Pathobiology of highly pathogenic H5 avian influenza viruses in naturally infected Galliformes and Anseriformes in France during 2015-2016.

In late 2015, an epizootic of Highly Pathogenic Avian Influenza (H5Nx) was registered in Southwestern France, including more than 70 outbreaks in commercial poultry flocks. Phylogenetic analyses suggested local emergence of H5 viruses which differed from A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage and shared a unique polybasic cleavage site in their hemagglutinin protein. The present work provides an overview of the pathobiological picture associated with this epizootic in naturally infected chickens, guinea fowls and ducks. Upon necropsy examination, selected tissues were sampled for histopathology, immunohistochemistry and quantitative Real Time Polymerase Chain Reaction. In Galliformes, HPAIVs infection manifested as severe acute systemic vasculitis and parenchymal necrosis and was associated with endothelial expression of viral antigen. In ducks, lesions were mild and infrequent, with sparse antigenic detection in respiratory and digestive mucosae and leukocytes. Tissue quantifications of viral antigen and RNA were higher in chickens and guinea fowls compared to duck. Subsequently, recombinant HA (rHA) was generated from a H5 HPAIV isolated from an infected duck to investigate its glycan-binding affinity for avian mucosae. Glycan-binding analysis revealed strong affinity of rHA for 3'Sialyl-LacNAc and low affinity for Sialyl-LewisX, consistent with a duck-adapted virus similar to A/Duck/Mongolia/54/2001 (H5N2). K222R and S227R mutations on rHA sequence shifted affinity towards Sialyl-LewisX and led to an increased affinity for chicken mucosa, confirming the involvement of these two mutations in the glycan-binding specificity of the HA. Interestingly, the rHA glycan binding pattern of guinea fowl appeared intermediate between duck and chicken. The present study presents a unique pathobiological description of the H5 HPAIVs outbreaks that occurred in 2015-2016 in Southwestern France.

Extrinsic factors, endocrine mechanisms, and behavioral indicators of migratory restlessness in wintering whooper swans (Cygnus cygnus).

Extrinsic factors, endocrine mechanisms, and behavioral indicators of migratory restlessness were studied in wintering whooper swans (Cygnus cygnus) in the Sanmenxia Swan National Wetland Park in western Henan Province, central China. First, the fecal glucocorticoid metabolite (FGM) concentration was established and related to mean air temperature or photo period (day length) using simple linear or non-linear regression models. After a model selection procedure, the best fitted model revealed that an increase of FGM concentration was associated with an increase in the squared mean air temperature (R2 = 0.88). Other models showed an increasing FGM concentration to correspond with increasing values of day length, squared day length, and mean air temperature-however without statistical support. In a second step, behavioral frequencies of seven behaviors were condensed into three behavioral principal components (PCs) using principal components analysis. Behavioral PCs largely corresponded to three activity phases described for wintering whooper swans in central China and were correlated with the FGM concentration using Spearman's rank-order correlations. Results revealed a significant correlation between FGM and behavioral PC2 (positive factor loadings from vigilance and preening, negative loading from foraging). Finally, we tested for an effect of behavioral PCs on changes in winter home range size using a set of multiple linear regression models. Results of averaged model parameter estimates showed only the behavioral PC3 (positive factor loadings from fighting and calling, negative loading from locomotion) had a marginal significant effect on home range size. Results confirmed findings of previous studies on migratory restlessness in whooper swans. However, due to the small sample size (N = 15 weeks) the effect of PC3 on home range size was weak and should be viewed with caution.

Convergent changes in muscle metabolism depend on duration of high-altitude ancestry across Andean waterfowl.

