Fecal Steroid Research Articles

Negligible hormonal response following dehorning in free-ranging white rhinoceros (Ceratotherium simum).

The white rhinoceros (Ceratotherium simum) is experiencing unsustainable poaching losses fuelled by a demand for horn. Increasingly, private and state reserves are dehorning their rhinoceros populations in an attempt to reduce poaching pressure. Rhinoceroses use their horns in social interactions as well as during resource access and so its partial removal as part of reserve management practices may adversely influence these behaviours. Physiological stress can correlate with animal welfare, reproductive state and health and thus acts as a useful indicator of these parameters. To establish whether dehorning causes a physiological stress response, glucocorticoid and gonadal steroid profiles of free-ranging white rhinoceroses were determined through the collection and analysis of faecal steroid metabolites before and after dehorning. Faecal corticoid profiles were not influenced by the number of occasions a rhinoceros had been dehorned or by the number of days that had elapsed since dehorning. Furthermore, there was no apparent suppression in the concentrations of testosterone or progesterone metabolites in males and females, respectively, after exposure to multiple dehorning procedures. These findings should increase wildlife managers' confidence that dehorning does not negatively impact white rhinoceros physiology as measured hormonally.

First evidence of terrestrial ambrein formation in human adipocere

To date, the only known occurrence of ambrein, an important perfumery organic molecule, is in coproliths found in about one in a hundred sperm whales. Jetsam ambergris coproliths from the whale are also found occasionally on beaches worldwide. Here we report on the surprising occurrence of ambrein in human adipocere. Adipocere is a waxy substance formed post-mortem during incomplete anaerobic decomposition of soft tissues. Adipocere samples obtained from grave exhumations were analysed using gas chromatography-mass spectrometry (GC-MS). In addition to the typical fatty acids of adipocere, lesser amounts of ambrein were identified in the samples, in abundances similar to those of the major accompanying faecal steroids. The distribution of these compounds suggests that ambrein was produced post-mortem during the microbial decomposition of faecal residues and tissues. It is assumed that the adipocere matrix of saturated fatty acidsaided the preservation of ambrein over extended periods of time, because adipocere is stable against degradation. The association of ambrein formation in ageing faecal material, under moist, oxygen-depleted conditions, now requires more attention in studies of other mammalian and geological samples. Indeed, ambrein and its transformation products may be useful novel chemical indicators of aged faecal matter and decomposed bodies.

Non-invasive Fecal Steroid Measurements for Monitoring the Reproductive Status of a Critically Endangered Yangtze Finless Porpoises (Neophocaena asiaeorientalis asiaeorientalis).

Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is a critically endangered freshwater cetacean dwelling in the Yangtze River and its adjoining lakes. Affected mainly by the various anthropogenic activities in this region, its population decreased dramatically in the past two decades. To protect this animal from extinction, captive breeding program is an important way to provide basic knowledge for wild population conservation. Non-invasive fecal steroid radioimmunoassay technique was validated in three captive Yangtze finless porpoises for the first time in this study. The seasonality of one captive male and the reproductive status of two females were investigated by longitudinal monitoring their fecal reproductive steroid hormone metabolites. Pregnancy could be diagnosed by an abrupt increase in fecal progesterone metabolites. In late pregnancy (4 months before birth), a significant decrease of fecal progesterone metabolites was observed, which might be referenced for the expectation of parturition date. Seven estrous cycles were recognized in one breeding season of a non-pregnant female judged by the variation of fecal progesterone metabolite levels. The fecal progesterone metabolite level was proved a reliable and precise indicator for estrus and pregnancy diagnosis.

