Comparative analysis of fecal microbiota and intestinal microbial metabolic activity in captive polar bears

The composition of the intestinal microbiota depends on gut physiology and diet. Ursidae possess a simple gastrointestinal system composed of a stomach, small intestine, and indistinct hindgut. This study determined the composition and stability of fecal microbiota of 3 captive polar bears by group-specific quantitative PCR and PCR-DGGE (denaturing gradient gel electrophoresis) using the 16S rRNA gene as target. Intestinal metabolic activity was determined by analysis of short-chain fatty acids in feces. For comparison, other Carnivora and mammals were included in this study. Total bacterial abundance was approximately log 8.5 DNA gene copies·(g feces)-1 in all 3 polar bears. Fecal polar bear microbiota was dominated by the facultative anaerobes Enterobacteriaceae and enterococci, and the Clostridium cluster I. The detection of the Clostridium perfringens α-toxin gene verified the presence of C.?perfringens. Composition of the fecal bacterial population was stable on a genus level; according to results obtained by PCR-DGGE, dominant bacterial species fluctuated. The total short-chain fatty acid content of Carnivora and other mammals analysed was comparable; lactate was detected in feces of all carnivora but present only in trace amounts in other mammals. In comparison, the fecal microbiota and metabolic activity of captive polar bears mostly resembled the closely related grizzly and black bears.     

Spatial patterns in brown bear Ursus arctos diet: the role of geographical and environmental factors

• 1 We reviewed worldwide spatial patterns in the food habits of the brown bear Ursus arctos in relation to geographical (latitude, longitude, altitude) and environmental (temperature, snow cover depth and duration, precipitation, primary productivity) variables.
• 2 We collected data from 28 studies on brown bear diet based on faecal analysis, covering the entire geographical range of this widely distributed large carnivore. We analysed separately four data sets based on different methods of diet assessment.
• 3 Temperature and snow conditions were the most important factors determining the composition of brown bear diet. Populations in locations with deeper snow cover, lower temperatures and lower productivity consumed significantly more vertebrates, fewer invertebrates and less mast. Trophic diversity was positively correlated with temperature, precipitation and productivity but negatively correlated with the duration of snow cover and snow depth. Brown bear populations from temperate forest biomes had the most diverse diet. In general, environmental factors were more explicative of diet than geographical variables.
• 4 Dietary spatial patterns were best revealed by the relative biomass and energy content methods of diet analysis, whereas the frequency of occurrence and relative biomass methods were most appropriate for investigating variation in trophic diversity.
• 5 Spatial variation in brown bear diet is the result of environmental conditions, especially climatic factors, which affect the nutritional and energetic requirements of brown bears as well as the local availability of food. The trade‐off between food availability on the one hand, and nutritional and energetic requirements on the other hand, determines brown bear foraging decisions. In hibernating species such as the brown bear, winter severity seems to play a role in determining foraging strategies. Large‐scale reviews of food habits should be based on several measures of diet composition, with special attention to those methods reflecting the energetic value of food.

     

Functional dietary response of Asian black bears to changes in sika deer density

Omnivores are generally opportunistic foragers and have a flexible dietary response to resource abundance and availability. Their populations may consist of individuals that differ from each other in terms of their trophic positions, which implies that the dietary response to resource fluctuations differs within a population. We investigated how changes in the abundance of sika deer (Cervus nippon) affected dietary variation and body condition in the Asian black bear (Ursus thibetanus). We used fecal analysis, nitrogen stable isotopes (δ¹⁵N), and body measurements to determine whether the variation in dietary meat content of Asian black bears is positively related to variations in the density of the sika deer population, whether male bears have a higher trophic position compared to females, and whether dietary meat content is positively related with body mass or body condition of bears. We found a positive correlation between the occurrence of deer remains in bear feces and deer density, suggesting that bears change their diet in response to temporal changes in deer density. Male bears had higher δ¹⁵N values than females, and neither values varied when deer density decreased. Males selectively consumed deer after a reduction in deer density, whereas females consistently consumed more plant-based diet. The δ¹⁵N values were positively related with body mass of adult (>4 yr) bears but had no relationship with body condition of bears of either sex or any age class. Deer seem to be an important food source for large adult males, which have an advantage in mating. Thus, increasing herbivore abundance and availability altered the foraging strategy of Asian black bears, but the importance of herbivore on bear diet differs within a population.     

