Using eggshell membranes as a DNA source for population genetic research

In the context of population genetic research, a faster and less invasive method of DNA sampling would allow large-scale assessments of genetic diversity and genetic differentiation with the help of volunteer observers. The aim of this study was to investigate the usefulness of eggshell membranes as a DNA source for population genetic research, by addressing eggshell membrane DNA quality, degeneration and cross-contamination. To this end, a comparison was made with blood-derived DNA samples. We have demonstrated 100% successful DNA extraction from post-hatched Black-tailed Godwit (Limosa limosa) eggshell membranes as well as from blood samples. Using 11 microsatellite loci, DNA amplification success was 99.1% for eggshell membranes and 97.7% for blood samples. Genetic information within eggshell membrane DNA in comparison to blood DNA was not affected (F-ST = -0.01735, P = 0.999) by degeneration or possible cross-contamination. Furthermore, neither degeneration nor cross-contamination was apparent in total genotypic comparison of eggshell membrane DNA and blood sample DNA. Our research clearly illustrates that eggshell membranes can be used for population genetic research

Eggshell pigment composition covaries with phylogeny but not with life history or with nesting ecology traits of British passerines

No single hypothesis is likely to explain the diversity in eggshell coloration and patterning across birds, suggesting that eggshell appearance is most likely to have evolved to fulfill many nonexclusive functions. By controlling for nonindependent phylogenetic associations between related species, we describe this diversity using museum eggshells of 71 British breeding passerine species to examine how eggshell pigment composition and concentrations vary with phylogeny and with life-history and nesting ecology traits. Across species, concentrations of biliverdin and protoporphyrin, the two main pigments found in eggshells, were strongly and positively correlated, and both pigments strongly covaried with phylogenetic relatedness. Controlling for phylogeny, cavity-nesting species laid eggs with lower protoporphyrin concentrations in the shell, while higher biliverdin concentrations were associated with thicker eggshells for species of all nest types. Overall, these relationships between eggshell pigment concentrations and the biology of passerines are similar to those previously found in nonpasserine eggs, and imply that phylogenetic dependence must be considered across the class in further explanations of the functional significance of avian eggshell coloration

Microfocus X-ray scattering investigations of eggshell nanotexture

The avian eggshell is a highly ordered calcitic bioceramic composite, with both inorganic and organic constituents. The interactions between the inorganic and organic components within the structure are poorly understood but are likely to occur at the nanometre level. Thus structural variation at this level may impinge on the overall structural integrity and mechanical performance of the eggshell, and therefore analysis at this level is fundamental in fully understanding this ordered structure. In this study, structural changes in the mineral crystallites were investigated by microfocus small-angle X-ray scattering (µSAXS) using synchrotron radiation. Small-angle X-ray scattering (SAXS) can be used to investigate structures on the nanometre scale such as size, shape, arrangement and internal porosity. A microfocused X-ray beam, 1.5 µm vertically by 7 µm, was used to produce vertical linear scans of the eggshell section. SAXS patterns were taken from the eggshell membrane (inner surface of the eggshell) to the cuticle (outer surface of the eggshell). This allowed textural variations within the eggshell to be mapped. The scattering intensity profile was then used to derive the dimension of scattering objects that define the nanotexture. The nanotexture observed may result from the presence of the organic matrix, which is embedded as intracrystalline particles producing voids within the calcified framework of large (>1 µm) calcite crystals. Porod analysis revealed the average size of a scattering interface to be approximately 4.5 nm with small changes that had a depth-dependent variation. These were largest at the mammillary layer/membrane boundary. The palisade layer displayed a small upward trend in size of scattering object. Parallel scans showed that the textural variations observed within the palisade layer are significant and indicate local subtextures. In addition, many of the patterns exhibit diffuse scattering streaks that could result from reflectivity from the larger crystallite interfaces. Changes in the orientation of diffuse streaks were observed within the different layers, the membranes, mammillary layer, palisade layer, vertical crystal layer and cuticle, indicating certain preferred orientations of the crystallites within the layers. The nanotextural variations that are apparent could have implications at the macroscopic level of the resulting eggshell

Use of chicken eggshell to improve dietary calcium intake in rural sub-Saharan Africa

Undernutrition resulting from inadequate access to high‐quality, nutritious food is a widespread issue in sub‐Saharan Africa impacting the health and survival of mothers and their children. Inadequate dietary intake leads to a deficiency in nutrients including calcium, required for growth and physiological functioning. This study investigated the potential of increasing dietary calcium intake by the addition of heat‐treated ground eggshell to locally prepared food. A mixed methods approach of literature review, Delphi expert survey and focus group discussions with women of childbearing age in rural Tanzania, were used to assess the practicality, safety, and acceptability of consumption of ground eggshell. Chicken eggshell has high calcium content (380 mg of calcium/gram) and bioavailability comparable to calcium carbonate (~39%) with 1 g sufficient to provide one half of a sub‐Saharan African adult female's dietary calcium needs. Salmonella was indicated as the most likely threat to human health through eggshell consumption. Experts agreed that eggshells boiled for 10 min when preparing hard‐boiled eggs with a further 20 min cooking of crushed eggshell in staple foods would eliminate identified egg‐associated pathogens. Five focus groups (n = 46) indicated eggshells were perceived as waste. However, there was an indication of general acceptance of the approach and a willingness to consider the incorporation of ground eggshells into their diets. Development of suitable communication methods are required to convey benefits and safe preparation methods. Ground eggshell could be a highly equitable method of increasing calcium intakes across rural sub‐Saharan Africa where calcium intake is low and village poultry ownership common

