Noninvasive assessment of adrenal activity associated with husbandry and behavioral factors in the North American clouded leopard population

The North American clouded leopard (Neofelis nebulosa) population is far from self‐sustaining. Breeding success is poor and behavioral problems (i.e., fur‐plucking, tail‐chewing, excessive hiding or pacing, and intersexual aggression that results in mate killing) are common. This study was undertaken to investigate whether some of these problems may be indicators of chronic stress (as reflected by persistently elevated glucocorticoid levels) and whether they are associated with specific management factors. A fecal corticoid metabolite assay was validated to monitor adrenal activity in clouded leopards. Adrenocorticotropic hormone (ACTH) challenges conducted in four clouded leopards established the biological relevance of the assay system. Fecal corticoid concentrations increased 14‐fold above baseline within 24 hours after ACTH administration. Adrenal activity then was monitored in 72 (36 males; 36 females) clouded leopards (65% of the North American Species Survival Plan population) during a 6‐week period and compared to husbandry and behavior data. There was a significant (P < 0.01) gender difference in fecal corticoid concentrations, with females producing higher concentrations than males. Multiple regression analyses revealed negative associations (P < 0.01) between enclosure height, number of hours keepers spent with each animal per week, and corticoid concentrations. A positive correlation (P < 0.001) was found between the number of keepers caring for an individual and corticoid concentrations. Higher fecal corticoid concentrations (P ≤ 0.05) were measured in clouded leopards kept on public display or near potential predators compared to individuals maintained off exhibit or in the absence of predators. Individuals that performed self‐injuring behaviors also had elevated fecal corticoids (P < 0.01). Spearman‐rank correlation analysis of keeper ratings and hormone data revealed positive associations (P ≤ 0.05) between some behaviors (pace, sleep, hide, and fearful/tense) and fecal corticoid concentrations. Overall these results indicate that noninvasive fecal corticoid monitoring has enormous potential for investigating how management and behavioral problems are related to animal well‐being. If conducted under carefully controlled experimental paradigms, this technique could allow researchers and managers to identify problem areas of captive management for clouded leopards (e.g., enclosure height, keeper time) and evaluate the efficacy of strategies designed to promote animal welfare and increased reproductive success. Zoo Biol 21:77–98, 2002. © 2002 Wiley‐Liss, Inc.     

Noninvasive monitoring of adrenocortical function in captive jaguars (Panthera onca)

Jaguars are threatened with extinction throughout their range. A sustainable captive population can serve as a hedge against extinction, but only if they are healthy and reproduce. Understanding how jaguars respond to stressors may help improve the captive environment and enhance their wellbeing. Thus, our objectives were to: (1) conduct an adrenocorticotrophic hormone (ACTH) challenge to validate a cortisol radioimmunoassay (RIA) for noninvasive monitoring of adrenocortical function in jaguars; (2) investigate the relationship between fecal corticoid (FCM) and androgen metabolite (FAM) concentrations in males during the ACTH challenge; and (3) establish a range of physiological concentrations of FCMs for the proposed protocol. Seven jaguars (3 M, 4 F) received 500 IU/animal of ACTH. Pre‐ and post‐ACTH fecal samples were assayed for corticoid (M and F) and androgen metabolites (M) by RIA. Concentrations of FCMs increased (P80.01) after ACTH injection (pre‐ACTH: 0.90 ± 0.12 µg/g dry feces; post‐ACTH: 2.55 ± 0.25 µg/g). Considering pre‐ and post‐ACTH samples, FCM concentrations were higher (P80.01) in males (2.15 ± 0.20 µg/g) than in females (1.30 ± 0.20 µg/g), but the magnitude of the response to ACTH was comparable (P>0.05) between genders. After ACTH injection, FAMs increased in two (of 3) males; in one male, FCMs and FAMs were positively correlated (0.60; P80.01). Excretion of FCMs was assessed in 16 jaguars (7 M, 9 F) and found to be highly variable (range, 80.11–1.56 µg/g). In conclusion, this study presents a cortisol RIA for monitoring adrenocortical function in jaguars noninvasively. Zoo Biol 31:426–441, 2012. © 2011 Wiley Periodicals, Inc.     

