A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

Comparative analysis of ACTH and corticosterone sampling methods in rats

A frequently debated question for studies involving the measurement of stress hormones in rodents is the optimal method for collecting blood with minimal stress to the animal. Some investigators prefer the implantation of indwelling catheters to allow for frequent sampling. Others argue that the implantation of a catheter creates a chronic stress to the animal that confounds stress hormone measures and therefore rely on tail vein sampling. Moreover, some investigators measure hormones in trunk blood samples obtained after anesthesia, a practice that may itself raise hormone levels. To address these controversies, we 1) compared plasma ACTH and corticosterone (Cort) concentrations in pre- and poststress rat blood samples obtained via previously implanted vena cava catheters, tail vein nicks, or clipping the tip off the tail and 2) compared plasma ACTH and Cort in rat blood samples obtained by decapitation with and without anesthesia. Rats sampled via indwelling catheters displayed lower prestress ACTH levels than those sampled by tail vein nick if the time to acquire samples was not limited; however, elevated basal ACTH was not observed in samples obtained by tail clip or tail nick when the samples were obtained within 3 min. Baseline Cort levels were similar in all groups. After restraint stress, the profile of the plasma ACTH and Cort responses was not affected by sampling method. Decapitation with prior administration of CO2 or pentobarbital sodium increased plasma ACTH levels approximately 13- and 2-fold, respectively, when compared with decapitation without anesthesia. These data indicate that tail vein nicking, tail clipping, or indwelling venous catheters can be used for obtaining plasma for ACTH and Cort during acute stress studies without confounding the measurements. However, the elevation in basal ACTH seen in the tail vein nick group at baseline suggests that sampling needs to be completed rapidly (<3 min) to avoid the initiation of the pituitary stress response. Death by CO2 and pentobarbital sodium injection before trunk blood collection cause significant stress to animals, as reflected in the elevated plasma ACTH levels. These results support the use of either chronic vascular cannulas or sampling from a tail vein. However, collection of blood under pentobarbital sodium or CO2 anesthesia is likely to confound the results of stress studies when ACTH is an important endpoint.     

Taking the stress out of blood collection: comparison of field blood‐sampling techniques for analysis of baseline corticosterone

Many ecological studies use stress hormones to assess the condition, health or disturbance levels of wild organisms. Common blood sampling protocols for this research involve trapping individuals and taking blood within three minutes to obtain a “baseline” for analysis of stress hormones (“conventional method”). In some situations it may be difficult to get an accurate measure of baseline values; therefore, alternative sampling techniques may be preferable. We compared corticosterone levels in samples taken via a newly developed, minimally invasive blood sampling technique with corticosterone levels in blood taken via the conventional method. We collected samples from incubating adult common terns Sterna hirundo via blood sucking bugs (Heteroptera, Triatominae) contained in “dummy eggs” (“bug method”) and compared measured corticosterone concentrations to concentrations in blood taken from the same birds using the conventional method. We found no significant differences in mean or variance of baseline corticosterone levels between samples collected via the different methods. This suggests that the bug method offers a viable alternative for hormone sampling.     

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.     

Utilization of dried blood spots within drug discovery: modification of a standard DiLab(®) AccuSampler(®) to facilitate automatic dried blood spot sampling

The use of dried blood spots (DBS) in preclinical studies has seen an enormous increase over the past two years. Despite its positive impact on the 3Rs (reduce, replace and refine), its uptake in exploratory drug discovery has been limited due mainly to protracted method development time in bioanalysis but also the need for small volumes (<20 μL) to be sampled manually. Automatic blood sampling technology such as the DiLab(?) AccuSampler(?) is widely used in drug discovery to facilitate exploratory rodent-based pharmacokinetic and pharmacokinetic/pharmacodynamic studies with minimal animal handling. Propranolol was orally administered to a Han-Wistar rat attached to either a standard DiLab(?) AccuSampler(?) or a retrofitted unit designed to directly collect the DBS samples. In all, 50 or 20 μL blood samples were then collected via the standard or retrofitted unit, respectively, at six timepoints over a 7 h period. After drying and storage the DBS samples were analysed for propranolol via liquid chromatography-mass spectrometry. In this report we demonstrate that a standard DiLab(?) AccuSampler(?) can be easily retrofitted to facilitate automatic dried blood spot sampling and that time-concentration data generated from these samples are equivalent to that from manually spotted samples.     

