Using meta-analysis to understand the impacts of dietary protein and fat content on the composition of fecal microbiota of domestic dogs (Canis lupus familiaris): A pilot study.

The interplay between diet and fecal microbiota composition is garnering increased interest across various host species, including domestic dogs. While the influence of dietary macronutrients and their associated microbial communities have been extensively reviewed, these reviews are descriptive and do not account for differences in microbial community analysis, nor do they standardize macronutrient content across studies. To address this, a meta-analysis was performed to assess the impact of dietary crude protein ("protein") and dietary crude fat ("fat") on the fecal microbiota composition in healthy dogs. Sixteen publications met the eligibility criteria for the meta-analysis, yielding a final data set of 314 dogs. Diets were classed as low, moderate, high, or supra in terms of protein or fat content. Sequence data from each publication were retrieved from public databases and reanalyzed using consistent bioinformatic pipelines. Analysis of community diversity indices and unsupervised clustering of the data with principal coordinate analysis revealed a small effect size and complete overlap between protein and fat levels at the overall community level. Supervised clustering through random forest analysis and partial least squares-discriminant analysis indicated alterations in the fecal microbiota composition at a more individual taxonomic level, corresponding to the levels of protein or fat. The Prevotellaceae Ga6A1 group and Enterococcus were associated with increasing levels of protein, while Allobaculum and Clostridium sensu stricto 13 were associated with increasing levels of fat. Interestingly, the random forest analyses revealed that Sharpea, despite its low relative abundance in the dog's fecal microbiome, was primarily responsible for the separation of the microbiome for both protein and fat. Future research should focus on validating and understanding the functional roles of these relatively low-abundant genera.

The anti-hyperlipidemic effects of Poria cocos (Schw.) Wolf extract: Modulating cholesterol homeostasis in hepatocytes via PPARα pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Poria cocos (Schw.) Wolf (Polyporaceae, P.cocos), which is born on the pine root, has a history of more than two thousand years of medicine in China. P.cocos was first recorded in the Shennong's Herbal Classic, studies have proved its lipid-lowering effect. AIM OF STUDY: The aim of study was to investigate the underlying mechanism of P.cocos extract on hyperlipidemia. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats aged 9-12 weeks were intraperitoneally (IP) injected with Triton-WR 1339 to establish an acute hyperlipidemia model. At 0 h and 20 h after the model was established, low and high doses of P.cocos extract or simvastatin were given twice. After 48 h, the rats were sacrificed, and liver and serum samples were collected for analysis. The cell model was constructed by treating L02 cells with 1% fat emulsion-10% FBS-RPMI 1640 medium for 48 h. At the same time, low and high doses of P.cocos extract and simvastatin were administered. Oil red O staining was used to evaluate the lipid accumulation in the cells, and H&E staining was used to evaluate the liver lesions of rats. Real-time quantitative PCR and western blotting were used to detect the expressions of lipid metabolism-related genes. RESULTS: P.cocos extract relieved lipid accumulation in vitro and alleviated hyperlipidemia in vivo. Both gene and protein expressions of peroxisome proliferator-activated receptor α (PPARα) were shown to be up-regulated by P.cocos extract. Additionally, P.cocos extract down-regulated the expressions of fatty acid synthesis-related genes sterol regulatory element-binding protein-1 (SREBP-1), Acetyl-CoA Carboxylase 1 (ACC1) and fatty acid synthase (FAS), while up-regulated the expressions of cholesterol metabolism-related genes liver X receptor-α (LXRα), ATP-binding cassette transporter A1 (ABCA1), cholesterol 7alpha-hydroxylase (CYP7A1) and low density lipoprotein receptor (LDLR), which were reversed by the treatment with the PPARα inhibitor GW6471. CONCLUSION: P.cocos extract ameliorates hyperlipidemia and lipid accumulation by regulating cholesterol homeostasis in hepatocytes through PPARα pathway. This study provides evidence that supplementation with P.cocos extract could be a potential strategy for the treatment of hyperlipidemia.

Metabolomics shows the Australian dingo has a unique plasma profile.

