Sexual discrimination and attraction through scents in the water vole, Arvicola terrestris.

In mammals, especially rodents, social behaviours, such as parenting, territoriality or mate attraction, are largely based on olfactory communication through chemosignals. These behaviours are mediated by species-specific chemosignals, including small organic molecules and proteins that are secreted in the urine or in various fluids from exocrine glands. Chemosignal detection is mainly ensured by olfactory neurons in two specific sensory organs, the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). This study aimed to characterise the olfactory communication in the fossorial ecotype of the water voles, Arvicola terrestris. We first measured the olfactory investigation of urine and lateral scent gland secretions from conspecifics. Our results showed that water voles can discriminate the sex of conspecifics based on the smell of urine, and that urinary male odour is attractive for female voles. Then, we demonstrated the ability of the VNO and MOE to detect volatile organic compounds (VOCs) found in water vole secretions using live-cell calcium imaging in dissociated cells. Finally, we evaluated the attractiveness of two mixtures of VOCs from urine or lateral scent glands in the field during a cyclical outbreak of vole populations.

Design of a structure-activity relationship model of vitamin K epoxide reductase (VKORC1) inhibitors combining chemical synthesis of new compounds, enzymatic assays and molecular modelling.

Vitamin K antagonists (VKAs) anticoagulants have been used since the 1950s as medicines and rodenticides. These molecules are mainly 4-hydroxycoumarin derivatives and act by inhibiting the vitamin K epoxide reductase (VKORC1), an endoplasmic reticulum membrane resident enzyme. However, many VKORC1 mutations have been reported over the last decade, inducing VKAs resistances and thus treatments failures. Although studies have reported experimental and computational investigations of VKAs based on VKORC1 structural homology models, the development of new effective anticoagulants has been quite complex due to the lack of structural data and reliable structure-activity relationships. However, the recent publication of VKORC1 crystal structure provides new information for further studies. Based on these findings, we combined chemical synthesis, enzymatic assays and molecular modelling methods to design a structure-activity relationship (SAR) model. Our results proved that the lipophilicity, the membrane permeability of inhibitors and their affinity towards human VKORC1 enzyme are the main characteristics for potent anticoagulants. Our SAR model managed to rank compounds according to their ability to inhibit the human VKORC1. Such a tool might constitute an alternative to evaluate new molecules potency before their chemical synthesis and biological assessment and might assist the development of new VKAs.

Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity.

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is a target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and are suspected to confer resistance to VKA and (or) affect its velocity. Nevertheless, the results of these studies have been conflicting, and the functional characterization of these mutations in the cell system is complex because of the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to evaluate the vitamin K cycle in HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when VKORC1 and VKORC1L1 were knocked out, the K/KO balance decreased significantly due to the accumulation of vitamin KO. In contrast, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating the limitation of VKOR activity. This limitation was shown to be due to insufficient expression of the activation partner of VKORC1, as overexpression of protein disulfide isomerase (PDI) overcomes this limitation. This study is the first to demonstrate the functional interaction between VKORC1 and PDI.

Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra- and inter-species model to predict the suitable formulation mix.

The ecotoxicity of anticoagulants used for rodent pests' management is a major concern, particularly with second generation anticoagulants, which are more persistent in the body of rodents and therefore more likely to cause secondary exposure in their predators. One of the solutions envisaged to mitigate this risk is to use stereoisomers of these anticoagulants, each of which has particular pharmacokinetics. However, the few studies published to date have considered only one species and one sex. Here, we study the pharmacokinetics of the 4 stereoisomers of 3.4 mg/kg of difethialone in rats (Rattus norvegicus) and 3 mg/kg in mice (Mus musculus) in both sexes and propose a model to choose the optimal stereoisomer efficacy/ecotoxicity mixture for the management of all these animals. Our results show that while the most persistent stereoisomer (E3-cis) is common to both species and sexes, the pharmacokinetics of the other stereoisomers show marked differences between sexes and species. Thus, the area under curve (AUC) of E4-trans in male rats is four times lower than in females or mice, making it a priori unusable in male rats. Conversely, our modeling seems to show that the E1-trans stereoisomer seems to offer the best compromise AUC persistence. In conclusion, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy and with regard to the ecotoxicity of these molecules.

Distribution of non-synonymous Vkorc1 mutations in roof rats (Rattus rattus) in France and in Spain - consequences for management.