A non-invasive method for detecting the metabolic stress response in rodents: characterization and disruption of the circadian corticosterone rhythm

Plasma corticosterone (CORT) measures are a common procedure to detect stress responses in rodents. However, the procedure is invasive and can influence CORT levels, making it less than ideal for monitoring CORT circadian rhythms. In the current paper, we examined the applicability of a non-invasive fecal CORT metabolite measure to assess the circadian rhythm. We compared fecal CORT metabolite levels to circulating CORT levels, and analyzed change in the fecal circadian rhythm following an acute stressor (i.e. blood sampling by tail veil catheter). Fecal and blood samples were collected from male adolescent rats and analyzed for CORT metabolites and circulating CORT respectively. Fecal samples were collected hourly for 24 h before and after blood draw. On average, peak fecal CORT metabolite values occurred 7-9 h after the plasma CORT peak and time-matched fecal CORT values were well correlated with plasma CORT. As a result of the rapid blood draw, fecal production and CORT levels were altered the next day. These results indicate fecal CORT metabolite measures can be used to assess conditions that disrupt the circadian CORT rhythm, and provide a method to measure long-term changes in CORT production. This can benefit research that requires long-term glucocorticoid assessment (e.g. stress mechanisms underlying health).     

Stability of Avian Fecal Corticosterone Metabolite Levels in Frozen Avian Feces

ABSTRACT Fecal corticosterone metabolites are commonly used in avian ecology as a measure of response to stress. Recent research on mammals suggested that the manner in which samples are stored could be critical to alleviating any storage handling bias. Cross-reacting metabolites can increase glucocorticoid metabolites even after samples are frozen and, thus, result in an overestimation of hormone levels as the time increases between when samples were collected and when levels are measured. We examined effects of sample storage time on fecal corticosterone metabolites for 2 avian species across 165 days. We observed no change in fecal corticosterone metabolites across the sampling periods in either fulvous whistling-ducks (Dendrocygna bicolor) or white ibis (Eudocimus albus). Results suggest that avian fecal corticosterone metabolite levels do not change when samples are frozen for long periods of time and that there were no differences in the response between the 2 species we compared. This study demonstrated that avian fecal corticosterone samples are accurate even after freezing and, thus, studies that seek to address conservation questions may rely on these data. Studies of additional bird species are needed to generalize our findings to other avian taxa.     

Measuring corticosterone in feathers using an acetonitrile/hexane extraction and enzyme immunoassay: feather corticosterone levels of food‐supplemented Atlantic Puffin chicks

Glucocorticoid levels measured in the blood of animals reflect hypothalamic‐pituitary‐adrenal (HPA) activity in response to predictable and unpredictable changes. In birds, circulating corticosterone is incorporated into growing feathers and provides an integrated measure of HPA activity over the period of feather growth. Measuring corticosterone in feathers can provide insight into the physiological state of birds during times when they are unavailable for blood sampling (e.g., during migratory or non‐breeding periods of the annual cycle). Building upon studies that used radioimmunoassay or liquid chromatography, we used a commercially available enzyme immunoassay kit to measure corticosterone in feathers of nestling Atlantic Puffins (Fratercula arctica) on Gull Island, Newfoundland, Canada, in 2012, and demonstrate the benefits of sample preparation via acetonitrile/hexane purification. We used this method to measure corticosterone in feathers of Atlantic Puffin chicks that experienced differences in mass gain in a supplementary feeding study. We found a positive relationship between feather corticosterone and mass gain, and a negative relationship between feather corticosterone and pre‐treatment body condition. Because feathers were growing prior to and during the supplementary feeding period, our results also suggest that extracting seabird feather samples with acetonitrile/hexane (in addition to methanol) prior to measuring corticosterone with enzyme immunoassay is beneficial, and, as reported in previous studies, blood and feather corticosterone values reflect different measures.     

