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The sustainability of endangered Asian elephants in human care is threatened in part by low breeding success and concerns over individual animal wellbeing. Male elephants have received less research attention compared to females, yet males deserve special consideration due to their unique reproductive biology (particularly the sexual state of “musth”) and the complex interaction of physiological, environmental, and social pressures they face. We measured fecal androgen metabolites (FAMs), fecal glucocorticoid metabolites (FGMs), and fecal triiodothyronine metabolites (FT3s) collected weekly over approximately 12 months from 26 male Asian elephants housed in zoos across the US, hypothesizing that FAM, FGM, and FT3 concentrations would be associated with temporal correlates of musth and would vary further with intrinsic (musth status, age, body condition) and extrinsic (social environment) factors. The duration of each musth episode was positively associated with exposure to male conspecifics and negatively associated with body condition. Further, elevated FAM concentrations were associated with social exposure, age, and body condition, and FGM concentrations also varied with age and body condition. FT3 concentrations were not associated with any factor we measured. We also identified periods of lower FAM concentration than confirmed musth episodes (but still higher than baseline FAM concentrations) that we termed “elevated FAM episodes.” The durations of these episodes were negatively correlated with exposure to other male elephants. Together, these results provide evidence that hormone profiles (including those that are predicted to change around musth) vary significantly between male Asian elephants in a way that may be attributed to intrinsic and extrinsic factors. Studies like these serve to enhance the sustainability of ex-situ populations by providing wildlife managers with information to enhance the health, welfare, and reproduction of threatened species like Asian elephants.

Understanding reproductive physiology in mysticetes has been slowed by the lack of repeated samples from individuals. Analysis of humpback whale baleen enables retrospective hormone analysis within individuals dating back 3–5 years before death. Using this method, we investigated differences in four steroid hormones involved in reproduction and mating during confirmed pregnant and non-pregnant periods in two female humpback whales (Megaptera novaeangliae) with known reproductive histories based on sightings and necropsy data. Cortisol, corticosterone, testosterone, and estradiol concentrations were determined via enzyme immunoassay using subsamples of each baleen plate at 2 cm intervals. There were no significant differences in cortisol or corticosterone during pregnancy when compared to non-pregnancy (inter-calving interval), but there were significant differences between the two whales in average glucocorticoid concentrations, with the younger whale showing higher values overall. For testosterone, levels for the younger female peaked at parturition in one pregnancy, but also had spikes during non-pregnancy. The older female had three large spikes in testosterone, one of which was associated with parturition. Estradiol had large fluctuations in both whales but had generally lower concentrations during non-pregnancy than during pregnancy. There were peaks in estradiol before each pregnancy, possibly coinciding with ovulation, and peaks coinciding with the month of parturition. Both estradiol and testosterone could be useful for determining ovulation or impending birth. Using baleen to investigate retrospective steroid hormone profiles can be used for elucidating long-term patterns of physiological change during gestation. Lay summary Case studies of two pregnant humpback whales whose hormones were analyzed in baleen may illuminate when humpback whales ovulate, gestate, and give birth. These physiological metrics could assist in accurate population growth assessments and conservation of the species. This study shows that baleen hormone analysis can be a useful tool for understanding whale reproductive physiology.

Made available in DSpace on 2022-05-01T08:15:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Sampling blood for endocrine analysis from some species may not be practical or ethical. Quantification of hormones extracted from nontypical sample types, such as keratinized tissues, offers a less invasive alternative to the traditional collection and analysis of blood. Here, we aimed to validate assays by using parallelism and accuracy tests for quantification of testosterone, corticosterone, progesterone, and triiodothyronine (T3) in shed skins of tegu lizards. We assessed whether hormone content of sheds varied across one year similar to what was previously detected in plasma samples. In addition, we aimed to identify the phase relationship between hormone levels of shed skin and plasma levels obtained from the same animals. High frequency of shedding occurred during the active season for tegus (spring/summer), while shedding ceased during hibernation (winter). All hormones measured in shed skins exhibited seasonal changes in concentration. Levels of testosterone in shed skins of male tegus correlated positively with plasma testosterone levels, while corticosterone in both males and females exhibited an inverse relationship between sample types for the same month of collection. An inverse relationship was found when accounting for a lag time of 3 and 4 months between sheds and plasma testosterone. These results indicate that endocrine content of sheds may be confounded by factors (i.e., seasons, environmental temperature, thermoregulatory behavior, among others) that affect frequency of molting, skin blood perfusion, and therefore hormone transfer from the bloodstream and deposition in sheds of squamates. Department of Physiology Institute of Biosciences University of São Paulo, Rua Do Matão, 14 - Butantã, SP Department of Animal Morphology and Physiology College of Agricultural and Veterinary Sciences São Paulo State University, SP Department of Biological Sciences Northern Arizona University Department of Biology and Smithsonian-Mason School of Conservation George Mason University Department of Animal Morphology and Physiology College of Agricultural and Veterinary Sciences São Paulo State University, SP FAPESP: 2014/16320-7 FAPESP: 2015/04849-6 FAPESP: 2017/01652-2 FAPESP: 2018/06192-2

Elevated noise from human activities in nature can impact animal behavior and physiology, with subsequent impacts on individual health and population dynamics. Baleen whale communication, navigation, habitat use, and ability to recognize and locate prey and predators may be impaired by anthropogenic activities that increase ocean noise within the whales' hearing frequency range. To understand the physiological impacts of noise disturbance on baleen whales, we investigated the adrenal stress response of gray whales (Eschrichtius robustus) to variable ambient noise levels through an assessment of fecal glucocorticoid metabolite concentrations. This analysis was conducted at the individual level, at multiple temporal scales (1-7 days), and accounted for factors that may confound glucocorticoid hormone concentrations: sex, age, nutritional status, and reproductive state. Data were collected along the Oregon coast, USA, from June to October of 2016-2018. Results indicate a significant positive correlation between underwater noise levels and vessel traffic. Vessel counts from the day prior to fecal sample collection and sex had significant positive relationships with glucocorticoid metabolite concentrations. This study increases knowledge of gray whale physiological response to variable ocean noise and may inform management decisions regarding regulations of anthropogenic noise activities and thresholds near critical whale habitats.

The short-beaked echidna (Tachyglossus aculeatus) is a monotreme endemic to Australia and New Guinea, and is the most widespread native mammal in Australia. Despite its abundance, there are considerable gaps in our understanding of echidna life history such as reproductive cycles in both sexes, patterns of stress physiology, and possible seasonal changes in metabolism. Slow-growing integumentary sample types comprised of keratin (hair, claw, etc.) have been used in other wildlife to assess these questions via analysis of longitudinal patterns in steroid and thyroid hormones that are deposited in these tissues as they grow. Hairs and spines comprise the pelage of echidnas, the spines being keratinized structures homologous to hair. Thus, echidna spines could be a viable sample type for hormone analysis contributing to a better understanding of the biology of echidnas. The aim of this work was to determine whether steroid hormones are detectable in echidna spines, to perform assay validations, and to establish a protocol for extracting and quantifying hormones in echidna spines using commercially available assay kits. We also inspected cross-sectioned spines using light and electron microscopy for any evidence of annual growth markers that might enable inferences about spine growth rate. Corticosterone, progesterone, estradiol, and testosterone were detectable in all samples, and echidna spine extract passed standard assay validations (parallelism and accuracy), indicating that commercially available assay kits can quantify hormones accurately in this sample type. No visible growth marks were identified in the spines and thus spine growth rate is currently unknown. Echidna spines show promise as a novel matrix from which hormones can be quantified; next steps should involve determination of spine annual growth rate, possible seasonal changes in growth rate, and persistence of spines over time in order to perform physiological validations, i.e., relationship between physiological status and hormone concentrations in spines.

