Fecal Testosterone Metabolites Research Articles

Testosterone concentrations in juvenile male polar bears (Ursus maritimus)

The ex situ polar bear population in the United States is not sustainable. Current management practices include pairing mates at five to six years of age, but there is evidence that wild bears have sired cubs as early as two years old. Timing acquisition of sexual maturity in male polar bears could aid in determining when mates should be paired in ex situ populations. To address this, non-invasive fecal testosterone metabolite monitoring was evaluated as a technique for establishing age of sexual maturity in 14 male polar bears (aged 1–6.9 years) for 1–6 years. Testosterone metabolite concentrations were monitored throughout the year and evaluated by breeding season (Jan. 1 – May 21) compared to non-breeding season (May 22 – Dec. 31). Sexual maturity was defined as when testosterone metabolite concentrations were significantly greater during breeding season than non-breeding season and bi-weekly averages were elevated above baseline for a minimum of ten weeks during breeding season mimicking patterns in adult male polar bears. Testosterone metabolite concentrations ranged from 5.9 to 8342 ng/g. One bear met the defined criteria at two years of age and an additional three bears met criteria at three years old. Due to the sampling strategy and the requirements set forth by the definition of sexual maturity, there were confounding results. In two cases, bears determined to be sexually mature the year prior, were not categorized as mature the next year. Another bear demonstrated greater testosterone metabolite values during the non-breeding season than breeding season. This study demonstrated the variability in fecal testosterone metabolites in juvenile polar bears and results suggest that hormone assessment alone is not adequate for determining sexual maturity in ex situ male polar bears. However, if used in tandem with behavioral monitoring and semen collection, when possible, could aid breeding management decisions for ex situ populations.

Testing the Challenge Hypothesis in Stumptail Macaque Males: The Role of Testosterone and Glucocorticoid Metabolites in Aggressive and Mating Behavior.

The "challenge hypothesis" predicts higher male-male aggressive behavior along with increases in testosterone levels during times of reproductive challenges and social instability. In addition, in some primate species, higher glucocorticoid levels can be observed as well, but this is usually modulated by dominance rank. We studied rank-related aggressive behavior, mating activity, and fecal testosterone and glucocorticoid metabolites (fTm and fGCm) in male stumptail macaques (Macaca arctoides) in order to test some predictions of the "challenge hypothesis". Over a 20-month period, we collected data on aggressive behavior and copulation, as well as fecal samples (n = 700) to quantify fTm and fGCm in seven adult stumptail males living in captivity. During periods of mating activity, male-to-male aggression increased in higher- and middle-ranking males. Neither fTm nor fGCm levels predicted male-to-male aggression. fGCm levels (but not fTm) were positively associated with male-to-female aggression; however, this association was pronounced during periods of mating activity. fGCm levels differed according to social rank, with middle-ranking males having the highest levels. Both hormones were higher during periods of mating activity, but only in higher- and middle-ranking males. Taken together, our findings partially support the challenge hypothesis in a non-seasonal primate and shed some light on the unique social and mating system of the stumptail macaque.

Non-Invasive Reproductive Hormone Monitoring in the Endangered Pygmy Hog (Porcula salvania).

Simple SummaryThe pygmy hog is one of the world’s rarest suids and classified as an endangered species. Efforts are being made to breed them in captivity and reintroduce them into the wild. In this study, we examined reproductive hormones in captive pygmy hogs using a non-invasive method by collecting 785 fecal samples from five females and two males for 12 months. High-pressure liquid chromatography was performed to examine the presence of immunoreactive progesterone and testosterone metabolites in the fecal samples. We standardized and validated enzyme immunoassays (EIA) for fecal progesterone and testosterone metabolites. Using progesterone EIA, we were able to detect pregnancies in four females and estimate the relevant gestation period. We also recorded 172 births from the captive breeding center and found strong seasonality patterns in births. In males, fecal testosterone metabolite concentrations were higher in the breeding season than in the non-breeding season as evidenced by elevated testosterone concentrations during breeding season. A significant difference in fecal progesterone metabolites concentration was observed between non-pregnant and pregnant females. This study can directly help in monitoring the reproductive status of reintroduced hogs both in the wild and in conservation breeding programs in India and elsewhere.The pygmy hog (Porcula salvania), until recently was classified as a critically endangered suid facing the threat of extinction due to habitat degradation. Efforts are being made to protect the pygmy hog from extinction and breed them in captivity under the Pygmy Hog Conservation Programme (PHCP). However, very little information is available on the reproductive physiology of pygmy hogs. Therefore, the present study aims to standardize enzyme immunoassays (EIAs) for monitoring pregnancy and reproductive status using progesterone and testosterone metabolites. A total of 785 fecal samples were collected from five females and two males over a period of one year from the PHCP Research and Breeding Centre, Guwahati, Assam. High-pressure liquid chromatography (HPLC) analysis revealed the presence of immunoreactive progesterone and testosterone metabolites in feces. Mating was observed in all five females, and four of them gave birth successfully. We were able to detect pregnancy using fecal progesterone metabolites. The mean gestation period, based on mating and parturition, was estimated to be 153.25 days from the four females studied. The breeding center recorded 172 births between 1996 and 2000 and found strong seasonal patterns in the birth rate, with most of the births occurring between May and June. In the males, fecal testosterone metabolites were significantly higher in the breeding season than in the non-breeding season. This is the first study on the subject and will help with future breeding programs in other captive breeding centers and with reproductive monitoring of reintroduced populations.