Physiological and Pathological Impact of Blood Sampling by Retro-Bulbar Sinus Puncture and Facial Vein Phlebotomy in Laboratory Mice

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals'' physiological condition, which should be considered whenever blood samples are obtained.     

Determination of corticosterone in mouse plasma by a sweeping technique using micellar electrokinetic chromatography

The separation and on-line concentration of corticosterone in mouse blood was achieved by means of capillary electrophoresis/UV absorbance using sodium dodecyl sulfate (SDS) as a surfactant. The procedure involved the use of an on-line sample concentration method by sweeping-micellar electrokinetic chromatography (sweeping-MEKC). Optimal on-line concentration and separation conditions were determined. The detection limit for this method was 5 ng/ml (S/N=3) and photodiode array detection at 247 nm was used for identification. For the analysis of actual samples, corticosterones from blood samples of a non-stressed and stressed mouse were determined. The results show that only a minor amount of corticosterone was produced by a non-stressed mouse, whereas a significant amount was present in the blood sample from a stressed mouse. The method developed here can be used to examine corticosterone levels as a marker of stress in test animals and may also be used for estimating the effect of stress-release medications.     

Comparison of clinical pathology parameters with two different blood sampling techniques in rats: retrobulbar plexus versus sublingual vein

Blood samples were taken from the retrobulbar venous plexus or the sublingual vein of male HamIbm:Wist rats to compare clinical pathology parameters between the two sampling techniques. By analogy with a pharmacokinetic study, blood was sampled six times during one day from unfasted animals. After 3 weeks of recovery, blood was taken from fasted animals on a single occasion. In addition, prolactin and corticosterone levels were determined to compare stress-related effects between the two sampling methods. Body weight development and food consumption were similar after single as well as after repeated blood sampling for the two blood sampling techniques. Haemotological evaluation showed a gradual decrease in erythrocyte count, haemoglobin concentration and haematocrit after repeated blood sampling. Repeated withdrawal of blood samples over 24 h corresponding to approximately 22% of the total blood volume resulted in a decrease in red blood cell parameters by up to 30%. The withdrawal of approximately 10% of the total blood volume was associated with a decrease in these parameters by up to 10% and should not be exceeded for animal welfare reasons and to allow a reliable evaluation of data in a study. Repeated blood sampling was associated with an initial decrease in the number of white blood cells, mainly due to a reduction in lymphocytes; white blood cell counts were slightly increased one day after. The decrease in lymphocytes and the increase in neutrophils after repeated sampling were generally slightly more pronounced in the blood from the retrobulbar plexus than from the sublingual vein. Comparison of serum clinical chemistry data showed significantly higher activities of creatine kinase and aspartate aminotransferase in samples from the retrobulbar plexus. These findings suggest a higher degree of tissue damage with blood sampling from the retrobulbar plexus than from the sublingual vein. Despite a large inter-individual variability, higher mean values of prolactin on each occasion and corticosterone after a single sample in fasted animals indicate a higher stress associated with blood sampling from the retrobulbar plexus.     

Adaptation of corticosterone-but not beta-endorphin-secretion to repeated blood sampling in rats

Effects of short-term repeated blood sampling on the secretion of corticosterone (CORT) and beta-endorphin (beta-END) were evaluated in male Wistar rats. Blood was drawn from the tail vein of conscious rats four times within 2 h both at the peak and trough period of the diurnal corticosterone secretion cycle. All rats were well accustomed to the procedure. The main findings were: (1) At both sampling intervals, CORT increased significantly in response to the first sampling and declined to baseline values in successive samples. (2) beta-END also increased significantly in response to the first sampling but remained elevated in successive samples. (3) Intensities of initial CORT and beta-END responses correlated positively with each other and with the baseline beta-END values. Feedback inhibition of CORT secretion with sustained elevation of beta-END titres suggests a moderate stress intensity of the repeated blood sampling procedures. In general, due to lack of short-term feedback inhibition, beta-END seems to reflect the effects of repeated administration of moderate intense stressors more closely than CORT.     