Feeding habits may explain the morphological uniqueness of brown bears on Etorofu Island,Southern Kuril Islands in East Asia

The brown bear (Ursus arctos) population on Etorofu Island, Southern Kuril Islands, has several unique morphological features: (1) the presence of white‐pelage bears within the population and (2) a larger body size than bears on a larger neighbour island, Hokkaido Island. Nevertheless, little ecological information is available about Etorofu brown bears. In the present study, we reveal the unique feeding habits of Etorofu brown bears and suggest that their unique morphological features and diet are related. The feeding habits of brown bears on Etorofu Island were assessed using carbon, nitrogen, and sulfur stable isotope analysis, and their feeding habits were compared with those of bears on the eastern side of Hokkaido Island. According to the stable isotope analysis, the dependence on salmon is great for bears on Etorofu but only slight for bears on Hokkaido. Our results suggest that the feeding habits of Etorofu brown bears may explain their unique morphological features because a white pelage colour confers an advantage when catching salmon, and a carnivorous diet can make their body size larger. The variation in feeding habits can be an important driver of the speciation and evolution of animals.     

Biomechanical consequences of rapid evolution in the polar bear lineage

The polar bear is the only living ursid with a fully carnivorous diet. Despite a number of well-documented craniodental adaptations for a diet of seal flesh and blubber, molecular and paleontological data indicate that this morphologically distinct species evolved less than a million years ago from the omnivorous brown bear. To better understand the evolution of this dietary specialization, we used phylogenetic tests to estimate the rate of morphological specialization in polar bears. We then used finite element analysis (FEA) to compare the limits of feeding performance in the polar bear skull to that of the phylogenetically and geographically close brown bear. Results indicate that extremely rapid evolution of semi-aquatic adaptations and dietary specialization in the polar bear lineage produced a cranial morphology that is weaker than that of brown bears and less suited to processing tough omnivorous or herbivorous diets. Our results suggest that continuation of current climate trends could affect polar bears by not only eliminating their primary food source, but also through competition with northward advancing, generalized brown populations for resources that they are ill-equipped to utilize.     

Non-invasive monitoring of glucocorticoid metabolites in brown hyaena (Hyaena brunnea) feces

The brown hyaena (Hyaena brunnea) is the least known of the large predators of southern Africa. The current IUCN status of the brown hyaena is “Near Threatened”, and there are conservation concerns related to a general lack of biological knowledge of the species. For instance, a better knowledge of the responses to environmental and social stressors would improve our abilities to sustainably manage brown hyaena populations in both captive and free‐ranging environments. We conducted adrenocorticotrophic hormone (ACTH) challenges in one female and one male adult brown hyaena at Lion Park Zoo, South Africa, to validate measurements of glucocorticoid metabolites (GCM) in brown hyaena feces via an enzyme immunoassay (EIA). We also measured gastrointestinal transit times (GIT times) and the GCM degradation in feces left in ambient temperature for up to 32 hr to more reliably assess the use of this assay as a tool for non‐invasive glucocorticoid measurements. Intramuscular injections of synthetic ACTH yielded GCM levels of 388% (female) and 2,682% (male) above baseline with peak increases occurring 25‐ to 40‐hr after injection. The time delay of fecal GCM excretion approximately corresponded with food transit time in the brown hyaenas. Fecal GCM levels declined significantly over time since defecation. Our results provided a good validation that fecal GCMs accurately reflect circulating glucocorticoid stress hormones in brown hyaenas, but we highlight that samples have to be frozen immediately after defecation to avoid bias in the measurements as a result of bacterial degredation. Zoo Biol 30:451–458, 2011. © 2010 Wiley‐Liss, Inc.     