High-altitude environments require that animals meet the metabolic O2 demands for locomotion and thermogenesis in O2-thin air, but the degree to which convergent metabolic changes have arisen across independent high-altitude lineages or the speed at which such changes arise is unclear. We examined seven high-altitude waterfowl that have inhabited the Andes (3812-4806 m elevation) over varying evolutionary time scales, to elucidate changes in biochemical pathways of energy metabolism in flight muscle relative to low-altitude sister taxa. Convergent changes across high-altitude taxa included increased hydroxyacyl-coA dehydrogenase and succinate dehydrogenase activities, decreased lactate dehydrogenase, pyruvate kinase, creatine kinase, and cytochrome c oxidase activities, and increased myoglobin content. ATP synthase activity increased in only the longest established high-altitude taxa, whereas hexokinase activity increased in only newly established taxa. Therefore, changes in pathways of lipid oxidation, glycolysis, and mitochondrial oxidative phosphorylation are common strategies to cope with high-altitude hypoxia, but some changes require longer evolutionary time to arise.

Comparative proteomic analysis revealed complex responses to classical/novel duck reovirus infections in the spleen tissue of Cairna moschata.

Duck reovirus (DRV), a member of the genus Orthoreovirus in the family Reoviridae, was first isolated from Muscovy ducks. The disease associated with DRV causes great economic losses to the duck industry. However, the responses of duck (Cairna moschata) to the classical/novel DRV (C/NDRV) infections are largely unknown. To reveal the relationship of pathogenesis and immune response, the proteomes of duck spleen cells under the control and C/NDRV infections were compared. In total, 5986 proteins were identified, of which 5389 proteins were quantified. The different accumulated proteins (DAPs) under the C/NDRV infections showed displayed various biological functions and diverse subcellular localizations. The proteins related to the serine protease system were siginificantly changed, suggesting that the activated serine protease system may play an important role under the C/NDRV infections. Furthermore, the differences in the responses to the C/NRDV infections between the duck liver and spleen tissues were compared. Only a small number of common DAPs were identified in both liver and spleen tissues, suggesting diversified pattern involved in the responses to the C/NRDV infections. However, the changes in the proteins involved in the serine protease systems were similar in both liver and spleen cells. Our data may give a comprehensive resource for investigating the responses to C/NDRV infections in ducks. SIGNIFICANCE: A newly developed MS/MS-based method involving isotopomer labels and 'tandem mass' has been applied to protein accurate quantification in current years. However, no studies on the responses of duck (Cairna moschata) spleen tissue to the classical/novel DRV (C/NDRV) infections have been performed. As a continued study of our previous report on the responses of duck liver tissue to the C/NDRV infections, the current study further compared the differences in the responses to the C/NRDV infections between the duck liver and spleen tissues. Our results will provide an opportunity to reveal the relationship of pathogenesis and immune response and basic information on the pathogenicity of C/NDRV in ducks.

Diel pattern of corticosterone metabolites in Arctic barnacle goslings (Branta leucopsis) under continuous natural light.

Here we describe the excretion pattern of corticosterone metabolites collected from droppings in barnacle goslings (Branta leucopsis) raised under 24 hours of continuous natural light in the Arctic. In lower latitudes, circulating corticosterone peaks around waking and shows a nadir between midnight and 4:00, whereas the peak and nadir are time-delayed slightly when measuring corticosterone metabolites from droppings. Photoperiod, along with other environmental factors, helps to entrain an animal's endogenous rhythm to that of the natural world. North of the Arctic Circle, photoperiod may not be a reliable cue as light is continuously absent during the winter and continuously present during the summer. Here, for the first time, we used droppings to describe a 24-hour excretion pattern of corticosterone metabolites (CORTm). By applying circular statistics for dependent data, we found a diel rhythmic pattern even under continuous natural light. We discuss potential alternative 'Zeitgeber' that may function even in the polar regions, focusing on melatonin. We propose a line of research to measure melatonin non-invasively from droppings. We also provide a validation of the adopted enzyme immunoassay (EIA) that was originally developed for greylag geese.

Heavy Metals in Liver and Brain of Waterfowl from the Evros Delta, Greece.