Reproductive assessment of the great hornbill (Buceros bicornis) by fecal hormone analysis

The population of great hornbills (Buceros bicornis) in the United States is rapidly aging, and captive breeding efforts have not met population managers' expectations for a sustainable captive group. Little is known about the reproductive physiology of these birds. This study reports the first data on the re‐productive endocrinology of the great hornbill. The hormone profiles of the only pair of these birds that hatched a chick in the 1999–2000 breeding season are compared to the profiles of six other pairs of hornbills, from different institutions in the United States, that did not reproduce successfully that season. The study investigates the estradiol, corticosterone, and testosterone profiles of these seven pairs of birds, establishing a base of knowledge from which endocrine data may be used to improve the success of captive breeding programs. The estradiol profiles from this study indicate a difference in hormonal patterns between laying and non‐laying female great hornbills. Egg‐laying females had significantly higher estradiol concentrations during the breeding season than the non‐laying females (P<0.003). Testosterone concentrations of the males were not significantly different between the mates of egg‐laying and non‐egg‐laying females. The corticosterone concentrations tended to be lower in the females that laid eggs vs. the non‐egg‐laying group. The males of the egg‐laying pairs showed a significantly lower (P<0.036) corticosterone concentration than the non‐egg‐laying male pairs. This, combined with the extremely low corticosterone levels (compared to the other birds in the study) of the pair of hornbills that hatched a chick in the 1999–2000 breeding season, suggests that adrenal activity may play a role in the reproductive failure of some captive great hornbills. Zoo Biol 22:135–145, 2003. © 2003 Wiley‐Liss, Inc.     

Reproductive endocrine patterns in captive female and male red wolves (Canis rufus) assessed by fecal and serum hormone analysis

Reproductive steroid profiles in female (n=13) and male (n=5) red wolves (Canis rufus) were characterized in fecal samples collected during the breeding season (December—May) and over a 1 year period, respectively. Blood samples from females (n=12) also were collected during the periovulatory period for luteinizing hormone (LH) and steroid analysis. High performance liquid chromatography (HPLC) of fecal extracts determined that estradiol and estrone constituted the major and minor forms, respectively, of fecal estrogen metabolites. Although native progesterone was present, pregnane metabolites predominated as the major forms of fecal progestins. HPLC analysis of fecal extracts from males revealed no native testosterone, but rather the predominance of more polar androgen metabolites. Based on hormone profiles and/or pup production, females were classified as pregnant (n=3), ovulatory‐nonpregnant (n=9), or acyclic (n=3). Longitudinal monitoring of females indicated no pregnancy‐specific differences in concentrations of either fecal progestagen or estrogen metabolites compared to ovulatory‐nonpregnant individuals; however, baseline progestagen concentrations were consistently elevated in acyclic females. There was good correspondence between serum and fecal steroid concentration during the periovulatory period. A rise in serum estrogens preceded the ovulatory LH surge which was then followed by a significant progesterone rise during the luteal phase. In males, changes in fecal androgen metabolite concentrations coincided with photoperiod fluctuations, increasing in late autumn and reaching peak concentrations during mid‐ to late winter just before the start of the breeding season. Collectively, these results serve as a database of ovarian and testicular endocrine events in this species, which can be utilized in population management and application of assisted reproductive technologies. Zoo Biol 21:321–335, 2002. © 2002 Wiley‐Liss, Inc.     