The seasonal sensitivity of brown bear denning phenology in response to climatic variability

Background

For brown bears (Ursus arctos), hibernation is a critical part of the annual life cycle because energy savings during hibernation can be crucial for overwintering, and females give birth to cubs at that time. For hibernation to be a useful strategy, timing is critical. However, environmental conditions vary greatly, which might have a negative effect on the functionality of the evolved biological time-keeping. Here, we used a long-term dataset (69 years) on brown bear denning phenology recorded in 12 Russian protected areas and quantified the phenological responses to variation in temperature and snow depth. Previous studies analyzing the relationship between climate and denning behavior did not consider that the brown bear response to variation in climatic factors might vary through a period preceding den entry and exit. We hypothesized that there is a seasonal sensitivity pattern of bear denning phenology in response to variation in climatic conditions, such that the effect of climatic variability will be pronounced only when it occurs close to den exit and entry dates.

Results

We found that brown bears are most sensitive to climatic variations around the observed first den exit and last entry dates, such that an increase/decrease in temperature in the periods closer to the first den exit and last entry dates have a greater influence on the denning dates than in other periods.

Conclusions

Our study shows that climatic factors are modulating brown bear hibernation phenology and provide a further structuring of this modulation. The sensitivity of brown bears to changes in climatic factors during hibernation might affect their ability to cope with global climate change. Therefore, understanding these processes will be essential for informed management of biodiversity in a changing world.

     

Genomic Evidence for Island Population Conversion Resolves Conflicting Theories of Polar Bear Evolution

Despite extensive genetic analysis, the evolutionary relationship between polar bears (Ursus maritimus) and brown bears (U. arctos) remains unclear. The two most recent comprehensive reports indicate a recent divergence with little subsequent admixture or a much more ancient divergence followed by extensive admixture. At the center of this controversy are the Alaskan ABC Islands brown bears that show evidence of shared ancestry with polar bears. We present an analysis of genome-wide sequence data for seven polar bears, one ABC Islands brown bear, one mainland Alaskan brown bear, and a black bear (U. americanus), plus recently published datasets from other bears. Surprisingly, we find clear evidence for gene flow from polar bears into ABC Islands brown bears but no evidence of gene flow from brown bears into polar bears. Importantly, while polar bears contributed <1% of the autosomal genome of the ABC Islands brown bear, they contributed 6.5% of the X chromosome. The magnitude of sex-biased polar bear ancestry and the clear direction of gene flow suggest a model wherein the enigmatic ABC Island brown bears are the descendants of a polar bear population that was gradually converted into brown bears via male-dominated brown bear admixture. We present a model that reconciles heretofore conflicting genetic observations. We posit that the enigmatic ABC Islands brown bears derive from a population of polar bears likely stranded by the receding ice at the end of the last glacial period. Since then, male brown bear migration onto the island has gradually converted these bears into an admixed population whose phenotype and genotype are principally brown bear, except at mtDNA and X-linked loci. This process of genome erosion and conversion may be a common outcome when climate change or other forces cause a population to become isolated and then overrun by species with which it can hybridize.     

Brown bear habitat selection in relation to anthropogenic structures in the Bieszczady Mountains,Poland

In Europe, brown bear Ursus arctos habitats frequently overlap with human settlements and infrastructure. We tested whether anthropogenic structures played an important role in habitat selection by brown bears in the Bieszczady Mountains, Poland. We analysed 668 signs of brown bear presence recorded during 6 counts along 246 km of transects (total 1,476 km) in spring, summer and autumn of 1993 and 1994. Habitat selection of bears was more related to habitat and altitude than to human factors. Avoidance of roads, settlements and forest clearings influenced habitat selection by brown bears in spring but less in summer and autumn.     