The concentrations of six heavy metals (cadmium [Cd], chromium [Cr], copper [Cu], manganese [Mn], lead [Pb], and zinc [Zn]) were determined in liver and brain of nine species of waterfowl from the Evros Delta, one of the most important wetlands in Greece, to assess metal contamination and potential risk to waterfowl. Significant differences among species were found for hepatic Cr, Cu, Pb, and Zn; the highest levels of all metals were found in liver of mute swans. Cd increased significantly with age in both organs of wigeon; some differences were also observed between males and females of the same species. Feeding habits were found to significantly affect hepatic Cr, Zn, and Cu concentrations with greater concentrations found in herbivorous than omnivorous species. Several significant positive correlations between metals were detected mainly in liver samples, which were attributable either to their association to metallothioneins or to common routes of exposure of waterfowl. Hepatic and cerebral concentrations of Cd and Pb correlated positively. Given the discrepancies in the thresholds or critical levels of metals in tissues of waterfowl, our survey showed that environmental exposure to increased levels of heavy metals may threaten, at least to some degree, the populations of waterfowl wintering in the Evros Delta. In a small percentage of birds, values indicative of severe adverse effects were determined. Swans that had been found dead showed some of the highest values, whereas accumulation levels did not follow a consistent pattern among the ducks examined.

DNA double-strand breaks in incubating female common eiders (Somateria mollissima): Comparison between a low and a high polluted area.

Alterations in the genetic material may have severe consequences for individuals and populations. Hence, genotoxic effects of environmental exposure to pollutants are of great concern. We assessed the impact of blood concentrations of persistent organic pollutants (POPs) and mercury (Hg) on DNA double-strand break (DSB) frequency, in blood cells of a high-exposed Baltic, and lower exposed Arctic population of common eiders (Somateria mollissima). Furthermore, we examined whether the genotoxic response was influenced by antioxidant concentration (plasma total glutathione (tGSH) and total antioxidant capacity) and female body mass. The DNA DSB frequency did not differ between the two populations. We found significant positive relationships between Hg and DNA DSB frequency in Baltic, but not in Arctic eiders. Although both p,p'-DDE and PCB 118 had a lesser effect than Hg, they exhibited a positive association with DNA DSB frequency in Baltic eiders. Antioxidant levels were not important for the genotoxic effect, suggesting alternative mechanisms other than GSH depletion for the relationship between Hg and DNA DSBs. Hence, the Baltic population, which is considered to be endangered and is under the influence of several environmental stressors, may be more susceptible to genotoxic effects of environmental exposure to Hg than the Arctic population.

Direct and indirect causes of sex differences in mercury concentrations and parasitic infections in a marine bird.

In many animal species, males and females differ in their levels of contaminants and/or parasitic infections. Most contaminants and gastro-intestinal parasites are obtained through prey ingestion, and thus the causes of sex differences in the distribution of these factors might follow similar pathways. We studied the northern common eider duck (Somateria molissima borealis) as an avian model, and used directed separation path analysis to explore the causes of sex differences in mercury (Hg) and gastro-intestinal helminths. Two trophically transmitted helminths were examined: a cestode (Lateriporus sp.) and an acanthocephalan (Polymorphus sp). We found that the number of Lateriporus sp. varied positively with stable isotope signature (as indicated by δ(15)N in eider breast muscle tissue), and negatively with crustaceans being present in the short term diet. We also found that Polymorphus sp. varied positively with eider tissue stable isotope signature. However, Polymorphus sp. varied negatively with sex indirectly through condition and liver mass. Similarly, Hg concentrations also varied negatively with sex indirectly through condition and liver mass, with both Polymorphus sp. intensity and Hg concentrations significantly higher in males. We found that model fit increased when a negative relationship between the two helminth species was included, suggesting a yet unknown causal mechanism linking these parasites. Our findings suggest that although Hg and gastro-intestinal parasites are both trophically transmitted through the eider's prey items, the factors that contribute towards bioaccumulation of these two burdens differ in source, likely caused by several different factors and may potentially influence each other.

Persistent organic pollutant levels and the importance of source proximity in Baltic and Svalbard breeding common eiders.

