The first record of Aelurostrongylus abstrusus (Angistrongylidae: Nematoda) in Eurasian lynx (Lynx lynx L.) from Poland based on fecal analysis

MATERIAL: Thirty eight fecal samples of Eurasian Lynx (Lynx lynx L.) collected in Bia?owieza Primeval Forest (E Poland) in years 2001-2004 were analysed. RESULTS: The presence of Aelurostrongylus abstrusus (L1) larvae was evidenced by use of decantation and flotation methods. The general prevalence of the infection recorded during the study was 21.1%, whereas mean intensity was 11,5 (1-33 larvae per sample). To our knowledge, this is the first case of Aelurostrongylus abstrusus recorded in Euroasian lynx from Poland.     

Comparative metabolism of gestagens and estrogens in the four lynx species, the Eurasian (Lynx lynx), the Iberian (L. pardinus), the Canada lynx (L. canadensis) and the bobcat (L. rufus)

With the increasing prevalence of faecal hormone metabolite analysis, it is important to develop a better understanding of the dynamics of faecal metabolite composition. The aim of this study was to compare the quantitative faecal gestagen and estrogen metabolite composition in the four lynx species: Eurasian lynx, Iberian lynx, Canada lynx and bobcats. Comparative HPLC immunograms were generated from faecal samples collected before, during, and after pregnancy from individual females of each lynx species. Gestagens and estrogens revealed three similar classes of immunoreactive faecal metabolites: (1) polar metabolites which were enzyme-hydrolysable and thus may be designated as conjugates, (2) non-hydrolysable polar metabolites, and (3) non-polar metabolites or free steroids. For both hormones, strong similarities in the HPLC immunograms across species suggests that steroid metabolism is relatively conserved among Lynx species. Gestagens were primarily excreted as polar conjugates or unknown metabolites, whereas estrogen metabolism revealed a huge proportion (∼50%) consisting of 17β-estradiol and estrone. These results are consistent with patterns of steroid metabolism in other felid species. Only two minor species-specific patterns emerged. In bobcats, we observed an exceptionally high proportion of gestagen conjugates, and in Iberian lynx, there was an exceptionally high proportion of estrone. The comparison of HPLC immunograms within individuals revealed that intra-individual variations in steroid metabolite composition are considerably high. However, changes in metabolite composition did not correlate with specific reproductive stages; rather, they seemed to occur at random. We assume that these differences may reflect changes in liver metabolism and/or qualitative and quantitative variations in gut bacteria composition, resulting in differences in faecal metabolite composition.     

Comparative aspects of the metabolism and excretion of cortisol in three individual nonhuman primates

A radiometabolism study is described to provide the first comparative data on the time course, route, and characteristics of excreted [3H]cortisol metabolites in three nonhuman primates: the common marmoset (Callithrix jacchus), the long-tailed macaque (Macacafascicularis), and the chimpanzee (Pan troglodytes). A low dose (40-100 microCi) of 3H-labeled cortisol was administered intravenously to one adult male of each species and the excreta collected over a 5-day period postinjection. The major proportion of radioactivity was excreted in the urine (>80%). Peak radioactivity in urine was recovered within 5.5 h following injection in all three species, while in the feces peak levels of radioactivity were recovered within 26 h postinjection. In all three species, urinary metabolites were primarily excreted as conjugates (61-87%), whereas the percentage of conjugated metabolites in feces was 50% or less. The number and relative abundance of urinary and fecal [3H]cortisol metabolites were determined by reverse-phase high-performance liquid chromatography (HPLC) and immunoreactivity of the radioactivity peaks was assessed by screening HPLC fractions with established cortisol, corticosterone, and 11-oxoetiocholanolone enzyme immunoassays (EIA), the latter being a group-specific assay for measuring 11,17-dioxoandrostanes. HPLC separation of urinary and fecal extracts revealed multiple peaks of radioactivity, several of which were common to all three species. The relative proportion of these peaks, however, differed considerably among species and between urine and feces. HPLC indicated that native cortisol was a major urinary excretory product in the marmoset, while comparatively small amounts were present in the urine of the macaque and chimpanzee. In contrast, in feces, cortisol was only detected in low amounts in the marmoset and was virtually absent in the macaque and chimpanzee. In all three species, one of the major radioactivity peaks showed a retention time comparable to 11-oxoetiocholanolone and high immunoreactivity in the 11-oxoetiocholanolone EIA. The measurement of urinary- and/or fecal-immunoreactive 11,17-dioxoandrostanes is therefore implicated for noninvasive assessment of adrenal function in Old World monkeys, New World monkeys, and great apes.     