Exposure of managed red river hogs (Potamochoerus porcus) to urine from males stimulates estrous cycling and modulates fecal sex steroid metabolites in males and females

For unknown reasons, reproductive success varies among zoos in managed red river hogs. In response to urine exposure from novel conspecifics, we hypothesized that males with low libido would exhibit increased concentrations of testosterone metabolites and that acyclic and/or non-breeding females would be induced to cycle or cycle more regularly. Estrous cycle length and progesterone metabolites in same-sex housed females were compared prior to and following exposure to novel red river hog male urine. Male testosterone metabolites and female progesterone metabolites as well as estrous cycle length were compared among: 1) proven-breeder females and males; 2) non-breeding females newly paired with novel males; 3) non-breeding females and males exposed to urine from novel females and males. Fecal samples were collected 3–5 times per week for eight to 12 months, lyophilized, extracted, and assayed for progesterone and testosterone metabolites with validated enzyme immunoassays. Introduction of female urine resulted in an increased number of estrous cycles per female per month, and decreased luteal and increased follicular progesterone metabolites in females. Introduction of male urine resulted in an increase in testosterone metabolites in males. Average progesterone metabolites for pregnant proven-breeder females were more than double that for pregnant females newly paired to novel males. An interaction between season and treatment group, as well as the acyclicity of females from July through November irrespective of treatment group, suggest that season may confound and warrant judicious interpretation of the results. Additionally, females housed with pregnant females were either acyclic or did not carry their pregnancies to term, indicating that reproductive suppression may occur in females. In conclusion, urine may be a cost-effective and efficient means to manipulate estrous cycling in managed red river hogs. Furthermore, careful consideration of the number of females in a managed herd is recommended to avoid reproductive suppression.

Evaluation of Steroid Sex Hormone Levels in Cockatiels During Their Reproductive Cycle

Sexual steroid hormone concentrations differ with respect to the different stages of animal life. For example, hormone concentrations in the reproductive stage show a periodic variation. Studies on variations in sexual hormones in parrots using non-invasive methods as well as the effect of nest-boxes in parrots are less. This study aimed to detect the levels of fecal steroid hormones during the cockatiel reproductive cycle and to investigate the effect of nest-box placement on reproduction. There were 24 mating pairs of cockatiels, and the study was conducted over 13 months. Based on their reproductive status, individual birds were divided into pre-reproduction (PR), incubation (IN), and parent period (PA) in the pre-test; then, the nest box was either temporarily removed for 20 days (RN) or continuously provided (pre-reproduction, PR). The estrogen concentrations measured in the last stage of the PR period and the initial stage of the IN period were significantly higher than those measured in the PA stage, and there were no significant differences in testosterone and progesterone concentrations throughout the reproductive cycle. The nest-box treatments (NB) indicated that eggs were laid 6-13 days after the new box was hung; however, in the pre-test, there was an interval of 30-40 days before the next mating pairs started the next reproductive stage or no reproduction. Estrogen concentrations in PR and testosterone concentrations in RN were significantly higher than those in the other periods during the nest-box treatments. In conclusion, estrogen showed the most significant change among the fecal steroid hormones measured. Nest boxes serve as a positive stimulus for parrot reproduction management and can help non-seasonal reproductive parrots to rapidly enter the next breeding cycle.

Behavioral and endocrine correlates of dominance in captive female Jackson's hartebeest (Alcelaphus buselaphus).

For species that form dominance hierarchies, such as group-living ungulates, aggressive interactions can pose a challenge to successful captive management. For example, Jackson's hartebeest (Alcelaphus buselaphus), a rare antelope of east Africa, can be difficult to maintain in captivity because aggression within female dominance hierarchies can lead to injury and death. We quantified behavioral and endocrine correlates of dominance in a captive herd of ten female hartebeest with the goal of understanding how to minimize dangerous interactions. We observed hartebeest for 18 months in a 4-ha enclosure on St. Catherines Island, Georgia. We quantified type of agonistic behavior, initiator and recipient of the behavior, who displaced whom, and age and relative size of participants. We also used fecal steroid analysis to take weekly measurements of corticoids, a hormonal correlate of stress. Hartebeest maintained a stable, linear dominance hierarchy. Only 5.9% of interactions were reversals of established rank. Most dominance interactions were low intensity, but 17.3% of interactions carried a high risk of injury (chase, horn butt, horn clash, horn clash on knees, and head push). High-ranking individuals usually initiated interactions. Most interactions were between animals of similar rank, and animals of intermediate rank participated in the highest number of interactions. The frequency of high-intensity interactions was unrelated to rank. Rank was correlated with age, but not size. There was no relationship between rank and fecal corticoid levels. Dominance interactions in hartebeest are not a function of social instability, and the frequency of dangerous interactions is not a function of rank.