Effects of Exposure,Diet, and Thermoregulation on Fecal Glucocorticoid Measures in Wild Bears

We examined fecal glucocorticoid (fGC) measures of nutrition and thermoregulatory demands on wild bears in Glacier National Park, Montana, and assessed how these measures changed in samples left in the field. Both ambient temperature and exposure can impact thermoregulation and sample degradation. Bear diets vary markedly with season, affecting body condition and thus fGC. We collected fecal samples during September and October, 2001, when ambient temperatures ranged from 30°C to −5°C. We collected half of each sample immediately and left the other half in its original location for 1–28 days. We used generalized linear models (GLM) to first predict fGC concentrations in fresh samples based on proxies of nutrition, ambient temperature, thermal exposure, and precipitation. These same covariates were then used to predict degradation-based differences in fGC concentrations between the paired sample halves. Variation in fGC was predicted by diet, Julian date, aspect, and the interaction between Julian date and aspect in both fresh and exposed samples. Cumulative precipitation was also a significant predictor of fGC concentrations in the exposed samples, independent of time, indicating that precipitation contributes to sample degradation but not enough to mask effects of other environmental factors on fGC concentrations. Differences between sample halves were only predicted by cumulative precipitation and exposure time; cumulative precipitation decreased, whereas exposure time increased, fGC concentrations in the exposed sample halves. Results indicate that fGC can provide reliable indices of nutrition and thermoregulatory demands in bears and that sample degradation impacts on these relations are minimal and can be virtually eliminated by controlling for cumulative precipitation over the estimated exposure times.     

青藏高原可可西里地区藏棕熊暖季食性及采食行为模式

藏棕熊(Ursus arctos pruinosus)是青藏高原特有的棕熊亚种,作者曾对该地区藏棕熊夏季食性进行了初步报道(Xu et al,2006),然而,藏棕熊的采食行为模式一直未见报道。2009年7-8月,作者又对可可西里地区藏棕熊食性及采食行为模式做了补充调查研究,对两次考察所获得的数据进行了整合分析,研究结果表明,该地区藏棕熊主要以动物性食物为主,其中,高原鼠兔(Ochotona curzoniae)的相对频次为37.3%和干物质量为44.7%,下同);野牦牛(Bos grunniens)分别为18.7%和30.2%;藏羚(Pantholops hodgsoni)分别为15.0%和16.2%。藏棕熊在可可西里地区有两种采食行为模式:主动捕食高原鼠兔和采食藏羚、藏原羚和野牦牛尸体。观察期间藏棕熊约用10%的时间挖掘高原鼠兔的洞穴捕捉高原鼠兔,但未见藏棕熊主动捕食大型哺乳动物。粪样分析结果发现,藏棕熊主动捕食的高原鼠兔和喜马拉雅旱獭(Marmota himalayana)和通过食腐方式获得的藏羚、藏原羚和野牦牛的生物量基本相等。     

Comparative endocrine investigations in three bear species based on urinary steroid metabolites and volatiles

In order to improve breeding of in situ populations of bears, a comprehensive study of reproductive physiology in Brown (Ursus arctos), Spectacled (Tremarctos ornatus) and Giant panda bears (Ailuropoda melanoleuca) was performed. The objective was to perform non-invasive analyses of urinary and fecal steroid metabolites. In addition, we investigated the presence of reproduction-related urinary volatile substances of these bears that might trigger the reproductive behavior. Urinary estrogen concentrations, routinely used to monitor follicular activity in Giant panda, were inappropriate for monitoring follicular activity in Spectacled bear. In addition, no estrogen peak related to mating activity was observed in Brown bear. Further contrasting Giant panda, although urinary pregnanediol analyses failed to indicate luteal activity in either Spectacled or Brown bears, urinary (Spectacled bear) and fecal (Brown bear) concentrations of progesterone were an appropriate indicator of luteal activity. The Giant panda had volatile components (medium-chain fatty acids) in their urine that increased simultaneously with the seasonal increase of estrogens. These fatty acids were also detected in the Brown during estrus and Spectacled bear. Further studies on the behavioral relevance of these fatty acids are required to determine if they are pheromones.     