Noninvasive assessment of stress in bank voles (<Emphasis Type="Italic">Myodes glareolus</Emphasis>, Cricetidae,Rodentia) by means of enzyme-linked immunosorbent assay (ELISA)

ELISA with antibodies to corticosterone was used to evaluate the possibility of estimating the level of adrenocortical activity in male bank voles, Myodes glareolus (Rodentia, Cricetidae), by determining the concentration of immunoreactive steroids (IRS) in their feces. The binding curves of dilutions of the corticosterone standard and the extracts from dried feces were shown to be parallel. The corticosteroid response was evoked by ACTH injection, blood sampling, or immobilization. The response to ACTH injection was highly significant both in the blood in and fecal samples (a delayed response after 4 h), with daily variation in the IRS level being insignificant. In the case of blood sampling, the increased level of fecal IRS was recorded after 4 h and remained high after 8 h. Immobilization did not result in any significant increase in blood corticosterone or fecal IRS level. Individual baseline concentrations of fecal IRS levels were found to be highly repeatable between days. Thus, the antibodies to corticosterone used in this study (IZW, Berlin, Germany) proved effective for the assessment of stress by measuring fecal IRS in bank voles.     

Fecal dehydroepiandrosterone (DHEA) immunoreactivity as a noninvasive index of circulating DHEA activity in young male laboratory rats

Evidence suggests that dehydroepiandrosterone (DHEA) plays a key role in stress and coping responses. Fecal sampling permits assessment of hormone-behavior interactions reliably and effectively, but no previous study has compared circadian- or stress-dependent alterations between serum DHEA and its fecal metabolites. In the current study, young (28 d of age) male rats were assigned to either an experimental (n = 6) or control (n = 6) group. Rats in the experimental group were exposed to a forced swim test to assess their behavioral and physiologic response to an environmental stressor; blood samples were drawn before the test (baseline), immediately after the test, and at 2 later time points. Only fecal samples were collected from control animals. Fecal DHEA and corticosterone metabolites were monitored in all animals for 24 h. DHEA metabolites in control rats exhibited significant diurnal variation, showing a similar temporal pattern as that of corticosterone metabolites. In addition, fecal and serum DHEA levels were highly correlated. Significant peaks in both DHEA and corticosterone metabolite levels were detected. These data suggest that measures of fecal DHEA can provide a complementary, noninvasive method of assessing adrenal gland function in rats.     

Tail docking in pigs: acute physiological and behavioural responses

Tail docking of piglets is a routine procedure on farms to control tail-biting behaviour; however, docking can cause an acute stress response. The objectives of this research were to determine the stress responses to tail docking in piglets and to compare two methods of tail docking; cautery iron (CAUT) and the more commonly used blunt trauma cutters (BT). At approximately 6 days of age, piglets were tail docked using CAUT (n = 20), BT (n = 20) or sham tail docked with their tails remaining intact (CON; n = 40). Blood samples were taken prior to tail docking and at 30, 60 and 90 min after tail docking to evaluate the effect of tail docking on white blood cell (WBC) measures and cortisol concentrations. The above experiment was repeated to observe behaviour without the periodic blood sampling, so as not to confound the effects of blood sampling on piglet behaviour. Piglet behaviour was recorded in the farrowing crate using 1 min scan-samples via live observations for 60 min prior to and 90 min after tail docking. Total WBC counts were reduced (P > 0.05) among BT and CAUT compared with CON piglets 30 min after tail docking. Cortisol concentrations were higher (P < 0.01) among BT compared with CON and CAUT piglets 60 min after tail docking. Cautery and BT-docked piglets spent more (P < 0.05) time posterior scooting compared with CON piglets between 0 and 15 min, and 31 and 45 min after tail docking. Piglets tail docked using CAUT and BT tended to spend more (P < 0.07) time sitting than CON piglets between 0 and 15 min post tail docking. Elevated blood cortisol can be reduced by the use of the CAUT rather than the BT method of tail docking. Although the tail docking-induced rise in cortisol was prevented by using CAUT, the behavioural response to BT and CAUT docking methods was similar.     

