Assessing reproductive status of right whales (Eubalaena glacialis) using fecal hormone metabolites

Long-term studies of the endangered North Atlantic right whale, Eubalaena glacialis, have revealed declining reproductive parameters over the past two decades, threatening recovery of this small population if current trends continue. Little is known about right whale reproductive physiology, and investigating this reproductive decline has been limited by a lack of non-lethal methods for assessing reproductive status (e.g., sexual maturation, ovarian activity, pregnancy, lactation, and reproductive senescence) in free-swimming whales. This paper describes validation of existing radioimmunoassay techniques to study reproduction in right whales by measuring estrogens, progestins, androgens, and their related metabolites in fecal samples. Over the past decade fecal steroid hormone assays have been used to assess reproductive status and function in a wide range of terrestrial wildlife species, but this is the first application of this methodology in wild cetaceans. Analysis of fecal hormone metabolite levels in combination with life history data from photographically identified whales shows that this non-invasive method can be used to determine gender, detect pregnancy and lactation, and to assess age at sexual maturity in right whales and potentially other endangered whale populations.     

Within-sample variation of fecal glucocorticoid measurements

Fecal glucocorticoid metabolite analysis is a useful tool for monitoring adrenocortical activity in captive and free-ranging wildlife. Glucocorticoid metabolites may not be evenly distributed within fecal samples and this variability could affect the interpretation of glucocorticoid metabolite measurements. Furthermore, the precision (i.e., repeatability of measurements) of fecal glucocorticoid measurements from well-mixed samples is unknown. We collected fresh white-tailed deer (Odocoileus virginianus) feces at various times pre- and post-adrenocorticotropin injection to provide samples with low (<75 ng/g), medium (75-90 ng/g), and high (>90 ng/g) glucocorticoid concentrations in case variability differs in samples of dissimilar hormone metabolite concentrations. We compared two sampling methods (selection of three pellet groups [one from each end of the fecal mass and one from the center] versus sampling three small portions of the thoroughly mixed fecal mass) to estimate within-sample variation of glucocorticoid metabolites. Glucocorticoid metabolite measures from pellet groups were higher than fecal glucocorticoid measures from mixed samples in the low (F=3.10; df = 1,56; P=0.08) and medium concentration (F=7.28; df = 1,50; P=0.01) groups. Fecal glucocorticoid metabolite estimates from mixed samples were less variable than glucocorticoid metabolite measures using pellet groups from the same fecal mass, although these differences were not statistically significant (low group: F=0.59; df = 1,38; P=0.45; medium group: F=0.13; df = 1,34; P=0.72; high group: F=2.30; df = 1,28; P=0.14). The mean coefficient of variation was <10% across all treatment groups and sampling methods. However, a power analysis indicated the mixed sub-sample technique requires fewer samples to detect statistically significant differences than pellet groups. Our results suggest that glucocorticoid metabolites may not be evenly distributed in white-tailed deer feces. Consequently, using only a few pellets from a fecal mass may bias assay interpretation. We suggest researchers homogenize the entire fecal mass before removing a sub-sample of fecal material for analysis. Also, other sources of variation must be considered when interpreting results of fecal glucocorticoid studies.     

Species-specific patterns in fecal glucocorticoid and androgen levels in zoo-living orangutans (Pongo spp.)

In contrast to most primate species, including the other great apes, orangutans maintain a fission-fusion social system in the wild without being part of a stable community. In zoos, however, they are kept in permanent groups, usually consisting of one adult male and several females. In zoo orangutans, we predict higher levels of glucocorticoids and androgens in the Bornean species compared to its congener from Sumatra, due to the much more solitary lifestyle of Bornean orangutans and the apparent higher frequency of male aggression directed towards females in this species in the wild. To compare hormone levels of the two orangutan species, we validated a fecal glucocorticoid and a fecal androgen assay. Subsequently, fecal samples from a total of 73 female and 38 male orangutans housed in 29 European zoos were analyzed to investigate the effect of species, social group size, age and (for female glucocorticoid levels) reproductive state and the presence of adult males on fecal hormone metabolite concentrations. The results of linear mixed effect models indicate that both male and female Bornean orangutans show a steeper increase in glucocorticoid levels with increasing group size than Sumatran orangutans. We therefore conclude that Sumatran zoo orangutans are better able to adjust to social housing conditions than their Bornean congeners. In addition, our analyses reveal higher glucocorticoid levels in lactating females of both species compared to non-lactating and juvenile females. Concerning androgen levels in males, our analyses revealed significantly higher concentrations in Bornean than Sumatran orangutans. These differences in both glucocorticoid and androgen output between the two species of orangutan are presumably linked to ecological and behavioral differences and could possibly be attributed to phenotypic plasticity. However, given that we found interspecific differences in hormone excretion in captivity, where both species live under very similar conditions, we conclude that this variation has a genetic basis.     