Fecal hormone analysis shows high potential for noninvasive assessment of population-level patterns in stress and reproduction of marine mammals. However, the marine environment presents unique challenges for fecal sample collection. Data are still lacking on collection methodology and assay validations for most species, particularly for those mysticete whales that have variable diets. In this study we tested collection techniques for fecal samples of free-swimming humpback whales (Megaptera novaeangliae), and validated immunoassays for five steroid and thyroid hormones. Resulting data were used for preliminary physiological validations, i.e., comparisons to independently confirmed sex and reproductive state. Pregnant females had significantly higher fecal progestins and glucocorticoids than did other demographic categories of whales. Two possible cases of previously undetected pregnancies were noted. Males had significantly higher fecal testosterone metabolites than nonpregnant females. Fecal glucocorticoids were significantly elevated in pregnant females and mature males compared to nonpregnant females. Calf fecal samples had elevated concentrations of all fecal hormones. Fecal thyroid hormones showed a significant seasonal decline from spring to summer. Though sample sizes were small, and sampling was necessarily opportunistic, these patterns indicate that noninvasive fecal hormone analysis may facilitate studies of reproduction, stress and potentially energetics in humpback whales.

Obtaining endocrine data from alternative sample types such as baleen and other keratinized tissues has proven a valuable tool to investigate reproductive and stress physiology via steroid hormone quantification, and metabolic stress via thyroid hormone quantification in whales and other vertebrates. These alternative sample types provide an integrated measure of plasma levels over the period that the structure was growing, thus capturing months or even years of an individual's endocrine history. Additionally, their robust and stable keratin matrix allows such samples to be stored for years to decades, enabling the analysis and comparison of endocrine patterns from past and modern populations. However, the extraction and analysis of hormones from baleen and other keratinized tissues remains novel and requires both biological and analytical validations to ensure the method fulfills the requirements for its intended use. We utilized baleen recovered at necropsy from southern right whales (Eubalaena australis) that died at Peninsula Valdes, Argentina, using a commercially available progesterone enzyme immunoassay (EIA) to address two methodological questions: 1) what is the minimum sample mass required to reliably quantify hormone content of baleen samples analyzed with commercially available EIAs, and 2) what is the optimal ratio of solvent volume to sample mass, i.e., the ratio that yields the maximum amount of hormone with high accuracy and low variability between replicates. We concluded that masses of at least 20 mg should be used whenever possible, and extraction is best performed using an 80:1 ratio of solvent to sample (volume of solvent to sample mass; μl:mg). These results can help researchers to make informed methodological decisions when using a destructive extraction method with rare or unique specimens.

In waters off Península Valdés (PV), Argentina, southern right whales (SRW, Eubalaena australis) are occasionally exposed to domoic acid (DA), a neurotoxin produced by diatoms of the genus Pseudo-nitzschia. Domoic acid toxicity in marine mammals can cause gastrointestinal and neurological clinical signs, alterations in hematologic and endocrine variables, and can be fatal in extreme cases. In this study, we validated an enzyme immunoassay to quantify fecal glucocorticoid metabolites (fGCm) in 16 SRW fecal samples from live and dead stranded whales in PV from 2013 to 2018 and assessed fGCm levels associated with DA exposure. Overall, fGCm levels were significantly lower in SRWs with detectable fecal DA (n = 3) as compared to SRWs with undetectable fecal DA levels (n = 13). The highest fecal DA was observed in a live lactating female, which had low fGCm compared to the other lactating females studied. The highest fGCm was observed in a lactating female with undetectable DA; interestingly, at the time of sample collection, this female was sighted with two calves, an extremely unusual occurrence in this species. Though the sample size of these exceptionally rare breeding-season fecal samples was unavoidably small, our study provides evidence of potential adrenal alterations in whales exposed to an environmental neurotoxin such as DA.

Monitoring the physiology of wild populations presents many technical challenges. Blood samples, long the gold standard of wildlife endocrinology studies, cannot always be obtained. The validation and use of non-plasma samples to obtain hormone data have greatly improved access to more integrated information about an organism’s physiological state. Keratinous tissues like skin, hair, nails, feathers, or baleen store steroid hormones in physiologically relevant concentrations, are stable across decades, and can be used to retrospectively infer physiological state at prior points in time. Most protocols for steroid extraction employ physical pulverization or cutting of the sample, followed by mixing with a solvent. Such methods do produce repeatable and useful data, but low hormone yield and detectability issues can complicate research on small or rare samples. We investigated the use of keratinase, an enzyme that breaks down keratin, to improve the extraction and yield of corticosterone from vertebrate keratin tissues. Corticosterone content of keratinase-digested extracts were compared to non-keratinase extracts for baleen from three species of whale (blue, Balaenoptera musculus; bowhead, Balaena mysticetus; southern right, SRW; Eubalaena australis), shed skin from two reptiles (tegu lizard, Salvator merianae; narrow-headed garter snake, Thamnophis rufipunctatus), hair from arctic ground squirrel (AGS; Urocitellus parryii), feathers from Purple Martins (PUMA; Progne subis), and spines from the short-beaked echidna (Tachyglossus aculeatus). We tested four starting masses (10, 25, 50, 100 mg) for each sample; digestion was most complete in the 10 and 25 mg samples. A corticosterone enzyme immunoassay (EIA) was validated for all keratinase-digested extracts. In all sample types except shed skin from reptiles, keratinase digestion improved hormone yield, with PUMA feathers and blue whale baleen having the greatest increase in apparent corticosterone content (100% and 66% more hormone, respectively). The reptilian shed skin samples did not benefit from keratinase digestion, actually yielding less hormone than controls. With further optimization and refinement, keratinase digestion could greatly improve yield of steroid hormones from various wildlife epidermal tissue types, allowing more efficient use of samples and ultimately improving understanding of the endocrine physiology of wild populations.

Baleen of two pregnant humpback whales contains areas of high progesterone that span the duration of pregnancy (determined from sighting records), while baleen from a non-pregnant female and juvenile male had low progesterone through the baleen. Thus, baleen hormone analysis can identify pregnancies and inter-calving intervals of female humpback whales., Understanding calving rates of wild whale populations is critically important for management and conservation. Reproduction of humpback whales (Megaptera novaeangliae) is difficult to monitor and, even with long-term sighting studies, basic physiological information such as pregnancy rates and calving intervals remain poorly understood in many populations. We hypothesized that pregnant whales have sustained elevations in baleen progesterone that temporally correlate with gestation. To test this hypothesis, baleen progesterone profiles from two adult female North Pacific humpbacks, both with extensive sighting records and documented pregnancies, were compared to those of a nulliparous female (adult female never seen with a calf) and a juvenile male. Baleen specimens recovered during necropsy were subsampled every 2 cm from the base to the tip of the plate, with each interval representing 30–45 days of growth. Homogenized baleen powder was assayed for progesterone using enzyme immunoassays. The date of growth of each sampling location on the baleen plate was estimated based on stable isotope analysis of annual δ15N cycles. Progesterone profiles from both pregnant whales showed sustained high progesterone content (>350 ng/g) in areas corresponding to known pregnancies, inferred from calf sightings and post-mortem data. The younger female, estimated to be 13 years old, had higher progesterone during pregnancy than the 44.5 year old, but levels during non-pregnancy were similar. The nulliparous female and the male had low progesterone throughout their baleen plates. Baleen hormone analysis can determine how progesterone concentrations change throughout gestation and has potential for estimating age at first reproduction, pregnancy intervals, failed pregnancies and early calf mortality. Understanding rates of calving and current and historic reproductive patterns in humpbacks is vital to continuing conservation measures in this species.

