Intestinal parasite infections and fecal steroid levels in wild chimpanzees

Immune-endocrine interactions have been evaluated much less frequently in nonhuman primates, and this may be due, in part, to logistical and ethical concerns regarding trapping and sampling of endangered species, especially apes. Using noninvasive fecal collection methods, the present study evaluates possible relationships between fecal steroid levels and gastrointestinal parasite infections in the Ngogo chimpanzee community in Kibale National Park, Uganda. Because both testosterone and cortisol exhibit immunosuppressive effects in vitro and in other animal models, it was hypothesized that both testosterone and cortisol would be positively associated with gastrointestinal parasite infections in these animals. When placed in a mixed model simultaneously, both testosterone (F = 4.98, df = 1, P = 0.033) and cortisol (F = 5.94, df = 1, P = 0.020) were positively associated with total (helminth and protozoan) parasite richness (the number of unique intestinal parasite species recovered from hosts' fecal samples). It is possible that androgens and corticoids alter the ability of a host to mount an effective immune response against concomitant infection with multiple parasitic species. The utility of fecal samples for assessing immune-endocrine interactions is discussed.     

Diurnal variation on the excretion patterns of fecal steroids in common marmoset (Callithrix jacchus) females

The use of fecal steroid analysis to assess gonadal and adrenal function in primates has rapidly increased in recent years due to the ability to collect feces from nonhuman primates living in wild conditions. These techniques offer an exciting new potential for enhancing our knowledge of the endocrine status of free-living animals. Prior to using these techniques under field conditions, it is important to determine the diurnal variation of fecal excreted steroids for assessing possible time limitations on fecal collections. The following study investigates the diurnal frequency of defecation and patterns of steroid levels excreted in feces from four female common marmosets, Callithrix jacchus, living in a family group. These females represented three reproductive conditions: early pregnancy, ovarian cycling, and noncycling (postpubertal). Cortisol, estradiol, and progesterone were extracted and analyzed by enzyme immunoassay. Diurnal variations in steroid levels were found by ANOVA for cortisol and progesterone but not for estradiol. Significantly higher levels of cortisol were found in the afternoon, while the reverse was found for progesterone. All females showed the same pattern of steroid level change, except for cortisol in the pregnant female. Since all females defecated within the first hour after they awoke in the morning, this time was determined to be the most effective time to collect feces. The consistency of our findings reinforces the usefulness of this approach for studying reproductive and adrenocortical function in marmosets and also indicates that fecal collection should be limited to either morning or afternoon collections. Am. J. Primatol. 46:105–117, 1998. © 1998 Wiley-Liss, Inc.     

Physiological and analytical validations of fecal steroid hormone measures in black howler monkeys

The measurement of hormones in fecal samples allows for the noninvasive assessment of the endocrine status of free-ranging primates. However, procedures and techniques for hormone analysis in feces must be validated, both analytically and physiologically. Few studies have addressed the endocrinology of black howler monkeys (Alouatta pigra). Due to its conservation status, direct handling of individuals from this species and invasive sample collection are highly regulated, and therefore traditional methods for the validation of hormone assays, such as pharmacological challenges, are not allowed. As a consequence, sometimes studies of the fecal hormones of free-ranging black howler monkeys do not report physiological validations and therefore the biological reliability of such measurements cannot be assessed. In order to stimulate future research with this species, the present study aimed at providing methodological bases for fecal endocrine monitoring. Specifically, we compared the validity of two immunoassays (radioimmunoassays, RIA; solid-phase chemiluminescent enzyme immunoassay, SPCEI) performed with commercial kits to measure cortisol, testosterone, estradiol, and progesterone; and demonstrate how the physiological functions of these steroid hormones can be determined through non-pharmacological validations. We found no differences between the analytical validity of RIA and SPCEI assays to measure cortisol and testosterone, whereas for estradiol and progesterone RIA showed better results. Concerning the physiological validation of our assays, we demonstrated that: (1) comparisons between pre- and post-stress situations may be used to assess cortisol response, (2) comparisons between females and males may be used to assess variation in testosterone levels, and (3) comparisons between pregnant and non-pregnant females may be used to determine variation in estradiol and progesterone activity. The analytical and physiological validations that we performed demonstrate that there are currently commercial kits that allow for correct endocrine monitoring of this species, and that there are non-pharmacological alternatives to assess the biological validity of hormone measurements.     