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

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

Noninvasive monitoring of adrenocortical activity in carnivores by fecal glucocorticoid analyses

Measurement of glucocorticoid metabolites in feces has become an accepted method for the noninvasive evaluation of adrenocortical activity. The objective of this study was to determine if a simple cortisol enzyme immunoassay (EIA) was suitable for monitoring adrenocortical activity in a variety of carnivore species. Performance of the cortisol EIA was gauged by comparison to a corticosterone radioimmunoassay (RIA) that has been used for measuring glucocorticoid metabolites in feces of numerous species. Tests for parallelism and extraction efficiency were used to compare the cortisol EIA and corticosterone RIA across eight species of carnivores (Himalayan black bear, sloth bear, domestic cat, cheetah, clouded leopard, black-footed ferret, slender-tailed meerkat, and red wolf). The biological relevance of immunoreactive glucocorticoid metabolites in feces was established for at least one species of each Carnivora family studied with an adrenocorticotropic hormone (ACTH) challenge. High performance liquid chromatography (HPLC) analysis of fecal extracts for each species revealed (1) the presence of multiple immunoreactive glucocorticoid metabolites in feces, but (2) the two immunoassays measured different metabolites, and (3) there were differences across species in the number and polarities of metabolites identified between assay systems. ACTH challenge studies revealed increases in fecal metabolite concentrations measured by the cortisol EIA and corticosterone RIA of approximately 228-1145% and approximately 231-4150% above pre-treatment baseline, respectively, within 1-2 days of injection. Concentrations of fecal glucocorticoid metabolites measured by the cortisol EIA and corticosterone RIA during longitudinal evaluation (i.e., >50 days) of several species were significantly correlated (P<0.0025, correlation coefficient range 0.383-0.975). Adrenocortical responses to physical and psychological stressors during longitudinal evaluations varied with the type of stimulus, between episodes of the same stimulus, and among species. Significant elevations of glucocorticoid metabolites were observed following some potentially stressful situations [anesthesia (2 of 3 subjects), restraint and saline injection (2 of 2 subjects), restraint and blood sampling (2 of 6 episodes), medical treatment (1 of 1 subject)], but not in all cases [e.g., gonadotropin injection (n=4), physical restraint only (n=1), mate introduction/breeding (n=1), social tension (n=1), construction (n=2) or relocation (n=1)]. Results reinforced the importance of an adequate baseline period of fecal sampling and frequent collections to assess adrenocortical status. The corticosterone RIA detected greater adrenocortical responses to exogenous ACTH and stressful exogenous stimuli in the Himalayan black bear, domestic cat (female), cheetah, clouded leopard, slender-tailed meerkat, and red wolf, whereas the cortisol EIA proved superior to resolving adrenocortical responses in the black-footed ferret and domestic cat (male). Overall results suggest the cortisol EIA tested in this study offers a practical method for laboratories restricted in the usage of radioisotopes (e.g., zoological institutions and field facilities) to integrate noninvasive monitoring of adrenocortical activity into studies of carnivore behavior and physiology.     

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

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

Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice

Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.     