The distance to sources and the long-range transport potential of persistent organic pollutants (POPs) are important in understanding the impact of anthropogenic pollution on natural seabird populations. The present study documented blood concentrations of POPs in the Baltic Sea (Tvärminne, Finland) population of common eiders (Somateria mollissima) in 2009 and in 2011 and compared the concentrations with the presumably less exposed Arctic population in Svalbard (Kongsfjorden, Norway). The Baltic population had 26, 10, and 5 times greater concentrations of hexachlorocyclohexane, polychlorinated biphenyls, and p,p'-dichlorodiphenyldichloroethylene than the Svalbard population. Unexpectedly, concentrations of chlordanes were higher in Svalbard eiders, whereas concentrations of hexachlorobenzenes (HCBs) did not differ between the 2 populations. Although the similar HCB levels may partly be explained by the high transport potential of HCBs, unknown factors may have been more important than distance to sources and long-range transport potential for the chlordanes. One plausible explanation may be that the fasting-related redistribution of POPs from fat to blood was greater throughout the incubation in Arctic eiders, causing them to have higher blood levels of these POPs at the end of incubation. The blood concentrations of POPs in Baltic eiders were higher than documented in any other eider population and were comparable to levels in seabirds feeding at higher trophic positions in the food chain. Environ Toxicol Chem 2016;35:1526-1533. © 2015 SETAC.

Trace element concentrations in feathers of five Anseriformes in the south of the Caspian Sea, Iran.

Birds are susceptible to environmental changes, which make them particularly important as indicators of environmental contamination, including metal concentrations. In the present study, the concentrations of arsenic (As), cadmium (Cd), chromium (Cr), iron (Fe), lead (Pb), manganese (Mn), nickel (Ni), and zinc (Zn) in the tail and flight feathers of greylag goose, mallard, pintail, pochard, and wigeon were investigated from the south of the Caspian Sea in Iran by the electrothermal atomic absorption spectrometry (ET-AAS) method. The trend of metal concentrations in both types of feathers of all the examined species was as follows: Fe < Zn < Mn < Cd ∼ Cr ∼ Ni ∼ Pb < As. The interspecies variation in concentrations of almost all the studied metals was observed, as well as the significant differences between feather types. The only exception was Ni, the medians of which were comparable in tail and primaries. Correlations of concentrations of Cd, Mn, Ni, Pb, and Zn in primaries and tail feathers were positive and statistically significant. Elevated concentrations of Cd, Cr, Mn, and Pb were noted so the possibility of environmental contamination of the area is suspected.

Monitoring of heavy metal burden in mute swan (Cygnus olor).

Concentrations of heavy metals (especially arsenic, cadmium, chromium, copper, mercury and lead) were measured in the contour (body) feathers of mute swans (Cygnus olor) and in its nutrients (fragile stonewort [Chara globularis], clasping leaf pondweed [Potamogeton perfoliatus], Eurasian watermilfoil [Myriophyllum spicatum], fennel pondweed [Potamogeton pectinatus]) to investigate the accumulation of metals during the food chain. The samples (17 feathers, 8 plants) were collected at Keszthely Bay of Lake Balaton, Hungary. Dry ashing procedure was used for preparing of sample and the heavy metal concentrations were analysed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Copper (10.24 ± 2.25 mg/kg) and lead (1.11 ± 1.23 mg/kg) were detected the highest level in feathers, generally, the other metals were mostly under the detection limit (0.5 mg/kg). However, the concentrations of the arsenic (3.17 ± 1.87 mg/kg), cadmium (2.41 ± 0.66 mg/kg) and lead (2.42 ± 0.89 mg/kg) in the plants were low but the chromium (198.27 ± 102.21 mg/kg) was detected in high concentration.

Composition, Shell Strength, and Metabolizable Energy of Mulinia lateralis and Ischadium recurvum as Food for Wintering Surf Scoters (Melanitta perspicillata).