Are baseline and short-term corticosterone stress responses in free-living amphibians repeatable?

Amphibians respond to environmental stressors by secreting corticosterone, a stress hormone which promotes physiological and behavioral responses. Capture handling can be used to stimulate physiological stress response in amphibians. The use of single blood sampling and presentation of mean data often limits the quantification of within and between individual variation in baseline and short-term corticosterone stress responses in amphibians. It is important for studies of amphibian physiological ecology to determine whether baseline and short-term corticosterone stress responses are consistent or not. We quantified repeatability (r), a statistical measure of consistency, in baseline and short-term corticosterone stress responses to a standard capture and handling stress protocol in free-living adult male cane toads (Rhinella marina). Corticosterone metabolite concentrations were measured entirely non-invasively in male toad urine samples via an enzyme-immunoassay. During the first sampling occasion, urine samples were collected manually from individual male toads (n = 20) immediately upon field capture. Toads were handled for 5 min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8 h in the field. The toads were resampled for baseline (0 h) urine corticosterone with hourly urine sampling over 8 h (for quantification of the stress induced corticosterone) at 14 day intervals on three consecutive occasions. Within and between sample variations in urinary corticosterone metabolite concentrations were also quantified. All toads expressed a corticosterone stress response over 8 h to our standard capture and handling stress protocol. Variations both within and between toads was higher for corrected integrated corticosterone concentrations than corticosterone concentrations at baseline, 3 or 6 h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r = 0.877) together with high statistical repeatabilities for 3 h (r = 0.695), 6 h (r = 0.428) and 8 h (r = 0.775) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r = 0.807; r = 0.743 respectively). This study highlights that baseline and short-term corticosterone stress responses are repeatable in free-living amphibians. Future studies should utilize this non-invasive tool to explore repeatability among seasons and across years, and determine its functional significance in relation to behavioral ecology and reproduction in amphibians generally.     

Effects of investigator disturbance on corticosterone concentrations of Black‐legged Kittiwake chicks

ABSTRACT Recent studies indicate that young seabird chicks exposed to relatively short periods of elevated levels of plasma corticosterone may suffer lifelong cognitive impairment that is detrimental to their survivorship and fitness as adults. We examined the chronic effects of investigator disturbance on the baseline and acute stress‐induced levels of plasma corticosterone of Black‐legged Kittiwake (Rissa tridactyla) chicks in Chiniak Bay, Kodiak Island, Alaska, in 2005. Kittiwake chicks were assigned to one of three disturbance treatments: (1) routine handling, (2) exposure to investigator presence, but not handled, and (3) neither handled nor exposed to investigator presence prior to sampling. At 12–15 d posthatching, blood samples were collected to determine baseline and stress‐induced concentrations of corticosterone. We found no significant differences in baseline or stress‐induced levels of corticosterone among the three disturbance treatments. Our results suggest that Black‐legged Kittiwake chicks do not perceive investigator presence as a stressor. However, investigators studying kittiwakes at other locations should proceed with caution because sampling protocols and environmental conditions may differ, potentially causing chicks to perceive disturbances differently as well.     