Rodent control is mainly done using anticoagulant rodenticides leading to the death of rodents through internal bleeding by targeting the VKORC1 protein. However, mutations in VKORC1 can lead to resistance to anticoagulant rodenticides that can cause treatment failure in the field. This study provides the first insight into the distribution, frequency and characterization of Vkorc1 mutations in roof rats (Rattus rattus) in France and in three administrative areas of Spain. The roof rat is present in France while it was thought to have almost disappeared with the expansion of the brown rat. Nevertheless, it has been found mainly in maritime areas. 151 roof rats out of 219 tested presented at least one missense mutation in the coding sequences of Vkorc1 gene (i.e. 69.0% of the rat). Nine Vkorc1 genotypes were detected (Y25F, A26P, R40G, S57F, W59C, W59R, H68N, Y25F/K152T and Y25F/W59R. Biochemical characterization of the consequences of these different genotypes proved that these various genotypes did not induce severe resistance to anticoagulant rodenticides. Even if many mutations of the Vkorc1 gene are present in roof rat populations in France, their management may be based in a first approach, considering the low levels of resistance induced, on the use of first-generation anticoagulants less dangerous for wildlife. The use of second-generation may be considered when treatment failure is observed or when bait consumption is limited.

Photoperiod is involved in the regulation of seasonal breeding in male water voles (Arvicola terrestris).

Mammals living at temperate latitudes typically display annual cyclicity in their reproductive activity: births are synchronized when environmental conditions are most favorable. In a majority of these species, day length is the main proximate factor used to anticipate seasonal changes and to adapt physiology. The brain integrates this photoperiodic signal through key hypothalamic structures, which regulate the reproductive axis. In this context, our study aimed to characterize regulations that occur along the hypothalamo-pituitary-gonadal (HPG) axis in male fossorial water voles (Arvicola terrestris, also known as Arvicola amphibius) throughout the year and to further probe the implication of photoperiod in these seasonal regulations. Our monthly field monitoring showed dramatic seasonal changes in the morphology and activity of reproductive organs, as well as in the androgen-dependent lateral scent glands. Moreover, our data uncovered seasonal variations at the hypothalamic level. During the breeding season, kisspeptin expression in the arcuate nucleus (ARC) decreases, while RFRP3 expression in the dorsomedial hypothalamic nucleus (DMH) increases. Our follow-up laboratory study revealed activation of the reproductive axis and confirmed a decrease in kisspeptin expression in males exposed to a long photoperiod (summer condition) compared with those maintained under a short photoperiod (winter condition) that retain all features reminiscent of sexual inhibition. Altogether, our study characterizes neuroendocrine and anatomical markers of seasonal reproductive rhythmicity in male water voles and further suggests that these seasonal changes are strongly impacted by photoperiod.

Seasonal diet-based resistance to anticoagulant rodenticides in the fossorial water vole (Arvicola amphibius).

Anticoagulant rodenticides (AR) resistance has been defined as "a major loss of efficacy due to the presence of a strain of rodent with a heritable and commensurately reduced sensitivity to the anticoagulant". The mechanism that supports this resistance has been identified as based on mutations in the Vkorc1 gene leading to severe resistance in rats and mice. This study evaluates the validity of this definition in the fossorial water vole and explores the possibility of a non-genetic diet-based resistance in a strict herbivorous rodent species. Genetic support was explored by sequencing the Vkorc1 gene and the diet-based resistance was explored by the dosing of vitamins K in liver of voles according to seasons. From a sample of 300 voles, only 2 coding mutations, G71R and S149I, were detected in the Vkorc1 gene in the heterozygous state with low allele frequencies (0.5-1%). These mutations did not modify the sensitivity to AR, suggesting an absence of genetic Vkorc1-based resistance in the water vole. On the contrary, vitamin K1 was shown to be 5 times more abundant in the liver of the water vole compared to rats. This liver concentration was shown to seasonally vary, with a trough in late winter and a peak in late spring/early summer related to the growth profile of grass. This increase in concentration might be responsible for the increased resistance of water voles to AR. This study highlights a non-genetic, diet-related resistance mechanism in rodents to AR. This diet-based resistance might explain the different evolution of the Vkorc1 gene in the fossorial water vole compared to rats and mice.

Field study reveals morphological and neuroendocrine correlates of seasonal breeding in female water voles, Arvicola terrestris.