贮存温度和时间对大熊猫粪便样品皮质醇浓度的影响

通过非损伤性取样比如粪便样品监测野生动物受胁迫的水平已成为一个重要的手段,然而针对从野外采集的样品,其运输和贮存温度条件是否干扰分析结果,尚未有过报道。为评估不同贮存温度和天数对野生动物粪便样品中皮质醇浓度的影响,我们将大熊猫的新鲜粪便置于3 种模拟条件(0℃ 、10℃ 和22℃ ),并进行连续6 d的检测分析。结果表明,贮存天数对粪样中皮质醇浓度有显著影响(F _5,107 = 7.501,P = 0.001),但温度则无显著影响(F _2,107 = 1.094,P = 0.366)。粪样在0℃ 、10℃ 和22℃ 条件下贮存6 d,皮质醇浓度与贮存前的基准值之间均不存在显著差异(0℃ :F _6,41 = 1.274,P = 0.290;10℃ :F _6,41 = 2.027,P = 0.084;22℃ :F _6,41 =1.009, P = 0.434)。结果提示在室温条件下短期运输和贮存(不超过6 d),不会引起大熊猫粪便样品中皮质醇浓度的显著变化。该结果对于野外所采集的粪便样品运输和贮存具有一定的指导意义。     

Diurnal patterns of urinary steroid excretion in wild chimpanzees

Urinary testosterone and cortisol concentrations were quantified in a large number of samples (>500) collected from wild male chimpanzees (n=11) over the course of 1 year. For both steroids, urinary concentrations were higher and more variable in the morning than in the afternoon. Urinary creatinine levels showed no such diurnal pattern. These patterns are consistent with studies of steroid excretion in humans and gorillas. This study emphasizes the importance of considering time of day as a confounding variable in field studies of primate endocrine function. It also suggests that if a small number of samples are to be used to characterize an individual's basal steroid levels, afternoon samples may be preferable because they show less intra-individual variability.     

Non‐invasive fecal hormone analysis and behavioral observations for monitoring stress responses in captive western lowland gorillas (Gorilla gorilla gorilla)

Non‐invasive techniques for monitoring the stress response in captive western lowland gorillas (Gorilla gorilla gorilla) were investigated. Fecal samples for cortisol measurement and concurrent behavioral data were collected from six individuals in a socially housed gorilla group (one adult male, three adult females and their three offspring) over a 7‐month period. Despite inter‐individual variation in the dynamics of fecal cortisol concentrations over time, several major secretory peaks coincided across individuals. High cortisol concentrations in feces were correlated with induced stressors or behavioral observations indicating high social tension, with a 1–2 day lag period. Entry progression order of the gorillas into a den complex and a supplant‐based dominance index were suitable indicators of overall dominance hierarchies, and fluctuations over time reflected periods of instability. Diurnal variation in fecal cortisol was not apparent when comparing afternoon and morning samples, however the sample collection interval was relatively short (3–5 hr). These results demonstrate the feasibility of monitoring stress responses based on the dynamics of both fecal cortisol excretion and behavior. This non‐invasive approach may be used for gauging responses to changes in husbandry, environment and group structure of captive gorillas. Zoo Biol 0:1–15, 2005. © 2005 Wiley‐Liss, Inc.     

Low-ranking individuals present high and unstable fecal cortisol levels in provisioned free-ranging adult male rhesus macaques (<Emphasis Type="Italic">Macaca mulatta</Emphasis>) during the birth season in a mountain area of northern China

Social hierarchy commonly exists in animal societies, affecting both the endocrine functioning and the behavior of animals. In nonhuman primates, the relationship between social rank and cortisol levels varies across species and even within species. Here, we assessed the relationships between social rank and fecal cortisol levels in adult male Taihangshan macaques (rhesus macaques, Macaca mulatta tcheliensis) from the provisioned, free-ranging Wulongkou-2 (WLK-2) group inhabiting Wulongkou Scenic Area, Jiyuan, China. From March to May 2014, we recorded 195 agonistic behaviors and collected 54 fresh fecal samples from eight adult male Taihangshan macaques. Males were assigned a social rank according to an agonistic behavior matrix, and enzyme-linked immunosorbent assay was then used to measure the cortisol concentration in the fecal samples. We found that social rank among the eight male Taihangshan macaques in WLK-2 group followed a strict linear hierarchy, and that fecal cortisol levels were significantly higher and more variable in low-ranking males than in more dominant individuals. Age was not significantly associated with social rank or fecal cortisol levels. Our results suggest that social rank and maintenance of the social hierarchy within the WLK-2 group is a chronic stressor, with low-ranking males maintaining heightened stress levels and enlarged reactive scope relative to dominant males. This provides new support for the theory that social environment can influence endocrine functioning.     