Non-invasive monitoring of fecal androgens in spotted hyenas (Crocuta crocuta)

Spotted hyenas (Crocuta crocuta) exhibit an array of behavioral and morphological characteristics that set them apart from other mammals: females are heavier and more aggressive than males, and females have external genitalia that closely resemble those of the male. Because androgenic hormones might mediate the expression of these traits, androgens are of great interest in this species. Past work on circulating androgens in wild hyenas has been limited, in part because of small sample sizes. In this study we validated a non-invasive method of monitoring variation in androgens by measuring total androgen metabolites in the feces of wild and captive spotted hyenas with an enzyme immunoassay. HPLC analysis revealed multiple immunoreactive androgen metabolites in fecal extracts from both males and females. LHRH challenge in three male and two female hyenas in captivity caused an increase in fecal androgens one to three days after LHRH injection. Furthermore, presence of bone in the diet did not affect fecal androgen concentrations in captive female hyenas. In wild spotted hyenas, time of day of fecal deposition, time elapsed between deposition and freezing of the sample, and time elapsed between freezing and extraction did not systematically affect fecal androgen concentrations. Finally, in wild hyenas, fecal androgen patterns mirrored plasma testosterone patterns in that adult immigrant males had higher concentrations than adult natal males, and pregnant females had higher concentrations than lactating females. These methods can therefore be used in future studies addressing relationships among fecal androgens, social status, reproductive state, and behavior in spotted hyenas.     

Annual profile of fecal androgen and glucocorticoid levels in free-living male American kestrels from southern mid-latitude areas

Fecal samples and behavioral data were collected at a fortnightly basis during 11 months period from free-living male American kestrels living in southeast Brazil (22°S latitude). The aim was to investigate the seasonal changes in testicular and adrenal steroidogenic activity and their correlation to reproductive behaviors and environmental factors. The results revealed that monthly mean of fecal glucocorticoid metabolites in May and June were higher than those estimated in November. In parallel, monthly mean of androgen metabolites in September was higher than those from January to April and from October to November. Molt took place from January to March, whereas copulation was observed from June to October but peaked in September. Nest activity and food transfer to females occurred predominantly in October, and parental behavior was noticed only in November. Territorial aggressions were rare and scattered throughout the year. Multiple regression analysis revealed that fecal androgen levels are predicted by photoperiod and copulation, while fecal glucocorticoid levels are only predicted by photoperiod. Bivariate correlations showed that fecal androgen metabolites were positively correlated with fecal glucocorticoid metabolites and copulation, but negatively correlated with molt. Additionally, copulation was positively correlated with food transfer to females and nest activity, but negatively correlated with molt. These findings suggest that male American kestrels living in southeast Brazil exhibit significant seasonal changes in fecal androgen and glucocorticoid concentrations, which seem to be stimulated by decreasing daylength but not by rainfall or temperature.     

Urinary androgens and cortisol metabolites in field-sampled bonobos (Pan paniscus)

Urinary metabolites of androgens and cortisol were measured in free-living male and female bonobos. Sex differences and correlations between adrenal and gonadal steroid excretion were investigated. The immunoreactive concentrations of androgens were measured with two different androgen assays. One assay used a testosterone (T) antibody raised with a 17beta-hydroxy group, and the other employed an antibody raised against a reduced form, 5alpha-androstane-17alpha-ol-3-one-CM (17alpha) with cross reactivity for epitestosterone and 5alpha-androstanedione. Both assays have been used in bonobo and chimpanzee studies where non-invasive techniques were employed. The levels of 17alpha-androgen metabolites were 1.7- and 3-fold higher than those of T-metabolites in males and females. The two androgen assay results correlated in males but not females. There was a sex difference in the T-metabolites measured. Male levels were significantly higher. Levels of 17alpha in the two sexes were similar. Cortisol metabolite levels (CORT) were similar between the sexes. The T-metabolites were significantly correlated with CORT in males but not in females. In females, the 17alpha-androgen metabolites correlated with CORT. This suggests that either androgen secretion or metabolism differs between the sexes. A parsimonious interpretation of the androgen assay cortisol/androgen correlation differences would be that larger components of dehydroepiandrosterone (DHEA), androstenedione or epitestosterone from the adrenal androgens were being excreted and measured in the females. The CORT/T metabolite interactions in males may reflect male-specific social or metabolic endocrine conditions.     