Estrogen Reduces 11β‐Hydroxysteroid Dehydrogenase Type 1 in Liver and Visceral,but Not Subcutaneous,Adipose Tissue in Rats

Following menopause, body fat is redistributed from peripheral to central depots. This may be linked to the age related decrease in estrogen levels. We hypothesized that estrogen supplementation could counteract this fat redistribution through tissue‐specific modulation of glucocorticoid exposure. We measured fat depot masses and the expression and activity of the glucocorticoid‐activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in fat and liver of ovariectomized female rats treated with or without 17β‐estradiol. 11βHSD1 converts inert cortisone, or 11‐dehydrocorticosterone in rats into active cortisol and corticosterone. Estradiol‐treated rats gained less weight and had significantly lower visceral adipose tissue weight than nontreated rats (P < 0.01); subcutaneous adipose weight was unaltered. In addition, 11βHSD1 activity/expression was downregulated in liver and visceral, but not subcutaneous, fat of estradiol‐treated rats (P < 0.001 for both). This downregulation altered the balance of 11βHSD1 expression and activity between adipose tissue depots, with higher levels in subcutaneous than visceral adipose tissue of estradiol‐treated animals (P < 0.05 for both), opposite the pattern in ovariectomized rats not treated with estradiol (P < 0.001 for mRNA expression). Thus, estrogen modulates fat distribution, at least in part, through effects on tissue‐specific glucocorticoid metabolism, suggesting that estrogen replacement therapy could influence obesity related morbidity in postmenopausal women.     

Comparative analysis of gonadal and adrenal activity in the black and white rhinoceros in North America by noninvasive endocrine monitoring

Patterns of fecal reproductive steroid metabolites and adrenal corticoids were characterized for 12‐ to 24‐month periods in black (n = 10 male, 16 female) and white (n = 6 male, 13 female) rhinoceroses at 14 institutions. All black rhinoceros females exhibited at least some ovarian cyclicity on the basis of fecal progestogen analysis (range, 2–12 cycles/yr). However, cycles often were erratic, with many being shorter (<20 days; 18% of cycles) or longer (>32 days; 21%) than the average of 26.8 ± 0.5 days (n = 104 cycles). Five females exhibited periods of acyclicity of 2–10‐month duration that were unrelated to season. One complete and seven partial pregnancies were evaluated in the black rhinoceros. Fecal progestogens increased over luteal phase concentrations after 3 months of gestation. Females resumed cyclicity within 3 months postpartum, before calves were weaned (n = 5). Approximately half of white rhinoceros females (6 of 13) showed no evidence of ovarian cyclicity. Of the cycles observed, 5 were “short” (32.8 ± 1.2 days) and 24 were “long” (70.1 ± 1.6 days). Only two females cycled continuously throughout the study. One had both long (n = 9) and short (n = 2) cycles, whereas the other exhibited long cycles only (n = 5). Fecal estrogen excretion was variable, and profiles were not useful for characterizing follicular activity or diagnosing pregnancy in either species. Males of both species showed no evidence of seasonality on the basis of fecal androgen profiles. Androgen metabolite concentrations were higher (P < 0.05) in the black (27.6 ± 6.9 ng/g) than in the white (16.8 ± 3.1 ng/g) rhinoceros. An adrenocorticotropin hormone challenge in four black rhinoceros males demonstrated that the clearance rate of corticoid metabolites into feces was ～24 hours. Fecal corticoid concentrations did not differ between males and females, but overall means were higher in the black (41.8 ± 3.1 ng/g) than in the white (31.2 ± 1.7 ng/g) rhinoceros. In summary, fecal steroid analysis identified a number of differences in hormonal secretory dynamics between the black and white rhinoceros that may be related to differences in reproductive rates in captivity. Most black rhinoceros females exhibited some cyclic ovarian activity. In contrast, few white rhinoceroses demonstrated evidence of regular estrous cyclicity, and those females that were active had comparatively long cycles. Results also suggest that fecal corticoid concentrations reflect adrenal activity and may be species specific. Continued studies are needed to determine whether fecal corticoid measurements will be useful for understanding the cause of inconsistent gonadal activity in these two species. Because all but three (15.8%) of the white rhinoceroses evaluated in this study were less than 20 years of age compared to 73.1% (19 of 26) of the black rhinoceroses, the impact of age on reproductive and adrenal activity also needs to be evaluated further. Zoo Biol 20:463–486, 2001. © 2002 Wiley‐Liss, Inc.     