Fecal cortisol levels in free-ranging female chacma baboons: relationship to dominance, reproductive state and environmental factors

Savannah baboons are one of the few mammalian species that do not exhibit seasonal reproduction patterns and are therefore ideally suited to study the effect of female reproductive states (cycling, pregnant, lactating) on cortisol levels independent of seasonal factors. Fecal samples from 10 free-ranging female chacma baboons (Papio hamadryas ursinus), collected during a period of 17 months, were analyzed using a steroid-extraction method. Reproductive state had a significant effect on fecal cortisol, with lowest levels found in estrous females. Fertility was not related to fecal cortisol levels; we found no significant differences between samples collected on conceptive and nonconceptive cycles. Environmental factors explained most of the variance of fecal cortisol levels. Cortisol measures were strongly correlated with seasonal differences such as daylight duration, temperature and the amount of time that baboons spent resting. We measured higher cortisol levels during winter months and suggest that this could be related to shorter resting periods and to the cold minimum ambient temperatures at this study site. Finally, we found no relationship between social rank nor the rate of agonistic interactions with basal fecal cortisol levels.     

Hormone measures in finger-prick blood spot samples: New field methods for reproductive endocrinology

Comparative endocrine studies have notably advanced understanding of ecological factors that contribute to variation in human reproductive function. Such research has relied on methodological advances that permit hormone determinations in samples that are easily and safely collected, stored, and transported, most recently on measurement of steroids in saliva. This report seeks to further expand the scope of endocrine research by demonstrating the value of blood spot samples collected by finger prick. As a sampling strategy, finger-prick blood spot collection offers the advantages of short collection time, low invasiveness, repeatability, absence of postcollection processing, low biohazard risk, and ease of sample storage and transport. We document good sample stability and present sensitive assay methods for a range of steroids and proteins (FSH, LH, PRL, T, E2, DHEAS, androstenedione, cortisol, SHGB) in blood spots that require sample volumes of 3–12 μl and display good reliability, specificity, precision, accuracy, and convertibility of results to plasma/serum equivalent concentrations. Laboratory evaluation was augmented by a feasibility study at a remote site in Papua New Guinea that confirmed validity and stability of blood spot collections under field conditions. Research applications of blood spot sampling are illustrated with a series of studies, including cross-sectional surveys for developmental and life span endocrinology, a longitudinal, population-based developmental epidemiologic study of puberty, and serial sampling in a dynamic study of neuroendocrine response to suckling. We conclude that the sampling features and wide range of measurable biomolecules of blood spots do constitute a methodological advance for endocrine research. Am J Phys Anthropol 104:1–21, 1997. © 1997 Wiley-Liss, Inc.     

School start time influences melatonin and cortisol levels in children and adolescents – a community-based study

School start time influences sleep parameters. Differences between circadian sleep parameters on weekends and weekdays have been associated with obesity, sleep, and psychiatric disorders. Moreover, circadian rhythm dysregulation affects the secretion of some hormones, such as melatonin and cortisol. In the current study, we investigate the effect of school start time on cortisol and melatonin levels in a community sample of Brazilian children and adolescents. This was a cross-sectional study of 454 students (mean age, 12.81 ± 2.56 years; 58.6% female). From this sample, 80 participants were randomly selected for saliva collection to measure melatonin and cortisol levels. Circadian sleep parameters were assessed by self-reported sleep and wake up schedules and the Morningness–Eveningness Questionnaire. The outcomes, salivary melatonin and cortisol levels, were measured in morning, afternoon and night saliva samples, and behavior problems were assessed using the Child Behavior Checklist (CBCL). The main results revealed that morning school start time decreased the secretion of melatonin. Morning melatonin levels were significantly positively correlated with the sleep midpoint on weekdays and on weekends. Afternoon melatonin levels were positively correlated with the sleep midpoint on weekends in the morning school students. Conversely, in the afternoon school students, night melatonin levels were negatively correlated with the sleep midpoint on weekdays. Cortisol secretion did not correlate with circadian sleep parameters in any of the school time groups. In conclusion, school start time influences melatonin secretion, which correlated with circadian sleep parameters. This correlation depends on the presence of psychiatric symptoms. Our findings emphasize the importance of drawing attention to the influence of school start time on the circadian rhythm of children and adolescents.     

