Comprehensive endocrine response to acute stress in the bottlenose dolphin from serum, blubber, and feces.

Several hormones are potential indicators of stress in free-ranging animals and provide information on animal health in managed-care settings. In response to stress, glucocorticoids (GC, e.g. cortisol) first appear in circulation but are later incorporated into other tissues (e.g. adipose) or excreted in feces or urine. These alternative matrices can be sampled remotely, or by less invasive means, than required for blood collection and are especially valuable in highly mobile species, like marine mammals. We characterized the timing and magnitude of several hormones in response to a stressor in bottlenose dolphins (Tursiops truncatus) and the subsequent incorporation of cortisol into blubber, and its metabolites excreted in feces. We evaluated the endocrine response to an acute stressor in bottlenose dolphins under managed care. We used a standardized stress protocol where dolphins voluntarily beached onto a padded platform and remained out of water for two hours; during the stress test blood samples were collected every 15 min and blubber biopsies were collected every hour (0, 60, and 120 min). Each subject was studied over five days: voluntary blood samples were collected on each of two days prior to the stress test; 1 and 2 h after the conclusion of the out-of-water stress test; and on the following two days after the stress test. Fecal samples were collected daily, each afternoon. The acute stressor resulted in increases in circulating ACTH, cortisol, and aldosterone during the stress test, and each returned to baseline levels within 2 h of the dolphin's return to water. Both cortisol and aldosterone concentrations were correlated with ACTH, suggesting both corticosteroids are at least partly regulated by ACTH. Thyroid hormone concentrations were generally unaffected by the acute stressor. Blubber cortisol increased during the stress test, and fecal GC excretion was elevated on the day of the stress test. We found that GCs in bottlenose dolphins can recover within hours of acute stress, and that cortisol release can be detected in alternate matrices within a few hours-within 2 h in blubber, and 3.5-5 h in fecal samples.

Modulation in Persistent Organic Pollutant Concentration and Profile by Prey Availability and Reproductive Status in Southern Resident Killer Whale Scat Samples.

Persistent organic pollutants (POPs), specifically PCBs, PBDEs, and DDTs, in the marine environment are well documented, however accumulation and mobilization patterns at the top of the food-web are poorly understood. This study broadens the understanding of POPs in the endangered Southern Resident killer whale population by addressing modulation by prey availability and reproductive status, along with endocrine disrupting effects. A total of 140 killer whale scat samples collected from 54 unique whales across a 4 year sampling period (2010-2013) were analyzed for concentrations of POPs. Toxicant measures were linked to pod, age, and birth order in genotyped individuals, prey abundance using open-source test fishery data, and pregnancy status based on hormone indices from the same sample. Toxicant concentrations were highest and had the greatest potential for toxicity when prey abundance was the lowest. In addition, these toxicants were likely from endogenous lipid stores. Bioaccumulation of POPs increased with age, with the exception of presumed nulliparous females. The exceptional pattern may be explained by females experiencing unobserved neonatal loss. Transfer of POPs through mobilization of endogenous lipid stores during lactation was highest for first-borns with diminished transfer to subsequent calves. Contrary to expectation, POP concentrations did not demonstrate an associated disruption of thyroid hormone, although this association may have been masked by impacts of prey abundance on thyroid hormone concentrations. The noninvasive method for measuring POP concentrations in killer whales through scat employed in this study may improve toxicant monitoring in the marine environment and promote conservation efforts.

Natural Variation in Stress Hormones, Comparisons Across Matrices, and Impacts Resulting from Induced Stress in the Bottlenose Dolphin.

Knowledge regarding stress hormones and how they vary in response to seasonality, gender, age, and reproductive status for any marine mammal is limited. Furthermore, stress hormones may be measured in more than one matrix (e.g., feces, blood, blubber), but the relationships between levels of a given hormone across these matrices are unknown, further complicating the interpretations of hormones measured in samples collected from wild animals. A study is underway to address these issues in a population of bottlenose dolphins trained for voluntary participation in sample collections from different matrices and across season and time of day.

Eliminating the artificial effect of sample mass on avian fecal hormone metabolite concentration.