First light for avian embryos: eggshell thickness and pigmentation mediate variation in development and UV exposure in wild bird eggs

Article first published online: 29 JUL 20141. The avian embryo's development is influenced by both the amount and the wavelength of the light that passes through the eggshell. Commercial poultry breeders use light of specific wavelengths to accelerate embryonic growth, yet the effects of the variably patterned eggshells of wild bird species on light transmission and embryonic development remain largely unexplored. 2. Here, we provide the first comparative phylogenetic analysis of light transmission, through a diverse range of bird eggshells (74 British breeding species), in relation to the eggshell's thickness, permeability, pigment concentration and surface reflectance spectrum (colour). 3. The percentage of light transmitted through the eggshell was measured in the spectral range 250–700 nm. Our quantitative analyses confirm anecdotal reports that eggshells filter the light of the externally coloured shell. Specifically, we detected a positive relationship between surface eggshell reflectance (‘brightness’) and the percentage of light transmitted through the eggshell, and this relationship was strongest at wavelengths in the human-visible blue-green region of the spectra (c. 435 nm). 4. We show that less light passes through thicker eggshells with greater total pigment concentrations. By contrast, permeability (measured as water vapour conductance) did not covary significantly with light transmission. Eggs of closed-nesting species let more light pass through, compared with open nesters. 5. We postulate that greater light transmission is required to assist embryonic development under low light exposure. Importantly, this result provides an ecological explanation for the repeated evolution of immaculate, white- or pale-coloured eggshells in species nesting in enclosed spaces. 6. Finally, we detected correlative support for the solar radiation hypothesis, in that eggshells of bird species with a longer incubation period let significantly less of the potentially harmful, ultraviolet (UV) light pass through the eggshell. In summary, we demonstrate suites of avian eggshell properties, including eggshell structure and pigmentation, which are consistent with an evolutionary pressure to both enhance and protect embryonic development.Golo Maurer, Steven J. Portugal, Mark E. Hauber, Ivan Mikšík, Douglas G. D. Russell and Phillip Casse

Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell’s thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality

Legacy pollutants are declining in Great Skuas (Stercorarius skua) but remain higher in Faroe Islands than in Scotland

To monitor environmental pollutants in Faroese biota, samples from a top predator were analysed and put into a spatial and temporal context. Analysis of 20 Great Skua eggs sampled in 2012 from the Faroe Islands showed >70 % lower concentrations of legacy persistent organic pollutants (POPs) than in samples analysed in 1977. The 2012 Faroese eggs showed higher concentrations than for eggs in Shetland from about the same period (2008). Eggshells were analysed for sub-lethal effects but there were no detectable effects of legacy POP levels on eggshell colour or thickness. A temporal decline in legacy POPs would indicate a reduction in the general pollutant levels present in the environment as has been shown in other areas of the North Atlantic, but there are significant geographic differences in POPs levels likely due to differences in diet resulting in significantly different exposures on a relatively limited spatial scale