Association of salivary steroid hormones and their ratios with time-domain heart rate variability indices in healthy individuals

BackgroundStress system consists of the hypothalamicpituitary-adrenal (HPA) axis and the locus caeruleus/norepinephrine-autonomic nervous system (ANS). Traditionally, HPA axis activity is evaluated by measuring its end-product cortisol, while the activity of ANS is assessed using heart rate variability (HRV) indices. Alterations in cortisol levels and HRV measures during laboratory-based stress tasks were extensively studied in previous research. However, scarce data exist on the associations of HRV measures with the levels of other adrenal steroid hormones under baseline conditions. Thus, we aimed to evaluate the activity of the HPA axis by measuring salivary cortisol, cortisone, dehydroepiandrosterone (DHEA) levels, and their ratios and to examine its association with HRV measures in a sample of healthy young and middle-aged adults.MethodsFor each participant (n=40), three data collection sessions taking place at the same time of the day were scheduled within five working days. Participants completed a self-reported questionnaire on sociodemographic and lifestyle characteristics, filled out t h e Perceived Stress Scale and State-Trait Anxiety Inventory. Also, saliva samples were collected, and physiological measures, including resting HR and HRV, were recorded during three data collection sessions.ResultsStatistically significant associations between diminished parasympathetic vagal tone evaluated by time domain HRV measures and higher salivary cortisol, lower DHEA levels, as well as decreased DHEA to cortisol ratio, were found. Also, physiological stress indicators (i.e., HRV) showed greater intraindividual stability compared with biochemical biomarkers (i.e., salivary steroid hormones) within five days.ConclusionsOur findings suggest that both cortisol and DHEA mediate the link between two stress-sensitive homeostatic systems.     

Seasonal changes in fecal testosterone concentrations and their relationship to the reproductive behavior, antler cycle and grouping patterns in free-ranging male Pampas deer (Ozotoceros bezoarticus bezoarticus)

The purpose of this study was to validate noninvasive endocrine monitoring techniques for Pampas deer and to evaluate seasonal changes in testicular steroidogenic activity and their correlation to reproductive behavior, antler cycle and group size. Thus, fecal samples, behavioral data and observations of antler status were collected at monthly intervals during 1 year from free-ranging Pampas deer stags (three radio-collared individuals and 15 random individuals) living in Emas National Park, Brazil (18 degrees S latitude). Fecal steroids were extracted using 80% methanol and steroid concentrations were quantified by a commercial enzyme immunoassay (EIA). Fecal testosterone concentrations peaked in December-January (summer), March (early autumn) and in August-September (winter-spring), with minimal values from April-July. Reproductive behavior had two peaks, the first in December-January, characterized by predominately anogenital sniffing, flehmen, urine sniffing, chasing and mounting behavior, and the second peak in July-September (behavior primarily related to gland marking). There were significant correlations between fecal testosterone and reproductive behavior (r=0.490), and between fecal testosterone and antler phases (r=0.239). Antler casting and regrowth occurred under low testosterone concentrations, whereas velvet shedding was associated with high concentrations of testosterone. We inferred that Pampas deer stags exhibited a seasonal cycle that modulated sexual behavior and the antler cycle, and we concluded that fecal steroid analysis was a practical and reliable non-invasive method for the evaluation of the endocrine status of free-ranging Pampas deer.     

