First Immunohistochemical Demonstration of the Expression of a Type-2 Vomeronasal Receptor, V2R2, in Wild Canids.

The mammalian vomeronasal system enables the perception of chemical signals crucial for social communication via the receptor families V1R and V2R. These receptors are linked with the G-protein subunits, Gαi2 and Gαo, respectively. Exploring the evolutionary pathways of V1Rs and V2Rs across mammalian species remains a significant challenge, particularly when comparing genomic data with emerging immunohistochemical evidence. Recent studies have revealed the expression of Gαo in the vomeronasal neuroepithelium of wild canids, including wolves and foxes, contradicting predictions based on current genomic annotations. Our study provides detailed immunohistochemical evidence, mapping the expression of V2R receptors in the vomeronasal sensory epithelium, focusing particularly on wild canids, specifically wolves and foxes. An additional objective involves contrasting these findings with those from domestic species like dogs to highlight the evolutionary impacts of domestication on sensory systems. The employment of a specific antibody raised against the mouse V2R2, a member of the C-family of vomeronasal receptors, V2Rs, has confirmed the presence of V2R2-immunoreactivity (V2R2-ir) in the fox and wolf, but it has revealed the lack of expression in the dog. This may reflect the impact of domestication on the regression of the VNS in this species, in contrast to their wild counterparts, and it underscores the effects of artificial selection on sensory functions. Thus, these findings suggest a more refined chemical detection capability in wild species.

Comparison of anticoagulant rodenticide concentrations in liver and feces from apparently healthy red foxes.

Exposure of wildlife and domestic animals to anticoagulant rodenticides (ARs) is a worldwide concern, but few methods exist to determine residue levels in live animals. Traditional liver detection methods preclude determining exposure in live wildlife. To determine the value of assessing AR exposure by fecal analysis, we compared fecal and liver residues of ARs in the same animals. We collected liver and fecal samples from 40 apparently healthy red foxes (Vulpes vulpes) potentially exposed to ARs, and quantified brodifacoum, bromadiolone, coumatetralyl, difenacoum, difethialone, and flocoumafen residues by liquid chromatography-tandem mass spectrometry. Residues of ARs were detected in 53% of the fecal samples and 83% of the liver samples. We found good concordance between AR residues in feces and liver for coumatetralyl, difenacoum, and difethialone. Bromadiolone occurred in significantly greater frequency in livers compared to feces, but no significant difference in concentration between feces and liver in individual foxes could be detected. Brodifacoum displayed a significant difference in concentration and occurrence of positive samples between liver and feces. Our findings demonstrate that fecal analysis of ARs provides a feasible and valuable non-lethal means of determine AR exposure in live wildlife.

The vomeronasal organ of wild canids: the fox (Vulpes vulpes) as a model.

The vomeronasal system (VNS) has been extensively studied within specific animal families, such as Rodentia. However, the study of the VNS in other families, such as Canidae, has long been neglected. Among canids, the vomeronasal organ (VNO) has only been studied in detail in the dog, and no studies have examined the morphofunctional or immunohistochemical characteristics of the VNS in wild canids, which is surprising, given the well-known importance of chemical senses for the dog and fox and the likelihood that the VNS plays roles in the socio-reproductive physiology and behaviours of these species. In addition, characterising the fox VNS could contribute to a better understanding of the domestication process that occurred in the dog, as the fox would represent the first wild canid to be studied in depth. Therefore, the aim of this study was to analyze the morphological and immunohistochemical characteristics of the fox VNO. Tissue dissection and microdissection techniques were employed, followed by general and specific histological staining techniques, including with immunohistochemical and lectin-histochemical labelling strategies, using antibodies against olfactory marker protein (OMP), growth-associated protein 43 (GAP-43), calbindin (CB), calretinin (CR), α-tubulin, Gαo, and Gαi2 proteins, to highlight the specific features of the VNO in the fox. This study found significant differences in the VNS between the fox and the dog, particularly concerning the expression of Gαi2 and Gαo proteins, which were associated with the expression of the type 1 vomeronasal receptors (V1R) and type 2 vomeronasal receptors (V2R), respectively, in the vomeronasal epithelium. Both are immunopositive in foxes, as opposed to the dog, which only expresses Gαi2. This finding suggests that the fox possesses a well-developed VNO and supports the hypothesis that a profound transformation in the VNS is associated with domestication in the canid family. Furthermore, the unique features identified in the fox VNO confirm the necessity of studying the VNS system in different species to better comprehend specific phylogenetic aspects of the VNS.