Avian endocrinology is a productive field that could benefit from increased application of non-invasive techniques. Although assay protocols vary, most studies that measure hormone metabolites in avian feces struggle with an artificial effect of sample mass on steroid metabolite concentration. Hormone metabolite concentrations measured in small samples are consistently higher than concentrations in larger samples, and this appears to be due to multiple methodological problems. We systematically tested several causal hypotheses for the mass effect. Based on results from these tests, we modified and validated our assay protocol to effectively eliminate the mass effect. Future studies should implement the following procedures when measuring hormone metabolites from small fecal samples (particularly of birds and reptiles): (1) remove urates from the fecal sample as completely as possible; (2) lyophilize the sample prior to extraction; (3) maximize accuracy of small mass measurements; (4) increase the volume of ethanol in the extraction to 15 ml per 0.05-0.1g of dried feces; and (5) eliminate ethanol from all samples prior to radioimmunoassay by drying down extract solutions and rehydrating in buffer. By applying these precautions we successfully eliminated the mass effect from fecal samples ranging in mass from 0.001 to 0.1 g using a radioimmunoassay commonly employed for studies of fecal glucocorticoid metabolites. These corrections also resulted in a more than 3-fold increase in effect size in glucocorticoid concentrations from a controlled test of the effects of 1h motorcycle exposure on northern spotted owls. These methods have important implications not only for avian studies, but for any study measuring hormone metabolites from small fecal samples.

Non-invasive measurement of thyroid hormone in feces of a diverse array of avian and mammalian species.

We developed and validated a non-invasive thyroid hormone measure in feces of a diverse array of birds and mammals. An I(131) radiolabel ingestion study in domestic dogs coupled with High Pressure Liquid Chromatography (HPLC) analysis, showed that peak excretion in feces occurred at 24-48h post-ingestion, with I(131)-labelled thyroid hormone metabolites excreted primarily as triiodothyronine (T3) and relatively little thyroxine (T4), at all excretion times examined. The immunoreactive T3 profile across these same HPLC fractions closely corresponded with the I(131) radioactive profile. By contrast, the T4 immunoreactive profile was disproportionately high, suggesting that T4 excretion included a high percentage of T4 stores. We optimized and validated T3 and T4 extraction and assay methods in feces of wild northern spotted owls, African elephants, howler monkeys, caribou, moose, wolf, maned wolf, killer whales and Steller sea lions. We explained 99% of the variance in high and low T3 concentrations derived from species-specific sample pools, after controlling for species and the various extraction methods tested. Fecal T3 reflected nutritional deficits in two male and three female howler monkeys held in captivity for translocation from a highly degraded habitat. Results suggest that thyroid hormone can be accurately and reliably measured in feces, providing important indices for environmental physiology across a diverse array of birds and mammals.

Fecal stress, nutrition and reproductive hormones for monitoring environmental impacts on tigers (Panthera tigris).

Non-invasive stress and nutritional hormone analysis in relation to ecological and other biological indices have tremendous potential to address environmental disturbance impacts on wildlife health. To this end, we examined the relation between glucocorticoid (GC) and thyroid (T3) hormone indices of disturbance and nutritional stress in response to ACTH and TSH challenges in captive tigers, as well as how reproductive hormones vary by sex and reproductive condition. Glucocorticoid, thyroid, progesterone and androgen assays conducted on high-performance liquid chromatography separated fractions of biologically relevant fecal extracts revealed high cross-reactivity of these assays for their respective biologically relevant fecal hormone metabolites. Both adrenal and thyroid hormone metabolites were elevated in response to ACTH and TSH challenges. However, the adrenal and thyroid hormone responses to ACTH challenge were concurrent, whereas the adrenal response to TSH challenge was delayed relative to thyroid hormone elevation in both males and females. The concurrently elevated T3 in response to ACTH may serve to raise metabolic rate to maximize use of GC-mobilized glucose, whereas the relatively delayed GC rise following TSH challenge may be a response to glucose depletion due to increased metabolic rate associated with elevated T3. Progesterone, testosterone and androstenedione hormone metabolites were significantly elevated during gestation compared to lactation in a female monitored from conception through early lactation. Results suggest that the glucocorticoid, thyroid and reproductive hormone assays we tested can accurately measure the stress, nutrition and reproductive response from tiger feces, providing useful non-invasive tools to assess physiological responses to environmental stressors and their reproductive consequences in the wild.

Pre-oil spill baseline profiling for contaminants in Southern Resident killer whale fecal samples indicates possible exposure to vessel exhaust.