Decline in surf scoter (Melanitta perspicillata) waterfowl populations wintering in the Chesapeake Bay has been associated with changes in the availability of benthic bivalves. The Bay has become more eutrophic, causing changes in the benthos available to surf scoters. The subsequent decline in oyster beds (Crassostrea virginica) has reduced the hard substrate needed by the hooked mussel (Ischadium recurvum), one of the primary prey items for surf scoters, causing the surf scoter to switch to a more opportune species, the dwarf surfclam (Mulinia lateralis). The composition (macronutrients, minerals, and amino acids), shell strength (N), and metabolizable energy (kJ) of these prey items were quantified to determine the relative foraging values for wintering scoters. Pooled samples of each prey item were analyzed to determine composition. Shell strength (N) was measured using a shell crack compression test. Total collection digestibility trials were conducted on eight captive surf scoters. For the prey size range commonly consumed by surf scoters (6-12 mm for M. lateralis and 18-24 mm for I. recurvum), I. recurvum contained higher ash, protein, lipid, and energy per individual organism than M. lateralis. I. recurvum required significantly greater force to crack the shell relative to M. lateralis. No difference in metabolized energy was observed for these prey items in wintering surf scoters, despite I. recurvum's higher ash content and harder shell than M. lateralis. Therefore, wintering surf scoters were able to obtain the same amount of energy from each prey item, implying that they can sustain themselves if forced to switch prey.

Potency of polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity in hepatocyte cultures from chicken, Pekin duck, and greater scaup.

The potency of tetrachlorodibenzo-p-dioxin (TCDD) and 18 polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity was assessed in primary hepatocyte cultures prepared from chicken (Gallus domesticus), Pekin duck (Anas platyrhynchos domesticus), and greater scaup (Aythya marila). TCDD and 8 of the PAHs induced EROD activity in a concentration-dependent manner. Seven of these were previously shown to be acutely toxic to avian embryos, while the 10 congeners that did not produce an EROD response caused limited mortality. The rank order potency of the EROD-active congeners in all three species was as follows: TCDD>dibenz[ah]anthracene>benzo[k]fluoranthene>indeno[1,2,3-cd]pyrene>benzo[a]pyrene>chrysene≈benz[a]anthracene≈benz[ghi]perylene>benzo[b]naphtho[2,3-d]thiophene. Chicken hepatoctyes were more sensitive than duck hepatocytes to EROD induction by all test compounds, but the gap in species sensitivity was 100-fold for TCDD, and generally ≤10-fold for PAHs. This study is the first to use in vitro methods to rank the AHR-mediated potency of PAHs in birds. These data may be useful for assessing risks associated with exposure to PAHs in the environment.

Pre-breeding energetic management in a mixed-strategy breeder.

Integrative biologists have long appreciated that the effective acquisition and management of energy prior to breeding should strongly influence fitness-related reproductive decisions (timing of breeding and reproductive investment). However, because of the difficulty in capturing pre-breeding individuals, and the tendency towards abandonment of reproduction after capture, we know little about the underlying mechanisms of these life-history decisions. Over 10 years, we captured free-living, arctic-breeding common eiders (Somateria mollissima) up to 3 weeks before investment in reproduction. We examined and characterized physiological parameters predicted to influence energetic management by sampling baseline plasma glucocorticoids (i.e., corticosterone), very-low-density lipoprotein (VLDL), and vitellogenin (VTG) for their respective roles in mediating energetic balance, rate of condition gain (physiological fattening rate) and reproductive investment. Baseline corticosterone increased significantly from arrival to the initiation of reproductive investment (period of rapid follicular growth; RFG), and showed a positive relationship with body mass, indicating that this hormone may stimulate foraging behaviour to facilitate both fat deposition and investment in egg production. In support of this, we found that VLDL increased throughout the pre-breeding period, peaking as predicted during RFG. Female eiders exhibited unprecedentedly high levels of VTG well before their theoretical RFG period, a potential strategy for pre-emptively depositing available protein stores into follicles while females are simultaneously fattening. This study provides some of the first data examining the temporal dynamics and interaction of the energetic mechanisms thought to be at the heart of individual variation in reproductive decisions and success in many vertebrate species.

Analysis of the serum concentrations of kisspeptin and neurokinin B in the geese during reproductive cycle and their localisation in the ovary.