Sampling blood for endocrine analysis from some species may not be practical or ethical. Quantification of hormones extracted from nontypical sample types, such as keratinized tissues, offers a less invasive alternative to the traditional collection and analysis of blood. Here, we aimed to validate assays by using parallelism and accuracy tests for quantification of testosterone, corticosterone, progesterone, and triiodothyronine (T

Baleen whales are subject to a myriad of natural and anthropogenic stressors, but understanding how these stressors affect physiology is difficult. Measurement of adrenal glucocorticoid (GC) hormones involved in the vertebrate stress response (cortisol and corticosterone) in baleen could help fill this data gap. Baleen analysis is a powerful tool, allowing for a retrospective re-creation of multiple years of GC hormone concentrations at approximately a monthly resolution. We hypothesized that whales that died from acute causes (e.g. ship strike) would have lower levels of baleen GCs than whales that died from extended illness or injury (e.g. long-term entanglement in fishing gear). To test this hypothesis, we extracted hormones from baleen plates of four humpback whales (Megaptera novaeangliae) with well-documented deaths including multiple and chronic entanglements (n = 1, female), ship strike (n = 2, male and female) and chronic illness with nutritional stress (n = 1, male). Over ~3 years of baleen growth and during multiple entanglements, the entangled whale had average corticosterone levels of 80–187% higher than the other three whales but cortisol levels were similar to two of the other three whales. The nutritionally stressed and chronically ill whale showed a slow increase in both cortisol and corticosterone spanning ~3 years, followed by a sharp decline in both hormones before death, possibly indicative of adrenal failure in this moribund individual. This whale’s correlation between cortisol and corticosterone was significant but there were no correlations in the other three whales. Our results show that cortisol and corticosterone concentrations vary according to the type and duration of illness or injury. Single-point GC concentrations should be interpreted with caution as low values can occur in whales experiencing pronounced stress and individual baselines can be highly variable. Baleen analysis is a promising tissue type for retrospective analyses of physiological responses to various stressors affecting baleen whales.

Understanding calving rates of wild whale populations is critically important for management and conservation. Reproduction of humpback whales (Megaptera novaeangliae) is difficult to monitor and, even with long-term sighting studies, basic physiological information such as pregnancy rates and calving intervals remain poorly understood in many populations. We hypothesized that pregnant whales have sustained elevations in baleen progesterone that temporally correlate with gestation. To test this hypothesis, baleen progesterone profiles from two adult female North Pacific humpbacks, both with extensive sighting records and documented pregnancies, were compared to those of a nulliparous female (adult female never seen with a calf) and a juvenile male. Baleen specimens recovered during necropsy were subsampled every 2 cm from the base to the tip of the plate, with each interval representing 30–45 days of growth. Homogenized baleen powder was assayed for progesterone using enzyme immunoassays. The date of growth of each sampling location on the baleen plate was estimated based on stable isotope analysis of annual δ15N cycles. Progesterone profiles from both pregnant whales showed sustained high progesterone content (>350 ng/g) in areas corresponding to known pregnancies, inferred from calf sightings and post-mortem data. The younger female, estimated to be 13 years old, had higher progesterone during pregnancy than the 44.5 year old, but levels during non-pregnancy were similar. The nulliparous female and the male had low progesterone throughout their baleen plates. Baleen hormone analysis can determine how progesterone concentrations change throughout gestation and has potential for estimating age at first reproduction, pregnancy intervals, failed pregnancies and early calf mortality. Understanding rates of calving and current and historic reproductive patterns in humpbacks is vital to continuing conservation measures in this species.

Conservation physiology tools can be difficult to employ in the wild. Here we discuss developments in conservation physiology research of large whales, a taxonomic group that is famously difficult to study with traditional tools. We focus on the North Atlantic right whale (Eubalaena glacialis) and southern right whale (Eubalaena australis), two closely related species that present similar logistical challenges for research, yet differ in population status and conservation pressures. Research has advanced via a suite of creative approaches including photo-identification, visual health assessment, remote methods of assessing body condition, and endocrine research on non-plasma sample types such as faeces, respiratory vapour, and baleen. These efforts have illuminated conservation-relevant physiological questions for both species, such as discrimination of acute from chronic stress, identification of likely causes of mortality, and monitoring causes and consequences of changes in body condition and reproduction.

An improved understanding of baleen whale hormone physiology is required to manage populations and reduce anthropogenic impacts. Gray whale fecal samples were assessed, and hormone metabolites quantified, which varied by demographic units and years. Two case studies are presented providing reference points for physiologically stressed individuals and adult breeding males., Baleen whale fecal samples have high potential for endocrine monitoring, which can be used as a non-invasive tool to identify the physiological response to disturbance events and describe population health and vital rates. In this study, we used commercial enzyme-linked immunosorbent assays to validate and quantify fecal steroid (progestins, androgens and glucocorticoids) and thyroid hormone metabolite concentrations in eastern North Pacific gray whales (Eschrichtius robustus) along the Oregon coast, USA, from May to October of 2016–2018. Higher mean progestin metabolite concentrations were observed in postweaning females, followed by pregnant females. Mean androgen, glucocorticoid and thyroid metabolites were higher in mature males. Progestin, glucocorticoids and thyroid fecal metabolites varied significantly by year, with positive correlations between progestin and androgen, and between glucocorticoid and thyroid metabolites. We also present two case studies of a documented injured whale and a mature male displaying reproductive competitive behavior, which provide reference points for physiologically stressed individuals and adult breeding males, respectively. Our methods and findings advance the knowledge of baleen whale physiology, can help guide future research on whale physiology and can inform population management and conservation efforts regarding minimizing the impact of anthropogenic stressors on whales.

SynopsisMany juvenile Kemp’s ridley (Lepidochelys kempii) and loggerhead (Caretta caretta) turtles strand during fall on the beaches of Cape Cod (MA, USA), with total stranding numbers sometimes exceeding 300 turtles per year. Once rehabilitated, turtles must be released at beaches with appropriate water temperatures, often requiring transportation to southeastern coastal states of the USA. These transportation events (transports) may approach or exceed 24 h in duration. Kemp’s ridley turtles are known to exhibit an adrenal stress response during such transports, but the effect of transport duration has been unclear, and no other sea turtle species has been investigated. To assess whether transport duration and/or species affects physiological reactions to transport, we studied pre- and post-transport physiological measures in Kemp’s ridley and loggerhead turtles transported by ground for

Research into stress physiology of mysticete whales has been hampered by difficulty in obtaining repeated physiological samples from individuals over time. We investigated whether multi-year longitudinal records of glucocorticoids can be reconstructed from serial sampling along full-length baleen plates (representing ∼10years of baleen growth), using baleen recovered from two female North Atlantic right whales (Eubalaena glacialis) of known reproductive history. Cortisol and corticosterone were quantified with immunoassay of subsamples taken every 4cm (representing ∼60d time intervals) along a full-length baleen plate from each female. In both whales, corticosterone was significantly elevated during known pregnancies (inferred from calf sightings and necropsy data) as compared to intercalving intervals; cortisol was significantly elevated during pregnancies in one female but not the other. Within intercalving intervals, corticosterone was significantly elevated during the first year (lactation year) and/or the second year (post-lactation year) as compared to later years of the intercalving interval, while cortisol showed more variable patterns. Cortisol occasionally showed brief high elevations ("spikes") not paralleled by corticosterone, suggesting that the two glucocorticoids might be differentially responsive to certain stressors. Generally, immunoreactive corticosterone was present in higher concentration in baleen than immunoreactive cortisol; corticosterone:cortisol ratio was usually >4 and was highly variable in both individuals. Further investigation of baleen cortisol and corticosterone profiles could prove fruitful for elucidating long-term, multi-year patterns in stress physiology of large whales, determined retrospectively from stranded or archived specimens.