100 Reproductive cycle and pregnancy monitoring in the common hippopotamus (Hippopotamus amphibius) through salivary steroid analyses and transabdominal ultrasonography

The common hippopotamus (Hippopotamus amphibius) is listed as vulnerable to extinction by the IUCN due to a significant decrease in population size, caused by habitat loss and poaching. Ex situ populations can help ensure against species loss, but careful reproductive management is essential to maintain sustainable populations. Hormone monitoring allows for characterisation of the reproductive cycle and gestation, offering insight into timing of receptivity and conception and facilitating pregnancy diagnosis and estimation of parturition date. Fecal steroid analysis has been validated for measuring progestogens in hippos. However, hippos are often housed in groups and frequently defecate in the water, making sample collection and source identification difficult. Salivary steroid analysis has been employed for monitoring reproductive activity in several species, but has not been tested in hippos. Additionally, transabdominal ultrasonography has proven valuable in diagnosing and monitoring pregnancy in many large mammals, but efficacy in the common hippo is unknown. The goals of this project were to (1) validate the use of an enzyme immunoassay to monitor progestogens in hippo saliva, (2) confirm that salivary progestogen profiles accurately reflect reproductive activity, (3) determine if transabdominal ultrasonography can be used to diagnose pregnancy, and, if so, (4) monitor and characterise fetal development via weekly examinations. Saliva (4-7 per week) and fecal (2-7 per week) samples were collected from 7 adult female hippos housed at 3 USA facilities over 3-7 months. Saliva and fecal samples were extracted in ethanol and extracts diluted (1:2 to 1:10 and 1:25 to 1:500, respectively) before evaluation by enzyme immunoassay (Progesterone mini-kit; Arbor Assays). Parallelism was confirmed between serially diluted fecal (r2=0.993) and saliva (r2=0.990) samples and the standard curve. Inter- and intra-assay coefficients of variation were maintained at <10%. Comparison of fecal and saliva progestogen concentrations revealed a strong correlation between the 2 sample types (r2=0.848) and suggested that saliva offers a comparable alternative. Both fecal and saliva extracts exhibited elevated progestogens during luteal phases and gestation. One nulliparous female housed at the Cincinnati Zoo & Botanical Garden (Cincinnati, OH, USA) was trained for voluntary transabdominal ultrasound exams. An Ibex Pro portable ultrasound machine (E.I. Medical Imaging, Loveland, CO, USA) with curvilinear probe (5-2.5MHz) was used at a scanning depth of 17.8 and 23.4cm. Intrauterine fluid and possible fetal tissue were observed 79 days following the last confirmed mating. Spine, rib cage, and beating heart were clearly visible at ~156 days of gestation. Ultrasound procedures were continued until the premature birth of a calf at ~181 days (normal hippo gestation ~231 days). Salivary progestogen monitoring and transabdominal ultrasonography appear suitable for tracking reproductive activity and diagnosing and monitoring pregnancy in the common hippo.

A NON-INVASIVE TECHNIQUE USING TO MONITOR REPRODUCTIVE HORMONES IN COMMON PALM CIVETS

Fecal steroid assays have been used to study and provide information on the estrous cycle, pregnancy, re-estrus, reproductive season and therapeutic treatments in an expanded list of species. The purpose of the present study was to monitor the reproductive status of the Commom Palm Civets (Paradoxurus hermaphroditus) by fecal steroid assays. The study was conducted to collect 2635 fecal samples from 12 adult female civets in Dong Nai Biotechnology Center. The fecal contents of Progesterone (P4) and Estradiol (E2) were determinned by using fully automatic ELISA Dynex DS2 (Dynex, USA), Progesterone and Estradiol ELISA Kit (DRG International, Inc., Germany). In non-pregnancy civets, the concentrations of fecal E2 ranged from 0.05 to 7.01 μg/g df, with an average of 1.07 ± 0.84 μg/g and a peak of 3.22 ± 0.64 μg/g. Fecal progesterone metabolites were from 0.15 to 12.32 μg/g, the overall mean of the samples was 1.72 ± 2.16 μg/g. The period of change in E2 content averaging was 28.6 ± 2.29 days. During pregnancy, the P4 content in the stool ranged from 6.21-23.12 μg/g, an average of 15.17 ± 5.22 μg/g and approximately 5 to 7 fold higher than non-pregnant (P