Sources of error associated with sample collection and preparation of nucleated blood cells for flow cytometric analysis

Analysis of cellular DNA content by flow cytometry has been used to detect genetic changes associated with exposure to environmental contaminants. In lower vertebrates, nucleated red blood cells can be collected for analysis without harm to the animal. Because erythrocytes sampled from an individual should have identical amounts of DNA, the coefficient of variation (CV) around the G₀/G₁ peak should be small. Increases in CV can indicate genetic aberrations, but may also be caused by sample handling and preparation or problems with instrumentation. To increase confidence in associating increases in CV with external causes, artifactual changes in CV due to sample treatment and instrument parameters should be identified and minimized. We assessed the effects of various sampling and handling protocols on the CV of nucleated blood cells collected from largemouth bass (Micropterus salmoides). We also compared the distribution of cells among the G₀/G₁, S, and G₂/M phases of the cell cycle to see whether these were affected by sampling or treatment protocols. Groups of 7 fish were bled on 7 consecutive days, and blood from each fish was analyzed by flow cytometry when freshly collected, and after freezing for 1 hour or 10 days. The same fish were bled again over a consecutive 7-day period, and the experiment was repeated. CV and cell cycle distribution were not affected by our freezing protocol. Repeat sampling from the same individual did not affect CV, but altered the distribution of cells in the cell cycle, suggesting increased hemopoiesis in response to blood sampling. Day-to-day variation in the CV occurred in both fresh and frozen samples, probably as the result of small variations in instrument adjustments. These results demonstrate the suitability of this freezing protocol for these blood samples, and illustrate the importance of assessing sources of variation when using flow cytometry to screen wild populations in genotoxicological studies.Abbreviations CV coefficient of variation - DNA deoxyribonucleic acid - DMSO dimethylsulfoxide - FCM flow cytometric analysis - PI propidium iodide - RNA ribonucleic acid     

Effects of delayed phlebotomy on plasma steroid hormone concentrations in two elasmobranch species

Measuring circulating concentrations of steroid hormones can be used as a method for determining reproductive maturity and cycles in elasmobranchs. However, it is unknown how long steroid hormones remain stable in elasmobranch blood following capture, and thus how quickly these samples should be collected for the results of subsequent steroid hormone analyses to be accurate. The objectives of this study were to determine if the sex steroid hormones progesterone, testosterone and estradiol would remain at stable concentrations in the blood of the Spiny Dogfish (Squalus acanthias Linnaeus, 1758) and the Atlantic Sharpnose Sharks (Rhizoprionodon terraenovae Richardson, 1836) that were captured, left on deck and un‐refrigerated for 24 hr. Blood samples were serially drawn from five initially live sharks over a period of 24 hr. While concentrations of all three hormones did significantly fluctuate over the sampling period in both species, the resulting hormone concentrations from each sampling period still fell within the range of previously reported values for each species in their respective reproductive stage. Additionally, no significant changes in hematocrit were detected in either species over the 24‐hr period. This research represents an extreme situation in which sharks were left on deck and un‐refrigerated, and suggests that even when subjected to these conditions steroid hormone concentrations may fluctuate, but the resulting values may still be useful for assessing reproductive stage.     

Corticosterone excretion patterns and affiliative behavior over development in ravens (Corvus corax)

Averse effects of social stress may be buffered by the presence of social allies, which mainly has been demonstrated in mammals and recently also in birds. However, effects of socio-positive behavior prior to fledging in relation to corticosterone excretion in altricial birds have not been investigated yet. We here monitored corticosterone excretion patterns in three groups of hand raised juvenile ravens (n=5, 6 and 11) in the nest, post-fledging (May-July) and when ravens would be independent from their parents (September-November). We related these corticosterone excretion patterns to socio-positive behavior. Behavioral data were collected via focal sampling in each developmental period considered. We analyzed amounts of excreted immunoreactive corticosterone metabolites (CM) using enzyme immuno assays. We collected fecal samples in each developmental period considered and evaluated the most appropriate assay via an isolation stress experiment. Basal CM was significantly higher during the nestling period than post-fledging or when birds were independent. The time nestlings spent allopreening correlated negatively with mean CM. Post-fledging, individuals with higher CM levels sat close to (distance <50 cm) conspecifics more frequently and tended to preen them longer. When birds were independent and a stable rank hierarchy was established, dominant individuals were preened significantly longer than subordinates. These patterns observed in ravens parallel those described for primates, which could indicate that animal species living in a complex social environment may deal with social problems in a similar way that is not restricted to mammals or primates.