Blubber cortisol qualitatively reflects circulating cortisol concentrations in bottlenose dolphins

Stress hormones, released into circulation as a consequence of disturbance, are classically assayed from blood samples but may also be detected in a variety of matrices. Blubber and fecal samples can be remotely collected from free‐ranging cetaceans without the confounding hormone elevations associated with chase, capture, and handling required to collect blood samples. The relationship between cortisol concentrations in circulation with that of blubber and feces, however, is unknown. To assess these associations, we elevated cortisol by orally administering hydrocortisone for five days in five bottlenose dolphins. Voluntary blood and fecal samples were collected daily; blubber biopsies were collected on day one, just prior to hydrocortisone administration, and days three and five of hydrocortisone administration. We evaluated subsequent changes in several circulating stress hormones as well as cortisol and glucocorticoid metabolites in blubber and feces, respectively. There was a significant association between cortisol levels in serum and in blubber (F_1,12.7 = 14.3, P < 0.01, mR² = 0.57) despite substantial variability in blubber cortisol levels. Counterintuitively, fecal cortisol metabolite levels were inversely related to serum cortisol. The relationship between serum and blubber cortisol levels suggests blubber samples from remote sampling may be useful to detect stress loads in this species.     

Migration and diving activity in three non‐breeding flesh‐footed shearwaters Puffinus carneipes

The flesh‐footed shearwater Puffinus carneipes is a medium‐sized shearwater and transequatorial migrant within the Pacific Ocean. We used archival data loggers to study the non‐breeding migration and diving behaviour of three flesh‐footed shearwaters following breeding in New Zealand. In early April, the birds migrated to the western North Pacific Ocean in 23±2 days, occupying core distributions within the Kuroshio/Oyashio transition system for 91±17 days. Subsequent movements were made into the Sea of Okhotsk prior to return migrations to New Zealand in mid September (19±1 days). Diving depths during migration (2.5±2.4 m), and in the western North Pacific (2.4±2.6 m) were shallower than during the onset of breeding (4.8±8.7 m). Non‐breeding flesh‐footed shearwaters occupy a region of high fisheries activity and the impact of these fisheries on adult survival in this declining species warrant further study.     

Influence of the hormonal forms of ACTH on the pattern of corticosteroid secretion

In the pituitaries of man, monkey, sheep, dog, cat and guinea pig the predominant component of immunoreactive ACTH has a molecular size resembling the usual 1–39 peptide (little ACTH). Cortisol is the predominant glucocorticoid in these species. Rabbit, rat and mouse pituitaries contain a newly described intermediate sized ACTH and corticosterone as their principal glucocorticoid. Bovine pituitaries contain intermediate and little ACTH in about equal amounts and their cortisol: corticosterone ratio is about 1. These findings are consistent with our hypothesis that the hormonal form of ACTH is an important factor regulating the cortisol: corticosterone ratio in mammalian adrenal corticoid secretion.     

Differences in Fecal Androgen Patterns of Breeding and Nonbreeding Kori Bustards (Ardeotis kori)