Dingoes occupy a wide range of the Australian mainland and play a crucial role as an apex predator with a generalist omnivorous feeding behaviour. Dingoes are ecologically, phenotypically and behaviourally distinct from modern breed dogs and have not undergone artificial selection since their arrival in Australia. In contrast, humans have selected breed dogs for novel and desirable traits. First, we examine whether the distinct evolutionary histories of dingoes and domestic dogs has lead to differences in plasma metabolomes. We study metabolite composition differences between dingoes (n = 15) and two domestic dog breeds (Basenji n = 9 and German Shepherd Dog (GSD) n = 10). Liquid chromatography mass spectrometry, type II and type III ANOVA with post-hoc tests and adjustments for multiple comparisons were used for data evaluation. After accounting for within group variation, 62 significant metabolite differences were detected between dingoes and domestic dogs, with the majority of differences in protein (n = 14) and lipid metabolites (n = 12), mostly lower in dingoes. Most differences were observed between dingoes and domestic dogs and fewest between the domestic dog breeds. Next, we collect a second set of data to investigate variation between pure dingoes (n = 10) and dingo-dog hybrids (n = 10) as hybridisation is common in regional Australia. We detected no significant metabolite differences between dingoes and dingo-dog hybrids after Bonferroni correction. However, power analysis showed that increasing the sample size to 15 could result in differences in uridine 5'-diphosphogalactose (UDPgal) levels related to galactose metabolism. We suggest this may be linked to an increase in Amylase 2B copy number in hybrids. Our study illustrates that the dingo metabolome is significantly different from domestic dog breeds and hybridisation is likely to influence carbohydrate metabolism.

Urinary proteome and metabolome in dogs (Canis lupus familiaris): The effect of chronic kidney disease.

Chronic kidney disease (CKD) is a progressive and irreversible disease. Although urine is an ideal biological sample for proteomics and metabolomics studies, sensitive and specific biomarkers are currently lacking in dogs. This study characterised dog urine proteome and metabolome aiming to identify and possibly quantify putative biomarkers of CKD in dogs. Twenty-two healthy dogs and 28 dogs with spontaneous CKD were selected and urine samples were collected. Urinary proteome was separated by SDS-PAGE and analysed by mass spectrometry, while urinary metabolome was analysed in protein-depleted samples by 1D 1H NMR spectra. The most abundant proteins in urine samples from healthy dogs were uromodulin, albumin and, in entire male dogs, arginine esterase. In urine samples from CKD dogs, the concentrations of uromodulin and albumin were significantly lower and higher, respectively, than in healthy dogs. In addition, these samples were characterised by a more complex protein pattern indicating mixed glomerular (protein bands ≥65 kDa) and tubular (protein bands <65 kDa) proteinuria. Urine spectra acquired by NMR allowed the identification of 86 metabolites in healthy dogs, belonging to 49 different pathways mainly involved in amino acid metabolism, purine and aminoacyl-tRNA biosynthesis or tricarboxylic acid cycle. Seventeen metabolites showed significantly different concentrations when comparing healthy and CKD dogs. In particular, carnosine, trigonelline, and cis-aconitate, might be suggested as putative biomarkers of CKD in dogs. SIGNIFICANCE: Urine is an ideal biological sample, however few proteomics and metabolomics studies investigated this fluid in dogs and in the context of CKD (chronic kidney disease). In this research, applying a multi-omics approach, new insights were gained regarding the molecular changes triggered by this disease in canine urinary proteome and metabolome. In particular, the involvement of the tubular component was highlighted, suggesting uromodulin, trigonelline and carnosine as possible biomarkers of CKD in dogs.

Concentrations of Selected Elements in Liver Tissue of Grey Wolves (Canis lupus) from Serbia.

The grey wolf (Canis lupus) is a large carnivore species and a top predator in the ecosystems that it inhabits. Considering its role in food webs, wolves may be exposed to high concentrations of potentially harmful elements. Therefore liver samples from 28 legally hunted wolves were analyzed for concentrations of 16 elements using inductively coupled plasma optical emission spectrometry. The Mann-Whitney U test showed a significant difference between the genders only for Li, and there were no differences between individuals caught in different years. The majority of statistically significant correlations between element levels were positive, except for three cases. Compliance with several criteria for suitable bioindicator organisms imply that wolves may serve for monitoring environmental contamination.

Dietary items as possible sources of (137)Cs in large carnivores in the Gorski Kotar forest ecosystem, Western Croatia.