Fear and Foxes: An Educational Primer for Use with "Anterior Pituitary Transcriptome Suggests Differences in ACTH Release in Tame and Aggressive Foxes".

The way genes contribute to behavior is complicated. Although there are some single genes with large contributions, most behavioral differences are due to small effects from many interacting genes. This makes it hard to identify the genes that cause behavioral differences. Mutagenesis screens in model organisms, selective breeding experiments in animals, comparisons between related populations with different behaviors, and genome-wide association studies in humans are promising and complementary approaches to understanding the heritable aspects of complex behaviors. To connect genes to behaviors requires measuring behavioral differences, locating correlated genetic changes, determining when, where, and how these candidate genes act, and designing causative confirmatory experiments. This area of research has implications from basic discovery science to human mental health.

Innervation of the pineal gland in the Arctic fox (Vulpes lagopus) by nerve fibres immunoreactive to substance P and calcitonin gene-related peptide.

BACKGROUND: The study demonstrates, for the first time, the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the nerve fibres supplying the pineal gland in the Arctic fox. MATERIALS AND METHODS: The expression and distribution pattern of the studied substances were examined by double-labelling immunofluorescence technique. RESULTS: The SP-positive fibres enter into the pineal gland through the capsule as the nervi conarii. The fibres formed thick bundles in the capsule and connective tissue septa, from where they penetrated into the pineal parenchyma. Inside the parenchyma, the nerve fibres created basket-like structures surrounding clusters of pinealocytes. The density of intrapineal SP positive fibres was slightly higher in the distal and middle parts of the gland than in the proximal one. Double immunostaining with antibodies against SP and CGRP revealed that the vast majority of SP positive fibres were also CGRP positive. The fibres showing a positive reaction to SP and negative to CGRP were scattered within the whole gland. The fibres immunopositive to CGRP and immunonegative to SP were not observed. In the habenular and posterior commissural areas adjoining to the pineal gland the immunoreactive nerve fibres were not found. Moreover, no immunopositive cell bodies were observed in both the pineal gland and the commissural areas. CONCLUSIONS: These results reveal that SP and CGRP are involved in the innervation of pineal gland in carnivores. In turn we suggest that these peptides can regulate/modulate melatonin secretion.

Effects of Vitamin E and Selenium on Growth Performance, Antioxidant Capacity, and Metabolic Parameters in Growing Furring Blue Foxes (Alopex lagopus).

The objective of this study was to determine whether different dietary vitamin E (VE) and selenium (Se) levels affect the nutrient digestibility, production performance, and antioxidant abilities of growing furring blue foxes. A 4 × 2 factorial arrangement that included 4 levels of VE (0, 100, 200, or 400 mg/kg diet from α-tocopherol acetate) and 2 levels of Se (0 or 0.2 mg/kg diet from glycine selenium) was performed from mid-September to pelting. A metabolism study was conducted for four days starting at the 30th day of the trial. Serum samples were collected at the last day of the study. The results showed that supplementation of growing furring blue fox diets with VE and Se significantly affected the average daily gain (ADG), average daily feed intake, and feed conversion ratio (F:G) (P < 0.05). Dietary Se supplementation enhanced protein and fat digestibility of male blue foxes. There were significant effects of different VE and Se levels in diets on serum antioxidant parameters and metabolic parameters of blue foxes (P < 0.05). In conclusion, this research indicated that dietary supplementation with VE improved ADG and F:G of blue foxes. Addition of VE and Se to blue fox diets increased the antioxidant capacity of blue foxes. The diet with high VE and Se supplementation reduced glucose and triglycerides concentrations in serum. The present study found that growing furring blue foxes had increased growth performance and antioxidant abilities when fed diets with 200 mg VE/kg and nearly 0.1 mg Se/kg.

Concentrations of cadmium and lead, but not zinc, are higher in red fox tissues than in rodents-pollution gradient study in the Malopolska province (Poland).