From the field to the lab: Muriqui endocrinology from a collaborative perspective.

The northern muriqui (Brachyteles hypoxanthus) is a critically endangered species endemic to the Atlantic Forest of southeastern Brazil. Long-term observational studies of wild muriquis have provided many insights into the behavioral ecology and life history of this species. However, nearly everything that is currently known about the northern muriqui's behavioral endocrinology has come from combining our respective expertise in noninvasive field and laboratory research. Here, we reflect on the history of our collaboration, focusing on major challenges, key scientific findings, and factors that contributed to its success. Challenges included insuring the reliable collection of frequent fecal samples from a large enough number of known individuals over extended periods of time, preserving the steroids in the field and transporting them, developing and validating the fecal steroid assays, and interpreting the hormonal profiles within the behavioral and ecological contexts of the study subjects. Major findings included our thorough description of the fecal progesterone and estradiol profiles associated with muriqui ovarian cycling and gestation, the seasonal resumption of cycling, its onset during puberty of dispersing females, and the differences between fertile and infertile cycles. We also documented the relationship between fecal cortisol and testosterone in sexually active males across breeding and nonbreeding seasons, and sex differences in cortisol levels across the mating and conception seasons. We attribute the success of our collaboration to a number of factors including our mutual appreciation for one another's high standards for ethics, data quality, and data interpretation.

Relationships between chemical and microbial faecal source tracking markers in urban river water and sediments during and post-discharge of human sewage

This study explores the relationships between faecal source tracking (FST) markers (quantitative Polymerase Chain Reaction (qPCR) markers and steroids), microbial indicators, the faecal ageing ratio of atypical colonies/total coliforms (AC/TC) and potential human pathogens (Giardia, Cryptosporidium and Campylobacter). Faecal source PCR markers tested were GenBac3, HumM3, HumBac (HF183-Bac708R); Bifidobacterium adolescentis, wildfowl and canine-associated markers. Sediment and water samples from the Avon River were collected during and post-discharge of untreated human sewage inputs, following a series of earthquakes, which severely damaged the Christchurch sewerage system.Significant, positive Spearman Ranks (rs) correlations were observed between human-associated qPCR markers and steroid FST markers and Escherichia coli and F-specific RNA bacteriophage (rs 0.57 to 0.84, p < 0.001) in water samples. These human source indicative FST markers demonstrated that they were also effective predictors of potentially pathogenic protozoa in water (rs 0.43–0.74, p ≤ 0.002), but correlated less well with Campylobacter. Human-associated qPCR and steroid markers showed significant, substantial agreement between the two FST methods (Cohen's kappa, 0.78, p = 0.023), suggesting that water managers could be confident in the results using either method under these contamination conditions.Low levels of fluorescent whitening agents (FWA) (mean 0.06 μg/L, range 0.01–0.40 μg/L) were observed in water throughout the study, but steroids and FWA appeared to be retained in river sediments, months after continuous sewage discharges had ceased. No relationship was observed between chemical FST markers in sediments and the overlying water, and few correlations in sediment between chemical FST markers and target microorganisms. The low values observed for the faecal ageing ratio, AC/TC in water, were significantly, negatively correlated with increasing pathogen detection. This study provides support for the use of the AC/TC ratio, and qPCR and steroid FST markers as indicators of health risks associated with the discharge of raw human sewage into a freshwater system.