Androgen and androgen metabolite levels in serum and urine of East African chimpanzees (Pan troglodytes schweinfurthii): comparison of EIA and LC-MS analyses

The primary male androgen testosterone (T) is often used as an endocrinological marker to investigate androgen-behaviour interactions in males. In chimpanzees and bonobos, studies investigating the relationship between T levels and dominance rank or aggressive behaviour have revealed contradictory results. The immunoassays used in these studies were originally developed for the measurement of steroids in serum. Their application to non-invasively collected samples, however, can lead to methodological problems due to cross-reacting metabolites, which might occur in urine or faeces but not in blood. The overall aim of this study, therefore, is to clarify whether a T enzyme immunoassay (EIA) is an applicable method to monitor testicular function in adult male chimpanzees. To estimate the impact of cross-reacting androgens on the used T EIA, we compared the results of an EIA measurement with a set of androgen metabolite levels measured by LC–MS. In urine from male chimpanzees, cross-reactivities appear to exist mainly with T and its exclusive metabolites, 5α-dihydrotestosterone (5α-DHT) and 5α-androstanediol (androstanediol). Both urinary and serum T levels of male chimpanzees were significantly higher than female T levels when measured with the T EIA, indicating a reliable measurement of testicular androgens and their exclusive metabolites with the used EIA. In urine from female chimpanzees, the comparison between LC–MS and T EIA results indicated a higher impact of cross-reactions with adrenal androgen metabolites. Therefore, the investigation of urinary T levels in female chimpanzees with a T EIA seems to be problematic. Overall our results show that a T EIA can be a reliable method to monitor testicular function in male chimpanzee urine and that LC–MS is a valuable tool for the validation of immunoassays.     

Patterns of ovarian and luteal activity in captive and wild Canada lynx (Lynx canadensis)

Canada lynx face some unique breeding restrictions, which may have implications for population viability and captive management. The goal of this study was to improve our understanding of basic reproductive physiology in Canada lynx. Using fecal hormone metabolite analysis, we established normative patterns of fecal estrogen (fE) and progestagen (fP) expression in captive and wild female Canada lynx. Our results indicate that Canada lynx have persistent corpora lutea, which underlie their uncharacteristic fP profiles compared to other felids. Thus, fP are not useful for diagnosing pregnancy in Canada lynx. We also found that Canada lynx are capable of ovulating spontaneously. Captive females had higher concentrations of fE and fP than wild females. Both populations exhibit a seasonal increase in ovarian activity (as measured by fE) between February and April. Finally, there was evidence of ovarian suppression when females were housed together.     

Patterns of testicular activity in captive and wild Canada lynx (Lynx canadensis)

Canada lynx are listed as a threatened species in the contiguous US. Understanding the reproductive characteristics (i.e., mating system, behavior, physiology) of a species is useful for ensuring effective in situ and ex situ management plans. The goal of this study was to describe patterns of androgen expression in both captive and wild male Canada lynx using fecal hormone metabolite analysis. Among captive lynx, juvenile and castrated males had lower concentrations of fecal androgens (fA) than intact males, thereby demonstrating that the assay detects biologically meaningful differences in testicular activity. We found that captive males in general had much higher fA levels than wild males. All males showed strong seasonal variation in fA concentrations, with significantly higher levels being expressed during the breeding season (February and March) than during the non-breeding season. Among captive males, variation in seasonal fA levels did not correlate with latitude. Finally, males housed with intact cage-mates (either male or female) had significantly higher fA levels than males housed alone or with a neutered cage-mate.     