Excreted steroids in primate feces over the menstrual cycle and pregnancy

Techniques were established for the extraction and measurement of 17 beta-estradiol (E2) and progestins (P4) from feces of Old World primates. Studies were conducted to show the sensitivity of these measures, means of preserving fecal samples in the field, effects of urinary contamination, and means to eliminate these effects. Our results show that excreted steroid measures can be used to distinguish between mid-follicular and luteal phases in the menstrual cycle, and to identify pregnancy by Day 20 of gestation; the steroid measures can also be used to identify ovulatory levels of E2 and to establish the length of the menstrual cycle. Urine was shown to contaminate the fecal sample and to confound the estimate of steroid levels in feces; prolonged storage (less than 6 h) was shown to change the steroid estimate. Both urinary contamination and storage-dependent changes were eliminated by the addition of ethanol to the sample. Preliminary results also suggest that effects of dietary fiber on steroid hormone levels are minimal when controlled quantitatively by adjusting for water content of the fecal sample. We conclude that these measurements of excreted steroids provide a valid, noninvasive measure of physiological state of the hypothalamic-pituitary-ovarian axis among free-ranging animals in the field.     

Validation of a field-friendly extraction and storage method to monitor fecal steroid metabolites in wild orangutans

Measuring hormone metabolites from feces is the most often used method to assess hormonal status in wildlife. Although immediate freezing of fecal samples collected in the field is the best method to minimize the risk of degradation of hormones over time, this is often not possible in remote field sites. Therefore, alternative storage and preservation methods for fecal samples are required in these conditions. We conducted an experiment to investigate if fecal glucocorticoid (FGCM) and progesterone metabolite (pregnanediol-3-glucuronide; PdG) levels measured from samples that were extracted with a simple, field-friendly methodology correlate with those generated from frozen samples. We also evaluated whether storing fecal samples in alcohol is a suitable alternative to preserve FGCM and PdG concentrations long-term (i.e. over a 9-month period) at locations where fecal extraction is not feasible. Finally, we tested if the hormone concentrations in unpreserved fecal samples of orangutans change over 14 h when stored at ambient conditions, representing the maximum duration between sample collection and return to the camp. FGCM and PdG levels measured from samples that were extracted with the field-friendly method showed strong correlations with those generated from frozen samples, and mean levels did not differ significantly between these methods. FGCM concentrations showed no significant change compared to control samples when fecal samples were stored for up to 6 months in alcohol at ambient temperature and PdG concentrations even remained stable for up to 9 months of storage. FGCM concentrations of fecal samples kept at ambient temperature for up to 14 h post-defecation did not significantly differ compared to control samples frozen immediately after collection. These results provide the basis for the successful monitoring of the physiological status of orangutans living in remote natural settings, like those included in the Indonesian reintroduction programs.     

Comparison of fecal storage methods for steroid analysis in black rhinoceroses (Diceros bicornis)

Many field studies and conservation programs for wildlife species include noninvasive endocrine monitoring of gonadal function. Freezing fecal samples immediately after collection until further analysis is often not a viable option for researchers in remote areas. Phase 1 of this study was designed to compare different methods of preserving fecal samples over several time periods (30, 90, or 180 days) in order to determine which method provided the most accurate and reliable technique for measuring fecal progestagens. Fecal samples were collected from two female black rhinoceroses (Diceros bicornis) housed at Disney's Animal Kingdom, Lake Buena Vista, FL. We compared three storage methods: 1) storing fecal samples without processing or preservatives (untreated), 2) storing an aliquot of fecal sample in 80% methanol (MeOH), and 3) drying the fecal sample in a solar box cooker prior to storage. Control samples (day 0) were collected and extracted, and then stored at ?20°C until they were analyzed. Phase 2 of the study was designed to examine the effects of long‐term storage (up to 180 days) on fecal progestagen profiles that reflect reproductive activity (pregnancy and estrous cycles). In samples obtained from a pregnant female and stored for 30 days, there were no significant differences in fecal progestagen concentrations between the three treatment conditions. However, the mean concentrations of progestagens (± SE) in untreated samples increased significantly from 8.3 ± 0.3 µg/g wet weight feces at day 0 to 17.7 ± 5.1 µg/g feces at day 90, and 17.8 ± 4.7 µg/g feces at day 180. Samples that were collected from a pregnant female and stored in 80% MeOH or dried in the solar box correlated with controls (r=0.86 and 0.87, respectively; P<0.05) at day 180. In contrast, samples that were stored without preservatives for 180 days did not correlate with controls (r=0.35, P>0.05). Progestagen concentrations from samples of the estrous cycling female showed similar results. In conclusion, fecal samples dried in a solar box cooker or stored in 80% MeOH maintained absolute and relative progestagen concentrations for at least 180 days when they were stored outdoors and exposed to the climatic conditions of central Florida. Both methods can have significant applications for the study of reproductive events in areas where access to electricity is limited. Zoo Biol 23:291–300, 2004. © 2004 Wiley‐Liss, Inc.     