Until now, physiological stress assessment of large whales has predominantly focused on adrenal glucocorticoid (GC) measures. Elevated GC concentrations in feces (fGC) are known to reflect stressful disturbances, such as fishing gear entanglement and human-generated underwater noise, in North Atlantic right whales (Eubalaena glacialis). However, there can be considerable variation in GC production as a function of sex and life history stage, which may confound the interpretation of fGC levels. Additionally, GC antibodies used in immunoassays can cross-react with other fecal metabolites (i.e., non-target steroids), potentially influencing fGC data. Here, aldosterone concentrations (fALD; aldosterone and related metabolites) were measured in fecal samples from right whales (total n=315 samples), including samples from identified individuals of known life history (n=82 individual whales), to evaluate its utility as a complementary biomarker to fGC for identifying adrenal activation. Concentrations of fALD were positively correlated with fGCs in right whales (r=0.59, P0.001), suggesting concurrent secretion of these hormones by the adrenal gland. However, fALD levels were less influenced by concentrations of reproductive steroids in feces, minimizing the potential confounder of assay cross-reactivity in samples with highly skewed hormone ratios. Across different life history states for right whales, fALD concentrations showed similar patterns to those reported for fGC, with higher levels in pregnant females (35.9±7.6ng/g) followed by reproductively mature males (9.5±0.9ng/g) (P0.05), providing further evidence of elevated adrenal activation in these groups of whales. The addition of fALD measurement as a biomarker of adrenal activation may help distinguish between intrinsic and external causes of stress hormone elevations in large whales, as well as other free-living wildlife species, providing a more comprehensive approach for associating adrenal activation with specific natural and anthropogenic stressors.

Raising nestlings to fledging is energetically demanding for songbirds, requiring parents to balance several major tradeoffs. Nestling growth rates are highly susceptible to variation in environmental conditions and parental investment; highly variable environments with short breeding seasons such as the Arctic magnify these tradeoffs. Arctic nesting passerines provide a good model system in which to explore variation within and between species in growth rates with regards to environmental conditions and the timing of clutch initiation. Here we investigated inter-annual and inter-species variation in nestling mass gain for two species of arctic-breeding passerine: Gambel’s White-crowned Sparrows (Zonotrichia leucophrys gambelii) and Lapland Longspurs (Calcarius lapponicus) across two years. The nestling period of 2014 was both colder (with lower minimum and maximum temperatures) and wetter (with 73% more rainfall) than 2013. Arthropod biomass was also reduced in shrub tundra in 2014 as compared to 2013. Both species showed reductions in rate of daily mass gain of nestlings in 2014 compared to 2013, but we observed no significant difference between species. Furthermore, we found that in 2014 early nesting birds had higher rates of nestling growth than those initiating clutches later in the season. These findings suggest that overall environmental conditions were more challenging for raising nestlings in 2014 as compared to 2013, and that these differences were manifested in a reduced rate of nestling mass gain in both species. Furthermore, both species showed a negative correlation between precipitation and growth rates, while only Lapland Longspurs showed a positive correlation between growth rates and temperature.

Physiological measurements are informative in assessing the relative importance of stressors that potentially impact the health of wildlife. Kelp Gulls, Larus dominicanus (KG), resident to the region of Peninsula Valdes, Argentina, have developed a unique behavior of landing on the backs of southern right whale adults and calves, Eubalaena australis (SRW), where they feed on their skin and blubber. This parasitic behavior results in large open wounds on the dorsal surface of the whale. Coincidently, the SRW population off the coast of Peninsula Valdes has experienced elevated calf mortality. We quantified levels of glucocorticoids and thyroid hormone extracted from baleen of dead calves to evaluate, retrospectively, the endocrine response of whale calves to gull wounding and harassment. Baleen accumulates hormones as it grows, allowing evaluation of long-term trends in physiological condition. While glucocorticoids (GCs) are known to increase in response to stressors such as disturbance, the metabolic hormone triiodothyronine (T3) has been shown to decrease under sustained food deprivation but is largely unaffected by disturbance stress. We quantified lifetime patterns of GCs and T3 in baleen recovered at necropsy from 36 southern right whale calves with varying severity of wounding from KGs. GC levels in baleen correlated positively with the degree of wounding, while T3 levels remained stable irrespective of the severity of the wounding. Our results suggest no evidence of malnutrition in low vs. severely wounded whales. However, the positive correlation of GCs with wound severity indicates that heavily wounded calves are suffering high levels of physiological stress before they die. This suggests that KG wounding may have contributed to the high southern right whale calf mortality observed in the Peninsula Valdes region of Argentina.

In many passerine birds, testosterone stimulates song and aggression but inhibits paternal care, but few studies have explored whether such effects can be reversed with testosterone blockers. We explored the effect of testosterone blockers on song, aggression and paternal care of Lapland longspurs (Calcarius lapponicus), an arctic passerine with a short breeding season. Twenty-one "blocker males" received implants containing an androgen receptor blocker and an aromatase inhibitor, compared to 27 control males with empty or no implants. Song, aggression and other behaviors were evaluated with simulated territorial intrusions (STI) during mate-guarding, and with focal observations (without STI) during mate-guarding and incubation. Nests were monitored and nestlings weighed as an indirect measure of paternal care. During STI, blocker males exhibited similar song rates, significantly lower aggression, and were significantly less likely to be found on territory than control males. Focal observations revealed no differences in spontaneous song, aggression, foraging, preening, or flight activity. Blocker males' nestlings had greater body mass on day 5 after hatching, but this difference disappeared by fledging, and both groups fledged similar numbers of young. Two blocker males exhibited unusual paternal care: incubation and brooding of young, or feeding of nestlings at another male's nest. In sum, testosterone blockers affected aggression but not song, contrasting with results from previously published testosterone implant studies. Effects on paternal care were concordant with testosterone implant studies. These patterns may be related to rapid behavioral changes characteristic of the short breeding season of the Arctic.

Exhaled breath analysis is a non-invasive assessment tool that has shown promise in human diagnostics, and could greatly benefit research, management, and conservation of large whales. However, hormone assessment of whale respiratory vapor (blow) has been challenged by variable water content and unknown total volume of collected samples. To advance this technique, we investigated urea (a compound present in narrow range in circulation) as a normalizing factor to correct for blow sample concentration. Normalized progesterone, testosterone, and cortisol concentrations of 100 blow samples from 46 photo-identified North Atlantic right whales (Eubalaena glacialis) were more biologically relevant compared to absolute estimates, varying by sex, age class, or individual. Progesterone was elevated in adult females compared with other cohorts and highest in one independently confirmed pregnant female. For both sexes, testosterone was two-fold higher in reproductively mature whales but studied adult females showed the widest variation. Cortisol was present in relatively low concentrations in blow and demonstrated variation between individual whales, suggesting potential for studies of individual differences in adrenal activity. Incorporation of methodologies that normalize sample concentration are essential for blow hormone analysis of free-swimming whales, and measurement of urea could be used to optimize non-invasive physiological assessment of whales.