Characterization of urinary and fecal metabolites of testosterone and their measurement for assessing gonadal endocrine function in male nonhuman primates

The aims of the present study were (i) to provide basic comparative data on the time course, route, and characteristics of excreted [14C]testosterone (T) metabolites in three nonhuman primates: the common marmoset (Callithrix jacchus), the long-tailed macaque (Macaca fascicularis) and the chimpanzee (Pan troglodytes) and (ii) to use this information to help validate the measurement of urinary and fecal testosterone metabolites for assessing androgen status in Anthropoid primates. Radiolabeled 14C-T (10-30 microCi) was injected intravenously into one adult male of each species and the excreta collected over the next 5 days. Peak radioactivity in urine was detected within 2h and accounted for 67% (Mf), 80% (Cj) and 91% (Pt) of the total radioactivity recovered. The time course of excretion of radioactivity in feces showed a higher variation between species (4-26 h to peak values). In all three species, the majority (>90%) of urinary metabolites were excreted as conjugates whereas the proportion of conjugated metabolites in feces was substantially lower and more variable. High pressure liquid chromatography (HPLC) analysis of urinary and fecal extracts revealed multiple peaks of radioactivity in all three individuals, but each with a distinctive pattern. Native T was excreted in only small amounts into the urine, whereas it was virtually absent in the feces of all three individuals. Three C17 group-specific enzymeimmunoassays using antisera against testosterone, 5alpha-androstane-17alpha-ol-3-one and androsterone were evaluated for their ability to discriminate immunoreactive androgen levels between intact males, castrated males and females based on measurements in urine and feces. In the marmoset, all assays (except for T in feces) clearly discriminated between test groups; in the chimpanzee significantly higher levels of androgen immunoreactivity in intact versus castrated males were measured in urine, but not feces. In the macaque, only the 5alpha-androstanolone measurement in feces discriminated between groups. Data on the results of a radiometabolism study using 3H-DHEA (a weak adrenal androgen) in a long-tailed macaque suggested that co-measurement of metabolites derived from T and DHEA in the assays tested might explain the difficulties in discriminating gonadal status in the two Old World primate species. Collectively, the data show that T metabolism in primates is highly complex and that no single method for noninvasive assessment of androgen status can be used for application across species. The importance of a proper validation of the methodology for each species is emphasised.     

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

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

Analysis of fecal glucocorticoids in the North Atlantic right whale (Eubalaena glacialis)

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

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.     

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

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

Non-invasive measurement of adrenocortical and gonadal activity in male and female guinea pigs (Cavia aperea f. porcellus)

Taking blood samples is a common method in biomedical and biological research using guinea pigs. However, most blood sampling techniques are complicated and highly invasive and may therefore not be appropriate for certain research topics concerning stress and reproduction. Thus, a non-invasive method to measure steroid hormones is critically needed. The aim of this study was the biological validation of corresponding enzyme immunoassays for the measurement of fecal cortisol, progesterone, estrogen, and testosterone metabolites in guinea pigs. We examined the effect of subcutaneous injections of ACTH or saline on fecal cortisol metabolites to investigate the suitability of fecal samples to monitor adrenocortical activity. Furthermore, we investigated whether fecal sex steroid metabolites accurately reflected endocrine changes observed in plasma samples during female estrous cycles and male puberty, respectively. In addition, we compared fecal testosterone metabolites of intact males, castrated males, and females to investigate the reliability of fecal samples in discriminating gonadal status of males. Concentrations of fecal cortisol metabolites were significantly increased following ACTH challenge, indicating that adrenocortical activity can be monitored via fecal samples. Secondly, in females, plasma and fecal gonadal steroids were significantly correlated in most subjects. The assay for testosterone metabolites, on the other hand, could not clearly discriminate between test groups. From these findings we conclude that fecal samples can be used for the non-invasive assessment of adrenocortical and female reproductive status in guinea pigs. Testosterone metabolism seems to be more complex and further investigations are needed to establish a more suitable assay.     