The main purpose of our research was to assess the chronic exposure of red foxes to Cd, Pb and Zn. We have determined concentrations of these metals in the kidney, liver and muscle of 36 red foxes hunted between December 2002 and March 2003 in differently polluted areas in southern Poland. Tissue concentrations of Pb and Cd in the red foxes significantly co-varied with concentrations of these elements in the soil, and differed between the tissues. We compared concentrations of Pb, Cd, and Zn in red foxes with two rodent species, Apodemus flavicollis and Myodes glareolus, trapped simultaneously in the same area. Concentrations of Pb and Cd appeared significantly higher in the red foxes than in the rodents, but the slopes of the relationship between metal concentrations in tissues and in soil were similar in all species. This indicates that extrapolation from results of monitoring studies conducted on rodents to mammalian carnivores is possible but requires applying an extrapolation factor.

Anterior Pituitary Transcriptome Suggests Differences in ACTH Release in Tame and Aggressive Foxes.

Domesticated species exhibit a suite of behavioral, endocrinological, and morphological changes referred to as "domestication syndrome." These changes may include a reduction in reactivity of the hypothalamic-pituitary-adrenal (HPA) axis and specifically reduced adrenocorticotropic hormone release from the anterior pituitary. To investigate the biological mechanisms targeted during domestication, we investigated gene expression in the pituitaries of experimentally domesticated foxes (Vulpes vulpes). RNA was sequenced from the anterior pituitary of six foxes selectively bred for tameness ("tame foxes") and six foxes selectively bred for aggression ("aggressive foxes"). Expression, splicing, and network differences identified between the two lines indicated the importance of genes related to regulation of exocytosis, specifically mediated by cAMP, organization of pseudopodia, and cell motility. These findings provide new insights into biological mechanisms that may have been targeted when these lines of foxes were selected for behavior and suggest new directions for research into HPA axis regulation and the biological underpinnings of domestication.

Concentrations and patterns of hydroxylated polybrominated diphenyl ethers and polychlorinated biphenyls in arctic foxes (Vulpes lagopus) from Svalbard.

Concentrations and patterns of hydroxylated (OH) polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) were investigated in liver from arctic foxes (Vulpes lagopus) sampled from Svalbard 1997-2011 (n = 100). The most important OH-PBDE in the arctic foxes was 6-OH-BDE47 detected in 24% of the samples. Relationships between 6-OH-BDE47, δ(13)C and BDE47 suggest that 6-OH-BDE47 residues in arctic foxes are related to marine dietary input, while the relative importance of the metabolic/natural origin of this compound remains unclear. 4-OH-CB187 and 4-OH-CB146 were the main OH-PCBs among the analyzed compounds. The OH-PCB pattern in the present arctic foxes indicates that arctic foxes have a capacity to biotransform a wide range of PCBs of different structures. Formation and retention of OH-PCBs was tightly related to PCB exposure. Furthermore, ΣOH-PCB concentrations were four times higher in the leanest compared to the fattest foxes. Concentrations of 4-OH-CB187 and 4-OH-CB146 among the highest contaminated arctic foxes were similar to the previously reported concentrations for polar bears. Given the high endocrine disruptive potential of OH-PCBs, we suggest that endocrine system may be affected by the relatively high OH-PCB residues in the Svalbard arctic fox population.

De novo assembly and characterization of farmed blue fox (Alopex lagopus) global transcriptome using Illumina paired-end sequencing.

The blue fox (Alopex lagopus), a coat-color variant of the Arctic fox, is a domesticated fur-bearing mammal. In the present study, transcriptome data generated from a pool of nine different tissues were obtained with Illumina HiSeq2500 paired-end sequencing technology. After filtering from raw reads, 32,358,290 clean reads were assembled into 161,269 transcripts and 97,252 unigenes by the Trinity fragment assembly software. Of the assembled unigenes, 37,967 were annotated in the National Center for Biotechnology Information (NCBI) Non-Redundant (NR) protein database and 26,264 in the Swiss-Prot database. Among the annotated unigenes, 24,839 and 24,267 were assigned using the Gene Ontology (GO) and euKaryotic Orthologous Groups (KOG) databases, respectively. Altogether, 17,057 unigenes were mapped onto 227 pathways using the Kyoto Encyclopedia of Genes and Genomes database. In addition, 6394 simple sequence repeats were identified by examining 12,965 unigenes (>1 kb), which could contribute to the development of molecular markers. This study generated transcriptome data for the blue fox that will promote further progress in expression profiling studies, and provide a good annotation basis for genomic studies.