The mountain forest ecosystem of Gorski Kotar is distant from any significant sources of environmental pollution, though recent findings have revealed that this region is among the most intense (137)Cs contaminated area in Croatia. Therefore, the aim of this study was to investigate (137)Cs and (40)K load in three large predator species in the mountain forest ecosystem. Radionuclides mass activities were determined by the gamma-spectrometric method in the muscle tissue of brown bear (47), wolf (7), lynx (1) and golden jackal (2). The highest (137)Cs mass activity was found in lynx (153 Bq kg(-1)), followed by brown bear (132 Bq kg(-1)), wolf (22.2 Bq kg(-1)), and golden jackal (2.48 Bq kg(-1)). Analysis of 63 samples of dietary items suggests that they are not all potentially dominant sources of (137)Cs for wildlife. The most important source of radionuclides for the higher parts of the food-chain from the study area were found to be the mushroom species wood hedgehog (Hydnum repandum), with a transfer factor TF of 5.166, and blueberry (Vaccinium myrtillus) as a plant species (TF=2.096). Food items of animal origin indicated higher mass activity of radionuclides and therefore are possible moderate bioindicators of environmental pollution. The results also revealed that possible unknown wild animal food sources are a caesium source in the study region, and further study is required to illuminate this issue.

Environmental and Intrinsic Correlates of Stress in Free-Ranging Wolves.

BACKGROUND: When confronted with a stressor, animals react with several physiological and behavioral responses. Although sustained or repeated stress can result in severe deleterious physiological effects, the causes of stress in free-ranging animals are yet poorly documented. In our study, we aimed at identifying the main factors affecting stress levels in free-ranging wolves (Canis lupus). METHODOLOGY/PRINCIPAL FINDINGS: We used fecal cortisol metabolites (FCM) as an index of stress, after validating the method for its application in wolves. We analyzed a total of 450 fecal samples from eleven wolf packs belonging to three protected populations, in Italy (Abruzzo), France (Mercantour), and the United States (Yellowstone). We collected samples during two consecutive winters in each study area. We found no relationship between FCM concentrations and age, sex or social status of individuals. At the group level, our results suggest that breeding pair permanency and the loss of pack members through processes different from dispersal may importantly impact stress levels in wolves. We measured higher FCM levels in comparatively small packs living in sympatry with a population of free-ranging dogs. Lastly, our results indicate that FCM concentrations are associated with endoparasitic infections of individuals. CONCLUSIONS/SIGNIFICANCE: In social mammals sharing strong bonds among group members, the death of one or several members of the group most likely induces important stress in the remainder of the social unit. The potential impact of social and territorial stability on stress levels should be further investigated in free-ranging populations, especially in highly social and in territorial species. As persistent or repeated stressors may facilitate or induce pathologies and physiological alterations that can affect survival and fitness, we advocate considering the potential impact of anthropogenic causes of stress in management and conservation programs regarding wolves and other wildlife.

Ecotoxicoparasitology: Understanding mercury concentrations in gut contents, intestinal helminths and host tissues of Alaskan gray wolves (Canis lupus).

Some gastrointestinal helminths acquire nutrients from the lumen contents in which they live; thus, they may be exposed to non-essential elements, such as mercury (Hg), during feeding. The objectives of this study were: 1) determine the total mercury concentrations ([THg]) in Gray wolves (Canis lupus) and their parasites, and 2) use stable isotopes to evaluate the trophic relationships within the host. [THg] and stable isotopes (C and N) were determined for helminths, host tissues, and lumen contents from 88 wolves. Sixty-three wolves contained grossly visible helminths (71.5%). The prevalence of taeniids and ascarids was 63.6% (56/88) and 20.5% (18/88), respectively. Nine of these 63 wolves contained both taeniids and ascarids (14.3%). All ascarids were determined to be Toxascaris leonina. Taenia species present included T. krabbei and T. hydatigena. Within the GI tract, [THg] in the lumen contents of the proximal small intestine were significantly lower than in the distal small intestine. There was a significant positive association between hepatic and taeniid [THg]. Bioaccumulation factors (BAF) ranged from <1 to 22.9 in taeniids, and 1.1 to 12.3 in T. leonina. Taeniid and ascarid BAF were significantly higher than 1, suggesting that both groups are capable of THg accumulation in their wolf host. δ13C in taeniids was significantly lower than in host liver and skeletal muscle. [THg] in helminths and host tissues, in conjunction with stable isotope (C and N) values, provides insight into food-web dynamics of the host GI tract, and aids in elucidating ecotoxicoparasitologic relationships. Variation of [THg] throughout the GI tract, and between parasitic groups, underscores the need to further evaluate the effect(s) of feeding niche, and the nutritional needs of parasites, as they relate to toxicant exposure and distribution within the host.

Trophodynamics of current use pesticides and ecological relationships in the Bathurst region vegetation-caribou-wolf food chain of the Canadian Arctic.