Baleen from stranded carcasses of five right whale calves (four southern and one North Atlantic) contain cortisol and corticosterone patterns that may represent longitudinal profiles of stress exposure over time. Calves with high wounding from Kelp Gulls had markedly higher glucocorticoids in postnatal baleen than did less wounded and unwounded calves., Baleen tissue accumulates stress hormones (glucocorticoids, GC) as it grows, along with other adrenal, gonadal and thyroid hormones. The hormones are deposited in a linear fashion such that a single plate of baleen allows retrospective assessment and evaluation of long-term trends in the whales’ physiological condition. In whale calves, a single piece of baleen contains hormones deposited across the lifespan of the animal, with the tip of the baleen representing prenatally grown baleen. This suggests that baleen recovered from stranded carcasses of whale calves could be used to examine lifetime patterns of stress physiology. Here we report lifetime profiles of cortisol and corticosterone in baleen of a North Atlantic right whale (‘NARW’—Eubalaena glacialis) calf that died from a vessel strike, as well as four southern right whale (‘SRW’—Eubalaena australis) calves that were found dead with varying severity of chronic wounding from Kelp Gull (Larus dominicanus) attacks. In all five calves, prenatally grown baleen exhibited a distinctive profile of elevated glucocorticoids that declined shortly before birth, similar to GC profiles reported from baleen of pregnant females. After birth, GC profiles in calf baleen corresponded with the degree of wounding. The NARW calf and two SRW calves with no or few gull wounds had relatively low and constant GC content throughout life, while two SRW calves with high numbers of gull wounds had pronounced elevations in baleen GC content in postnatal baleen followed by a precipitous decline shortly before death, a profile suggestive of prolonged chronic stress. Baleen samples may present a promising and valuable tool for defining the baseline physiology of whale calves and may prove useful for addressing conservation-relevant questions such as distinguishing acute from chronic stress and, potentially, determining cause of death.

Made available in DSpace on 2019-10-04T12:35:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) University of Alaska Anchorage's Environment and Natural Resources Institute Northern Arizona University's Center for Bioengineering Innovation The tegu lizard Salvator merianae is a large, widely distributed teiid lizard endemic to South America that exhibits annual cycles of high activity during the spring and summer, and hibernation during winter. This pattern of activity and hibernation is accompanied by profound seasonal changes in physiology and behavior, including endothermy during the austral spring. The unusual combination of seasonal endothermy, hibernation and oviparity, in a non-avian, non-mammalian species, makes S. merianae an interesting subject for study of comparative aspects of endocrine regulation of seasonal changes in physiology. In the present study, we first validated commercially available immunoassay kits for quantification of hormone concentrations of the reproductive (testosterone, estradiol and progesterone), adrenal (corticosterone), and thyroid [thyroxine (T-4) and triiodothyronine (T-3)] axes in plasma of an outdoor, captive adult male and female S. merianae in southeastern Brazil. All assays exhibited parallelism and accuracy with S. merianae plasma. We next assessed patterns of concentration of these hormones across the annual cycle of S. merianae. Testosterone in males and estradiol in females peaked in spring coincident with the peak in reproductive behavior. Progesterone in females was significantly elevated in October coincident with putative ovulation when gravid females build nests. Thyroid hormones, known for regulating energy metabolism, varied seasonally with some sex-dependent differences. T-4 gradually increased from an annual nadir during pre-hibernation and hibernation to high concentrations during spring in both sexes. In contrast, T-3 did not vary seasonally in males, but females showed a two-fold increase in T-3 during the spring reproductive season. T-3 may be involved in energy investment during the seasonal production of large clutches of eggs. Corticosterone was significantly elevated during the active season in both sexes, suggesting its involvement in mobilization of energy stores and modulation of behavior (territoriality) and physiology. Ours is the first investigation of concurrent changes in reproductive, thyroid and adrenal hormone concentrations in this endemic and physiologically unique South American lizard. Our findings set the stage for future investigations to determine the extent to which these hormones influence activity and thermoregulation in S. merianae. Univ Sao Paulo, Inst Biosci, Dept Physiol, BR-05508090 Sao Paulo, SP, Brazil Sao Paulo State Univ, Coll Agr & Vet Sci, Dept Anim Morphol & Physiol, BR-14884900 Jaboticabal, SP, Brazil Natl Inst Sci & Technol Comparat Physiol, INCT Fisiol Comparada, Jaboticabal, Brazil No Arizona Univ, Dept Biol Sci, Ctr Bioengn Innovat, Box 5640, Flagstaff, AZ 86011 USA Sao Paulo State Univ, Coll Agr & Vet Sci, Dept Anim Morphol & Physiol, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 2017/01652-2 FAPESP: 2015/04849-6 FAPESP: 2014/16320-7

Progesterone, testosterone, 17β-estradiol, cortisol, corticosterone, aldosterone, thyroxine and tri-iodothyronine are detectable in baleen of eight whale species. The presence of a broad array of hormones in baleen of multiple species indicates that baleen hormone analysis may be widely applicable for studies of reproduction, stress and metabolism of the large whales., Recent studies have demonstrated that some hormones are present in baleen powder from bowhead (Balaena mysticetus) and North Atlantic right (Eubalaena glacialis) whales. To test the potential generalizability of this technique for studies of stress and reproduction in large whales, we sought to determine whether all major classes of steroid and thyroid hormones are detectable in baleen, and whether these hormones are detectable in other mysticetes. Powdered baleen samples were recovered from single specimens of North Atlantic right, bowhead, blue (Balaenoptera [B.]musculus), sei (B. borealis), minke (B. acutorostrata), fin (B. physalus), humpback (Megaptera novaeangliae) and gray (Eschrichtius robustus) whales. Hormones were extracted with a methanol vortex method, after which we tested all species with commercial enzyme immunoassays (EIAs, Arbor Assays) for progesterone, testosterone, 17β-estradiol, cortisol, corticosterone, aldosterone, thyroxine and tri-iodothyronine, representing a wide array of steroid and thyroid hormones of interest for whale physiology research. In total, 64 parallelism tests (8 species × 8 hormones) were evaluated to verify good binding affinity of the assay antibodies to hormones in baleen. We also tested assay accuracy, although available sample volume limited this test to progesterone, testosterone and cortisol. All tested hormones were detectable in baleen powder of all species, and all assays passed parallelism and accuracy tests. Although only single individuals were tested, the consistent detectability of all hormones in all species indicates that baleen hormone analysis is likely applicable to a broad range of mysticetes, and that the EIA kits tested here perform well with baleen extract. Quantification of hormones in baleen may be a suitable technique with which to explore questions that have historically been difficult to address in large whales, including pregnancy and inter-calving interval, age of sexual maturation, timing and duration of seasonal reproductive cycles, adrenal physiology and metabolic rate.

Immunoassay of hormone metabolites extracted from faecal samples of free-ranging large whales can provide biologically relevant information on reproductive state and stress responses. North Atlantic right whales (Eubalaena glacialis Muller 1776) are an ideal model for testing the conservation value of faecal metabolites. Almost all North Atlantic right whales are individually identified, most of the population is sighted each year, and systematic survey effort extends back to 1986. North Atlantic right whales number

We examined the potential influences of existing social housing arrangements on captive sun bear female reproductive cycling. Three social conditions were studied: 1.2, 1.1, and 0.2. Fecal hormone metabolites of total estrogens, progestins and glucocorticoids were compared between the three social conditions and were analyzed along with vaginal cytology data in individuals that experienced a change in social condition. Behavioral data were collected on females in each of the social conditions and summarized into agonistic, affiliative and sexual categories. Results indicated that sun bears are spontaneous ovulators, but that the presence of a male does influence hormone metabolite concentrations and cytological profiles. Male presence was also associated with a greater proportion of females cycling. In most female pairs, only one female cycled, typically the younger, subordinate female. The presence of a second female appeared to have a suppressive influence on both cycling and mating behavior. Agonistic behavior and associated stress may be a mechanism for lowering progesterone. In contrast, high estrogen levels were associated with low levels of agonistic interactions; thus, reproductive cycle monitoring could facilitate social introductions with either sex. Females in 1.2 social groupings had significantly higher GC metabolite concentrations and agonistic behavior, suggesting that 1.2 social groupings may not be advisable for captive breeding programs. Data from the North American historical captive population indicate that at most 32% of all sun bear pairs and only 18.5% of females have successfully reproduced. Implications of these social and reproductive patterns for captive management are discussed.

