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Understanding how fecal glucocorticoid (GC) concentration is associated with reproduction in wild animals allows us to associate physiological stress with the costs of reproduction. Glucocorticoids are among the main stress-related hormones, and their secretion is strongly associated with reproductive seasonality. Using non-invasive methods (thereby avoiding causing stress), we used fecal GC metabolites (FGC) to test the hypothesis that the reproductive phase (mating, non-mating, gestation, and lactation) influences stress levels of the seasonally reproductive Pampas Deer (Ozotoceros bezoarticus). Furthermore, we compared FGC between sexes and between males of different antler statuses (velvet, hard, cast). During 1 year, in the Pantanal of Brazil, we collected 621 fresh fecal samples (327 from females, 294 from males) from which we estimated FGC using enzyme immunoassay (EIA). FGC concentrations varied by reproductive phase and antler status. Agonistic and courtship interactions associated with mating (i.e., fights between males, attempts to mount females), suggest that they influence FGC concentrations most strongly in both sexes. Females also had greater FGC concentrations during lactation, suggesting that this phase and parental care are also associated with increased physiological stress. In males, the association of FGC concentrations with antler status may be, in part, associated with photoperiod and testosterone secretion, both of which can trigger reproductive and agonistic behaviors. Finally, reproductive synchrony of the sexes causes similar FGC over time and suggests that environmental factors contribute as well. We show that non-invasive monitoring of glucocorticoid metabolites levels is an effective tool for detecting changes in the physiological stress response in Pampas Deer, suggesting that this tool will be useful for detecting changes in physiological stress caused by human disturbance, such as cattle ranching in Pantanal and similar disturbances elsewhere. [ABSTRACT FROM AUTHOR]

• Validated water-borne hormone assay for cortisol and corticosterone in tadpoles. • Corticosterone release rates begin to decline 2 h post stressor in tadpoles. • Corticosterone release rates show moderate to high repeatability in tadpoles. • Rearing tadpoles in groups or individually are viable methods for repeated measures. Amphibian populations are declining globally, so understanding how individuals respond to anthropogenic and environmental stressors may aid conservation efforts. Using a non-invasive water-borne hormone assay, we measured the release rates of two glucocorticoid hormones, corticosterone and cortisol, in Rio Grande Leopard frog, Rana berlandieri , tadpoles. We validated this method pharmacologically and biologically using an adrenocorticotropic hormone (ACTH) challenge, exposure to exogenous corticosterone, and an agitation test. We calculated the repeatability of hormone release rates, the recovery time from an acute stressor, and explored rearing methods for tadpoles. Tadpole corticosterone release rates increased following an ACTH challenge, exposure to exogenous corticosterone, and agitation, validating the use of water-borne hormone methods in this species. After exposure to an acute stressor via agitation, corticosterone release rates began to decline after 2 h and were lowest after 6 h, suggesting a relatively rapid recovery from an acute stressor. Tadpoles reared in groups had higher corticosterone release rates than tadpoles reared individually, and lost mass by Day 7, while tadpoles reared individually did not show a stress response, therefore either rearing method is viable, but have differing physiological costs for tadpoles. Repeatability of corticosterone release rates was moderate to high in R. berlandieri tadpoles, indicating that this species can show a response to selection and potentially respond to rapid environmental change. Our results show that the water-borne hormone assay is a viable way to measure glucocorticoids in this species and is useful in the field of conservation physiology for rare and endangered species. [ABSTRACT FROM AUTHOR]

Minimally invasive methods for estimating hormone concentrations in wild vertebrates offer the opportunity to repeatedly measure behavior and hormone concentrations within individuals while minimizing experimenter interference during sample collection. We examined three steroid hormones (corticosterone, CORT; 17-β estradiol, E 2 ; progesterone, PROG) in túngara frogs ( Physalaemus pustulosus ) using non-invasive water-borne methods. Using solid-phase extraction of water samples and liquid extraction of plasma and homogenate samples, coupled with enzyme immunoassays, we complimented the conventional validation approaches (parallelism, recovery determination) with dose-response assays that incorporated pharmacological challenges with adrenocorticotropic hormone (ACTH) and human chorionic gonadotropin (HCG). We also compared steroid concentrations in water to those observed in plasma and whole body homogenates. Lastly, we identified the constituent steroids in each sample type with a panel targeting 30 steroid species using high performance liquid chromatography-mass spectrometry (HPLC-MS). We found that a 60-min water-bath captures physiologically relevant changes in concentrations of CORT, E 2 and PROG. Peak levels of water-borne CORT were found at approximately 2 h after ACTH injection. Water-borne CORT and E 2 concentrations were positively correlated with their plasma and homogenate equivalents, while water-borne PROG was uncorrelated with homogenate PROG concentrations but negatively correlated with homogenate E 2 concentrations. Together, our findings indicate that sampling water-borne hormones presents a non-invasive and biologically informative approach that will be useful for behavioral endocrinologists and conservation physiologists. [ABSTRACT FROM AUTHOR]