To better understand breeding conditions to promote reproduction in captive kori bustards, fundamental endocrine studies measuring fecal androgen metabolites in male and female kori bustards were conducted. Feces collected weekly from males and females were analyzed for testosterone using enzyme‐linked immunoassay. Results from adult males (n = 5), adult females (n = 10), immature males (n = 10), and immature females (n = 10) revealed seasonally elevated testosterone concentrations in fertile, but not nonfertile adult males and females (P > 0.05). Adult females that were not maintained in a breeding group, or that did not produce eggs, did not demonstrate increases in testosterone compared to egg laying counterparts. In males, but not females, seasonal testosterone increases were accompanied by weight gain. Peaks in male fecal androgen metabolites ranged from 10‐ to 22‐fold higher than nonbreeding season (181.5 ± 19.1 vs. 17.0 ± 0.94 ng/g; P < 0.05). Mean breeding season values for adult males were 83.6 ± 6.1 ng/g vs. nonbreeding season values of 12.3 ± 0.73 ng/g (P < 0.05). In females, average breeding season testosterone concentrations were approximately 4‐fold higher than nonbreeding season (55.9 ± 6.0 vs. 14.5 ± 1.8 ng/g), with peaks 10‐ to 30‐fold higher. Results show that noninvasive fecal androgen metabolite analysis can provide a means of predicting fertility potential of male and female kori bustards and might be utilized to assess effects of modifying captive environments to promote reproduction in this species. Zoo Biol. 32:54‐62, 2013. © 2012 Wiley Periodicals, Inc.     

Behavioral and physiologic responses to environmental enrichment in the maned wolf (Chrysocyon brachyurus)

The ex situ population of maned wolves is not self‐sustaining due to poor reproduction, caused primarily by parental incompetence. Studies have shown that environmental enrichment can promote natural parental behaviors in zoo animals. The objective of this study was to determine the effects of environmental enrichment on behavioral and physiological responses of maned wolves. During an 8‐week experimental period, daily behavior observations and fecal sample collection were conducted on four adult wolves (2.2) individually housed in environments without enrichment. After 2 weeks, the wolves were chronologically provided with 2‐week intervals of hiding dead mice around the exhibit, no enrichment, and introduction of boomer balls. Responses of the wolves to enrichment were assessed based on activity levels and exploratory rates, as well as the level of corticoid metabolites in fecal samples collected daily throughout the study period. Providing wolves with environmental enrichment significantly increased exploratory behaviors (P<0.05), especially when mice were hidden in the enclosure. Fecal corticoid concentrations were increased during periods of enrichment in males (P<0.05), but not in females. Overall, there were no correlations between behavioral responses to enrichment and fecal corticoid levels. Behavioral results suggest that environmental enrichment elicits positive effects on the behavior of captive maned wolves. There is evidence suggesting that providing animals with ability to forage for food is a more effective enrichment strategy than introducing objects. There is need for a longer term study to determine the impact of environmental enrichment in this species. Zoo Biol 26:331–343, 2007. © 2007 Wiley‐Liss, Inc.     

Efficacy of levonorgestrel when administered as an irradiated,slow‐release injectable matrix for feline contraception

Reliable and safe methods of reversible contraception are needed for use in zoo felids. The efficacy of levonorgestrel (LNG) as a contraceptive, when delivered as a cesium‐irradiated, slow‐release, injectable matrix, was tested in domestic cats as a model for exotic cats. An increase (P = 0.0017) in body weight was observed in treated but not control queens (P = 0.2146). All control queens (n = 6), which received injections of matrix only, but none of the LNG‐treated queens (n = 6) became pregnant during the trial. Levonorgestrel was effective in preventing pregnancy for at least 36 weeks after two injections of drug‐loaded formulations (40 mg/kg body weight), administered 68 days apart. Throughout the study, all control queens displayed luteal activity and fluctuating fecal estradiol concentrations, whereas the LNG‐treated queens displayed lower estradiol concentrations and no luteal activity after treatment. We conclude that LNG, when delivered as a cesium‐irradiated, slow‐release, injectable matrix, is an effective contraceptive in domestic cats, reducing follicular activity, and thus, preventing mating and luteal activity. Zoo Biol 20:407–421, 2001. © 2001 Wiley‐Liss, Inc.     