Genomic evidence of geographically widespread effect of gene flow from polar bears into brown bears

Polar bears are an arctic, marine adapted species that is closely related to brown bears. Genome analyses have shown that polar bears are distinct and genetically homogeneous in comparison to brown bears. However, these analyses have also revealed a remarkable episode of polar bear gene flow into the population of brown bears that colonized the Admiralty, Baranof and Chichagof islands (ABC islands) of Alaska. Here, we present an analysis of data from a large panel of polar bear and brown bear genomes that includes brown bears from the ABC islands, the Alaskan mainland and Europe. Our results provide clear evidence that gene flow between the two species had a geographically wide impact, with polar bear DNA found within the genomes of brown bears living both on the ABC islands and in the Alaskan mainland. Intriguingly, while brown bear genomes contain up to 8.8% polar bear ancestry, polar bear genomes appear to be devoid of brown bear ancestry, suggesting the presence of a barrier to gene flow in that direction.     

Longitudinal fecal hormone analysis for monitoring reproductive activity in the female polar bear (Ursus maritimus)

The objective was to identify suitable enzyme immunoassays to monitor gonadal and placental function in the female polar bear. Immunoreactive progesterone, progesterone metabolite (PdG), estrogen, and androgen metabolite (T) concentrations were measured in fecal samples collected over 24 mo from captive female bears (N = 20). Whereas fecal extracts produced displacement curves parallel to the standard curve for each respective steroid, T and PdG more accurately reflected reproductive events. Concentrations of fecal T increased (P < 0.05) during the breeding season, and brief spikes were associated with estrus and mating. A postovulatory increase in PdG was not always detected, but sustained baseline T after mating appeared consistent with ovulation. Parturient bears excreted higher PdG concentrations (P < 0.05) during expected time of embryo implantation in Fall, and a late gestational rise in fecal T occurred 30 days prepartum. Many nonparturient bears also had a PdG rise in the Fall, suggesting they experienced either pregnancy loss or a pseudopregnancy. Differentiating pregnant and pseudopregnant states was not achieved using fecal PdG alone, but when combined with fecal T, comprehensive diagnoses could be made. Nonparturient bears demonstrated elevated (P < 0.05) fecal T during summer months, whereas parturient bears did not. In summary, noninvasive hormone monitoring techniques were established for the female polar bear. Although this study was directed at facilitating management and breeding efforts of captive polar bears, the methods could be applied to studies of reproductive function in wild populations.     

Efficacy of Bear Deterrent Spray in Alaska

Abstract: We present a comprehensive look at a sample of bear spray incidents that occurred in Alaska, USA, from 1985 to 2006. We analyzed 83 bear spray incidents involving brown bears (Ursus arctos; 61 cases, 74%), black bears (Ursus americanus; 20 cases, 24%), and polar bears (Ursus maritimus; 2 cases, 2%). Of the 72 cases where persons sprayed bears to defend themselves, 50 (69%) involved brown bears, 20 (28%) black bears, and 2 (3%) polar bears. Red pepper spray stopped bears' undesirable behavior 92% of the time when used on brown bears, 90% for black bears, and 100% for polar bears. Of all persons carrying sprays, 98% were uninjured by bears in close-range encounters. All bear—inflicted injuries (n = 3) associated with defensive spraying involved brown bears and were relatively minor (i.e., no hospitalization required). In 7% (5 of 71) of bear spray incidents, wind was reported to have interfered with spray accuracy, although it reached the bear in all cases. In 14% (10 of 71) of bear spray incidents, users reported the spray having had negative side effects upon themselves, ranging from minor irritation (11%, 8 of 71) to near incapacitation (3%, 2 of 71). Bear spray represents an effective alternative to lethal force and should be considered as an option for personal safety for those recreating and working in bear country. (JOURNAL OF WILDLIFE MANAGEMENT 72(3):640–645; 2008)     