Immune challenge of mating effort: steroid hormone profile, dark ventral patch and parasite burden in relation to intrasexual competition in male Iberian red deer.

Testosterone secretion may regulate the reproductive effort and the development of sexual traits, but it may also involve costs at the immunological and metabolic levels. However, the evidence for this trade-off in wild populations is scarce. Cortisol also plays an important role in mediating the reproductive and immune functions. In this study, we analyzed whether the endoparasite burden relates to hormonal levels (fecal testosterone and cortisol metabolites) and/or morphological sexual traits (size of the dark ventral patch, a trait that indicates reproductive effort in males) in male Iberian red deer. For this purpose, we sampled male red deer harvested during hunting actions in 2 types of populations in south western Spain that differed in structure, affecting the level of male-male competition for mates. We used coprological analyses to estimate the parasite burden mainly of gastrointestinal and bronchopulmonary nematodes and of protozoa, and assessed testosterone and cortisol metabolite levels from fecal pellets. We found a positive relationship of host parasitation with both testosterone levels and the size of the dark ventral patch, but these relationships depended on the intensity of male-male competition in the population, being only found under the high-competition scenario. These results are discussed under the hypothesis of the testosterone immunocompetence handicap, suggesting a cost at the immunological level, and, therefore, higher susceptibility to parasite infection in males that make a greater reproductive effort. However, this effect seems to be modulated by the social environment (male-male competition) that might lead to different optima in testosterone production and sexual trait development.

Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques.

Simple SummaryTestosterone is the main reproductive hormone in male vertebrates. Conventional methods to assess testosterone rely on invasive blood sampling procedures, which can induce pain and distress to the animals. Here we successfully validated a non-invasive method to determine testosterone levels by assessing testosterone metabolites (TMs) in excreta of mice. We investigated the effects of sex and daytime on the metabolism and the excretion of TMs and validated the applied EIA to measure TMs. Further, we assessed diurnal fluctuations in TM excretions in both sexes and across strains. We found that males excreted more radiolabeled TMs via their feces compared to females. TM excretion patterns did not differ between sexes but TM excretion occurred faster in urinary than fecal samples. Animals excreted TM faster during the night than during the day. Daytime had no effect on the formed TMs; however, males and females formed different TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences between mouse strains. Our non-invasive method to assess fecal TMs can be widely used and will benefit various research disciplines.Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of ³H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17β-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be widely used in various research disciplines like animal behavior, reproduction, animal welfare, ecology, conservation, and biomedicine.

The role of steroid hormones and individual traits in food intake in the wood mouse (Apodemus sylvaticus).

Availability of food resources affects animal survival and reproduction. Thus, coping with changes in food availability is one of the most crucial behavioural and physiological processes in wildlife. Food intake is a key concept in animal ecology that is directly conditioned by food quality and abundance or diet choice, but may also vary according to individual-related factors (e.g. foraging behaviours, social rank or energy-demanding periods) and the influence of the endocrine system on energy metabolism. Here, we studied food intake in relation to individual characteristics (sex, breeding condition and age) and whether steroid hormones (testosterone and corticosterone metabolites) mediate food intake in wild wood mouse (Apodemus sylvaticus). Field work was carried out in February-March 2014 in Monte de Valdelatas (Madrid, Spain). Wood mice were live-trapped for 10 consecutive days in four independent plots. Traps were baited with 4g of toasted corn and food intake was calculated by subtracting the remaining bait found inside traps. Fresh faecal samples from 130 different wood mice were collected and faecal testosterone and corticosterone metabolites (FTM and FCM, respectively) were analysed by enzyme immunoassays. Food intake was higher in females than males, probably due to greater energy requirements. Non-breeders and young individuals also showed a higher food intake. These individuals usually hold a lower social rank which is associated to limited food resources because of dominants; thus, increased food intake may be a result of freely exploit food bait inside traps while avoiding risky competition. In addition, food intake negatively correlated with FTM levels and positively with FCM levels indicating that both hormones have an active role mediating food intake in the wood mouse. Our data suggest that food intake is a function of both individual traits and the endocrine system that accordingly respond throughout different energy-demanding periods.