Measurement of plasma corticosterone and fecal glucocorticoid metabolites in the chicken (Gallus domesticus), the great cormorant (Phalacrocorax carbo), and the goshawk (Accipiter gentilis)

A method for the non-invasive measurement of glucocorticoid metabolites in feces of chickens was established and validated. After high-performance liquid chromatography (HPLC) the presence of at least two fecal immunoreactives was demonstrated, one co-eluting with authentic corticosterone, whereas the second substance migrates close to corticosterone sulphate. We investigated the relationship between corticosterone in blood plasma obtained by a vena brachialis catheter and fecal samples in groups of five chickens after an ACTH and a dexamethasone injection to stimulate and to suppress adrenal activity. A control group received a saline injection. After ACTH plasma cortisol concentrations increased 16-fold after 1.5 h to levels between 19 and 38 ng/ml and dropped to pre-treatment levels (1.1-2.5 ng/ml) 4h after stimulation. Dexamethasone did not result in a distinct suppression of adrenocortical activity and plasma corticosterone dropped only slightly below pre-treatment levels. The concentrations in fecal metabolites corresponded to the changes in the levels of biological active hormone in plasma. Fecal peak excretion (105-295 ng/g) was obtained with a delay of approximately 4 h compared to plasma. The profile obtained after ACTH challenge reflected a broader and dampened pattern of glucocorticoid secretion and provided a more integrated measure of adrenal activity. Dexamethasone treatment did not induce a measurable decrease in fecal metabolites and concentrations fluctuated around a mean of 30.0+/-9.9 ng/g, almost identical to those obtained from the saline treatment group (29.4+/-13.9 ng/g). In a separate experiment the effect of an alternative capture method (remote-controlled injection system) was investigated in cormorants. Plasma corticosterone measurements revealed a significantly diminished stress reaction compared to traditional trapping (1.24+/-0.78 vs. 10.9+/-12.1 ng/ml). Investigations whether goshawk nestlings infected with Trichomas gallinae differ in fecal corticosterone metabolite concentrations compared to healthy subjects revealed no significant changes. However, a significant correlation was found between the glucocorticoid metabolite concentrations and the number of nestlings per nest. The demonstration that adrenal activity can be detected by the assay is a prerequisite that ecologically meaningful levels of imposed stress can be validated. Therefore, non-invasive measurements of fecal metabolites are a promising perspective to monitor stress in birds.     

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 0.05-0.1 g 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 1 h 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.     

Effects of simulated environmental conditions on glucocorticoid metabolite measurements in white-tailed deer feces

Environmental conditions may influence fecal glucocorticoid metabolite measurements if feces cannot be collected immediately after deposition. To evaluate the influence of environmental conditions on fecal glucocorticoid metabolite concentrations, we exposed fresh fecal samples to 1 of 5 simulated conditions: (1) room temperature (22 degrees C), (2) high heat (38 degrees C), (3) alternating high heat and room temperature cycle, (4) alternating freezing (-20 degrees C) and room temperature cycle, and (5) simulated rainfall (0.85 cm every other day at 22 degrees C) for 7 days. We collected fresh white-tailed deer (Odocoileus virginianus) feces at various times pre- and post-adrenocorticotropin injection to provide samples with initially low (n=5), medium (n=5), and high (n=5) glucocorticoid concentrations. Feces were mixed thoroughly and then allocated into five 10-g samples. Also, a 5-g sub-sample was taken from each fecal mass prior to treatment and stored at -20 degrees C until assayed. We subsampled from all treatments once every 24-h for 7 days. Fecal samples were assayed using [125I]corticosterone radioimmunoassay kits. Fecal glucocorticoid metabolites in all three groups in the simulated rainfall treatment and the low group in the alternating freezing and room temperature treatment increased significantly over the 7-day period. We believe increased microbial metabolism of fecal glucocorticoids may partly explain these results. Other biochemical processes (e.g., cleavage of conjugate side groups from hormone metabolites by non-microbial action or release of glucocorticoids from lipid micelles) may also have increased fecal glucocorticoid measurements. Our findings suggest that fecal samples exposed to rainfall for one week may artificially inflate fecal glucocorticoid measurements. Thus, researchers should recognize the potential bias when collecting fecal samples exposed to rainfall. Non-fresh samples may prove useful when care is taken to address the elevation in immunoreactive glucocorticoid concentrations.     