We compared known calving events to longitudinal baleen progesterone profiles from baleen plates of two stranded adult female North Atlantic right whales. Reproductive history of the previous ten years was recorded accurately in baleen, with pronounced elevations in progesterone in regions of baleen grown during known pregnancies., Reproduction of mysticete whales is difficult to monitor, and basic parameters, such as pregnancy rate and inter-calving interval, remain unknown for many populations. We hypothesized that baleen plates (keratinous strips that grow downward from the palate of mysticete whales) might record previous pregnancies, in the form of high-progesterone regions in the sections of baleen that grew while the whale was pregnant. To test this hypothesis, longitudinal baleen progesterone profiles from two adult female North Atlantic right whales (Eubalaena glacialis) that died as a result of ship strike were compared with dates of known pregnancies inferred from calf sightings and post-mortem data. We sampled a full-length baleen plate from each female at 4 cm intervals from base (newest baleen) to tip (oldest baleen), each interval representing ∼60 days of baleen growth, with high-progesterone areas then sampled at 2 or 1 cm intervals. Pulverized baleen powder was assayed for progesterone using enzyme immunoassay. The date of growth of each sampling location on the baleen plate was estimated based on the distance from the base of the plate and baleen growth rates derived from annual cycles of stable isotope ratios. Baleen progesterone profiles from both whales showed dramatic elevations (two orders of magnitude higher than baseline) in areas corresponding to known pregnancies. Baleen hormone analysis shows great potential for estimation of recent reproductive history, inter-calving interval and general reproductive biology in this species and, possibly, in other mysticete whales.

Birds breeding at high latitudes can be faced with extreme weather events throughout the breeding season. In response to environmental perturbations, vertebrates activate the hypothalamic-pituitary-adrenal (HPA) axis and synthesize corticosterone, which promotes changes in behavior and physiology to help the animal survive. The parental care hypothesis suggests that the HPA axis activity should be downregulated during the parental stage of breeding to prevent nest abandonment. However, it is unknown what happens to HPA axis activity in response to severe weather at the transition from the pre-parental to parental stages of breeding. We sampled baseline corticosterone levels and the time course of corticosterone elevation over 60min of restraint stress and assessed body condition and fat stores in Lapland longspurs (Calcarius lapponicus) breeding in the Low Arctic in the presence and absence of snowstorms. The results showed that during the pre-parental stage, HPA axis activity was up-regulated in response to snowstorms, with corticosterone levels continuing to increase through 60min of restraint. However, once birds were parental, HPA axis activity was unaffected by snowstorms and levels peaked at 10min. Fat levels and body condition did not change in response to snowstorms but fat levels declined in males during the pre-parental stage. These data suggest that the parental care hypothesis can be applied to severe storm events; parental birds restrained the activity of the HPA axis, likely to focus on the reproductive effort that is already underway, while pre-parental birds greatly upregulated HPA axis activity in response to snowstorms to maximize self-preservation.

The potential benefits of a physiological approach to conservation are well-established. Here we present a cross-section of conservation physiology success stories and a discussion of their shared characteristics to illustrate how the discipline has tangibly contributed to conservation and management across a diversity of topics, taxa, and spatial scales., The potential benefits of physiology for conservation are well established and include greater specificity of management techniques, determination of cause–effect relationships, increased sensitivity of health and disturbance monitoring and greater capacity for predicting future change. While descriptions of the specific avenues in which conservation and physiology can be integrated are readily available and important to the continuing expansion of the discipline of ‘conservation physiology’, to date there has been no assessment of how the field has specifically contributed to conservation success. However, the goal of conservation physiology is to foster conservation solutions and it is therefore important to assess whether physiological approaches contribute to downstream conservation outcomes and management decisions. Here, we present eight areas of conservation concern, ranging from chemical contamination to invasive species to ecotourism, where physiological approaches have led to beneficial changes in human behaviour, management or policy. We also discuss the shared characteristics of these successes, identifying emerging themes in the discipline. Specifically, we conclude that conservation physiology: (i) goes beyond documenting change to provide solutions; (ii) offers a diversity of physiological metrics beyond glucocorticoids (stress hormones); (iii) includes approaches that are transferable among species, locations and times; (iv) simultaneously allows for human use and benefits to wildlife; and (v) is characterized by successes that can be difficult to find in the primary literature. Overall, we submit that the field of conservation physiology has a strong foundation of achievements characterized by a diversity of conservation issues, taxa, physiological traits, ecosystem types and spatial scales. We hope that these concrete successes will encourage the continued evolution and use of physiological tools within conservation-based research and management plans.

Kemp's ridley sea turtles (Lepidochelys kempii), a critically endangered species, frequently strand on the shores of Cape Cod (Massachusetts, USA) in late autumn in a state of "cold-stunning" exhibiting low body temperature and related clinical issues. Stranded turtles are transported to the New England Aquarium (Boston, Massachusetts, USA) for treatment and rehabilitation. This study tested the hypothesis that cold-stunned sea turtles might exhibit high corticosterone ("stress hormone") or low thyroxine (which is often affected by temperature), or both, and that monitoring of both hormones may be useful for assessing recovery. In a retrospective analysis, 87 archived plasma samples were assayed from 56 cold-stunned juvenile Kemp's ridley sea turtles for corticosterone and free thyroxine (fT4). Upon admission, mean corticosterone was the highest yet reported for a population of sea turtles (39.3 +/- 2.5 ng/ml; mean +/- standard error of the mean [SEM]) and fT4 was usually undetectable. On admission, corticosterone was negatively correlated with white blood cell count but was not correlated with blood glucose. There were no differences in either hormone between survivors and nonsurvivors on admission. After 18+ days in recovery, surviving turtles' corticosterone dropped significantly to levels typical of baseline in other species (0.9 +/- 1.0 ng/ml) while fT4 increased significantly (1.3 +/- 1.5 pg/ml). During recovery, corticosterone was positively correlated with blood glucose and was not correlated with white blood cell count. Turtles that showed persistent deficits in feeding, activity, or both during recovery had significantly lower fT4 than did turtles with no such deficits. The "high corticosterone, low fT4" endocrine profile seen on admission may be a useful marker of cold-stunning in this and other species. Further studies are necessary to determine whether low thyroid hormones play a causal role in deficits in feeding and activity during recovery. Monitoring of both hormones may be useful for triage, monitoring of recovery, and assessing readiness for release.

