect on the CA activity in the eggshell gland (P < 0.05). Shell thickness was greater in the organic Zn-added groups (P < 0.05), and its relationship with the supplemental level of Zn showed linearly and quadratically, increasing with the organic Zn and with the inorganic Zn at wk 4, while linearly increasing with the inorganic Zn at wk 6 (P < 0.05). At wk 4, the supplemental level of inorganic Zn had a linear effect on shell weight, and linear and quadratic effects on shell index and ratio (P < 0.05), while shell weight, the index, and ratio increased linearly and quadratically with the organic Zn level in the diet (P < 0.05), with more obvious effects in the organic Zn-added groups (P < 0.05). Overall, dietary Zn supplementation, up to 140 mg/kg feed, could increase eggshell thickness by enhancing CA activity in the plasma and eggshell gland of aged layers; thicker eggshells were found in the organic Zn-added groups, but the breaking strength did not increase despite the eggshell thickness increasing.

Effect of dietary supplementation of organic or inorganic manganese on eggshell quality, ultrastructure, and components in laying hens.

This study evaluated the effects of dietary supplemental levels and sources of manganese (Mn) on performance, eggshell quality, ultrastructure, and components in laying hens. A total of 1,080 46-wk-old Jing Brown hens were fed a basal diet (Mn, 32.7 mg/kg) for 2 wks laying and then randomly allocated to 9 groups that were fed a basal diet (control) or the basal diet supplemented with inorganic (MnSO4·H2O) or organic (amino-acid-Mn, 8.78%) Mn at 40, 80, 120, or 160 mg per kg of feed for 8 wks. Each group had 8 replicates of 15 hens. The results showed that dietary Mn supplementation did not affect the performance of hens (P > 0.05). Dietary Mn supplementation resulted in linear and quadratic increases of breaking strength and thickness in both inorganic and organic forms (P < 0.05), but fracture toughness increased quadratically only in organic groups (P < 0.05). Linear and quadratic effects on effective and mammillary thickness were observed with Mn supplementation from inorganic and organic sources (P < 0.05), and lower mammillary thickness was observed in organic groups (P < 0.05). However, the width of mammillary knobs decreased quadratically only with the supplementation of organic Mn (P < 0.05). Dietary Mn supplementation had a quadratic effect on the shell Mn content in both inorganic and organic forms (P < 0.05). Linear and quadratic effects on the content of sulfated glycosaminoglycans (GAGs) were observed only in calcified eggshell with inorganic Mn supplementation (P < 0.05), while the supplementation of organic Mn had a quadratic effect on sulfated GAGs content in both calcified eggshell and membranes (P < 0.05). Overall, dietary Mn supplementation, regardless of the source, could increase breaking strength and thickness by improving the ultrastructure, which partly results from increased sulfated GAGs content in the eggshell. Moreover, the supplementation of organic Mn could increase fracture toughness by decreasing the width of mammillary knobs, which is partially due to increased sulfated GAGs content in the membranes.

Effects of S6-strain Mycoplasma gallisepticum inoculation at ten, twenty-two, or forty-five weeks of age on the performance characteristics of commercial egg laying hens.

Experimental inoculation of commercial laying hens, maintained under controlled conditions, with the S6-strain of Mycoplasma gallisepticum (S6MG) at 10 wk of age has previously been shown to affect the lengths and weights of various portions of the reproductive tract without affecting subsequent performance. Two trials were conducted to compare the effects of S6MG inoculation at 10 wk of age (prior to lay), 22 wk of age (onset of lay), and 45 wk of age (during lay) on performance characteristics in commercial layers housed and maintained under controlled conditions, as in previous studies. In each trial, BW, mortality, egg production, egg weight, eggshell weight per unit of surface area, percentage eggshell weight, percentage albumen weight, percentage yolk weight, and yolk weight per albumen weight ratio were examined at various ages throughout an entire laying cycle. Across wk 47 and 58 (age periods after the last 45 wk inoculation), eggshell weight per unit of surface area and percentage eggshell weight were significantly reduced in birds that had received an S6MG inoculation at 45 wk of age when compared with birds that had not received an S6MG inoculation or had been inoculated with S6MG at either 10 or 22 wk of age. Alterations in eggshell quality in response to S6MG may become evident only in older birds that are experiencing declines in production when housed under controlled conditions.

Egg shell quality in Japanese quail: characteristics, heritabilities and genetic and phenotypic relationships.

The objective of the present study was to estimate heritabilities as well as genetic and phenotypic correlations for egg weight, specific gravity, shape index, shell ratio, egg shell strength, egg length, egg width and shell weight in Japanese quail eggs. External egg quality traits were measured on 5864 eggs of 934 female quails from a dam line selected for two generations. Within the Bayesian framework, using Gibbs Sampling algorithm, a multivariate animal model was applied to estimate heritabilities and genetic correlations for external egg quality traits. The heritability estimates for external egg quality traits were moderate to high and ranged from 0.29 to 0.81. The heritability estimates for egg and shell weight of 0.81 and 0.76 were fairly high. The genetic and phenotypic correlations between egg shell strength with specific gravity, shell ratio and shell weight ranging from 0.55 to 0.79 were relatively high. It can be concluded that it is possible to determine egg shell quality using the egg specific gravity values utilizing its high heritability and fairly high positive correlation with most of the egg shell quality traits. As a result, egg specific gravity may be the choice of selection criterion rather than other external egg traits for genetic improvement of egg shell quality in Japanese quails.