Noninvasive monitoring of adrenocortical activity in roe deer (Capreolus capreolus) by measurement of fecal cortisol metabolites

A method for measuring glucocorticoids noninvasively in feces of roe deer was established and validated. The enzyme immunoassay (EIA) measures 11,17-dioxoandrostanes (11,17-DOA), a group of cortisol metabolites. Such measurement avoids blood sampling and reflects a dampened pattern of diurnal glucocorticoid secretion, providing an integrated measure of adrenocortical activity. After high-performance liquid chromatography, the presence of at least three different immunoreactive 11,17-DOA in the feces of roe deer was demonstrated. The physiological relevance of these fecal cortisol metabolites to adrenocortical activity was evaluated with an adrenocorticotropic hormone challenge test: cortisol metabolite concentrations exceeded pretreatment levels (31-78 ng/g) up to 13-fold (183-944 ng/g) within 8-23 h. Starting from basal levels between 13 and 71 ng/g, a suppression of adrenocortical activity after dexamethasone administration, indicated by metabolite levels close to the detection limit, was obtained 36-81 h after treatment, whereas unmetabolized dexamethasone was detectable in feces 12 h after its injection. Fecal glucocorticoid metabolite assessment via EIA is therefore of use in the monitoring of adrenocortical activity in roe deer. In a second experiment, capture, veterinary treatment, and transportation of animals were used as experimental stresses. This resulted in a 7.5-fold increase of fecal metabolites (1200 +/- 880 ng/g, mean +/- SD) compared to baseline concentrations. The administration of a long-acting tranquilizer (LAT), designed to minimize the physiological stress response, 2 days prior to a similar stress event led to a reduced stress response, resulting in only a 4-fold increase of fecal metabolites (650 +/- 280 ng/g; mean +/- SD). Therefore, LATs should be further investigated for their effectiveness in reducing stress responses in zoo and wild animals, e.g., when translocations are necessary.     

Non-invasive methods to measure androgen metabolites in excrements of European stonechats,Saxicola torquata rubicola

Traditionally androgen concentrations are measured invasively in blood plasma. However, non-invasive methods to detect androgens are desirable, as this reduces interference with the natural behavior of the study species and multiple samples can be obtained relatively easy. The aim of this study was to validate a method to measure androgens non-invasively in excrements of male European stonechats (Saxicola torquata rubicola). Extracts of excrements of a male stonechat injected with [3H]testosterone ([3H]T) were chromatographically separated using high performance liquid chromatography (HPLC). The resulting HPLC fractions were then analyzed with a radioimmunoassay against testosterone (T-RIA). The results showed that the assay picked up major metabolites of [3H]T. The physiological relevance of excreted androgen metabolites was further validated by showing that injection of exogenous GnRH to seven males led to a significant increase in excreted androgen metabolites. In contrast, androgen metabolite levels of six saline-injected control males did not increase. Furthermore, excrements from nine males were collected from January until April to see whether the typical seasonal increase in testosterone levels can also be traced when measuring excreted androgen metabolites. As expected, there was a significant seasonal increase in androgen metabolite concentrations. Thus, the T-RIA measures androgen metabolites in droppings of male European stonechats and to our knowledge this study represents the first validation of a non-invasive androgen assay in a passerine bird.     

Sex, stress and social status: Patterns in fecal testosterone and glucocorticoid metabolites in male Ethiopian wolves