The Southern Resident killer whale population (Orcinus orca) was listed as endangered in 2005 and shows little sign of recovery. Exposure to contaminants and risk of an oil spill are identified threats. Previous studies on contaminants have largely focused on legacy pollutants. Here we measure polycyclic aromatic hydrocarbons (PAHs) in whale fecal (scat) samples. PAHs are a diverse group of hazardous compounds (e.g., carcinogenic, mutagenic), and are a component of crude and refined oil as well as motor exhaust. The central finding from this study indicates low concentrations of the measured PAHs (<10 ppb, wet weight), as expected; however, PAHs were as high as 104 ppb prior to implementation of guidelines mandating increased distance between vessels and whales. While causality is unclear, the potential PAH exposure from vessels warrants continued monitoring. Historical precedent similarly emphasizes the importance of having pre-oil spill exposure data available as baseline to guide remediation goals.

Effects of oral megestrol acetate administration on the hypothalamic-pituitary-adrenal axis of male bottlenose dolphins (Tursiops truncatus).

OBJECTIVE To evaluate the impact of oral megestrol acetate (MA) administration on adrenal function in male bottlenose dolphins (Tursiops truncatus). DESIGN Serial cross-sectional study. ANIMALS 8 adult male dolphins, all of which were receiving MA at various daily doses (range, 0 to 60 mg, PO) for the control of reproductive behavior. PROCEDURES Blood samples were collected every 2 weeks for 1 year from dolphins trained to voluntarily provide them. Cortisol, ACTH, and other hormone concentrations were measured in serum or plasma via radioimmunoassay or ELISA. Fecal samples, also provided by dolphins voluntarily, were assayed for glucocorticoid metabolite concentrations. Effects of daily MA dose on hormone concentrations were evaluated. RESULTS Daily MA doses as low as 10 mg strongly suppressed cortisol secretion in nearly all dolphins, and except for a single measurement, no dolphin had measurable serum concentrations at doses ≥ 20 mg. Variations in serum cortisol concentration were unrelated to season but were directly related to ACTH concentrations, suggesting primary effects upstream of the adrenal gland. Cessation of MA administration resulted in almost immediate restoration of measurable serum cortisol concentrations, although concentrations continued to rise in a few dolphins over the following weeks to months. CONCLUSIONS AND CLINICAL RELEVANCE Caution should be exercised when administering MA to control reproductive behavior in male dolphins. Because the hypothalamic-pituitary-adrenal axis appeared to be sensitive to even small doses of MA in dolphins, duration of treatment may be the most critical consideration.

Using detection dogs to conduct simultaneous surveys of northern spotted (Strix occidentalis caurina) and barred owls (Strix varia).

State and federal actions to conserve northern spotted owl (Strix occidentalis caurina) habitat are largely initiated by establishing habitat occupancy. Northern spotted owl occupancy is typically assessed by eliciting their response to simulated conspecific vocalizations. However, proximity of barred owls (Strix varia)-a significant threat to northern spotted owls-can suppress northern spotted owl responsiveness to vocalization surveys and hence their probability of detection. We developed a survey method to simultaneously detect both species that does not require vocalization. Detection dogs (Canis familiaris) located owl pellets accumulated under roost sites, within search areas selected using habitat association maps. We compared success of detection dog surveys to vocalization surveys slightly modified from the U.S. Fish and Wildlife Service's Draft 2010 Survey Protocol. Seventeen 2 km × 2 km polygons were each surveyed multiple times in an area where northern spotted owls were known to nest prior to 1997 and barred owl density was thought to be low. Mitochondrial DNA was used to confirm species from pellets detected by dogs. Spotted owl and barred owl detection probabilities were significantly higher for dog than vocalization surveys. For spotted owls, this difference increased with number of site visits. Cumulative detection probabilities of northern spotted owls were 29% after session 1, 62% after session 2, and 87% after session 3 for dog surveys, compared to 25% after session 1, increasing to 59% by session 6 for vocalization surveys. Mean detection probability for barred owls was 20.1% for dog surveys and 7.3% for vocal surveys. Results suggest that detection dog surveys can complement vocalization surveys by providing a reliable method for establishing occupancy of both northern spotted and barred owl without requiring owl vocalization. This helps meet objectives of Recovery Actions 24 and 25 of the Revised Recovery Plan for the Northern Spotted Owl.

Evaluation of circulating eosinophil count and adrenal gland function in California sea lions naturally exposed to domoic acid.