The bioaccumulation of current use pesticides (CUPs) and stable isotopes of carbon and nitrogen were investigated in vegetation-caribou-wolf food chain in the Bathurst region (Nunavut, Canada). Volumetric bioconcentration factors (BCF(v)) in vegetation were generally greatest for dacthal (10-12) ≥ endosulfan sulfate (10-11) > ß-endosulfan (>9.0-9.7) ≥ pentachloronitrobenzene (PCNB; 8.4-9.6) > α-endosulfan (8.3-9.3) > chlorpyrifos (8.0-8.7) >chlorothalonil (7.6-8.3). The BCF(v) values in vegetation were significantly correlated with the logarithm of the octanol-air partition coefficients (log K(OA)) of CUPs (r(2) = 0.90, p = 0.0040), although dacthal was an outlier and not included in this relationship. Most biomagnification factors (BMFs) for CUPs in caribou:diet comparisons were significantly less than 1. Similarly, the majority of wolf:caribou BMFs were either significantly less than 1 or were not statistically greater than 1. Significant trophic magnification factors (TMFs) were all less than 1, indicating that these CUPs exhibit trophic dilution through this terrestrial food chain. The log K(OA) reasonably predicted bioconcentration in vegetation for most CUPs but was not correlated with BMFs or TMFs in mammals. Our results, along with those of metabolic studies, suggest that mammals actively metabolize these CUPs, limiting their biomagnification potential despite entry into the food chain through effective bioconcentration in vegetation.

Polonium-210 and Caesium-137 in lynx (Lynx lynx), wolverine (Gulo gulo) and wolves (Canis lupus).

Wolves, lynx and wolverines are on the top of the food-chain in northern Scandinavia and Finland. (210)Po and (137)Cs have been analysed in samples of liver, kidney and muscle from 28 wolves from Sweden. In addition blood samples were taken from 27 wolves. In 9 of the wolves, samples of muscle, liver and blood were analysed for (210)Po. Samples of liver and muscle were collected from 16 lynx and 16 wolverines from Norway. The liver samples were analysed for (210)Po and (137)Cs. Only (137)Cs analyses were carried out for the muscle samples. The wolves were collected during the winter 2010 and 2011, while the samples for lynx and wolverines were all from 2011. The activity concentrations of (210)Po in wolves were higher for liver (range 20-523 Bq kg(-1) d.w.) and kidney (range 24-942 Bq kg(-1) d.w.) than muscle (range 1-43 Bq kg(-1) d.w.) and blood (range 2-54 Bq kg(-1) d.w.). Activity ratios, (210)Po/(210)Pb, in wolf samples of muscle, liver and blood were in the ranges 2-77, 9-56 and 2-54. Using a wet weight ratio of 3.8 the maximal absorbed dose from (210)Po to wolf liver was estimated to 3500 µGy per year. Compared to wolf, the ranges of (210)Po in liver samples were lower in lynx (range 22-211 Bq kg(-1) d.w.) and wolverine (range16-160 Bq kg(-1) d.w.). Concentration of (137)Cs in wolf samples of muscle, liver, kidney and blood were in the ranges 70-8410 Bq kg(-1) d.w., 36-4050 Bq kg(-1) d.w., 31-3453 Bq kg(-1) d.w. and 4-959 Bq kg(-1) d.w., respectively. (137)Cs in lynx muscle and liver samples were in the ranges 44-13393 Bq kg(-1) d.w. and 125-10260 Bq kg(-1) d.w. The corresponding values for (137)Cs in wolverine were 22-3405 Bq kg(-1) d.w. for liver and 53-4780 Bq kg(-1) d.w. for muscle. The maximal absorbed dose from (137)Cs to lynx was estimated to 3000 µGy per year.

Mercury in gray wolves (Canis lupus) in Alaska: increased exposure through consumption of marine prey.

Mercury (Hg) bioaccumulates in the tissues of organisms and biomagnifies within food-webs. Gray wolves (Canis lupus) in Alaska primarily acquire Hg through diet; therefore, comparing the extent of Hg exposure in wolves, in conjunction with stable isotopes, from interior and coastal regions of Alaska offers important insight into their feeding ecology. Liver, kidney, and skeletal muscle samples from 162 gray wolves were analyzed for total mercury (THg) concentrations and stable isotopic signatures (δ(13)C, δ(15)N, and δ(34)S). Median hepatic THg concentrations were significantly higher in wolves with coastal access compared to wolves from interior Alaska. Stable isotope ratios, in conjunction with THg concentrations, provide strong evidence that coastal wolves are utilizing marine prey representing several trophic levels. The utilization of cross-ecosystem food resources by coastal wolves is clearly contributing to increased THg exposure, and may ultimately have negative health implications for these animals.

