Assessment of oxidative stress is an important but technically challenging procedure in medical and biological research. The reactive oxygen metabolites (d-ROMs) test is a simple assay marketed for analyzing the total amount of hydroperoxides in serum via the Fenton's reaction. Earlier reports have raised a suspicion that a part of the signal detected in the assay comes from sources other than metabolites generated by oxidative stress. The aim of this study was to identify which serum components interfere with the d-ROMs signal. By application of sodium azide, ethylenediaminetetraacetic acid, sodium dodecylsulphate, varying temperature, and spiking endogenous substances we demonstrate that in the case of mammalian sera the assay determines ceruloplasmin (CP) activity with potential interferences from hydroperoxides, iron level, thiols, and albumin. In sera of avian species hydroperoxides contribute more to the test outcome, but the CP part is insensitive to inhibition by azide. In conclusion, this assay has deficiencies in terms of detecting realistic concentrations of hydroperoxides, is mostly measuring CP and is also interfered with other serum components, making it very difficult to interpret in most biological systems.

SUMMARYOxidative stress (OS) is widely believed to be responsible for the generation of trade-offs in evolutionary ecology by means of constraining investment into a number of components of fitness. Yet, progress in understanding the true role of OS in ecology and evolution has remained elusive. Interpretation of current findings is particularly hampered by the scarcity of experiments demonstrating which of the many available parameters of oxidative status respond most sensitively to and are relevant for measuring OS. We addressed these questions in wild-caught captive greenfinches (Carduelis chloris) by experimental induction of OS by administration of the pro-oxidant compound paraquat with drinking water. Treatment induced 50% mortality, a significant drop in body mass and an increase in oxidative DNA damage and glutathione levels in erythrocytes among the survivors of the high paraquat (0.2gl -1 over 7days) group. Samples taken 3days after the end of paraquat treatment showed no effect on the peroxidation of lipids (plasma malondialdehyde), carbonylation of proteins (in erythrocytes), parameters of plasma antioxidant protection (total antioxidant capacity and oxygen radical absorbance), uric acid or carotenoids. Our findings of an increase in one marker of damage and one marker of protection from the multitude of measured variables indicate that detection of OS is difficult even under the most stringent experimental induction of oxidative insult. We hope that this study highlights the need for reconsideration of over-simplistic models of OS and draws attention to the limitations of detection of OS due to time-lagged and hormetic upregulation of protective mechanisms. This study also underpins the diagnostic value of measurement of oxidative damage to DNA bases and assessment of erythrocyte glutathione levels.

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Animals’ capability to absorb energy and nutrients from food poses a major internal constraint that affects the amount of resources available for allocation to maintenance, growth, signaling, and reproduction. Intestinal surface is the largest area of contact between immune system and microbial antigens; gut thus appears the main arena where trade‐offs between immune function and other components of fitness arise. Assessment of the integrity of digestive machinery should therefore be of high priority in ecophysiological research. Traditional methods of digestive physiology, however, appear unsuitable for most ecological applications due to lethality or complexity of the procedure.Here, we test the reliability of a simple, cheap, and noninvasive procedure, an acid steatocrit that assesses fat content in feces. It is based on centrifugation of a fecal sample, diluted in acid medium, in hematocrit capillary tube and quantifying the percentage of fat in fecal matter. The method has been previously validated in humans and mice; here, we apply it for the first time in birds.When applied to captive wild‐caught greenfinches, the method showed reasonable internal consistency (r s = 0.71 for steatocrit values, sampled from the same fecal aliquot in duplicate but processed separately). Individual steatocrit values were significantly repeatable in time in different intervals from eight to at least 20 days (r s = 0.32–0.49). The relationship between intestinal health and steatocrit values was tested by experimental manipulations. Medication against coccidiosis (a naturally pervasive intestinal infection) reduced, and experimental infection with heterologous coccidian strains increased steatocrit. Individual changes in steatocrit correlated negatively with changes of two markers of nutritional state—plasma triglyceride levels and body mass.Findings of this study suggest that steatocrit has a wide application potential as a marker of intestinal health in ecophysiological research. In particular, we see the perspective of this method for increasingly popular immunoecological research, conservation medicine, and studies of animal coloration.