Kisspeptin and neurokinin B (NKB) have various functions. Their expression has been demonstrated in rat and human ovary, and estrogen affects their activity, suggesting a role for these molecules in the control of ovary function. However, whether these signaling systems are present in geese ovary, and the associated with serum estrogen remains largely unexplored. In this study we investigated the expression of kisspeptin and NKB in the ovary, and analysed their changes in the serum during the reproductive cycle and their association with serum estradiol in the geese. The results showed both kisspeptin and NKB immunoreactivity was found in the ovary, with marked expression in the granular layer and theca of the follicle, where intense coexpression of kisspeptin and NKB was also detected. The serum concentrations of kisspeptin and NKB in geese were significantly higher (P<0.05) in broody period than in laying period and laying cessation stage. However, the level of estradiol was markedly higher (P<0.05) in laying period than in broody and laying cessation stage. Serum kisspeptin was positively correlated with NKB (r=0.866, P<0.001), serum estradiol was negatively correlated with kisspeptin (r=-0.977, P<0.05) and NKB (r=-0.887, P<0.05). Overall, the existence of kisspeptin and NKB in geese ovary, and the difference of their serum concentrations during reproductive cycle and the inverse correlation with serum estradiol are highly suggestive of a role for kisspeptin and NKB in the regulation of geese reproductive function.

[Preparation and characterization of polyclonal antibody against Ia-associated invariant chain of Muscovy Duck (Cairina moschata)].

OBJECTIVE: To prepare polyclonal antibody against invariant chain of Muscovy duck (Cairina moschata) (MDIi) and identify its reaction with MDIi extracted from tissues of Muscovy duck. METHODS: MDIi was amplified by PCR and used to construct the prokaryotic expression vector of pET-32a/MDIi by linking with the plasmid of pET-32a. Then pET-32a/MDIi was transformed into E.coli Rosetta to induce the prokaryotic expression. After identified by SDS-PAGE, prokaryotic expression products were further purified from running gel of SDS-PAGE and injected into mice to prepare polyclonal antibody against MDIi. The titer and specificity of the polyclonal antibody against MDIi were analyzed by indirect ELISA and Western blotting, respectively. The intensity of reaction between the polyclonal antibody and MDIi extracted from tissues of Muscovy duck was also identified by indirect ELISA. RESULTS: The prokaryotic expression vector pET-32a/MDIi was successfully constructed. About 40 kD recombinant proteins of MDIi were confirmed to be expressed in the form of inclusion body in Rosetta. Polyclonal antibody against MDIi with a titer of 1:128 000 was obtained from the immunized mice and its high specificity was demonstrated by Western blotting. The titer of reaction between the polyclonal antibody and MDIi was 1:32 000. CONCLUSION: The polyclonal antibody against MDIi was successfully prepared with a high titer and specificity. It has a strong immune reaction with MDIi extracted from tissues of Muscovy duck.

DNA double-strand breaks in relation to persistent organic pollutants in a fasting seabird.

Lipophilic persistent organic pollutants (POPs) are released from fat reserves during fasting, causing increased blood concentrations. Thus, POPs represent a potential anthropogenic stressor during fasting periods. We analysed the blood of female common eiders (Somateria mollissima) by using agarose gel electrophoresis and image data analysis to quantify the DNA-fraction, of total DNA, that migrated into the gel (DNA-FTM) as a relative measure of DNA double strand-breaks (DSBs) during the fasting incubation period in the high arctic. In 2008 and in 2009 blood samples were obtained for analysis of 9 POPs and DNA-FTM at day 5 of the incubation period, and then in the same individuals at day 20. This unique study design gave us the opportunity to analyse the same individuals throughout two points in time, with low and high stress burdens. During the incubation period the body mass (BM) decreased by 21-24%, whereas the POP levels increased by 148-639%. The DNA-FTM increased by 61-67% (being proportional to the increase in DSBs). At day 5, but not day 20, DNA-FTM was positively correlated with most analysed POPs. The increase in DNA-FTM was positively correlated with the decrease in BM (g) during incubation. Thus, we suggest that fasting stress (BM loss) decreases DNA integrity and that stress caused by fasting on BM loss appeared to override the additional stress caused by concurrent increase in levels of the analysed POPs in the eiders. Blood levels of POPs in the eiders in Svalbard were relatively low, and additive and/or synergistic genotoxic effects of fasting stress and POP exposure may occur in populations with higher POP levels.