Role of brown bears (Ursus arctos) in the flow of marine nitrogen into a terrestrial ecosystem

We quantified the amount, spatial distribution, and importance of salmon (Oncorhynchus spp.)-derived nitrogen (N) by brown bears (Ursus arctos) on the Kenai Peninsula, Alaska. We tested and confirmed the hypothesis that the stable isotope signature (δ¹⁵N) of N in foliage of white spruce (Picea glauca) was inversely proportional to the distance from salmon-spawning streams (r=–0.99 and P<0.05 in two separate watersheds). Locations of radio-collared brown bears, relative to their distance from a stream, were highly correlated with δ¹⁵N depletion of foliage across the same gradient (r=–0.98 and –0.96 and P<0.05 in the same two separate watersheds). Mean rates of redistribution of salmon-derived N by adult female brown bears were 37.2±2.9 kg/year per bear (range 23.1–56.3), of which 96% (35.7±2.7 kg/year per bear) was excreted in urine, 3% (1.1±0.1 kg/year per bear) was excreted in feces, and <1% (0.3± 0.1 kg/year per bear) was retained in the body. On an area basis, salmon-N redistribution rates were as high as 5.1±0.7 mg/m² per year per bear within 500 m of the stream but dropped off greatly with increasing distance. We estimated that 15.5–17.8% of the total N in spruce foliage within 500 m of the stream was derived from salmon. Of that, bears had distributed 83–84%. Thus, brown bears can be an important vector of salmon-derived N into riparian ecosystems, but their effects are highly variable spatially and a function of bear density. Received: 11 February 1999 / Accepted: 7 July 1999     

Mitogenetic structure of brown bears (Ursus arctos L.) in northeastern Europe and a new time frame for the formation of European brown bear lineages

We estimated the phylogenetic relationships of brown bear maternal haplotypes from countries of northeastern Europe (Estonia, Finland and European Russia), using sequences of mitochondrial DNA (mtDNA) control region of 231 bears. Twenty-five mtDNA haplotypes were identified. The brown bear population in northeastern Europe can be divided into three haplogroups: one with bears from all three countries, one with bears from Finland and Russia, and the third composed almost exclusively of bears from European Russia. Four haplotypes from Finland and European Russia matched exactly with haplotypes from Slovakia, suggesting the significance of the current territory of Slovakia in ancient demographic processes of brown bears. Based on the results of this study and those from the recent literature, we hypothesize that the West Carpathian Mountains have served either as one of the northernmost refuge areas or as an important movement corridor for brown bears of the Eastern lineage towards northern Europe during or after the last ice age. Bayesian analyses were performed to investigate the temporal framework of brown bear lineages in Europe. The molecular clock was calibrated using Beringian brown bear sequences derived from radiocarbon-dated ancient samples, and the estimated mutation rate was 29.8% (13.3%-47.6%) per million years. The whole European population and Western and Eastern lineages formed about 175,000, 70,000 and 25,000 years before present, respectively. Our approach to estimating the time frame of brown bear evolution demonstrates the importance of using an appropriate mutation rate, and this has implications for other studies of Pleistocene populations.     

Sudden expansion of a single brown bear maternal lineage across northern continental Eurasia after the last ice age: a general demographic model for mammals?

The brown bear has proved a useful model for studying Late Quaternary mammalian phylogeography. However, information is lacking from northern continental Eurasia, which constitutes a large part of the species' current distribution. We analysed mitochondrial DNA sequences (totalling 1943 bp) from 205 bears from northeast Europe and Russia in order to characterize the maternal phylogeography of bears in this region. We also estimated the formation times of the sampled brown bear lineages and those of its extinct relative, the cave bear.
Four closely related haplogroups belonging to a single mitochondrial subclade were identified in northern continental Eurasia. Several haplotypes were found throughout the whole study area, while one haplogroup was restricted to Kamchatka. The haplotype network, estimated divergence times and various statistical tests indicated that bears in northern continental Eurasia recently underwent a sudden expansion, preceded by a severe bottleneck. This brown bear population was therefore most likely founded by a small number of bears that were restricted to a single refuge area during the last glacial maximum. This pattern has been described previously for other mammal species and as such may represent one general model for the phylogeography of Eurasian mammals. Bayesian divergence time estimates are presented for different brown and cave bear clades. Moreover, our results demonstrate the extent of substitution rate variation occurring throughout the phylogenetic tree, highlighting the need for appropriate calibration when estimating divergence times.     