OBJECTIVE: To determine the effect of natural exposure to domoic acid (DA) on eosinophil counts and adrenal gland function in California sea lions (Zalophus californianus). DESIGN: Cross-sectional prospective study. ANIMALS: 39 California sea lions. PROCEDURES: Adult female sea lions admitted to a rehabilitation hospital during 2009 were classified into 1 of 3 groups (acute DA toxicosis, chronic DA toxicosis, or no DA exposure) on the basis of clinical signs, DA concentration in urine or feces, and hippocampal morphology. Endoparasite burden, eosinophil count, and serum cortisol and plasma ACTH concentrations were determined for each sea lion. For a subset of 8 sea lions, fecal glucocorticoid concentration after IM administration of cosyntropin was determined. RESULTS: Sea lions exposed to DA (acute DA toxicosis, n = 11; chronic DA toxicosis, 19) had higher eosinophil counts and lower serum cortisol concentrations, compared with values for sea lions with no DA exposure (9). Eosinophil count was not associated with endoparasite burden. Serum cortisol concentration was associated with plasma ACTH concentrations in sea lions from the no DA exposure group but not in sea lions in the acute or chronic DA toxicosis groups. Following cosyntropin injection, fecal glucocorticoid concentrations increased in all sea lions evaluated except 1. CONCLUSIONS AND CLINICAL RELEVANCE: In adult sea lions, eosinophilia may be a cost-effective biomarker for DA exposure and may reflect alterations in hypothalamic, pituitary gland, or adrenal gland function. Domoic acid exposure may have subtle health effects on marine animals in addition to induction of neurologic signs.

Distinguishing the impacts of inadequate prey and vessel traffic on an endangered killer whale (Orcinus orca) population.

Managing endangered species often involves evaluating the relative impacts of multiple anthropogenic and ecological pressures. This challenge is particularly formidable for cetaceans, which spend the majority of their time underwater. Noninvasive physiological approaches can be especially informative in this regard. We used a combination of fecal thyroid (T3) and glucocorticoid (GC) hormone measures to assess two threats influencing the endangered southern resident killer whales (SRKW; Orcinus orca) that frequent the inland waters of British Columbia, Canada and Washington, U.S.A. Glucocorticoids increase in response to nutritional and psychological stress, whereas thyroid hormone declines in response to nutritional stress but is unaffected by psychological stress. The inadequate prey hypothesis argues that the killer whales have become prey limited due to reductions of their dominant prey, Chinook salmon (Oncorhynchus tshawytscha). The vessel impact hypothesis argues that high numbers of vessels in close proximity to the whales cause disturbance via psychological stress and/or impaired foraging ability. The GC and T3 measures supported the inadequate prey hypothesis. In particular, GC concentrations were negatively correlated with short-term changes in prey availability. Whereas, T3 concentrations varied by date and year in a manner that corresponded with more long-term prey availability. Physiological correlations with prey overshadowed any impacts of vessels since GCs were lowest during the peak in vessel abundance, which also coincided with the peak in salmon availability. Our results suggest that identification and recovery of strategic salmon populations in the SRKW diet are important to effectively promote SRKW recovery.

Synthesis and characterisation of luminescent rhenium tricarbonyl complexes with axially coordinated 1,2,3-triazole ligands.

A series of 1-alkyl-4-aryl-1,2,3-triazoles (1-methyl-4-phenyl-1,2,3-triazole (1a); 1-propyl-4-phenyl-1,2,3-triazole (1b); 1-benzyl-4-phenyl-1,2,3-triazole (1c); 1-propyl-4-p-tolyl-1,2,3-triazole (1d)) have been prepared through a one-pot procedure involving in situ generation of the alkyl azide from a halide precursor followed by copper catalysed alkyne/azide cycloaddition (CuAAC) with the appropriate aryl alkyne. Cationic Re(I) complexes [Re(bpy)(CO)(3)(1a-d)]PF(6) (2a-d) were then prepared by stirring [Re(bpy)(CO)(3)Cl] with AgPF(6) in dichloromethane in the presence of ligands 1a-d. X-ray crystal structures were obtained for 2a and 2b. In the solid state, 2a adopts a highly distorted geometry, which is not seen for 2b, in which the plane of the triazole ligand tilts by 13° with respect to the Re-N bond as a result of a π-stacking interaction between the Ph substituent and one of the rings of the bpy ligand. This π-stacking interaction also results in severe twisting of the bpy ligand. Infrared spectra of 2a-d exhibit ν(CO) bands at ∼2035 and ∼1926 cm(-1) suggesting that these ligands are marginally better donors than pyridine (ν(CO) = 2037, 1932 cm(-1)). The complexes are luminescent in aerated dichloromethane at room temperature with emission maxima at 542 to 552 nm comparable to that of the pyridine analogue (549 nm) and blue shifted relative to the parent chloride complex. Long luminescent lifetimes are observed for the triazole complexes (475 to 513 ns) in aerated dichloromethane solutions at room temperature.