Steroid fingerprints: Efficient biomarkers of human decomposition fluids in soil

The decomposition of animal and human cadavers can lead to comprehensive chemical and biochemical changes in soil, which can provide helpful information for the analysis of archaeological and crime scenes. The current study focused specifically on the concentration and distribution patterns of steroids in soil with the aim of assessing their suitability for demonstrating the presence of human decomposition products. Soil samples were collected from a forensic site where a human corpse had been lying on the soil surface for 18 days. Total organic carbon (TOC) and steroid concentrations were analysed in soil samples taken from beneath the body at the time the corpse was removed and also one year later. High concentrations of TOC and steroids were detected close to the soil surface at the time the body was removed, and the concentration of cadaver-derived cholesterol was considerably higher than that of the plant-derived sitosterol. The soil beneath the cadaver contained higher concentrations of faeces-derived 5β-stanol coprostanol than the control soil. Different concentrations and distribution patterns of steroid compounds in the soil upon removal of the corpse and one year later indicate that significant transformation, degradation and translocation processes had taken place during this period of time. The depth distribution of steroids beneath a decomposing body along with changes in the human steroidal fingerprints in soil over time were found to provide information that could help resolve forensic and archaeological issues. The combined analysis of tissue steroids (cholesterol), the reduction product of cholesterol in soil (5α-cholestanol) as well as faecal steroids (coprostanol, 5β-stigmastanol and their conversion products epicoprostanol and epi-5β-stigmastanol) represent excellent additions to the forensic toolbox for determining whether a soil of interest has ever been affected by a human cadaver or not.

Characterizing estrus by trans-abdominal ultrasounds, fecal estrone-3-glucuronide, and vaginal cytology in the Steller sea lion (Eumetopias jubatus)

The ability to monitor the estrus cycle in wild and captive marine species is important for identifying reproductive failures, ensuring a successful breeding program, and monitoring animal welfare. Minimally invasive sampling methods to monitor estrus in captive populations have been developed, but results suggest these tools can be species-specific in their precision and accuracy. Therefore, the minimally invasive sampling methods of trans-abdominal ultrasounds, a fecal steroid analysis (estrone-3-glucuronide, E1G), and vaginal cytology, were evaluated for their efficacy to characterize and monitor estrus in a captive breeding population of Steller sea lions (Eumetopias jubatus). Three adult females were sampled over five breeding seasons, resulting in six estrus profiles characterized by trans-abdominal ultrasounds, five by fecal E1G, and four by vaginal cytology. Animals were trained to allow trans-abdominal ultrasounds, fecal samples, and vaginal swabs to be collected approximately daily. Of the 76 trans-abdominal ultrasound sessions attempted, 8 successfully visualized both ovaries. From these scans, the chronology of ovarian changes during proestrus and estrus was estimated. The time from the detection of developing follicles to the identification of a dominate follicle occurred in 2–5 days and a corpus hemorrhagicum formed approximately 4 days later. However, because visualization of the ovaries was prevented by the gastrointestinal system in 88% of scans, this tool was overall unreliable for monitoring changes associated with estrus. To detect fine scale physiological changes associated with estrus, we analyzed changes in fecal E1G (n = 62) and vaginal cytology (n = 157) 15 days before and after each female's single copulation event (Day = 0). Changes in fecal E1G had the highest accuracy at detecting Day = 0. Fecal E1G increased leading up to estrus, peaked at Day = 0, and then declined. Although we did observe the characteristic increase in superficial cells associated with impending estrus, the type of cell which peaked closest to Day = 0 was intermediate. The uncertainty around the peak in intermediate cells, indicating estrus, was greater than the uncertainty associated with detecting estrus from fecal E1G. Collectively, these results suggest that changes in fecal E1G and vaginal cytology are viable tools to detect estrus in Steller sea lions, but require daily sampling to detect gradual changes, limiting their applicability to studies of wild populations.