Levels of metals in kidney, liver and muscle tissue and their relation to the occurrence of parasites in the red fox in the Lower Silesian Forest in Europe.

Together with the occurrence of parasites, increased concentrations of xenobiotics, to which scavengers are greatly exposed, may significantly influence the physiology of red foxes. It is also suspected that these two factors interact. The accumulation of various metals (Ca, Cd, Cu, Fe, Hg, K, Mg, Ni, Pb, Zn) in kidney, liver and muscle tissue was investigated, as well as the occurrence of parasites, and the potential link to the presence of metals. Generally speaking, neither sex nor age influenced these concentrations. K, Mg and Fe were found in the highest concentrations and Hg was found in the lowest. Various relationships between the concentrations of metals were observed in the tissues. 34% of the specimens studied were hosts to parasites. No clear, significant connection between the concentrations and the occurrence of parasites was noted, but the discernible trend confirmed by the logistic regression, needs further study.

Perfluoroalkyl Acid Concentrations in Livers of Fox (Vulpes vulpes) and Chamois (Rupicapra rupicapra) from Germany and Austria.

The concentrations of 11 perfluorinated alkyl acids (PFAAs) were measured in the livers of foxes (Vulpes vulpes) from Germany, a primarily carnivorous species, and chamois (Rupicapra rupicapra) from Austria, an herbivorous species. Perfluorooctanesulfonate (PFOS) at concentrations [all results refer to wet weight (ww)] of 3.2-320 µg/kg were detected in all 40 fox livers tested, yielding an arithmetic mean of 46.6 µg/kg and a median of 29.8 µg/kg. Long-chain PFAAs were detected at concentrations of 1.7 µg/kg perfluorononanoic acid (PFNA) to 2.4 µg/kg perfluorodecanoic acid (PFDA) and perfluorododecanoic acid (PFDoDA). Of the short-chain PFAAs tested, only perfluorohexanoic acid (PFHxA) was found in 1 fox liver at a concentration of 1.4 µg/kg, and perfluorohexane sulfonate (PFHxS) was found in 2 fox livers at a concentration of 1 µg/kg each. PFOS and PFNA concentrations higher than limit of quantification (LOQ) were detected in 90.9 and 81.8 % of chamois livers, respectively. The arithmetic mean for PFOS concentrations was 2.2 µg/kg (median 2.4 µg/kg), a factor of 21 (median factor of 12) lower than in fox livers. The arithmetic mean for PFNA concentrations was 2.0 µg/kg (median 1.9 µg/kg). Perfluorobutanoic acid, PFHxA, perfluorooctanoic acid, perfluorobutanesulfonate, and PFHxS were not detected at concentrations higher than the LOQ in any of the samples. The various results are compared with one another and with the results of other studies of herbivorous, carnivorous, and omnivorous wild animals. The highest concentrations of PFAA, in particular PFOS, were found in omnivorous animals followed by carnivores. The lowest levels were present in herbivores.

Hair and bone as predictors of tissular mercury concentration in the western Alaska red fox, Vulpes vulpes.

We evaluated if total mercury (THg) concentrations of keratin-based and bone-based tissues can predict THg concentrations in skeletal muscle, renal medulla, renal cortex, and liver. The THg concentration in matched tissues of 65 red foxes, Vulpes vulpes, from western Alaska was determined. Hair THg concentration had a significant positive correlation with liver, renal medulla, renal cortex, and muscle. The THg concentration for males and females is moderately predictive of THg concentration in the renal cortex and liver for these foxes based on R(2) values (R(2)=0.61 and 0.63, respectively). Bone is weakly predictive of THg concentration in muscle (R(2)=0.40), but not a reliable tissue to predict THg concentration in liver (R(2)=0.24), renal cortex (R(2)=0.35), or renal medulla (R(2)=0.25). These results confirm the potential use of trapped animals, specifically foxes, as useful Arctic sentinel species to inform researchers about patterns in THg levels over time as industrialization of the Arctic continues.