Effect of ACTH (tetracosactide) on steroid hormone levels in the mare. Part B: effect in ovariectomized mares (including estrous behavior)

The mare is the only non-primate species known to display estrous signs after ovariectomy and adrenal hormones have been implicated as a possible cause. Moreover, in several species, estradiol seems to have a stimulatory effect on the hypothalamic-pituitary-adrenal axis. The aim of the present study was to compare the effect of ACTH (tetracosactide) on pertinent hormones [cortisol, progesterone, androstenedione, testosterone (intact and ovariectomized mares) and estradiol (ovariectomized mares only)] in intact mares in estrus with the same mares after ovariectomy (n=5). Blood samples were collected hourly from 12:00 until 14:00 h the following day (half-hourly between 14:00 and 17:00 h) on two occasions, with saline or ACTH treatment at 14:00 h (saline treatment day or ACTH treatment day). The mares, both when intact and after ovariectomy, showed a significant increase in all measured hormones, except estradiol (not measured in intact mares), after ACTH treatment, lasting at least 3h post-treatment (P<0.001). On the saline treatment day, cortisol levels in ovariectomized mares were lower than in intact mares in the evening (18:00-23:00 h), but higher at night (24:00-05:00 h). No differences in cortisol response between mares, when intact and after ovariectomy, were found after ACTH treatment (P=0.3). Androstenedione levels were lower (P<0.001) and increased less after ACTH treatment in ovariectomized mares, as compared to when intact (P<0.05). Progesterone concentrations were lower in the ovariectomized mares at night (24:00-05:00 h) on the saline treatment day and at all times on the ACTH treatment day (P<0.05). Testosterone concentrations were lower in ovariectomized mares on both treatment days, as compared to when intact (P<0.001). It was concluded that ovariectomy affected basal cortisol pattern. Ovarian androstenedione and testosterone contributed to the basal circulating levels and, in the case of androstenedione, was stimulated by ACTH. Endogenous estradiol did not act stimulatory on adrenal gland hormone production in the mare.     

Fecal cortisol radioimmunoassay to monitor adrenal gland activity in the bottlenose dolphin (Tursiops truncatus) under human care

Fecal glucocorticoid measurement is an important noninvasive tool to monitor animal health. A radioimmunoassay (RIA) method was developed to measure fecal cortisol in bottlenose dolphins under human care. The method was used to measure baseline hormone levels and evaluate the adrenal response to environmental challenges in a small number of individual dolphins. The method was validated by precision and accuracy tests and by comparison with liquid chromatography‐mass spectrometry (LC‐MS). The parallelism test suggested few matrix interferences. The assay showed a good degree of precision within assay (CV = 5.4%) and between assays (CV = 4.1%). The RIA significantly correlated with the LC‐MS method (r = 0.838, P < 0.01). The recovery test and the comparison between RIA and LC‐MS suggested that the RIA slightly underestimates fecal cortisol concentrations, although the degree of accuracy was good. This study established that bottlenose dolphins excrete appreciable amounts of fecal cortisol (healthy subjects: 0.2–9.5 ng/g). Therefore, chronic HPA axis activation may be monitored in fecal samples by immunoassays after validating a suitable extraction protocol. The RIA could discriminate conditions of stimulation (pregnancy, parturition, isolation, transportation) and inhibition (diazepam administration) of the HPA axis and may, therefore, be useful for monitoring dolphin well‐being.     

Annual pattern of fecal corticoid excretion in captive Red-tailed parrots (<Emphasis Type="Italic">Amazona brasiliensis</Emphasis>)

Annual patterns of fecal corticoid excretion were analyzed in the threatened Red-tailed parrot (Amazona brasiliensis) in captivity. Corticoid concentration over the 15 months of the study (mean ± standard error, 12.6 ± 0.32 ng g⁻¹, n = 585) was lowest around May (the southern Fall), and greatest around September (late winter), just prior to their normal breeding period. Corticoid excretion follows a seasonal pattern best explained by reproductive cycles rather than climate, although climate may be involved in the timing of corticoid excretion. Fecal corticoids also show promise as a tool to measure stress levels. We demonstrate that fecal corticoid measurement is a simple, yet efficient method for monitoring adrenocortical activity in captive, and perhaps wild, parrots. Monitoring adrenocortical activity can inform researchers about imposed stress in captivity, whether pair-bonds are forming in captive birds, and of the timing of breeding both in captivity and in nature.     