Noninvasive heavy metal pollution assessment by means of Iberian wolf (Canis lupus signatus) hair from Galicia (NW Spain): a comparison with invasive samples.

The current study represents the first investigation of the suitability of wolf hair as indicator tissue for metal exposure and accumulation within NW Spanish ecosystems. Dead animals, which were not specifically killed for these purposes, were necropsied, and further toxicological analyses were performed in order to establish the heavy metal content (Pb, Cd, and Zn) in liver, kidney, and hair, by means of inductively coupled plasma optical emission spectrometry. Two different factors, gender and age, were considered in order to determine their influence on heavy metal accumulation. Mean liver, kidney and hair concentrations of both toxic element, Cd (0.528, 2.692, and 0.026 ppm) and Pb (4.108, 0.031, and 0.196 ppm) considered on a dry weight basis, were situated below the established as acute toxicity levels for mammals. The highest concentrations were quantified for Zn, with means ranging from a maximum in hair samples (150.9 ppm) to a minimum in kidney samples (25.81 ppm). When the variable of gender and age were considered, female and adult wolves had higher concentrations of Cd in all the considered organs, although differences were only statistically significant for kidney. Neither the sex nor the age had a significant effect on Pb and Zn concentrations. A significant positive correlation (p < 0.05) was identified between hair and liver Pb concentrations, as well as between hair and kidney Cd concentrations, thus suggesting the interest of the noninvasive sample for future ecotoxicological biomonitoring studies.

Sex, stress and social status: patterns in fecal testosterone and glucocorticoid metabolites in male Ethiopian wolves.

Ethiopian wolves, Canis simensis, live in large multi-male family packs, where males are philopatric and do not disperse. Within a pack, mating and breeding is largely monopolized by the dominant male and female, although extra-pack copulations are common, and subordinate males may sire pups in neighboring packs. Regardless of paternity, all males in a pack help rear the pups. We non-invasively studied patterns in fecal testosterone and glucocorticoid metabolite concentrations using radioimmunoassays of fecal samples collected from nine wild male Ethiopian wolves between August 2007 and February 2008. We tested the predictions of the Challenge Hypothesis, namely that fecal testosterone metabolite concentrations would be higher during the annual mating season, which is the portion of the reproductive cycle when mating and increased aggression typically occur, and lower when there were pups in the pack for which to care. Contrary to the predictions of the Challenge Hypothesis, we did not detect patterns in fecal testosterone metabolite concentrations associated with reproductive stage during our study period. Similarly, we found no patterns associated with reproductive stage in male fecal glucocorticoid metabolite concentrations. Dominant males had higher average fecal testosterone and glucocorticoid metabolite concentrations than did subordinates, which may be related to higher rates of aggression and mate guarding in dominant males of group-living canids, a pattern also reported in African wild dogs, Lycaon pictus.

Biomagnification of perfluorinated compounds in a remote terrestrial food chain: Lichen-Caribou-wolf.

The biomagnification behavior of perfluorinated carboxylates (PFCAs) and perfluorinated sulfonates (PFSAs) was studied in terrestrial food webs consisting of lichen and plants, caribou, and wolves from two remote northern areas in Canada. Six PFCAs with eight to thirteen carbons and perfluorooctane sulfonate (PFOS) were regularly detected in all species. Lowest concentrations were found for vegetation (0.02-0.26 ng/g wet weight (ww) sum (Σ) PFCAs and 0.002-0.038 ng/g ww PFOS). Wolf liver showed highest concentrations (10-18 ng/g ww ΣPFCAs and 1.4-1.7 ng/g ww PFOS) followed by caribou liver (6-10 ng/g ww ΣPFCAs and 0.7-2.2 ng/g ww PFOS). Biomagnification factors were highly tissue and substance specific. Therefore, individual whole body concentrations were calculated and used for biomagnification and trophic magnification assessment. Trophic magnification factors (TMF) were highest for PFCAs with nine to eleven carbons (TMF = 2.2-2.9) as well as PFOS (TMF = 2.3-2.6) and all but perfluorooctanoate were significantly biomagnified. The relationship of PFCA and PFSA TMFs with the chain length in the terrestrial food chain was similar to previous studies for Arctic marine mammal food web, but the absolute values of TMFs were around two times lower for this study than in the marine environment. This study demonstrates that challenges remain for applying the TMF approach to studies of biomagnification of PFCAs and PFSAs, especially for terrestrial animals.