Most species of bears exhibit a reproductive strategy that includes a highly defined breeding season, delayed implantation, pseudopregnancy, and no postpartum estrus. Sun bears (Helarctos malayanus), a tropical lowland species, represent a potential departure from the typical ursid reproductive pattern. We studied reproductive timing in captive sun bears by examining fecal hormones and behavior of 13 female and 5 male sun bears, the entire North American population of H. m. euryspilus at the time of the study. Fecal estrogens and progestins in females did not vary by season or month, nor did fecal androgens in males. Estrus occurred in 11 of 12 months; breeding and births occurred year-round. However, birth rates were lower in the spring and higher in the fall and winter. Our study documented 1 complete pregnancy, in which delayed implantation did not occur; the delay between estrus and the progesterone rise (i.e., implantation) in the pregnant bear was similar to that seen in nonpregnant bears. All bears, whether nonpregnant or pregnant, exhibited a prolonged period of high progesterone after estrus, indicating obligate pseudopregnancy. Studbook records show that estrus can occur within 6 months after parturition; these cases were typically associated with loss of the previous cub. Our findings suggest that sun bears are unique among ursids, being polyestrous, nonseasonal breeders that do not appear to exhibit delayed implantation and that are capable of initiating estrus in the event of early loss of a cub.

Limited data on stress physiology exist for leatherback turtles. Examination of 32 leatherback turtles showed that individuals exposed to entanglement, stranding, or entrapment in a weir net had elevated corticosterone and thyroxine compared to healthy controls., Leatherback turtles (Dermochelys coriacea) are exposed to many anthropogenic stressors, yet almost no data on stress physiology exist for this species. As a first step toward understanding the physiological responses of leatherback turtles to stress, and with the particular goal of assessment of the effect of capture, we quantified corticosterone (an adrenal stress hormone) and thyroxine (a regulator of metabolic rate, often inhibited by chronic stress) in 17 healthy leatherback turtles captured at sea for scientific study, with comparisons to 15 ‘distressed’ leatherbacks that were found entangled in fishing gear (n = 8), confined in a weir net (n = 1) or stranded on shore (n = 6). Distressed leatherbacks had significantly elevated corticosterone (mean ± SEM 10.05 ± 1.72 ng/ml, median 8.38 ng/ml) and free thyroxine (mean 0.86 ± 0.37 pg/ml, median 0.08 pg/ml) compared with healthy leatherbacks sampled immediately before release (after ∼40 min of handling; corticosterone, mean 4.97 ± 0.62 ng/ml, median 5.21 ng/ml; and free thyroxine, mean 0.05 ± 0.05 pg/ml, median 0.00 pg/ml). The elevated thyroxine in distressed turtles compared with healthy turtles might indicate an energetic burden of entanglement and stranding. Six of the healthy leatherbacks were sampled twice, at ∼25 and ∼50 min after the time of first disturbance. In all six individuals, corticosterone was higher in the later sample (earlier sample, mean 2.74 ± 0.88 ng/ml, median 2.61 ng/ml; later sample, mean 5.43 ± 1.29 ng/ml, median 5.38 ng/ml), indicating that capture and handling elicit an adrenal stress response in this species. However, the corticosterone elevation after capture appeared relatively mild compared with the corticosterone concentrations of the entangled and stranded turtles. The findings suggest that capture and handling using the protocols described (e.g. capture duration

The objective was to explore multiple methods for detecting and characterizing the reproductive cycle of the sun bear (Helarctos malayanus). Thirteen H. m. euryspilus females, loaned from the Malaysian government to US zoos, were used. Fecal metabolite concentrations of estrogen and progesterone were compared to vaginal cytology, changes in genital appearance, and behavior (videotapes and zookeeper observations). Cytology and video behavior were characterized during five hormonally defined states: high, low, and baseline progesterone, estrus, and high estrogen. Among states, there were significant differences in cytology and behavior. Sexual, affiliative, and stereotypic behaviors were highest during estrus, whereas affiliative and social behaviors were lowest during high progesterone. In this captive breeding population, 30.8% of females cycled two or three times a year, 30.8% cycled once a year, and 38.5% did not cycle during this study. Inter-estrus intervals were (mean ± SEM) 115.7 ± 6.3 d (range, 101-131). Spearman rank correlations were significant between both ordinal sexual and affiliative behaviors and vulva swelling and color. Sexual behavior was significantly positively correlated with superficial and keratinized cells, but negatively correlated with parabasal and basophilic cells in cycling females (opposite pattern for appetitive behavior). In conclusion, data for cytology, vulva changes and behavior were consistent with, and complementary to, hormonal data; collectively, they delineated estrus and identified specific reproductive types.

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.

The North Atlantic right whale, Eubalaena glacialis (NARW), a critically endangered species that has been under intensive study for nearly four decades, provides an excellent case study for applying modern methods of conservation physiology to large whales. By combining long-term sighting histories of known individuals with physiological data from newer techniques (e.g., body condition estimated from photographs; endocrine status derived from fecal samples), physiological state and levels of stress can be estimated despite the lack of any method for nonlethal capture of large whales. Since traditional techniques for validating blood assays cannot be used in large whales, assays of fecal hormones have been validated using information on age, sex, and reproductive state derived from an extensive NARW photo-identification catalog. Using this approach, fecal glucocorticoids have been found to vary dramatically with reproductive state. It is therefore essential that glucocorticoid data be interpreted in conjunction with reproductive data. A case study correlating glucocorticoids with chronic noise is presented as an example. Keys to a successful research program for this uncatchable species have included: consistent population monitoring over decades, data-sharing across institutions, an extensive photo-identification catalog that documents individual histories, and consistent efforts at noninvasive collection of samples over years. Future research will require flexibility to adjust to changing distributions of populations.

Very little is known about the endocrinology of the baleen whales. The highly endangered North Atlantic right whale (NARW; Eubalaena glacialis) is a good model species, because most NARW individuals are photo-identiWed with known histories. We used an 125 I corticosterone assay, shown to reliably measure cortisol metabolites, to determine glucocorticoid metabolite concentrations in 177 NARW fecal samples collected between 1999‐2004 in the Bay of Fundy, Canada. Fecal glucocorticoid metabolite concentrations varied signiWcantly with sex and reproductive category, being highest in pregnant females (mean §SE: 238.14 §74.37 ng/g) and mature males (71.6 §11.36), intermediate in lactating females (39.33 §5.82), and lower in non-reproducing females (23.11 §4.25) and immature males (34.33 §5.01) and females (14.0 §0.41). One case also suggests that glucocorticoids rise markedly in response to severe entanglement in Wshing lines. Whales with fecal glucocorticoid content over 100 ng/g (termed “high-cort” samples) were rare, and included most pregnant females, some mature males, a fatally entangled whale, and several very young animals. Glucocorticoid concentrations were highly correlated with androgen concentrations in males and pregnant females. We analyzed the elution proWles of glucocorticoid and androgen metabolites in 13 samples with high-performance liquid chromatography (HPLC) to determine the extent to which androgen metabolites cross-react with our glucocorticoid assay. Males, pregnant females, non-pregnant females, and “high-cort” whales each had distinctly diVerent immunoreactive HPLC proWles of glucocorticoid and androgen metabolites. A major glucocorticoid metabolite was prominent in all “high-cort” whales including the fatally entangled whale. The major fecal androgen was not testosterone but was instead a more nonpolar steroid (possibly dihydrotestosterone), which may be diagnostic of males. Androgen metabolites showed only minor cross-reactivity to our glucocorticoid assay, having a slight inXuence on glucocorticoid results in particular individuals. We conclude that fecal glucocorticoid analysis appears to be a useful measure of adrenal activity and reproductive condition for NARW. ! 2006 Published by Elsevier Inc.