Ethiopian wolves, Canis simensis, live in large multi-male family packs, where males are philopatric and do not disperse. Within a pack, mating and breeding is largely monopolized by the dominant male and female, although extra-pack copulations are common, and subordinate males may sire pups in neighboring packs. Regardless of paternity, all males in a pack help rear the pups. We non-invasively studied patterns in fecal testosterone and glucocorticoid metabolite concentrations using radioimmunoassays of fecal samples collected from nine wild male Ethiopian wolves between August 2007 and February 2008. We tested the predictions of the Challenge Hypothesis, namely that fecal testosterone metabolite concentrations would be higher during the annual mating season, which is the portion of the reproductive cycle when mating and increased aggression typically occur, and lower when there were pups in the pack for which to care. Contrary to the predictions of the Challenge Hypothesis, we did not detect patterns in fecal testosterone metabolite concentrations associated with reproductive stage during our study period. Similarly, we found no patterns associated with reproductive stage in male fecal glucocorticoid metabolite concentrations. Dominant males had higher average fecal testosterone and glucocorticoid metabolite concentrations than did subordinates, which may be related to higher rates of aggression and mate guarding in dominant males of group-living canids, a pattern also reported in African wild dogs, Lycaon pictus.     

Noninvasive fecal monitoring of glucocorticoids in spotted hyenas, Crocuta crocuta.

The aim of this study was to validate a method for measuring glucocorticoids noninvasively in feces of spotted hyenas (Crocuta crocuta). Three established enzyme immunoassays (EIA) for cortisol, corticosterone, and 11-oxoetiocholanolone were tested, but proved unsatisfactory. A new EIA using another corticosterone antibody was established and was used for all subsequent analyses; this EIA was validated by demonstrating parallelism between serial dilutions of spotted hyena fecal extracts and dilutions of standard corticosterone and by the recovery of corticosterone added to fecal extracts. High-performance liquid chromatography (HPLC) fractions analyzed by EIA showed various immunoreactive substances with polarities of unconjugated steroids. The physiological relevance of fecal glucocorticoid metabolites was further validated by demonstrating that (1) injection of exogenous ACTH to four males and two females led to a significant increase in fecal glucocorticoid metabolites within 24-50 h, (2) the translocation of a male spotted hyena to a new enclosure resulted in a fivefold increase compared to baseline concentrations, and (3) agonistic social interactions and physical conflict resulted in large increases of fecal glucocorticoid metabolites in both protagonists. Fecal steroid assessment is therefore of use in monitoring adrenal activity in spotted hyenas.     

Effects of season,sex, and sample collection on concentrations of fecal cortisol metabolites in red deer (Cervus elaphus)

Seasonal variation, sex differences, and invasive sample collection may confound glucocorticoid measures as indices of stress. We investigated the effects of sex and season on glucocorticoid production on a non-invasive basis by measuring concentrations of cortisol metabolites in feces of undisturbed red deer (Cervus elaphus). Although feces can be collected easily, assignment to individuals is difficult. Anonymous fecal samples may cause overrepresentation of particular individuals thus introducing a source of error when estimating mean hormone levels within a population. We therefore examined the effects of collecting anonymous fecal samples on mean fecal cortisol metabolite levels. Neither sex nor sample collection mode significantly affected mean fecal cortisol metabolite concentrations in the studied population of red deer. Fecal glucocorticoid excretion varied seasonally with a peak during December and January. Out of several potential predictor variables investigated, minimum ambient temperature and snow proved to be the only factors exerting a significant effect on fecal glucocorticoid excretion. We suggest that high winter glucocorticoid levels may act via catabolic function during adaptation of deer to cold winter month when resources are limited.     

Fecal progesterone, estrogen, and androgen metabolites for noninvasive monitoring of reproductive function in the female Indian rhinoceros, Rhinoceros unicornis