The interests of males and females in mating contexts often conflict, and identifying the information conveyed by sexual signals is central to understanding how signalers manage such conflicts. Research into the information provided by female primate sexual signals has focused on exaggerated anogenital swellings as either reliable-indicators of reproductive quality (reliable-indicator hypothesis) or probabilistic signals of fertility (graded-signal hypothesis). While these morphological signals are mostly confined to catarrhine primates, these hypotheses are potentially widely applicable across primates, but have not been tested in taxa that lack such morphological signals. Here, we tested these hypotheses in wild black capuchins ( Sapajus nigritus ), a species in which females lack morphological sexual signals but produce conspicuous behavioral estrous displays. Specifically, we examined the proportion of time different females spent producing these signals with respect to measures of female quality (dominance rank, parity, age-related fecundity and cycle type) and in relation to the timing of fertility, as determined by analysis of fecal progesterone. Time spent displaying did not vary across females based on measures of female quality, but increased with the approach of ovulation. Further, male mating effort varied according to the timing of female fertility. Proceptive behaviors in this species thus meet predictions of the graded-signal hypothesis, providing the first support for this hypothesis based solely on behavioral signals. [ABSTRACT FROM AUTHOR]

• Dietetic fiber affects fecal androgen metabolites detection. • High dietetic fiber enhances fecal androgen metabolites detection. • A rich-fiber diet increases fecal production. • Defecation rate, and passage and retention times are similar in experimental diets. Non-invasive endocrinology is an important tool for animal conservation, but its success depends on many factors (e.g. adequate hormonal extraction, diet, antibody used in the assay). Dietary fiber is one of the main sources that can lead to erroneous interpretation of the endocrine status provided by EIA analysis. This study aimed to evaluate the dietary fiber effect on the fecal androgen metabolites (FAM) detection, on the daily defecation rate and fecal production, as well as to analyze the gastrointestinal passage and retention time of the experimental diets. Eight brown brocket deer (Mazama gouazoubira) males were randomly assigned to two groups and submitted to both isocaloric and isoproteic experimental diets for 10 days, in a crossover system: low fiber percentage feed (LF, 7% fiber) and high fiber percentage feed (HF, 19% fiber). Such groups were alternated in the middle of this period, with an interval of 10 days between them. In addition, there was a five-day adaptation phase at the beginning of each diet. Fecal collection for FAM measurement was performed during 10 days of treatment, whereas, the defecation rate and fecal production were performed every two hours, for 6 days. The mean FAM level in the HF group was 5038.0 ± 1529.1 ng/g, while for LF, 2178.7 ± 824.9 ng/g (p < 0.05). The mean HF fecal production was 182.6 ± 36.2 g DM/day and 117.5 ± 12.6 g DM/day for LF (p < 0.05). There were no differences in terms of mean defecation rate, passage, and retention times between groups. The results suggest that dietary fiber affects the FAM detection, and this should be taken into consideration before conducting experiments using fecal samples as a source of reproductive hormones profiling. [ABSTRACT FROM AUTHOR]

Abstract: Non-invasive endocrine monitoring is an important conservation tool frequently used to support the management of wildlife species. Investigating ovarian activity and pregnancy rates of free-ranging African elephants can enhance our knowledge about the physiology of this highly social and long-lived species. The utility of a new, field-friendly progestagen enzymeimmunoassay (EIA) method was tested by monitoring ovarian activity of 40 females in Addo Elephant National Park (AENP), South Africa. Fecal samples were collected opportunistically from known individuals (June 2007–September 2009) and fecal progestagen metabolites (FPM) were extracted in the field and analyzed on-site using a visual, qualitative EIA. Those results were compared to the laboratory quantitative EIA assessment of hormone concentrations in the U.S. Qualitative FPM concentrations were strongly correlated with quantitative concentrations (rs =0.72). Fecal samples collected from pregnant elephants (n =22) had significantly higher FPM concentrations than non-pregnant individuals, and month of gestation was the strongest predictor of FPM concentrations. Endocrine profiles suggested that five older females were no longer cycling normally. Simplified field endocrine techniques could transform endangered species research under field conditions and provide reliable and economical new tools for making sensible conservation management decisions based on real-time hormone results. [Copyright &y& Elsevier]