Evaluation of fecal hormones for noninvasive research on reproduction and stress in humpback whales (Megaptera novaeangliae)

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

Measuring fecal testosterone metabolites in spotted hyenas: Choosing the wrong assay may lead to erroneous results

ABSTRACTEnzyme-immunoassays (EIA) that detect fecal testosterone metabolites (fTM) are powerful tools to monitor gonadal activity non-invasively. However, a challenge with testosterone EIAs might be their potential for cross-reactivities with structurally similar glucocorticoid metabolites. Therefore, we aimed to verify the capability of four different testosterone EIAs to monitor fTM without reflecting changes in adrenocortical activity in spotted hyenas by analyzing fecal samples following testosterone and ACTH challenge tests. We demonstrated that none of the testosterone EIAs is appropriate to measure fTM as all of them showed substantial cross-reactivities to unknown metabolites. Our study underlines the importance of validating androgen EIAs.

Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus)

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs.

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an “in-house” epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using β-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because β-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used β-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.

Harem-holding males do not rise to the challenge: androgens respond to social but not to seasonal challenges in wild geladas.

The challenge hypothesis has been enormously successful in predicting interspecific androgen profiles for vertebrate males. Nevertheless, in the absence of another theoretical framework, many researchers ‘retrofit’ the challenge hypothesis, so that its predictions also apply to intraspecific androgen comparisons. We use a wild primate, geladas (Theropithecus gelada), to illustrate several considerations for androgen research surrounding male contests that do not necessarily fit within the challenge hypothesis framework. Gelada society comprises harem-holding males (that can mate with females) and bachelor males (that cannot mate with females until they take over a harem). Using 6 years of data from known males, we measured androgens (i.e. faecal testosterone (fT) metabolites) both seasonally and across specific male contests. Seasonal androgen variation exhibited a very different pattern than variation resulting from male contests. Although harem-holding males had higher testosterone levels than bachelors across the year, bachelors had higher testosterone during the annual ‘takeover season’. Thus, harem-holding males did not ‘rise to the challenge’ exactly when needed most. Yet, androgen profiles across male contests indicated that both sets of males exhibit the expected fT rise in response to challenges. Results from male geladas also support the idea that the context before (e.g. male condition) and after (e.g. contest outcome) a contest are critical variables for predicting hormones and behaviour.

Sexual maturation in the sumatran rhinoceros (Dicerorhinus sumatrensis)

To help save the Sumatran rhino from extinction, the captive breeding program must capitalize on each rhino's reproductive lifespan. Doing so requires knowing when calves are sexually mature. The goal of this study was to monitor physiological changes associated with sexual maturation in two captive born calves (one male and one female) to determine the approximate age of maturity for both sexes of this species. Fecal testosterone metabolites were monitored in the male calf from 6 months to 7 years of age, and fecal pregnane metabolites were measured in the female calf from 6 months to 5.5 years of age. In addition, rectal ultrasonography was employed to monitor changes in ovarian activity from 2 to 5.5 years of age. The male calf's fecal testosterone concentrations reached levels comparable to those detected in samples from adult males when he was 6-6.5 years of age. The first pre-ovulatory sized follicle was observed on the ovaries of the female calf when she was 4.75 years old, but fecal pregnane metabolite concentrations only reached maximum mean concentrations and variability when she was 5-5.5 years of age. Results from this study indicate that male and female Sumatran rhino calves are sexually mature at 6-6.5 and 5-5.5 years of age, respectively.

Factors Influencing Annual Fecal Testosterone Metabolite Profiles in Captive Male Polar Bears (Ursus maritimus)

The objectives of this study were to assess the effects of season, breeding activity, age and latitude on fecal testosterone metabolite concentrations in captive, adult male polar bears (Ursus maritimus). Fourteen polar bears from 13 North American zoos were monitored for 12-36 months, producing 25-year-long testosterone profiles. Results indicated that testosterone was significantly higher during the breeding season (early January through the end of May) compared with the non-breeding season with the highest concentrations excreted from early January through late March. Variations in excretion patterns were observed among individuals and also between years within an individual, with testosterone peaks closely associated with breeding activity. Results indicate that fecal testosterone concentrations are influenced by season, breeding activity and age, but not by latitude. This is the first report describing longitudinal fecal testosterone metabolite concentrations in individual adult male polar bears.