This investigation aimed to establish noninvasive methods for endocrine monitoring of estrous cycles and pregnancy in the Indian rhinoceros. Fecal samples were collected 1-3 times per week from nonpregnant and pregnant captive females (n = 7). Enzyme immunoassays for fecal progesterone, androgen, and estrogen metabolites, respectively, were tested for their ability to determine follicular and luteal phases and to characterize endocrine profiles during pregnancy. Antibodies used were raised against pregnanediol (20 alpha-OH-pregnanes), 20-oxo-pregnanes, epiandrosterone (17-oxo-androstanes), and total estrogens. Androgens and estrogens were found to be reliable indicators of the follicular phase, whereas 20 alpha-OH- and 20-oxo-pregnanes were reliable indicators of luteal function. Progesterone metabolites were also reliable indicators of pregnancy, whereas 17-oxo-androstanes and estrogens were basal throughout gestation. Estrous cycles were regular throughout the year, with an average cycle length of 43.4 +/- 1.5 (n = 27) days; the length of the follicular phase, as indicated by elevated estrogen levels, was 15.9 +/- 1.0 days, whereas the luteal phase, as indicated by elevated 20-oxo-pregnane levels, was 19.1 +/- 0.4 days. Fecal pregnane values were already increasing while follicular estrogen values were still decreasing. The length of the diestrus, indicated by basal steroid levels between declining 20-oxo-pregnanes and subsequently increasing estrogens, was 11.4 +/- 1.2 days. Pregnane levels increased from the 3rd month of gestation onward and levels exceeded luteal phase concentrations approximately 10 times by the 7th month of gestation onward. HPLC separation of immunoreactive fecal metabolites indicated the presence of estrone, estradiol-17beta, and several 17-oxo-androstanes, 20 alpha-OH-pregnanes, and 20-oxo-pregnanes. Concentrations of a peak with an elution profile similar to that of pregnanediol increased as pregnancy progressed. Postpartum fecal estrogen and 17-oxo-androstane concentrations in one animal indicated follicular development comparable to the follicular phase of the estrous cycle, but this was not followed by a subsequent luteal phase. In conclusion, estrous cycle and pregnancy in Indian rhinoceroses can be monitored using fecal steroid analysis. Pregnane metabolites were reliable indicators of the corpus luteum and pregnancy, whereas fecal 17-oxo-androstanes and estrogens were indicators of the follicular phase.     

Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth

Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.     

Diagnosing pregnancy in free-ranging dugongs using fecal progesterone metabolite concentrations and body morphometrics: a population application

Assessing reproductive status and monitoring reproductive rates is important in the effective management of vulnerable marine mammal species such as the dugong (Dugong dugon). Knowledge of the reproductive physiology of this species is limited, and determining reproductive parameters (e.g., sexual maturation, pregnancy, and reproductive senescence) has been restricted by a lack of non-lethal methods for assessing reproductive status in free-ranging individuals. The aim of this study was to develop a method to identify pregnant individuals in a wild dugong population. Using an enzymeimmunoassay, we quantified concentrations of fecal progesterone metabolites (fP) in 322 dugongs, including confirmed pregnant females (n=10), presumed non-pregnant adult females (n=25), juvenile females (n=24), subadult females (n=41), adult females of unknown pregnancy state (n=63), and males of all sizes (n=159). External body morphometrics of each dugong were measured, and confirmation of pregnancy in adult female dugongs was determined by ultrasonography or observation of subsequent neonates. Concentrations of fP were different between sexes and reproductive size classes (P<0.001), and ～30-fold higher in confirmed pregnant dugongs (2017-7760 ng/g) compared to presumed non-pregnant females (30-221 ng/g), juvenile females (29-195 ng/g), and males (24-261 ng/g) (P<0.001). Body measures of maximum and anal girths, and teat length were all greater in confirmed pregnant females than presumed non-pregnant females (all P<0.05). We evaluated a Discriminant Function Analysis (DFA) to provide a model for predicting pregnant and non-pregnant dugongs. Cross-validated results showed that the DFA correctly classified 100% of pregnant and non-pregnant females using fP concentrations, body length, fineness ratio (an index of body shape), and teat length (a female reproductive trait). Using the DFA model, we classified the pregnancy status of all female dugongs and identified a total of 30 females as pregnant and 133 females as non-pregnant from the sampled population over the sample period. Pregnant dugongs in the Moreton Bay population are characterized by fecal progesterone metabolite concentrations > 1000 ng/g, body length ≥ 260 cm, maximum girth ≥ 215 cm, anal girth ≥ 126 cm, and teat length ≥ 5 cm long. In summary, analysis of fP concentrations in combination with body morphometrics may be used to diagnose pregnancy in free-ranging dugongs, and provides a new tool to monitor breeding rates of wild sirenian populations.     