Lactation‐associated infertility in Japanese monkeys (Macaca fuscata) during the breeding season

To investigate the endocrine factors in Japanese monkeys (Macaca fuscata) responsible for the suppression of the estrous cycle during the first reproductive season after delivery (150–360 days postpartum), peripheral blood was taken to measure plasma concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone, estradiol‐17β, immunoreactive (ir)‐inhibin, and cortisol. The results demonstrated that during the breeding season of lactating Japanese monkeys, circulating concentrations of FSH (1.7–2.7 ng/ml), LH (308.5–461.0 pg/ml), estradiol‐17β (<62.6 pg/ml), and progesterone (145.0–453.0 pg/ml) remained low and were similar to the nadir levels observed during both the normal menstrual cycles and the nonbreeding season. Concentrations of ir‐inhibin, which is secreted from both follicles and corpus luteum in female Japanese monkeys, were also low (300.5–585.0 pg/ml). This strongly suggests that no follicular development occurs during lactation. Serum concentrations of cortisol (261.0–519 ng/ml) were higher during lactation than during the nonbreeding season. Since babies were often seen suckling their mothers during the study, the results indicate that the increased cortisol levels were associated with suckling‐induced secretion of corticotrophin‐releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). The results of this study indicate that a long period of postpartum infertility in lactating Japanese monkeys, with apparent inhibition of follicle growth and anovulation, is due to weak gonadotropin stimulation, which may occur as the result of a suckling stimulus. Zoo Biol 22:65–76, 2003. © 2003 Wiley‐Liss, Inc.     

Comparison of different drying and storage methods on quantifiable concentrations of fecal steroids in the cheetah

Fecal steroid analysis is a powerful tool that can provide important information on the health, physiology, and reproductive status of nondomestic species. However, studying free‐ranging animals requires that feces be stored and transported from the collection site to the laboratory in a manner that prevents degradation or alteration of steroid metabolites. To determine the effects of different handling and storage methods on fecal steroids, 30 fresh fecal samples from five captive cheetahs were collected, thoroughly mixed, separated into aliquots, and processed (stored or dried) under different conditions. Concentrations of gonadal and adrenal steroid hormones were analyzed in feces stored frozen at –20°C or at room temperature in 95% ethanol. Both frozen and ethanol‐stored aliquots were desiccated using a lyophilizer, solar oven, or conventional oven. The steroid values from aliquots stored and desiccated using the different methods were compared to those obtained using the optimal storage method of freezing at –20°C and desiccating in a lyophilizer (control). Concentrations of corticoid, estrogen, progestagen, and androgen metabolites in fecal extracts were quantified by radioimmunoassay. Androgen metabolite concentrations were not significantly affected (P > 0.05) by storage or drying methods. Fecal samples stored at room temperature in ethanol and lyophilized also had steroid concentrations that did not differ (P > 0.05) from controls. However, the concentrations of corticoid and estrogen metabolites were significantly lower (P < 0.05), and progestagen metabolites were significantly higher (P < 0.05) in samples desiccated in solar and conventional ovens without regard to storage method. These results suggest that storage of fecal samples at room temperature in ethanol is the best alternative to freezing for subsequent analysis of steroid hormone concentrations. Differences in measured concentrations of hormones in oven‐desiccated samples could be due to hormone degradation or shifts in the immunodominant metabolite. Therefore, validation of storage and processing techniques should be included in the development of any new fecal steroid analysis methodology. Zoo Biol 21:215–222, 2002. © 2002 Wiley‐Liss, Inc.     