BackgroundImmunoecology aims to explain variation among hosts in the strength and efficacy of immunological defences in natural populations. This requires development of biomarkers of the activation of the immune system so that they can be collected non-lethally and sampled from small amounts of easily obtainable tissue. We used transcriptome profiling in wild greenfinches (Carduelis chloris) to detect whole blood transcripts that most profoundly indicate upregulation of antimicrobial defences during acute phase response. The more general aim of this study was to obtain a functional annotation of a substantial portion of the greenfinch transcriptome that would enable to gain access to more specific genomic tools in subsequent studies. The birds received either bacterial lipopolysaccharide or saline injections and RNA-seq transcriptional profiling was performed 12 h after treatment to provide initial functional annotation of the transcriptome and assess whole blood response to immune stimulation.ResultsA total of 66,084 transcripts were obtained from de novo Trinty assembly, out of which 23,153 could be functionally annotated. Only 1,911 of these were significantly upregulated or downregulated. The manipulation caused marked upregulation of several transcripts related to immune activation. These included avian-specific antimicrobial agents avidin and gallinacin, but also some more general host response genes, such as serum amyloid A protein, lymphocyte antigen 75 and copper-transporting ATPase 1. However, links with avian immunity for most differentially regulated transcripts remained rather hypothetical, as a large set of differentially expressed transcripts lacked functional annotation.ConclusionsThis appears to be the first large scale transcriptional profiling of immune function in passerine birds. The transcriptomic data obtained suggest novel markers for the assessment of the immunological state of wild passerines. Characterizing the function of those possible novel infection markers would assist future vertebrate genome annotation. The extensive sequence information collected enables to identify possible target and housekeeping genes needed to gain access to more specific genomic tools in future studies.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-533) contains supplementary material, which is available to authorized users.

BackgroundAim of this study is to describe the relationship between anthropometric traits and educational attainment among Estonian schoolchildren born between 1937 and 1962. We asked whether height, cranial volume and face width (a testosterone-dependent trait), measured in childhood predict later educational attainment independently of each other, family socioeconomic position (SEP) and sex. Associations between morphometric traits and education and their interactions with biosocial variables are of scholarly importance because higher education is nearly universally associated with low fertility in women, and often with high fertility in men. Hence, morphometric traits associated with educational attainment are targeted by natural selection and describing the exact nature of these associations is relevant for understanding the current patterns of evolution of human body size.MethodsData on morphometric measurements and family background of 11,032 Estonian schoolchildren measured between seven and 19 years of age were obtained from the study performed by Juhan Aul between 1956 and 1969. Ordinal logistic regression was used for testing the effects of morphometric traits, biosocial variables and their interaction on the cumulative probability of obtaining education beyond primary level.ResultsOf biosocial variables, family SEP was the most important determinant of educational attainment, followed by the sex, rural vs urban origin and the number of siblings. No significant interactions with morphometric traits were detected, i.e., within each category of SEP, rural vs urban origin and sex, taller children and those with larger heads and relatively narrower faces were more likely to proceed to secondary and/or tertiary education. The effect of height on education was independent of cranial volume, indicating that taller children did not obtain more educations because their brains were larger than those of shorter children; height per se was important.ConclusionsOur main finding – that adjusting for other morphometric traits and biosocial variables, morphometric traits still robustly predicted educational attainment, is relevant for understanding the current patterns of evolution of human body size. Our findings suggest that fecundity selection acting on educational attainment could be partly responsible for the concurrent selection for smaller stature and cranial volume in women and opposite trends in men.

Age-related declines in life-history traits have been widely observed in free-living animals. Several theories link senescence to oxidative stress. The aim of this study was to measure several widely used markers of oxidative and nutritional state in a long-lived seabird, the common gull (Larus canus), in order to assess the suitability of these markers for describing deterioration in physiological condition associated with chronological age and survival. Associations with longevity and individual consistency of these parameters over the years (repeatability) were also assessed. Senescence in fitness parameters was observed during the study period: in females, laying date and clutch mass were related to bird age in a curvilinear manner, with middle-aged birds breeding earlier and laying heavier eggs. The only parameter associated with aging processes was glutathione concentration in erythrocytes, which was lower in female birds with longer life spans. Of indexes of nutritional state, plasma triglyceride concentration showed a between-individual increase with age, suggesting selective mortality of birds with low levels. Additionally, total plasma protein levels of individual males increased with age. The mostly negative results of this study hint that the commonly used parameters of physiological condition and oxidative state used in this study do not adequately reflect an individual's long-term health condition. Alternatively, it is possible that in common gulls, senescence occurs in reproductive mechanisms but not in mechanisms responsible for maintaining an organism's redox balance, consistent with the idea that different aspects of an organism's physiological condition age at different rates. Significant interannual repeatability was detected in three plasma constituents-carotenoids, uric acid, and total protein-all of which can possibly be linked to variation in dietary habits.

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