Diurnal and annual changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca

Until present, fundamental studies on cortisol secretory patterns have not been conducted in cetaceans. The objectives of this study were: (1) to examine diurnal changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca, (2) to investigate annual cortisol changes in killer whales, and (3) to investigate the relationship between cortisol and sex steroids (testosterone and progesterone) concentrations in killer whales. Diurnal changes in serum cortisol concentrations were investigated at various intervals in the two species. In Indo-Pacific bottlenose dolphins, serum cortisol levels exhibited the same episodic fluctuations for 24 h as did diurnal terrestrial mammals: cortisol levels were lower at 18:00 h and higher in the early morning. In killer whales, cortisol concentrations continued to decrease until 18:00 h, after which they fluctuated, and then increased in the next morning. Annual changes in cortisol levels were investigated by collecting blood samples every two weeks from two male killer whales and a pregnant female one twice per day (during 09:00-10:00 and 16:00-17:00 h) throughout a one-year period. Regarding sera collected during 09:00-10:00 h from the female, cortisol concentrations showed cyclic changes having about 4-month intervals. In males, cortisol showed higher concentrations in winter and lower concentrations during the summer season. There was a negative correlation between cortisol and progesterone levels in the female and a negative correlation was also observed between cortisol and testosterone in male no. 2. In the female and male no. 1, cortisol levels during 09:00-10:00 h were significantly higher than those during 16:00-17:00 h, and their data are considered to support observations regarding diurnal changes in cortisol levels in the two cetacean species.     

Fecal cortisol metabolite levels in free-ranging North American red squirrels: Assay validation and the effects of reproductive condition

Patterns in stress hormone (glucocorticoid: GC) levels and their relationship to reproductive condition in natural populations are rarely investigated. In this study, we (1) validate an enzyme-immunoassay to measure fecal cortisol metabolite (FCM) levels in North American red squirrels (Tamiasciurus hudsonicus), and (2) examine relationships between FCM levels and reproductive condition in a free-ranging red squirrel population. Injected radiolabeled cortisol was entirely metabolized and excreted in both the urine (mean ± SE; 70.3 ± 0.02%) and feces (29.7 ± 0.02%), with a lag time to peak excretion in the feces of 10.9 ± 2.3 h. Our antibody reacted with several cortisol metabolites, and an adrenocorticotropic injection significantly increased FCM levels above baseline levels at 8 h post-injection. Relative to baseline levels, manipulation by handling also tended to increase FCM levels at 8 h post-manipulation, but this difference was not significant. FCM levels did not differ significantly between samples frozen immediately and 5 h after collection. Reproductive condition significantly affected FCM levels in free-ranging females (pregnant > lactating > post-lactating > non-breeding) but not males (scrotal testes vs. abdominal testes). Among females with known parturition dates, FCM levels increased during gestation, peaked at parturition, and declined during lactation. The difference between pregnant and lactating females was therefore dependent upon when the fecal samples were obtained during these periods, suggesting caution in categorizing reproductive stages. This study demonstrates the utility of fecal hormone metabolite assays to document patterns of glucocorticoid levels in free-ranging animals.     

Metabolism of cortisol in anorexia nervosa

In patients with anorexia nervosa 24-h mean plasma concentration of cortisol were 0.44 +/- 0.09 mumol/l (normal less than 0.28 mumol/l). Following stimulation by ACTH (1-24) urinary excretion rates of cortisol were stimulated from 0.22 +/- 0.08 to 4.85 +/- 2.78 mumol/24 h. Similarly, plasma concentrations of the glucocorticoid metabolite, tetrahydrocortisone, increased from 23.3 +/- 9.0 to 47.3 +/- 30.2 nmol/l; urinary excretion rates of tetrahydrocortisone increased from 3.61 +/- 0.90 to 8.40 +/- 1.72 mumol/24 h. The relative share of the sulphate, glucuronide and free fractions of tetrahydrocortisone in the patients' urine did not indicate any defect in metabolization of this steroid metabolite. Excretion rates of the four glucocorticoid tetrahydro-metabolites, tetrahydrocortisone, allotetrahydrocortisone, tetrahydrocortisol, and allo-tetrahydrocortisol, expressed as percent of total steroid excretion, were similar in patients with anorexia and in healthy women under basal conditions (24 +/- 6 vs 23 +/- 6%) and during stimulation by ACTH (1-24) (36 +/- 10 vs 45 +/- 6%). The share of the two androgen metabolites, androsterone and etiocholanolone, was 24 +/- 5% of total steroid excretion (basal; healthy women: 27 +/- 8%) and 13 +/- 2% (ACTH stimulation; healthy women: 12 +/- 4%) in patients with anorexia nervosa. Thus, analysis of urinary steroid excretion rates did not indicate a shift in adrenocortical function. The results confirmed enhanced secretion of cortisol in patients with anorexia nervosa under basal conditions and during/following stimulation by ACTH. The ACTH-induced increase in the concentrations of the tetrahydro-glucocorticoid metabolites in urine was less pronounced than that of cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)