[The influence of stinging nettle (Urtica dioica L.) extracts on the activity of catalase in THP1 monocytes/macrophages]. / Wplyw ekstraktów z pokrzywy zwyczajnej (Urtica dioica L.) na aktywnosc katalazy w monocytach/makrofagach THP1.

INTRODUCTION: Stinging nettle (Urtica dioicd L.) is one of the most valuable plants used in phytotherapy. The herbal raw material is a herb (Urticae herba), leaves (Urticae folium), roots (Urticae radix) and seeds (Urticae semina). This plant is a good source of vitamins, minerals, fibre, protein and biologically active compounds with antioxidant properties. The literature provides limited information about the chemical composition and properties of the seed heads. No papers are available on the effect of extracts of this plant on catalase activity in human cells. The aim of this study was to investigate the impact of stinging nettle (Urtica dioica L.) extracts on the antioxidant activity of catalase in THP1 macrophages. MATERIAL AND METHODS: Two types of extracts: water and alcohol, at two different concentrations, were used in experiments. Nettle was collected in September and October in 2012 in the area of Szczecin. The collected plant material was frozen and lyophilized. After those procedures water and alcohol extracts of nettle were prepared and then added to THP1 cells. RESULTS AND CONCLUSIONS: The antioxidant activity of catalase was established with the spectrophotometric method. The study showed that both extracts (water and alcohol) significantly increased the antioxidant activity of catalase in THP1 cells. The increase in catalase was directly proportional to the concentration of the added alcohol extract.

Accumulation and potential health effects of organohalogenated compounds in the arctic fox (Vulpes lagopus)--a review.

This review addresses biological effects of anthropogenic organohalogenated compounds in the arctic fox (Vulpes lagopus). When considering the current levels, spatial and tissue distributions of selected organic pollutants in arctic fox subpopulations, especially the Svalbard based populations accumulate high levels. The dominating contaminant groups are the polychlorinated biphenyls (PCBs) and chlordanes (CHLs), which reach high levels in adipose tissues, adrenals and liver. Recent controlled exposure studies on domesticated arctic fox and Greenland sledge dogs, show adverse health effects associated with OC concentrations lower than those measured in free-ranging populations. This indicates that especially populations at Svalbard may be at risk of experiencing OC related effects. The arctic fox as such may be an overlooked species in the Arctic Monitoring and Assessment Programs and it would add further information about pollution in the Arctic to include this species in the monitoring program.

Heavy metal concentrations in the small intestine of red fox (Vulpes vulpes) with and without Echinococcus multilocularis infection.

Heavy metal (Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) levels in red fox small intestine samples with or without Echinococcus multilocularis infection were studied. The red foxes were taken from the open countryside of northwest Bohemia (CR). Red foxes with E. multilocularis infection had lower levels of toxic metals (Cd, Pb); cadmium levels in infected foxes (0.0052 mg/kg) were twice as low as in uninfected foxes (0.0106 mg/kg). This was the same case for lead: 0.0288 mg/kg infected red foxes (inf.) and 0.0413 mg/kg uninfected (uninf.). Conversely, red foxes with E. multilocularis infection yielded higher concentrations in comparison to their uninfected counterparts: Cr (0.0087 mg/kg uninf. and 0.0116 mg/kg inf.), Cu (0.2677 mg/kg uninf. and 0.3205 mg/kg inf.), Fe (6.46 mg/kg uninf. and 10.89 mg/kg inf.), Mn (0.1966 mg/kg uninf. and 0.2029 mg/kg inf.), Ni (0.0415 mg/kg uninf. and 0.064 mg/kg inf.) and Zn (16.71 mg/kg uninf. and 20.25 mg/kg inf). This could support the hypothesis that tapeworms are able to absorb toxic heavy metals from the host body into their tissues, as well as to modify other element concentrations in the host body.

Levels and temporal trends of persistent organic pollutants (POPs) in arctic foxes (Vulpes lagopus) from Svalbard in relation to dietary habits and food availability.