Hematological parameters in relation to age, sex and biochemical values for mute swans (Cygnus olor).

The knowledge of the correct morphological and biochemical parameters in mute swans is an important indicator of their health status, body condition, adaptation to habitat and useful diagnostic tools in veterinary practice and ecological research. The aim of the study was to obtain hematological parameters in relation to age, sex and serum biochemistry values in wild-living mute swans. We found the significant differences in the erythrocyte count, hematocrit, hemoglobin concentration and erythrocyte sedimentation rate in relation to age of mute swans. There were no differences in hematological values between males and females. The leukogram and H/L ratio did not vary by age and sex in swans. Among of biochemical parameters the slightly increased AST, ALP, CK, K, urea, decreased CHOL and TG values were recorded. As far as we know, this is the first study in which the morphometric parameters of blood cells in mute swans were presented. We found extremely low concentration of lead in blood (at subthreshold level). No blood parasites were found in blood smears. The analysis of body mass and biometric parameters revealed a significant differences dependent on age and sex. No differences in the scaled mass index were found. Our results represent a normal hematologic and blood chemistry values and age-sex related changes, as reference values for the mute swan.

Improvements and application of a modified gas chromatography atomic fluorescence spectroscopy method for routine determination of methylmercury in biota samples.

Improvements to the application of a combined solid-phase microextraction followed by gas chromatography coupled to pyrolysis and atomic fluorescence spectrometry method (SPME-GC-AFS) for methylmercury (MeHg) determination in biota samples are presented. Our new method includes improvements in the methodology of determination and the quantification technique. A shaker instead of a stirrer was used, in order to reduce the possibility of sample contamination and to simplify cleaning procedures. Then, optimal rotation frequency and shaking time were settled at 800 rpm and 10 min, respectively. Moreover, the GC-AFS system was equipped with a valve and an argon heater to eliminate the effect of the decrease in analytical signal caused by the moisture released from SPME fiber. For its determination, MeHg was first extracted from biota samples with a 25% KOH solution (3h) and then it was quantified by two methods, a conventional double standard addition method (AC) and a modified matrix-matched calibration (MQ) which is two times faster than the AC method. Both procedures were successfully tested with certified reference materials, and applied for the first time to the determination of MeHg in muscle samples of goosander (Mergus merganser) and liver samples of white-tailed eagle (Haliaeetus albicilla) with values ranging from 1.19 to 3.84 mg/kg dry weight (dw), and from 0.69 to 6.23 mg kg(-1) dw, respectively.

Context dependency of baseline glucocorticoids as indicators of individual quality in a capital breeder.

Identifying markers of individual quality is a central goal of life-history theory and conservation biology. The 'corticosterone (CORT)-fitness hypothesis' postulates that low fitness signals impaired ability to cope with the environment, resulting in elevated baseline CORT levels. CORT can, however, be negatively, positively or neutrally related to fitness, depending on the context. In order to clarify this controversial issue, we elucidate the utility of using baseline CORT as a correlate of individual fitness in incubating female eiders across variable environments. An increase in serum CORT with decreasing body condition was evident in older, more experienced breeders, while increased clutch mass was associated with elevated serum CORT in females breeding late in the season. For faecal CORT, the expected negative association with body condition was observed only in early breeders. We found a strong increase in faecal CORT with increasing baseline body temperature, indicating the utility of body temperature as a complementary stress indicator. Females in good body condition had a lower baseline body temperature, but this effect was only observed on open islands, a harsher breeding habitat less buffered against weather variability. Females with higher reproductive investment also maintained a lower baseline body temperature. Nest success strongly decreased with increasing serum and faecal CORT concentrations, and individual stress hormone and body temperature profiles were repeatable over years. Although our data support the tenet that baseline CORT is negatively related to fitness, the complex context-dependent effects call for cautious interpretation of relationships between stress physiology and phenotypic quality.