Patterns of fecal hormones in a fenced population of white‐tailed deer

AbstractAnalysis of voided feces has become a popular method of monitoring physiological parameters of many wildlife species. During the breeding seasons of 2006–2007 and 2007–2008, we collected fecal samples to determine concentrations of fecal glucocorticoid (FG) and fecal testosterone (FT) metabolites in a fenced population of white‐tailed deer (Odocoileus virginianus). The purpose of the study was to determine how concentrations of FG and FT metabolites fluctuated throughout the breeding season, whether there was a difference between males and females, and whether there was a relationship between the two hormones. FG and FT metabolites of males peaked during the rut, while female glucocorticoid levels remained relatively stable throughout the breeding season. We also observed a significant positive correlation between FG and FT metabolites. During the pre‐rut and rut, males that exhibit high testosterone also had elevated glucocorticoid metabolite concentrations, but in the post‐rut there was no relationship between FT and FG concentrations. We hypothesize that the effect of testosterone on glucocorticoid secretion is partially a result of testosterone‐induced behavior, and not strictly a physiological link between the two hormones. © 2012 The Wildlife Society.

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.

Sociodemographic Correlates of Fecal Androgen Levels in Wild Male White-Handed Gibbons (Hylobates lar)

Despite the central role of testosterone in influencing many aspects of the male life cycle, information on the influence of social and behavioral factors associated with androgen output is available for only a few primate species, mainly those living in multimale–multifemale societies. We collected 322 fecal samples and measured fecal testosterone metabolite levels in 19 adult male white-handed gibbons (Hylobates lar) from 13 groups living in the Khao Yai National Park, Thailand, to examine the extent to which androgen output is related to sociodemographic variables (social organization, social status, presence of infants, age) in a socially flexible species, i.e., pair-living/multimale groups. We predicted that androgen levels would be higher in males 1) that live in potentially more unstable single-male/single-female units; 2) that are primary (dominant) vs. secondary; 3) that live in groups with dependent infants; and 4) that are of prime vs. senior age. We found marked differences in androgen levels among males and a significant effect of time of the year. Males living in pairs exhibited significantly higher androgen concentrations vs. males living in multimale groups, and males residing in groups with infants showed a significant increase in androgen levels throughout the sampling year. However, we found no relationship with male age or social status. The increased androgen output found in both pair-living males and those residing with infants might be a physiological mechanism that facilitates male aggression to cope with potential threats posed by social challenges and the demand for infant/group defense, respectively. More generally, our data indicate that in socially flexible species male androgen output is less strongly linked to key life-history variables than in species that live permanently in multimale–multifemale groups.

Testosterone and tusks: maturation and seasonal reproductive patterns of live, free-ranging male dugongs (Dugong dugon) in a subtropical population

Knowledge of male reproductive status and activity in free-ranging animals is vital to understanding reproductive patterns and population dynamics. Until now, almost all information regarding reproductive behavior of the dugong, a cryptic marine mammal, has relied on post-mortem examination. We examined the relationships between body length, tusk eruption (secondary sexual characteristic), seasonality, and group association on fecal testosterone metabolite concentrations in 322 free-ranging dugongs (159 males, 163 females) in subtropical Moreton Bay, Australia. Fecal testosterone concentrations demonstrated biologically meaningful differences in testicular activity between sexes and across reproductive/age classes, and were correlated with circulating concentrations in serum. Male dugongs have a pre-reproductive period that persists until a body length of 240 cm is achieved. Puberty apparently occurs between 240 and 260 cm body length when fecal testosterone levels increase fourfold (>500 ng/g) over juvenile levels, and is associated with tusk eruption. However, social maturity may be delayed until male dugongs are larger than 260 cm with well-developed tusks. In mature males, the lowest (<500 ng/g) fecal testosterone concentrations occur in the austral autumn months with maximal concentrations in September-October, coincident with the onset of a spring mating season. During spring, solitary mature males had fecal testosterone concentrations double those of mature males sampled within groups, potentially suggesting a mating strategy involving roving of reproductively active males. This study demonstrates that single-point physiological data from individuals across a population have value as indicators of reproductive processes. Our approach provides an efficacious non-lethal method for the census of reproductive status and seasonality in live male dugongs.