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum.

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.

Cadmium, copper, iron, and zinc concentrations in kidneys of grey wolves, Canis lupus, from Alaska, Idaho, Montana (USA) and the Northwest Territories (Canada).

Cadmium, copper, iron, and zinc levels were measured in the kidneys of 115 grey wolves (Canis lupus) from Idaho, Montana and Alaska (United States), and from the Northwest Territories (Canada). No significant differences in the levels of iron or copper were observed between locations, but wolf kidneys from more northern locations had significantly higher cadmium levels (Alaska > Northwest Territories > Montana ≈ Idaho), and wolves from Alaska showed significantly higher zinc than other locations. Additionally, female wolves in Alaska had higher iron levels than males, and adult wolves in Montana had higher copper levels than subadults.

Relationships between sex and stress hormone levels in feces and marking behavior in a wild population of Iberian wolves (Canis lupus signatus).

Feces deposited by the breeding alpha pair on exposed substrates and/or zones may act as visual and olfactory marks associated with social dominance in wolves. The aim of this study was to determine if there was a correlation between marking behavior, sex hormone levels, and physiological stress in a wild population of Iberian wolves in Northwest Spain. The glucocorticoid and sex hormone levels were measured in feces collected as a function of exposure (conspicuous/inconspicuous), height (above ground level/at ground level), and strategic location in the habitat (at crossroads/off crossroads), as well as the frequency of re-marking. The feces, believed to serve as marking cues, had higher glucocorticoid levels (cortisol) and sex hormones (testosterone, progesterone, and estradiol). The results suggest that in Iberian wolves, the alpha pair is subject to higher social stress than subordinate individuals, and that the reproductive suppression of subordinates is not mediated by chronic glucocorticoid elevation.

Quantification of sexual steroid hormones in faeces of Iberian wolf (Canis lupus signatus): a non-invasive sex typing method.

The determination of gender in wild animals is essential for behavioural and ecological studies, and also for conservation. The objectives of this study were (i) the determination of gender in faecal samples of Iberian wolf based on the differential concentrations of sexual steroid hormones (SSH) and (ii) to analyse the profiles of SSH in males and females (considering the gender determination carried out previously) during the non-reproductive and reproductive periods. The quantification of androgens (testosterone, T), progestin (progesterone, P) and oestrogen (oestradiol, E) was conducted by means of enzyme immunoassay. The k-means conglomerate analysis showed that the 59 faecal samples grouped into three different conglomerates, considering SSH levels. Groups 1 and 2 showed higher levels of T than group 3. Therefore, the faecal samples included in groups 1 and 2 (17 samples) corresponded to males and those of group 3 (42 samples) to females. The levels of T + P + E and T/P were higher in the group of males than in the group of females. The results of this study also showed that levels of T in males were higher during the reproductive period than in the non-reproductive period. However, the concentrations of P and E turned out to be higher during the non-reproductive season. In females, the levels of the three hormones (T, P and E) were higher during the reproductive period.

Stable isotopes, ecological integration and environmental change: wolves record atmospheric carbon isotope trend better than tree rings.

Large-scale patterns of isotope ratios are detectable in the tissues of organisms, but the variability in these patterns often obscures detection of environmental trends. We show that plants and animals at lower trophic levels are relatively poor indicators of the temporal trend in atmospheric carbon isotope ratios (delta13C) when compared with animals at higher trophic levels. First, we tested how differences in atmospheric delta13C values were transferred across three trophic levels. Second, we compared contemporary delta13C trends (1961-2004) in atmospheric CO2 to delta13C patterns in a tree species (jack pine, Pinus banksiana), large herbivore (moose, Alces alces) and large carnivore (grey wolf, Canis lupus) from North America. Third, we compared palaeontological (approx. 30000 to 12000 14C years before present) atmospheric CO2 trends to delta13C patterns in a tree species (Pinus flexilis, Juniperus sp.), a megaherbivore (bison, Bison antiquus) and a large carnivore (dire wolf, Canis dirus) from the La Brea tar pits (southern California, USA) and Great Basin (western USA). Contrary to previous expectations, we found that the environmental isotope pattern is better represented with increasing trophic level. Our results indicate that museum specimens of large carnivores would best reflect large-scale spatial and temporal patterns of carbon isotopes in the palaeontological record because top predators can act as ecological integrators of environmental change.

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.