Introgressive hybridization: brown bears as vectors for polar bear alleles

The dynamics and consequences of introgression can inform about numerous evolutionary processes. Biologists have therefore long been interested in hybridization. One challenge, however, lies in the identification of nonadmixed genotypes that can serve as a baseline for accurate quantification of admixture. In this issue of Molecular Ecology, Cahill et al. (2015) analyse a genomic data set of 28 polar bears, eight brown bears and one American black bear. Polar bear alleles are found to be introgressed into brown bears not only near a previously identified admixture zone on the Alaskan Admiralty, Baranof and Chichagof (ABC) Islands, but also far into the North American mainland. Elegantly contrasting admixture levels at autosomal and X chromosomal markers, Cahill and colleagues infer that male‐biased dispersal has spread these introgressed alleles away from the Late Pleistocene contact zone. Compared to a previous study on the ABC Island population in which an Alaskan brown bear served as a putatively admixture‐free reference, Cahill et al. (2015) utilize a newly sequenced Swedish brown bear as admixture baseline. This approach reveals that brown bears have been impacted by introgression from polar bears to a larger extent (up to 8.8% of their genome), than previously known, including the bear that had previously served as admixture baseline. No evidence for introgression of brown bear into polar bear is found, which the authors argue could be a consequence of selection. Besides adding new exciting pieces to the puzzle of polar/brown bear evolutionary history, the study by Cahill and colleagues highlights that wildlife genomics is moving from analysing single genomes towards a landscape genomics approach.     

Comparison of fecal preservation and extraction methods for steroid hormone metabolite analysis in wild crested macaques

Since the non-invasive field endocrinology techniques were developed, several fecal preservation and extraction methods have been established for a variety of species. However, direct adaptation of methods from previous studies for use in crested macaques should be taken with caution. We conducted an experiment to assess the accuracy and stability of fecal estrogen metabolite (E1C) and glucocorticoid metabolite (GCM) concentrations in response to several preservation parameters: (1) time lag between sample collection and fecal preservation; (2) long-term storage of fecal samples in 80% methanol (MeOH) at ambient temperature; (3) different degrees of feces drying temperature using a conventional oven; and (4) different fecal preservation techniques (i.e., freeze-drying, oven-drying, and field-friendly extraction method) and extraction solvents (methanol, ethanol, and commercial alcohol). The study used fecal samples collected from crested macaques (Macaca nigra) living in the Tangkoko Reserve, North Sulawesi, Indonesia. Samples were assayed using validated E1C and GCM enzyme immunoassays. Concentrations of E1C and GCM in unprocessed feces stored at ambient temperature remained stable for up to 8 h of storage after which concentrations of both E1C and GCM changed significantly compared to controls extracted at time 0. Long-term storage in 80% MeOH at ambient temperature affected hormone concentrations significantly with concentrations of both E1C and GCM increasing after 6 and 4 months of storage, respectively. Drying fecal samples using a conventional oven at 50, 70, and 90 °C did not affect the E1C concentrations, but led to a significant decline for GCM concentrations in samples dried at 90 °C. Different fecal preservation techniques and extraction solvents provided similar results for both E1C and GCM concentrations. Our results confirm previous studies that prior to application of fecal hormone analysis in a new species, several preservation parameters should be evaluated for their effects on hormone metabolite stability. The results also provide several options for fecal preservation, extraction, and storage methods that can be selected depending on the condition of the field site and laboratory.