Low serum testosterone/dihydrotestosterone ratio in patients with pancreatic carcinoma

Serum testosterone, its metabolite 5 alpha-dihydrotestosterone, and the testosterone/dihydrotestosterone ratio were investigated in 22 male patients with proven pancreatic cancer, and compared with values from male patients with chronic pancreatitis (n = 21) and with nonpancreatic gastrointestinal tumors (n = 19). Testosterone and the testosterone/dihydrotestosterone ratio were significantly lower (p less than 0.001) in the pancreatic cancer group when they were compared with the other two groups. There was no significant difference in the dihydrotestosterone values between cancer groups. A testosterone/dihydrotestosterone ratio of less than 5 clearly distinguished most of the patients (20/22) with cancer of the pancreas from those with other tumors or chronic pancreatitis. The results suggest an alteration in the serum androgen profile in these patients. Therefore, the testosterone/dihydrotestosterone ratio could be a useful marker in the diagnosis of pancreatic carcinoma in male patients.     

Snow conditions may create an invisible barrier for lynx

The dynamics of Canadian lynx (Lynx canadensis) abundance are geographically structured according to the influence of large-scale climatic regimes. Here we demonstrate that this structuring matches zones of differential snow conditions, in particular surface hardness, as determined by the frequency of winter warm spells. Through a modified functional response curve, we show that various features of the snow may influence lynx interaction with its main prey species, the snowshoe hare (Lepus americanus). This study highlights the importance of snow, and exemplifies how large-scale climatic fluctuations can mechanistically influence population biological patterns.     

Noninvasive monitoring of ovarian endocrine activity in the chinchilla (Chinchilla lanigera)

Reproductive endocrinology information is limited for Chinchilla lanigera, a South American species characterized by extremely long gestation and estrus cycle compared with others rodents. This study was designed to validate a non-invasive technique for monitoring ovarian endocrine activity. Animals were exposed indoors to natural photoperiod (31 degrees S-64 degrees W, Argentina); temperature range: 17-26 degrees C, with food and water ad libitum. Radiolabelled infusion (n=4): (3)H-estradiol ((3)H-E(2)) and (14)C-progesterone ((14)C-P(4)) were injected (i.p). Biochemical validation: HPLC-UV detector was employed to determine natural steroids in urine and fecal extracts and to determine immunoreactive metabolites. Physiological validation: (1) pregnancy (n=5): body weight and urinary and fecal steroidal metabolites were measured until birth; (2) Seasonality (n=9): urine and feces were collected in May, August, November, and February. Total (3)H-E(2) and (14)C-P(4) radioactivity recovered was 60.5+/-15.5 and 74.5+/-19.4%, respectively. After (3)H-E(2) injection, urinary radioactivity peaked at 7.0+/-0.6 hr; in contrast, urinary (14)C-P(4) excretion peaked at 44.0+/-4.0 hr (p=0.000). Peak radioactivity in feces occurred between 24-48 hr for both hormones. Several correlations were detected during pregnancy between body weight vs. urinary progestagens/day (r=0.44, p<0.03); vs. urinary progestagens/creatinine (r=0.73, p=2.9 x 10(-5)); vs. urinary estrogens/day (r=0.74; p<0.2); and vs. urinary estrogens/creatinine (r=0.74; p<2.0 x 10(-5)). On the other hand, urinary and fecal progestagen excretion exhibited significant seasonal fluctuations and urinary estrogen concentrations showed a similar pattern (p=0.062 for winter-spring vs. summer-autumn). This methodology proved to be useful for monitoring ovary endocrine activity in urine of chinchilla female.     