How does diet affect fecal steroid hormone metabolite concentrations? An experimental examination in red squirrels

A growing number of longitudinal studies in free-ranging animals are measuring fecal steroid hormone metabolite concentrations (FHM). Free-ranging animals can exhibit major seasonal changes in their diet, yet we know relatively little about how diet affects FHM. We experimentally manipulated the diets of female and male North American red squirrels ( Tamiasciurus hudsonicus) to determine how diet affected fecal cortisol (FCM) and androgen (FAM) metabolite concentrations. We measured FCM using an enzyme immunoassay (EIA) that we have previously validated and measured FAM using an assay we have previously validated for use in females and validate for males herein. We validated our EIA to measure FAM in males by identifying that 44.5 ± 0.05% of recovered radiolabeled testosterone was excreted in the feces, our EIA antibody detected the fecal testosterone metabolites, and males with scrotal testes had significantly higher FAM (3.02 ± 0.06 ln ng/g dry feces) than those with abdominal testes (2.73 ± 0.06). We initially fed all squirrels the same diet, but then switched one group of squirrels to a diet consisting of conifer seed ( n = 4 squirrels) whereas the other group was switched to peanut butter ( n = 7). FCM and FAM in squirrels fed conifer seed significantly increased from 0 to 94 h after their diets were changed. FCM in squirrels fed peanut butter significantly declined, whereas FAM declined but not significantly. This demonstrates that change in dietary fiber consumption (peanut butter versus conifer seed) or even slight differences in diet (conifer versus sunflower seeds) can strongly influence FHM.

Effects of season and social interaction on fecal testosterone metabolites in wild male giant pandas: implications for energetics and mating strategies

In the first-ever study of reproductive endocrinology in wild male giant pandas (Ailuropoda melanoleuca), we provide new insights into the reproductive ecology of the species. We tracked and observed pandas in Foping Nature Reserve of the Qinling Mountains for 3 years, collecting fecal samples for testosterone metabolite analysis and data on reproductive activity. Males encountered multiple potential mates and competed for reproductive access to females. Male testosterone metabolites increased in February, peaked in March and April, and fell back to baseline after the mating season. However, males did not maintain a high testosterone level throughout the mating season. Male testosterone instead peaked during encounters with potential mates and declined between encounters. These results indicate that testicular activity is typically dormant until mobilized by interactions with females and potentially by interactions with male competitors. This suggests that male pandas may be energetically constrained, elevating testosterone levels only when necessary to meet the demands of intrasexual competition and courtship and fertilization of females. Maintaining a high testosterone level is metabolically expensive and male pandas enter the mating season during a period of low food availability. If this hypothesis is correct, male panda body condition may be an important determinant of reproductive outcome, and anthropogenic activities that diminish foraging resources may have significant impacts on the mating ecology of the species, affecting its conservation.

The reproductive endocrinology and behavior of Vancouver Island marmot (Marmota vancouverensis)

The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America's most endangered species with fewer than 150 individuals remaining in the wild. A captive breeding program was established across four facilities in Canada as an insurance population and source of animals for reintroduction to the wild. The purpose of this study was to gather information about the basic reproductive biology and behavior of this species, which is essential to improve captive breeding programs. Regular fecal samples were obtained from adult female (n = 14) and male (n = 10) marmots, 2 years of age and older, over 1-3 breeding seasons (2-3 months duration posthibernation) for steroid hormone analysis. Enzyme immunoassays were validated for quantifying fecal testosterone metabolite concentrations for males, and fecal estrogen and progesterone metabolite concentrations for females. Results indicated that fecal progesterone metabolite concentrations can be used to monitor ovulation and pregnancy. Behavioral monitoring through infrared video surveillance was conducted in four breeding pairs over a 2-year period (n = 7 behavioral profiles). Breeding behaviors correlated strongly with changes in reproductive endocrine profiles. A high frequency of play behavior or "wrestling" was observed in conjunction with breeding activity before an elevation in progesterone metabolite concentrations. Impending parturition was associated with increased aggression and exclusion of the male from the maternal nestbox as well as an increase in nesting activity. Observational data combined with hormonal analysis suggest that female VIMs are induced ovulators and that multiple breeding attempts may be required for ovulation and conception. Gestation appears to be approximately 34 days from peak breeding activity (32 days from estimated ovulation). Fecal testosterone concentrations suggest that testicular activity is seasonal with the reproductive activity occurring immediately posthibernation. Monitoring breeding behavior is a useful means of indicating estrus, conception and pregnancy, which can also be supported by the hormonal analysis of daily fecal samples of individual animals.