Ancient DNA & stable isotope profiling of fossil eggshell

Eggshell is frequently encountered within palaeontological and archaeological deposits and due to various chemical and physical properties, it is highly valued as a substrate for geochronology, palaeodietary and palaeoecological applications. Despite the wellrecognised preservation of biomolecules in fossil eggshell, the substrate had never been evaluated for ancient DNA (aDNA) preservation. Herein, this thesis describes the first exploration involving the recovery and characterisation of aDNA from fossil eggshell. The primary aim of this thesis research is to explore eggshell aDNA and how genetic data, when combined with stable isotope profiles, provides new insights into hunting practices, zooarchaeology, palaeodiets, palaeobiology and extinction processes. The visualisation of fluorescently-stained DNA in fossil eggshell by confocal microscopy revealed that genetic material was present in the eggshell of the extinct New Zealand moa (Aves: Dinornithiformes) and Madagascan elephant bird (Aves: Aepyornithiformes). The immediate task was to determine how to go about characterising the aDNA encapsulated in this previously unrecognised substrate. Quantitative PCR assays were employed to thoroughly evaluate different methods to maximise the recovery and amplification of aDNA from powdered eggshell. This study resulted in the successful characterisation of both mitochondrial and nuclear DNA isolated from 19,000 year-old emu eggshell and the first ever DNA sequence from the elephant bird, Aepyornis. All data pointed toward fossil eggshell as a reliable source of relatively pure aDNA. This work culminated in a formal description of the methodology that should be employed to maximise recovery of aDNA from eggshell. Next, this thesis investigates whether eggshell aDNA could be applied to ‘first-contact’ archaeological sites in New Zealand, to gain new insights into the Moa-hunter period of prehistory. The abundance of moa eggshell within the ‘first-contact’ Polynesian archaeological sites is well recognised from previous excavations. With nine species of moa contained within the New Zealand archipelago however, the scientific value of these remains has been limited by the lack of definitive species assignments. Genetic species assignments of moa eggshell combined with morphological identification of bone (literature and museum catalogued specimens) has enabled the most comprehensive audit of zooarchaeology assemblages from several significant 13th-15th century AD deposits. In total, mitochondrial DNA (mtDNA) was amplified from 251 of 323 (78 %) eggshell fragments and 22 of 27 (88 %) bone samples, and the analyses revealed the presence of four moa species: Anomalopteryx didiformis; Dinornis robustus; Emeus crassus; and Euryapteryx curtus. The mtDNA, together with highly polymorphic microsatellite markers, were used in combination to individualise eggshell fragments. These data revealed over 50 individual eggs from Wairau Bar – a number that likely represents a considerable proportion of the total reproductive output of moa in the area. This emphasises that human predation of all life stages of moa was intense. In addition, an excess of male bones at the Wairau Bar site (determined by molecular sexing) suggests that males might have been preferential targets, perhaps while incubating eggs. Lastly, moa eggshell genetics and stable isotope profiles were combined to examine the palaeodiets, palaeobiology, palaeoecology and the human mediated extinction of the moa. Organic (δ13Corg and δ15N) and carbonate (δ13Ccarb and δ18O) stable isotope profiles were analysed from 247 genetically identified eggshell fragments. The combination of ancient DNA and stable isotope profiles revealed moa consumed a diet consisting exclusively of C3 vegetation and occupied the varied landscape that surrounded the archaeological deposits. Given that moa were foraging in such diverse landscapes suggests that the first colonisers hunted these moa away from the homogenous landscapes immediately surrounding these archaeological sites and thus engaged in ‘central point foraging’. Discriminant functional analyses indicate moa eggshells can be assigned with good precision to their parent species based purely on their stable isotope profiles. In conclusion, this thesis research has revealed a ‘new’ aDNA substrate that has a number of useful applications in archaeology, palaeogenomics and palaeoecology. The research on New Zealand moa eggshell demonstrated, first-hand, how this substrate can provide valuable insights into the biology, ecology and extinction of a megafaunal species. It is hoped the research will have a lasting influence on eggshell discovered across the globe

Computation of forces in strongly nonlinear magnetic fields using higher-order eggshell algorithm

A novel version of the eggshell-based procedure for numerical computation of magnetic forces and torques acting on ferromagnetic bodies in highly nonlinear magnetic fields is presented. The procedure works with a fully adaptive higher-order finite element method developed for years in our research group, that is implemented in own code Agros2D and library Hermes. The power of the methodology and both codes is demonstrated on the solution of two typical examples: computation of the static characteristic of a magnetic actuator and torque characteristic of a flux-switched permanent-magnet machine. The results obtained are compared with data calculated by several other available codes

Egg Production and Quality of Magelang Duck Fed Diets Containing Different Ratio of Omega 3 : Omega 6 and Organic Zn

The aims of this study were to determine the feeding effect of diets containing different ratio of ω-3 and ω-6 fatty acids and organic Zn on egg production and quality of Magelang duck. A total of 90 ducks of 20 weeks old were randomly divided into 18 experimental units by assigning a completely randomized design with 6 treatments and 3 replications. There were 6 dietary treatments, namely 1) ration without palm oil, fish oil and organic Zn with the ratio of ω-3 : ω-6 was 1: 5 (R0), 2) ration with the ratio of ω-3 : ω-6 was 1: 1.5 + 200 ppm organic Zn (R1), 3) ration with the ratio of ω-3 : ω-6 was 1: 3 + 200 ppm organic Zn (R2), 4) ration with the ratio of ω-3 : ω-6 was 1: 4.5 + 200 ppm organic Zn (R3), 5) ration with the ratio of ω-3 : ω-6 was 1: 6 + 200 ppm organic Zn (R4), 6) ration with the ratio of ω-3 : ω-6 was 1 : 7.5 + 200 ppm organic Zn (R5). Observation was carried out for 9 wk. The results showed that the ration containing the ratio of ω-3 : ω-6 = 1 : 3 and organic Zn (R2) produced significantly (P<0.05) the highest egg production and the lowest egg production was resulted by the dietary treatment without oil and organic Zn. Ration containing the ratio of ω-3 : ω-6 = 1: 3 and organic Zn (R2) significantly (P<0.05) decreased feed conversion ratio to the lowest value and it was significantly different as compared to R0 and R5. It can be concluded that feeding diet containing ω-3 and ω-6 with the ratio of 1:3 + 200 ppm organic Zn improves egg production without decreasing egg quality