There is an urgent need for noninvasive methods to study reproduction and environmental stress in at-risk species such as the northern spotted owl (Strix occidentalis caurina). Two related owl species (barred owl and great horned owl) were used as surrogates to validate hormone assays for fecal metabolites of progesterone, 17beta-estradiol, testosterone, and corticosterone. Infusions of radiolabeled hormones showed that the owls excreted most hormone within 6 h. Feces and urine contained roughly equal amounts of hormone, and most fecal hormone metabolites were quite polar. The testosterone and corticosterone assays in this study bound to the major excreted metabolites of these hormones, but two progesterone assays did not appreciably bind to the major progesterone metabolites. All assays showed excellent parallelism with hydrolyzed and unhydrolyzed samples and with previously dried or undried fecal samples. Thus, samples do not require hydrolysis or prior drying. Samples from a female barred owl had significantly higher fecal estrogen, lower fecal testosterone, and higher fecal estrogen/testosterone ratio than samples from two male barred owls. The fecal estrogen/testosterone ratio was the most accurate predictor of owl gender, particularly if two or more samples are available from the same individual. Fecal corticosterone metabolites also demonstrated considerable utility for wild northern spotted owls. Fecal glucocorticoid levels varied by gender and breeding stage, being highest in male northern spotted owls early in the breeding season and highest in females when nestlings were fledging. Collectively, these studies show that noninvasive fecal hormone measurements show great promise for noninvasive assessment of reproduction and stress in wild owls.

The role of estrogens in reproductive behavior of free-living birds has been little investigated, despite the importance of estrogens in coordinating reproductive behavior and the need for information on potential effects of estrogenic contaminants on wildlife. We gave subcutaneous implants of estradiol (E2) to 15 female Lapland longspurs (Calcarius lapponicus), an arctic bird with a six-week breeding season. Controls were 9 females with empty implants and 14 unimplanted females. After implantation, E2 females had greater E2 levels and greater body mass than controls. We did not detect any increases in, or prolongation of, sexual or nest-building behavior. The incubation period was longer for E2 nests than for control nests. Nest videotapes showed that E2 females and their mates exhibited normal nestling care, and nestling growth, fledging success, and overwinter survival were also unaffected. However, whenever the males arrived at the nests, E2 females showed a significantly greater likelihood of begging from the male rather than leaving the nest. Courtship or incubation feeding have been considered atypical for this species, and it is not clear what role E2 normally plays in promoting begging behavior. Overall, elevated E2 levels did not result in any obvious costs for the female, save perhaps for slightly reduced incubation efficiency. The weak response to hormonal implants is similar to patterns seen in male Lapland longspurs, and may be related to the short breeding season. However, since little is presently known about the role of estrogens in behavior of wild female birds, it remains to be seen whether these patterns are typical of female passerines in general, or of short-season breeders. Further research on effects of estrogens (both exogenous and endogenous) on free-living female birds is needed.

The aims of this study were to validate a radioimmunoassay (RIA) for quantifying glucocorticoid metabolite concentrations in the feces of Alaskan brown bears (Ursus arctos horribilis) and to investigate whether any of the following factors are associated with those concentrations: the presence of humans or other bears, fishing difficulty, sex-age class, diet, and season. We tested an established corticosterone RIA for assay sensitivity, similarity, precision, and sample matrix effects of brown bear feces, and it proved satisfactory. We collected fecal samples from brown bears along salmon-spawning streams and assessed fecal glucocorticoid (FG) concentrations. We observed that the factors explaining the most variation in measured concentrations were date and diet type and that there was a significant interaction between the two. We did not observe a significant effect of human and bear activities or sex-age class on FG concentrations. This study demonstrates that although FG concentrations may be assessed in brown bears, complex dietary patterns and seasonal variations must be taken into consideration in the study design in order to make inferences regarding stress.

The long-term goal of this research is to broaden the existing panel of endocrine stress assessment techniques for large whales. Few methods exist for the assessment of physiological stress levels of free-swimming cetaceans (Amaral 2010, ONR 2010, Hunt et al. 2013a). We have previously demonstrated that respiratory vapor (blow) sampling is practical and feasible for large whales, and that blow samples contain detectable steroid and thyroid hormones (Hunt et al. 2013b). We also have developed a suite of fecal hormone assays for reproductive and stress-related hormones in North Atlantic right whales (Hunt et al. 2006, Rolland et al. 2005, 2012). However, blow sampling needs further testing before it can enter widespread use, and some additional stress-related hormones have not yet been tested in either feces or blow, particularly aldosterone. Our aim in this project is to further develop both techniques -- respiratory hormone analysis and fecal hormone analysis -- for use in stress assessment of large whales. We have two specific objectives in this project: (1) further development of respiratory sampling methodology, via modifications to our sampling apparatus and testing of internal controls to control for water content; and (2) development of a noninvasive aldosterone assay (for both feces and blow) that can be used as an alternative measure of adrenal activation to complement existing glucocorticoid assays.

Baleen samples from sixteen bowhead whales contained measurable cortisol and progesterone, and both hormones demonstrated significant correlations with sex, age class and reproductive state. Analysis of hormones in baleen shows promise as a novel tool for retrospective analysis of stress and reproductive physiology of large mysticete whales., Arctic marine mammals are facing increasing levels of many anthropogenic stressors. Novel tools are needed for assessment of stress physiology and potential impacts of these stressors on health, reproduction and survival. We have investigated baleen as a possible novel tissue type for retrospective assessment of stress and reproductive hormones. We found that pulverized baleen powder from bowhead whales (Balaena mysticetus) contained immunoreactive cortisol and progesterone that were detectable with commercially available enzyme immunoassay kits. Both assays passed parallelism and accuracy validations using baleen extracts. We analysed cortisol and progesterone at the base of the baleen plate (most recently grown baleen) from 16 bowhead whales of both sexes. For a subset of 11 whales, we also analysed older baleen from 10, 20 and 30 cm distal to the base of the baleen plate. Immunoreactive cortisol and progesterone were detectable in all baleen samples tested. In base samples, females had significantly higher concentrations of cortisol and progesterone compared with males. Cortisol concentrations in older baleen (10, 20 and 30 cm locations) were significantly lower than at the base and did not exhibit correlations with age-class or sex. Progesterone concentrations were significantly higher in females than in males at all baleen locations tested and were significantly higher in pregnant females than in non-pregnant females. Four of five mature females showed dramatic variation in progesterone concentrations at different locations along the baleen plate that may be indicative of previous pregnancies or luteal phases. In contrast, all males and all immature females had uniformly low progesterone. Baleen hormone analysis is a novel approach that, with further methodological development, may be useful for determining individual longitudinal profiles of reproductive cycles and stress responses.

Noninvasive fecal glucocorticoid analysis has tremendous potential as a means of assessing stress associated with environmental disturbance in wildlife. However, interspecific variation in excreted glucocorticoid metabolites requires careful selection of the antibody used in their quantification. We compared four antibodies for detecting the major fecal cortisol metabolites in yellow baboons following 3 H cortisol administration, ACTH challenge, and HPLC separation of fecal glucocorticoid metabolites. The most effective antibody (ICN corticosterone RIA; Cat. No. 07-120102) demonstrated relatively high cross-reactivities to the major cortisol metabolites present in feces during peak excretion, following both radiolabel infusion and ACTH challenge. This same antibody also detected increased fecal glucocorticoid metabolites after ACTH administration in the African elephant, black rhinoceros, Roosevelt elk, gerenuk, scimitar-horned oryx, Alaskan sea otter, Malayan sun bear, cheetah, clouded leopard, longtailed macaque, and northern spotted owl. Results suggest that (1) fecal glucocorticoid assays reliably detect endogenous changes in adrenal activity of a diverse array of species and (2) where comparisons were made, the ICN corticosterone antibody generally was superior to other antibodies for measuring glucocorticoid metabolites in feces.