Abstract: This study provides endocrine data in relation to behavioral events during the transition of the non-breeding into the breeding season in American bison (Bison bison). Fecal progesterone metabolite patterns (20-oxo-P) were obtained in 13 adult female American bison and hormonal data were correlated with behavioral observations; i.e. copulation, male tending, female tail-up behavior and gestation length. Based on fecal progesterone metabolite patterns, the breeding season started between the middle of July and early August. Predictable short cycles reflected the transition from non-breeding to the breeding season; the luteal phase of these cycles was 4.10±0.86 days. Copulations and female tail-up behavior were reliably associated with the hormonally detected ovulation. Male tending behavior was more loosely associated with hormonally detected ovulation. The observed hormonal pattern in the study females indicated that 9 of 10 pregnant cows conceived during the second ovulatory period in the breeding season. One other cow conceived during her third ovulatory period, and one cow did not conceive until later in the breeding season by beginning of October. Gestation duration was on average 266.30±1.00 days. In summary, this study confirmed that the bison is a seasonally polyestrous species; the transition from the non-breeding into the breeding season was characterized by short cycles with low progesterone metabolite values. [Copyright &y& Elsevier]

• Wild tiger faeces showed high and variable amount of inorganic matter (IOM). • IOM negatively influenced tiger fGCM, fT3M and lion fT3M measures. • Excluding samples with ≥80% IOM content improves quantification of fGCM and fT3M. • Calculating fGCM/fT3M as per gram organic dry matter further reduces IOM influence. • IOM-driven variations changed the ecological interpretation of tiger fT3M data. Faecal glucocorticoid (GC) and triiodothyronine (T3) metabolites and their interactions are increasingly used to monitor perceived stress and nutritional challenges in free-ranging animals. However, a number of extrinsic and intrinsic factors including hormone-inert dietary materials, inorganic matters etc. are known to affect reliable hormone metabolite quantifications. In this study, the impacts of inorganic matter (IOM) on faecal GC (fGCMs) and T3 (fT3Ms) metabolite measure were addressed in wild tiger (n = 193 from Terai Arc landscape, India) and captive lion (n = 120 from Sakkarbaug Zoological Garden, Gujarat, India) and possible corrective measures were evaluated. The wild tiger samples contained highly variable IOM content (9–98%, mostly with > 40% IOM) compared to captive Asiatic lion (17–57%, majority with < 40% IOM). Significant correlations were observed between IOM content and tiger fGCM (r = -0.46, p = 0.000), fT3M (r = -0.58, p = 0.000) and lion fT3M measures (r = -0.43, p = 0.003). Two corrective measures viz. removing samples with ≥ 80% IOM and subsequently expressing concentrations as per gram of organic dry matter (instead of total dry matter) reduced IOM influence on tiger fGCM, fT3M and lion fT3M, without affecting lion fGCM measures. The corrective measures changed the interpretations of fT3M data of field-collected tiger samples with no significant changes in fGCM (both tiger and lion) and fT3M (lion) data. As faecal IOM content is common in many wild species, the results emphasize the need to reduce IOM-driven hormone data variation for ecologically relevant interpretations towards species conservation. [ABSTRACT FROM AUTHOR]

Unlike in terrestrial animals, the boundary between internal (e.g., hormones) and external (e.g., social) stimulation can be blurred for aquatic and amphibious species. When chemicals such as hormones and glandular secretions leach into the water, they can further interact with other signaling systems, creating multimodal stimuli. It is unclear, however, whether water-borne chemical secretions from courting male frogs affect the physiology and behavior of their rivals. In order to address this question we first established non-invasive, continuous sampling methods for simultaneously measuring both hormones and behavior in amphibious species. Then, we examined whether interactions between water-borne chemical secretions and conspecific calls affect reproductive behavior and physiology (testosterone and corticosterone) of courting male túngara frogs. Our results demonstrate that conspecific acoustic stimulation alone increases locomotor activity, decreases latency to call, and increases calling behavior but does not alter the amount of hormones excreted. In response to water containing chemical secretions from rivals, but in the absence of calls from other males, males excrete more testosterone. Interestingly, the combined acoustic and chemical stimulus causes a multiplicative increase in both calling behavior and hormonal excretion. Taken together, our results suggest that a multimodal chemical-acoustic stimulus physiologically primes males for aggressive behavior. • Male frogs perceive and respond to water-borne chemical secretions from rivals. • Acoustic stimulation enhances locomotor activity, motivation to call, and calling. • Chemical stimulation enhances androgen excretion. • Multimodal stimulation multiplicatively increases behavioral and hormonal responses. [ABSTRACT FROM AUTHOR]