Influence of Radiotransmitters on Fecal Glucocorticoid Levels of Free-Ranging Male American Kestrels

ABSTRACT Although radiotelemetry is considered a valuable technique for ornithological field studies, several assumptions have been made about the impact that transmitters may have on the estimation of behavioral, ecological, and reproductive parameters. To assess the potential effects of backpack radiotransmitters, we captured and assigned 8 male American kestrels (Falco sparverius) into 2 groups: radiotagged (n = 6) and control individuals (leg-banded, n = 2). Thereafter, we collected feces approximately 2 hours after capture (day −1), and subsequently during days 0 (releasing day), 4, 7, 15, 30, 40, and 55. Prior to fecal analysis, we validated the corticosterone enzyme immunoassay using standard procedures (e.g., parallelism, dose-response curve), and we confirmed physiological significance of fecal glucocorticoid metabolites through adrenocorticotropin challenge, which induced an increase of 4-fold (446.10 ± 60.73 ng/g) above baseline (114.27 ± 15.23 ng/g) within 4 hours (P < 0.001). Both groups exhibited a significant increase in fecal glucocorticoids during day 0 (P < 0.001), but concentrations returned to preattachment values within 4 days. Fecal glucocorticoid concentrations did not differ between samples of radiotagged and leg-banded kestrels (P > 0.05). In spite of the small number of monitored subjects, these findings suggested that radiotransmitters did not affect adrenocortical activity in these male American kestrels.     

Steroid metabolism and validation of noninvasive endocrine monitoring in the African wild dog (Lycaon pictus)

The purpose of this study was to validate noninvasive endocrine monitoring techniques for African wild dogs (Lycaon pictus) and to establish physiological validity of these methods by evaluating longitudinal reproductive-endocrine profiles in captive individuals. To determine the primary excretory by-products of ovarian steroid metabolism, [¹⁴C]-progesterone and [³H]-estradiol were co-administered to a female and all excreta were collected for 80 hr postinjection. Radiolabel excretion peaked ≤ 18 hr postinfusion, and progesterone and estradiol metabolites were excreted in almost equivalent proportions in urine (39.7 and 41.1%, respectively) and feces (60.3 and 58.9%, respectively). Most of the urinary metabolites were conjugated (estradiol, 94.3 ± 0.3%; progesterone, 90.4 ± 0.5%), so that immunoassays for pregnanediol-3α-glucuronide (PdG) and estrogen conjugates (EC) were effective for assessing steroid metabolites. Two immunoreactive estrogens (estradiol and estrone) and at least one immunoreactive progesterone metabolite (3α-hydroxy-5α, pregnan-20-one) were detected in feces. Urine and fecal samples were collected (1–3 times per week) for 1.5 yr from one adult female and two adult males to assess longitudinal steroid metabolite excretion. Overall correlation of urinary PdG to matched, same-day fecal progesterone metabolites immunoreactivity was 0.38 (n = 71, P < 0.05). Similarly, urinary EC was correlated (P < 0.05) with same-day fecal estrogen immunoreactivity (r = 0.49, n = 71). During pregnancy and nonpregnant cycles, copulation occurred at the time of peak (or declining) estrogen metabolites and increasing progesterone metabolites concentrations. Estrus duration was 6–9 days and gestation lasted 69 days with parturition occurring coincident with a drop in progesterone metabolites. Males exhibited seasonal trends in fecal testosterone excretion with maximal concentrations from July to September coincident with peak mating activity. Although these limited longitudinal hormone profiles should be interpreted cautiously, noninvasive gonadal steroid monitoring suggests that: (1) both female and male wild dogs may exhibit reproductive seasonality in North America, (2) females are monoestrous, and (3) peak testicular activity occurs between August and October coincident with mating behavior. From a conservation perspective, noninvasive endocrine monitoring techniques should be useful for augmenting captive breeding programs, as well as for developing an improved understanding of the physiological mechanisms underlying reproductive suppression in response to social and ecological pressures. Zoo Biol 16:533–548, 1997. © 1997 Wiley-Liss, Inc.     

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

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