Bacteria in the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva

Bacteria load in saliva was experimentally manipulated, and the consequences for the measurement of salivary testosterone (T), dehydroepiandrosterone (DHEA), and cortisol (C) were examined. Healthy adults (n = 19) donated the first saliva sample upon rising after which they rinsed their mouths with water, waited 10 min, and donated a second sample. Samples were either left untreated or passed through a 0.22-microm filter and then frozen at -80 degrees C or incubated at room temperature (RT) for 10 days. Aliquots of each sample were cultured on agar to determine baseline and post-incubation (or freezing) bacteria load. Bacteria counts were not significantly influenced by rinsing (with water), were substantially reduced by filtration, and increased by incubation at RT. Average levels of salivary T and C, but not DHEA, were significantly lower in samples stored at RT than samples frozen the day of collection. The change in bacteria count induced by storing samples at RT was associated with a change in testosterone but not cortisol or DHEA. When samples were passed through a 0.22-microm filter bacteria counts were reduced, and the association between bacteria and testosterone was reduced to non-significant. These findings contribute to a growing body of literature revealing that the process of sample collection, storage, and handling can dramatically influence the accuracy of information generated when salivary biomarkers are integrated into research and clinical diagnostics.     

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.     

Fecal corticoid metabolite measurement in the cheetah (Acinonyx jubatus)

This longitudinal study addresses the relationship of cortisol excretion to ovarian activity in captive female cheetahs. A radioimmunoassay was developed and validated to measure corticoid metabolite concentrations in feces. A restraint experiment was used to demonstrate that fecal cortisol is detectable following stressful episodes. In studies of 7 females, fecal cortisol output indicated that they could be placed into 3 different categories. Females of the high-in-cortisol category (∼200 ng/g feces, n = 2) were independently rated by caretakers as the most nervous individuals in the collection (n = 24). These females appeared to be compromised in their ovarian cycling, as indicated by fecal estrogen measurements. In contrast, reproducing females fell into the low and intermediate cortisol excretion categories. A non-cycling high-cortisol female had an episodic cycle following a period of relatively low (intermediate) cortisol levels, followed by resumption of acyclicity and high cortisol excretion. Stress and reproductive failure may, therefore, be associated in the female cheetah. The close proximity of conspecifics as a potential source of stress and, consequently, suppressed ovarian activity in some females is suggested by these results. Zoo Biol 16:133–147. 1997 © 1997 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.     

A non-invasive technique for analyzing fecal cortisol metabolites in snowshoe hares (<Emphasis Type="Italic">Lepus americanus</Emphasis>)

To develop non-invasive techniques for monitoring steroid stress hormones in the feces of free-living animals, extensive knowledge of their metabolism and excretion is essential. Here, we conducted four studies to validate the use of an enzyme immunoassay for monitoring fecal cortisol metabolites in snowshoe hares (Lepus americanus). First, we injected 11 hares with radioactive cortisol and collected all voided urine and feces for 4 days. Radioactive metabolites were recovered predominantly in the urine (59%), with only 8% recovered in the feces. Peak radioactivity was detected an average of 3.5 and 5.7 h after injection in the urine and feces, respectively. Second, we investigated diurnal rhythms in fecal cortisol metabolites by measuring recovered radioactivity 2 days after the radioactive cortisol injection. The total amount of radioactivity recovered showed a strong diurnal rhythm, but the amount of radioactivity excreted per gram of feces did not, remaining constant. Third, we injected hares with dexamethasone to suppress fecal cortisol metabolites and 2 days later with adrenocorticotropic hormone to increase fecal cortisol metabolites. Dexamethasone decreased fecal cortisol metabolites concentrations by 61% and adrenocorticotropic hormone increased them by 1,000%, 8–12 h after injection. Fourth, we exposed hares to a simulated predator (dog). This increased the fecal cortisol metabolites concentrations by 175% compared with baseline concentrations 8–12 h after exposure. Thus, this enzyme immunoassay provides a robust foundation for non-invasive field studies of stress in hares.