Assessing puberty in ex situ male cheetahs (Acinonyx jubatus) via fecal hormone metabolites and body weights

Cheetahs are one of the most heavily studied felid species, with numerous publications on health, disease, and reproductive physiology produced over the last 30 years. Despite this relatively long history of research, there is a paucity of crucial biological data, such as pubertal onset, which has direct and significant applications to improved management of ex situ cheetah populations. This study aimed to determine age of pubertal onset in ex situ male cheetahs using non-invasive fecal steroid hormone monitoring and body weights. Fecal samples from 12 male cheetahs from four institutions were collected 2–3 times weekly from 1 to 42 months of age. Fecal androgen and glucocorticoid metabolites were analyzed using enzyme immunoassays previously validated for use with cheetah feces. Animal body weights were recorded monthly. Fecal hormone and body weight data were analyzed using generalized linear mixed models. Androgen concentrations exhibited an increase to levels similar to those observed in adult males by 18–24 months of age, and males attained adult body weights by 21 months of age. Based on these weight data and the initial increase in androgens toward adult concentrations, males were considered pubertal from 18 to 24 months of age. Glucocorticoid concentrations and amplitude of concentration over baseline were also increased during this period. Knowledge about the physiological changes associated with puberty is useful for management and improving reproductive success of cheetah populations under human care, particularly for determining timing of litter separation from dam, littermate dispersal and when to introduce potential breeding pairs.

Nasty, Brutish and Short?; The Life Cycle of an Iron Age Roundhouse at Black Loch of Myrton, SW Scotland

ABSTRACTExcavations at Black Loch of Myrton, Dumfries & Galloway are revealing the very well-preserved remains of an Iron Age settlement, the wetland context ensuring that the timber structures have remained intact and that the detritus of daily occupation survives for us to pick apart and understand. One of the structures in this settlement is an exceptionally well-preserved roundhouse, the material remains of which have been subjected to a barrage of analyses encompassing the insect, macroplant, bone and wood assemblages, soil micromorphology, faecal steroids, radiocarbon-dating and dendrochronology. These will enable us to address some of the key issues regarding the life cycles of Iron Age roundhouses, from conception and construction, use of internal space, nature of occupation and likely function, through to abandonment. Critically, we are now able to view that life cycle through the lens of a tightly-defined chronology bringing us close to the ‘ … short-term timescales of lived reality’ [Foxhall, L. 2000. “The Running Sands of Time: Archaeology and the Short-Term.” World Archaeology 31 (3): 484–498].

Changes in African Elephant (Loxodonta africana) faecal steroid concentrations post-defaecation

Background: Faecal hormone metabolite measurement is a widely used tool for monitoring reproductive function and response to stressors in wildlife. Despite many advantages of this technique, the delay between defaecation, sample collection and processing may influence steroid concentrations, as faecal bacterial enzymes can alter steroid composition post-defaecation.Objectives: This study investigated changes in faecal glucocorticoid (fGCM), androgen (fAM) and progestagen (fPM) metabolite concentrations in faeces of a male and female African elephant (Loxodonta africana) post-defaecation and the influence of different faeces-drying regimes.Method: Subsamples of fresh faeces were frozen after being dried in direct sun or shade for 6, 20, 24, 48 and 72 h and 7 and 34 days. A subset of samples for each sex was immediately frozen as controls. Faecal hormone metabolite concentrations were determined using enzyme immunoassays established for fGCM, fAM and fPM monitoring in male and female African elephants.Results: Hormone metabolite concentrations of all three steroid classes were stable at first, but changed distinctively after 20 h post-defaecation, with fGCM concentrations decreasing over time and fPM and fAM concentrations steadily increasing. In freeze-dried faeces fGCM concentrations were significantly higher than respective concentrations in sun-dried material, which were in turn significantly higher than fGCM concentrations in shade-dried material. In contrast, fAM concentrations were significantly higher in sun- and shade-dried faeces compared to freeze-dried faeces. Higher fPM concentrations were also found in air-dried samples compared to lyophilised faeces, but the effect was only significant for sun-dried material.Conclusion: The revealed time restriction for collecting faecal material for hormone monitoring from elephants in the wild should be taken into account to assure reliable and comparable results. However, if logistics allow a timely collection, non-invasive hormone measurement remains a powerful and reliable approach to provide information about an elephant’s endocrine status.