Temporal trends of persistent organic pollutants (POPs) in arctic foxes (Vulpes lagopus) from Svalbard, Norway, were investigated in relation to feeding habits and seasonal food availability. Arctic foxes from Svalbard forage in both marine and terrestrial ecosystems and the availability of their food items are impacted by climatic variability. Concentrations of polychlorinated biphenyls (PCBs), organochlorinated pesticides (OCPs) and brominated flame retardants (polybrominated diphenyl ethers [PBDEs] and hexabromocyclododecane [HBCDD]) were analyzed in the liver of 141 arctic foxes collected between 1997 and 2013. Stable carbon isotope values (δ13C) were used as a proxy for feeding on marine versus terrestrial prey. The annual number of recovered reindeer carcasses and sea ice cover were used as proxies for climate influenced food availability (reindeers, seals). Linear models revealed that concentrations of PCBs, chlordanes, p,p'-DDE, mirex and PBDEs decreased 4-11% per year, while no trends were observed for hexachlorobenzene (HCB) or ß-hexachlorocyclohexane (ß-HCH). Positive relationships between POP concentrations and δ13C indicate that concentrations of all compounds increase with increasing marine dietary input. Increasing reindeer mortality was related to lower HCB concentrations in the foxes based on the linear models. This suggests that concentrations of HCB in arctic foxes may be influenced by high mortality levels of Svalbard reindeer. Further, ß-HCH concentrations showed a positive association with sea ice cover. These results in addition to the strong effect of δ13C on all POP concentrations suggest that climate-related changes in arctic fox diet are likely to influence contaminant concentrations in arctic foxes from Svalbard.

Use of biochemical markers to evaluate the quality of fresh and cryopreserved semen from the arctic fox (Vulpes lagopus).

The aim of this study was to use biochemical markers to evaluate the quality of fresh and cryopreserved semen from the arctic fox (Vulpes lagopus). Twenty-three manually collected ejaculates were analysed for the main indicators of semen quality (sperm concentration and ejaculate volume). Sperm motility and percentage of morphologically normal and abnormal spermatozoa were determined according to the stage of cryopreservation (fresh--measurement A; equilibrated--measurement B; frozen/thawed--measurement C). Furthermore, the seminal plasma and supernatants were analysed after equilibration and freeze/thawing for the activity of the enzymes alkaline phosphatase (ALP), acid phosphatase (AcP), lactate dehydrogenase (LDH) and aspartate aminotransferase (AspAT), and for the activity of acrosin inhibitors (AP). The mean concentration of sperm was 625.1 million/cm3, and ejaculate volume averaged 1.6 cm3. Seminal plasma was characterized by the highest activity of alkaline phosphatase (3.43 x 10(3) U/l) and lowest activity of acrosin inhibitors (4.55 x 10(3) U/l). After equilibration, the supernatants showed the highest activity of acid phosphatase (94.9 U/l) and after freeze-thawing, they showed a high activity of lactate dehydrogenase (535.8 U/l) and aspartate aminotransferase (577.1 U/l), which indicates that these proteins had leaked from spermatozoa into the extracellular medium during the biotechnique of semen cryopreservation. In addition, several significant relationships were found between some indicators of semen quality and plasma and/or supernatant enzyme activity.

Fluoride in the bones of foxes (Vulpes vulpes Linneaus, 1758) and raccoon dogs (Nyctereutes procyonoides Gray, 1834) from North-Western Poland.

Assessment of exposure to fluoride (F(-)) is increasingly focused on mineralized tissues, mainly bones. Their periodic growth and continuous reconstruction make them a good material for studying long-term F(-) accumulation. In this study, F(-)concentrations were determined in the bones of foxes and raccoon dogs from north-western Poland and relationships between bone F(-) and the age categories of the animals were attempted to be identified. Bone samples were collected from femurs of 32 foxes (15 males and 17 females) and 18 raccoon dogs (10 males and 8 females) from polluted, medium-polluted, and unpolluted by F(-) areas. Bone F(-) was determined by potentiometric method, and results were expressed per dry weight (dw); they ranged from 176 to 3,668 mg/kg dw in foxes and from 84 to 1,190 mg/kg dw in raccoon dogs. Foxes from north-western Poland accumulated much more F(-) in their bones than raccoon dogs. Our study shows that the assessment of hazards created by industrial emitters can be conducted conveniently by the measurements of fluorine content in hard tissues of wild animals. Due to availability of such type of material for studies, it seems that the analysis of fluoride content in bones can be a good tool in the development of ecotoxicology.