Purging of deleterious burden in the endangered Iberian lynx

Deleterious mutations continuously accumulate in populations, building up a burden that can threaten their survival, particularly in small populations when inbreeding exposes recessive deleterious effects. Notwithstanding, this process also triggers genetic purging, which can reduce the deleterious burden and mitigate fitness inbreeding depression. Here, we analyzed 20 whole genomes from the endangered Iberian lynx and 28 from the widespread Eurasian lynx, sister species which constitute a good model to study the dynamics of deleterious mutation burden under contrasting demographies, manifested in the consistently smaller population size and distribution area of the Iberian lynx. We also derived analytical predictions for the evolution of the deleterious burden following a bottleneck. We found 11% fewer derived alleles for the more putatively deleterious missense category in the Iberian lynx than in the Eurasian lynx, which, in light of our theoretical predictions, should be ascribed to historical purging. No signs of purging were found in centromeres nor in the X chromosome, where selection against recessive deleterious alleles is less affected by demography. The similar deleterious burden levels for conspecific populations despite their contrasting recent demographies also point to sustained differences in historical population sizes since species divergence as the main driver of the augmented purging in the Iberian lynx. Beyond adding to the ongoing debate on the relationship between deleterious burden and population size, and on the impact of genetic factors in endangered species viability, this work contributes a whole-genome catalog of deleterious variants, which may become a valuable resource for future conservation efforts.