Changes in African Elephant (Loxodonta africana) faecal steroid concentrations post-defaecation

Background: Faecal hormone metabolite measurement is a widely used tool for monitoring reproductive function and response to stressors in wildlife. Despite many advantages of this technique, the delay between defaecation, sample collection and processing may influence steroid concentrations, as faecal bacterial enzymes can alter steroid composition post-defaecation.Objectives: This study investigated changes in faecal glucocorticoid (fGCM), androgen (fAM) and progestagen (fPM) metabolite concentrations in faeces of a male and female African elephant (Loxodonta africana) post-defaecation and the influence of different faeces-drying regimes.Method: Subsamples of fresh faeces were frozen after being dried in direct sun or shade for 6, 20, 24, 48 and 72 h and 7 and 34 days. A subset of samples for each sex was immediately frozen as controls. Faecal hormone metabolite concentrations were determined using enzyme immunoassays established for fGCM, fAM and fPM monitoring in male and female African elephants.Results: Hormone metabolite concentrations of all three steroid classes were stable at first, but changed distinctively after 20 h post-defaecation, with fGCM concentrations decreasing over time and fPM and fAM concentrations steadily increasing. In freeze-dried faeces fGCM concentrations were significantly higher than respective concentrations in sun-dried material, which were in turn significantly higher than fGCM concentrations in shade-dried material. In contrast, fAM concentrations were significantly higher in sun- and shade-dried faeces compared to freeze-dried faeces. Higher fPM concentrations were also found in air-dried samples compared to lyophilised faeces, but the effect was only significant for sun-dried material.Conclusion: The revealed time restriction for collecting faecal material for hormone monitoring from elephants in the wild should be taken into account to assure reliable and comparable results. However, if logistics allow a timely collection, non-invasive hormone measurement remains a powerful and reliable approach to provide information about an elephant’s endocrine status.

Fecal steroid hormones reveal reproductive state in female blue whales sampled in the Gulf of California, Mexico

Steroid hormone assessment using non-invasive sample collection techniques can reveal the reproductive status of aquatic mammals and the physiological mechanisms by which they respond to changes in their environment. A portion of the eastern North Pacific blue whale (Balaenoptera musculus) population that seasonally visits the Gulf of California, Mexico has been monitored using photo-identified individuals for over 30 years. The whales use the area in winter-early spring for nursing their calves and feeding and it therefore is well suited for fecal sample collection. Using radioimmunoassays in 25 fecal samples collected between 2009 and 2012 to determine reproductive state and stress, we validated three steroid hormones (progesterone, corticosterone and cortisol) in adult female blue whales. Females that were categorized as pregnant had higher mean fecal progesterone metabolite concentrations (1292.6 ± 415.6 ng·g-1) than resting and lactating females (14.0 ± 3.7 ng·g-1; 23.0 ± 5.4 ng·g-1, respectively). Females classified as pregnant also had higher concentrations of corticosterone metabolites (37.5 ± 9.9 ng·g-1) than resting and lactating females (17.4 ± 2.0 ng·g-1; 16.8 ± 2.8 ng·g-1, respectively). In contrast, cortisol metabolite concentrations showed high variability between groups and no significant relationship to reproductive state. We successfully determined preliminary baseline parameters of key steroid hormones by reproductive state in adult female blue whales. The presence of pregnant or with luteal activity and known lactating females confirms that the Gulf of California is an important winter-spring area for the reproductive phase of these blue whales. The baseline corticosterone levels we are developing will be useful for assessing the impact of the increasing coastal development and whale-watching activities on the whales in the Gulf of California.