A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling.

Cysteine proteinases are involved in a variety of important biological processes and have been implicated in molting and tissue remodeling in free living and parasitic nematodes. We show that in the lymphatic filarial nematode Brugia pahangi molting of third-stage larvae (L3) to fourth-stage larvae is dependent on the activity of a cathepsin L-like cysteine protease (CPL), which can be detected in the excretory/secretory (ES) products of molting L3. Directed cloning of a cysteine protease gene in B. pahangi and analysis of the expressed sequence tag (EST) and genomic sequences of the closely related human lymphatic filarial nematode Brugia malayi have identified a family of CPLs. One group of these enzymes, Bm-cpl-1, -4, -5 and Bp-cpl-4, is highly expressed in the B. malayi and B. pahangi infective L3 larvae. Immunolocalization indicates that the corresponding enzymes are synthesized and stored in granules of the glandular esophagus of L3 and released during the molting process. Functional analysis of these genes in Brugia and closely related CPL genes identified in the filarial nematode Onchocerca volvulus and the free living model nematode Caenorhabditis elegans indicate that these genes are also involved in cuticle and eggshell remodeling.

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.

Effect of dietary calcium level on medullary bone calcium reserves and shell weight of Leghorn hens.

Two experiments were conducted to determine the effect of different levels of dietary Ca and subsequent feeding of a very low level of Ca on the medullary bone Ca reserves of laying hens. In Experiment 1, a total of 30 40-wk-old Single Comb White Leghorn hens were offered a diet with 2.5, 3.5, or 4.5% Ca for a period of 21 days with 10 birds per dietary treatment. On Day 21, five hens from each dietary treatment were euthanatized. The remaining birds were offered a .46% Ca diet for 5 days. In Experiment 2, 25 Single Comb White Leghorn hens were fed a 3.5% Ca diet for 21 days. Hens were then offered a .46% Ca diet and five hens euthanatized on Days 0, 2, 3, 5, and 7 of feeding the low-Ca diet. In Experiment 1, dietary Ca level had a significant (P less than .05) effect on total medullary Ca reserves of laying hens. Previous dietary Ca level had no significant (P greater than .05) effect upon medullary bone Ca reserves after subsequently feeding the low-Ca diet for 5 days. There was a (P less than .05) significant reduction in medullary bone Ca reserves of hens, regardless of previous level of calcium fed. In Experiment 2, feeding a low level of dietary Ca resulted in a significant (P less than .01) reduction in medullary bone Ca reserves of all bones measured, except the humerus. Although there was a significant (P less than .05) reduction in medullary bone Ca during the depletion period, hens appeared to make some attempt to conserve medullary bone Ca reserves.

The effect of 1 alpha,25-dihydroxyvitamin D3 added to a layer diet containing adequate amounts of vitamin D3 on the performance of layers.

The effect of adding 1 alpha,25-dihydroxyvitamin D3 (calcitriol) to a well-balanced, commercial layer diet containing 1,900 IU vitamin D3/kg on eggshell quality and laying performance of 56-wk-old White Leghorn hens was investigated. After 8 wk of treatment, calcitriol at 1.5 micrograms/kg diet had no discernible effect on egg weight, egg deformation, percentage shell, shell thickness, feed consumption, and egg production. At 3.0 and 4.5 micrograms/kg, feed consumption, feed production, and egg weight were significantly reduced, whereas egg deformation, percentage shell, and shell thickness were not affected. The results show that calcitriol added to a diet already adequately supplemented with vitamin D3 does not improve eggshell quality and may impair laying performance.

Is there lipid peroxidation induced malondialdehyde production during egg shell formation?

The marked increase observed in malondialdehyde concentration of the blood plasma and liver of laying hens during egg shell formation in a previous experiment (Mézes and Lencsés, 1985) were explained as a possible consequence of physiologically controlled lipid peroxidation. In this experiment, 54-week-old laying hens were treated intrauterinally with indomethacin (1.0 and 2.0 mg/kg body mass) after oviposition. Two other groups of hens were treated per os with excess amounts of vitamin E (100 and 200 mg/bird). The higher dose of indomethacin significantly decreased the PGF2 alpha production and malondialdehyde content of the sell gland, as well as the malondialdehyde concentration of the plasma. The excess amount of vitamin E had the same effect. The results suggest that during egg shell formation malondialdehyde is derived from prostanoid biosynthesis rather than from a free-radical initiated lipid peroxidative process. On the other hand, the excess amount of vitamin E inhibited prostaglandin biosynthesis as well as malondialdehyde production.