In the last few centuries, the ever-growing anthropogenic pressures (1) have significantly accelerated the extinction of populations and species across a wide spectrum of taxa (2–4). Small populations are particularly susceptible to environmental and demographic stochastic events (5), as well as to negative genetic processes such as genetic drift, inbreeding depression, the accumulation of deleterious variation, and the loss of adaptive potential, which jeopardize their persistence (6). Most worryingly, these genetic hazards may persist even when the original cause of population decline is removed (7), creating a positive feedback loop as the population spirals toward extinction (8).Albeit highly relevant for conservation and evolutionary biology, the dynamics of genetic load remain poorly understood. Classically, this load has been defined in terms of consequences on population fitness, although it can also be defined at the individual level. It includes the reduction of expected fitness due to segregating deleterious alleles (i.e., the expressed load), the load from recessive deleterious effects hidden in heterozygosis (i.e., the inbreeding load) responsible for inbreeding depression, and the slow fitness decline due to the continuous fixation of deleterious mutations (9). Characterizing these components of the genetic load is not an easy task, since measuring fitness in wild populations is extremely difficult (10). However, nowadays, whole-genome sequencing offers the opportunity to gain insights into the load of wild populations using a number of genomic summaries, such as the average number of (segregating plus fixed) derived alleles per individual (10), hereafter the derived count. This is possible provided there exists a good reference annotation that allows arranging the identified mutations into rough deleteriousness categories according to the genomic feature they lay in, their predicted effect on protein function, and the degree of conservation of the site across taxa (11). For deleterious categories, the derived count in individuals provides an appropriate and robust measure of the deleterious burden that can be interpreted at the individual or population level. Since it takes into account the burden from alleles occurring in heterozygosis, this measure is related to the whole fitness load, including the hidden inbreeding load. Therefore, the more recessive the deleterious effects, the looser the relationship between this count and the fitness load expressed in the individuals becomes (see ref. 10). When applied to populations with a common origin but contrasting demographies, such a genomic empirical approach can improve our understanding of how genetic and demographic factors shape the deleterious burden of small, endangered populations (12), which, in turn, may lead to more effective management. In these populations, genetic purging, that is, the increased selective pressure against (partially) recessive deleterious alleles due to increased homozygosis, is expected to translate into a reduction in both the inbreeding load (13) and the derived count in deleterious categories with prevailing recessive fitness effects. At the same time, the reduced efficacy of purifying selection caused by drift is expected to increase the derived count of alleles with small deleterious effects (14, 15). Therefore, genomic analysis can help to understand the relative roles of the purging of deleterious alleles and the drift-driven relaxation of natural selection in the evolution of the deleterious burden after a reduction in population size, and provide insights into their fitness consequences. In recent years, several studies focused mainly on the genetic consequences of the Out-Of-Africa bottleneck have contributed to the ongoing debate on whether human populations differ in their genetic load (10, 16–20). Only a few studies, however, have seized upon the power of genomics to assess the deleterious burden in small, endangered wild populations of nonmodel organisms (21–23).The Iberian lynx (Lynx pardinus) and the Eurasian lynx (Lynx lynx) are the two extant species of the Lynx genus in Eurasia. After their divergence, dated around 1.1 million years ago (24, 25), they followed quite parallel demographic trajectories; however, Iberian lynx population sizes remained consistently smaller than those of the Eurasian lynx (26). Nowadays, they show highly contrasting distribution areas and conservation statuses. The Iberian lynx, a habitat and prey specialist endemic to the Iberian Peninsula, went through several bottlenecks and has maintained small population sizes throughout its recent history. The extreme and well-documented decline during the second half of the 20th century (27–29) resulted in the extirpation of all populations except for two in southern Spain: 1) a central population in Sierra de Andújar in the Sierra Morena range (AND), which maintained a comparatively larger population size and remained well connected to other populations until the mid-20th century, and 2) a smaller, peripheral population in the protected area of Doñana (DON), which has been effectively isolated for around two centuries (30, 31). The species was classified as “critically endangered” in the 2002 and 2008 International Union for Conservation of Nature (IUCN) red lists (32) after its global population dwindled to less than 100 individuals. Nonetheless, active conservation efforts, which include in situ, ex situ, and reintroduction programs (33, 34), have since kick-started a remarkable recovery which led to the downlisting of the species to “endangered” in the 2015 IUCN red list (32), and have propelled its population to 1,100 free-living lynxes in 2020 (35). Genetic erosion in the species is well documented (26, 31, 36, 37), with studies on mitochondrial, microsatellite, and genome-wide data providing evidence of a high inbreeding rate, high differentiation between the two populations, and a whole-genome species-wide nucleotide diversity (π = 0.026%) that is among the lowest reported thus far, and comparable to the single most genetically eroded population of the island fox (Urocyon littoralis) (38). Furthermore, there are documented signs of high inbreeding load in the species (39) and inbreeding depression in the DON population (36).In contrast, the Eurasian lynx is one of the most widespread felids in the world, with a distribution area that extends from eastern Asia to central Europe and covers a wide range of habitats. It is currently classified at the global level as “least concern” by the IUCN. However, recently inferred demographic trajectories have revealed a generalized decline across its range during the last millennia (40). This decline has been particularly intense in the European part as a consequence of higher anthropogenic pressure (40), to the point where many of the western-most populations became extirpated during the 19th and 20th centuries, and some of the remaining ones, such as those in Norway (NOR) and the Białowieża and Knyszyn Primeval Forests in northeast Poland (POL), underwent severe bottlenecks and complete genetic isolation for decades (41, 42). Meanwhile, the Kirov (European Russia) population (KIR), which is part of a large continuous patch of the species in eastern Europe, has maintained a relatively large size and good connectivity with neighboring populations (40, 43). Even so, genetic diversity for KIR (π = 0.046%) is barely twice as high as that of the Iberian lynx and still below that of severely bottlenecked populations of other species, such as the Apennine brown bear, the Scandinavian wolverine, and the Amur tiger (40).The contrasting demographic histories of the Iberian and the Eurasian lynx, coupled with the availability of a reference genome for the former species, showcases this dyad as a suitable model for carrying out genome-wide studies of the dynamics of the deleterious burden in bottlenecked populations. We specifically intend to address four questions: 1) whether there are empirically measurable differences in the amount or the patterns of the derived count between the Iberian lynx and the Eurasian lynx; 2) whether such differences extend to populations within each species; 3) whether differences in the derived count are dominated by the relaxation of purifying selection or by genetic purging; and 4) how these differences vary across genomic features, mutation effect categories, and genomic regions with contrasting efficiency of natural selection. To better interpret the observed patterns, we also extended the previous theoretical model on the purging of the inbreeding genetic load (13, 44) to develop analytical expressions predicting the evolution of the derived count, and we obtained theoretical projections under demographic scenarios similar to those inferred for the two lynx species. Beyond broadening our understanding of genetic processes in populations at the verge of extinction, such insights could be critical to predict the fate of small lynx populations, and to improve the efficacy of ongoing conservation programs.