One year daily changes in fecal sexual steroids of two captive female cheetahs (Acinonyx jubatus) in Italy

The present study evaluated changes of fecal sexual steroids in two female cheetahs (Geijsha and Duchessa) in Northern Italy throughout one year. Wet feces were collected daily from two sibling animals of the same age, housed with conspecific males and managed in the same conditions, and estrogens and progestogens concentrations were analyzed by radioimmunoassay (RIA).Evidence of ovarian activity based on regular fluctuation in estrogen excretion was demonstrated in both females. None of the animals was continuously cycling, as follicular activity was interrupted by anestrous periods, during the spring and early winter. No significant increases of progestogens were recorded after the estrogen peaks, indicating that induced or spontaneous ovulations did not occur during the observation period. The wavelet decomposition evidenced the temporal pattern of ovarian activity in the two females, underlying throughout the year a more pronounced rhythmical ovarian estrogenic activity in Geijsha than in Duchessa. However, this statistical approach had a smoothing effect in depicting the hormonal patterns and the number of follicular phases might be lower than that revealed by the iterative method.In this study, RIA on wet feces performed very well to determine sexual steroid concentrations, and an ovarian activity interrupted by anestrous periods along the year in captive cheetahs co-housed in a small group was demonstrated.More information on estrous behavior of captive cheetahs were obtained in this study, but the effects of husbandry and management conditions on natural reproductive physiology of this species remain to elucidate.

Organic and inorganic contamination in sediments from Araçá Bay, São Sebastião, Brazil

The aim of this work is to verify the organic and inorganic contamination in Araçá Bay, which contains one of the last remnants of mangrove forest on the northern coast of São Paulo State, Brazil, to improve the environmental management of this area, which is part of the Marine Protected Area of the Northern Coast of São Paulo State (APAMLN) and the Environmental Protected Area of the Alcatrazes Municipality (APAMA). Surface sediments were analyzed to provide information on the levels, spatial trends and sources of trace elements, aliphatic hydrocarbons (AH), polycyclic aromatic hydrocarbons (PAH), and fecal steroids. Low levels of trace elements were observed, and the enrichment factors showed no evidence of contamination in the area. The highest concentrations of all organic contaminants were recorded in the samples collected in the external sublittoral stations, mainly in the São Sebastião Channel, which had the finest grain sizes and higher OC contents. With the exception of the samples collected in the inner part of the bay, most of the samples presented unresolved complex mixtures (UCM) of AH, which are indicators of petroleum and its derivative inputs. PAH concentrations were higher in the external stations, but the levels were below the limits of the sediment quality guidelines (SQG) recommended in Brazilian federal legislation on dredged sediments. The major sources of these compounds were from pyrolytic sources. Sewage contamination was detected mainly in the inner part of the bay, especially near the northern stream and the sewage outfall. The contaminant distributions were related to the hydrodynamics of the intertidal flat, with the most central dynamic areas presenting lower contaminant levels.

Enzyme immunoassays for water-soluble steroid metabolites in the urine and feces of Japanese macaques (Macaca fuscata) using a simple elution method.

A simple, non-alcoholic extraction method for measuring estrogen and progesterone metabolites in excreta using enzyme immunoassays (EIAs) was developed in Japanese macaques. The obtained detection limits of EIAs using estrone conjugates (E1C), pregnanediol glucuronide (PdG), and estriol glucuronide (E3G) polyclonal antibodies with cross-reactivity to urinary and fecal steroid metabolites were 6.6 pg/ml, 2.1 ng/ml and 0.35 ng/ml, respectively. These assays allowed the determination of E1C, PdG, and E3G from the excreta with good reproducibility and accuracy. Thereafter, urine and fecal samples of two menstrual cycles and six pregnancies from eight female Japanese macaques were assayed. A typical increase in urinary and fecal E1C in follicular phase and PdG in luteal phase were shown during non-conceptive menstrual cycles. Urinary E3G levels also showed a preovulatory increase; however, fecal E3G levels were very low throughout the non-conceptive menstrual cycles. Levels of E1C and PdG in the urine and feces of pregnant females were gradually increased until parturition, while fecal E3G levels were low and reached detectable levels after the mid-pregnancy period. Although the extraction rate of estrogen and progestogen metabolites by our method was lower compared to those of the previous extraction method using an alcohol-containing buffer, our method was simple, and the correlation coefficients for the relationship between two methods were found to be statistically significant. The results presented here are of great practical value for a non-invasive method of monitoring ovarian function and pregnancy in Japanese macaques.