Seasonally breeding mammals display timely physiological switches between reproductive activity and sexual rest, which ensure synchronisation of births at the most favourable time of the year. These switches correlate with seasonal changes along the hypothalamo-pituitary-gonadal axis, but they are primarily orchestrated at the hypothalamic level through environmental control of KISS1-dependent GnRH release. Our field study shows that births of fossorial water voles, Arvicola terrestris, are concentrated between March and October, which indicates the existence of an annual reproductive cycle in this species. Monthly field monitoring for over a year further reveals dramatic seasonal changes in the morphology of the ovary, uterus and lateral scent glands, which correlate with the reproductive status. Finally, we demonstrate seasonal variation in kisspeptin expression within the hypothalamic arcuate nucleus. Altogether, this study demonstrates a marked rhythm of seasonal breeding in the water vole and we speculate that this is governed by seasonal changes in photoperiod.

Structural Investigation of the Vitamin K Epoxide Reductase (VKORC1) Binding Site with Vitamin K.

The vitamin K epoxide reductase (VKORC1) enzyme is of primary importance in many physiological processes, i.e., blood coagulation, energy metabolism, and arterial calcification prevention, due to its role in the vitamin K cycle. Indeed, VKORC1 catalyzes reduction of vitamin K epoxide to quinone and then to hydroquinone. However, the three-dimensional VKORC1 structure remains experimentally undetermined, because of the endoplasmic reticulum membrane location of this enzyme. Here we present a molecular modeling investigation of the VKORC1 enzymatic site structure and function, supported by in vitro enzymatic assays. Four VKORC1 mutants were designed in silico (F55G, F55Y, N80G, and F83G) based on a previous study that identified residues F55, N80, and F83 as being crucial for vitamin K epoxide binding. F55G, N80G, and F83G nonconservative mutants were all predicted to be inactive by molecular modeling analyses. However, the F55Y conservative mutant was expected to be active compared to wild-type VKORC1. In vitro enzymatic assays performed on recombinant proteins assessed our molecular modeling hypotheses and led us to describe the role of accurate VKORC1 active site residues with respect to VKORC1. Residues F55, N80, and F83 appeared to act in a concerted manner to keep vitamin K epoxide close to the C135 catalytic residue. Residues F55 and N80 prevent naphthoquinone head rotation away from the active site, assisted by residue F83 that prevents vitamin K from sliding outside the enzymatic pocket, through hydrophobic tail stabilization. Our results thus highlighted the specific functions of VKORC1 catalytic pocket residues and evidenced the ability of our structural model to predict biological effects of VKORC1 mutations.

Comparative biological properties of the four stereoisomers of difethialone, a second-generation anticoagulant rodenticide, in rats: development of a model allowing to choose the appropriate stereoisomeric ratio.

The current management of rodent pest populations is based on second-generation anticoagulant rodenticides (SGAR). These molecules, of which difethialone is part, are much more efficient than the first generation. Nevertheless, this efficiency comes with a major drawback, SGARs are tissue persistent that increases the exposure of rodent predators to them. According to its chemical structure, difethialone has four stereoisomers, whose specific inhibition potency and pharmacokinetic have never been described and might be useful to design new eco-friendly rodenticides. The study aimed to investigate the ability to inhibit anticoagulant target enzyme (VKORC1) and the pharmacokinetics in rats of the four difethialone stereoisomers in rats. We show that stereoisomers are all highly efficient to inhibit VKORC1 activity, but they have distinct initial half-life with 6.0 h, 25.4 h, 69.3 h, and 82.3 h for, respectively, E4-trans, E2-cis, E1-trans, and E3-cis stereoisomer. These results open the way of the development of eco-friendly and efficient rodenticide by mixing some of these stereoisomers. Preferential incorporation of the E4-trans stereoisomer (high inhibitory VKORC1 potency, relatively shorter liver half-life) into difethialone rodenticides baits might result in a more eco-friendly product than current commercially available difethialone formulations. In addition, we put forward modelling to help design bait according to the circumstance of use (presence of non-target species, food competition, etc.) by modulating the theorical AUC and and the theorical concentration of the product at the death of the rodent pest. Thus, this modeling might allow to diminish the use of laboratory animal in assay.

Thrombin generation test: A reliable tool to evaluate the pharmacodynamics of vitamin K antagonist rodenticides in rats.

Vitamin K antagonist rodenticide pharmacodynamics (PD) is studied in rodents with traditional laboratory tests. We wondered if thrombin generation test (TGT) could add value. Difethialone (10 mg/kg) was administered per os to 97 OFA-Sprague Dawley rats. PD was studied over a 72 h-period using the Calibrated Automated Thrombogram on platelet poor plasma before and after intoxication (3 female and 3 male rats for each 13 time points) and TGT parameters were compared with the prothrombin time (PT) and vitamin K dependent factor activities previously reported. Following intoxication, preliminary tests evidenced rapid and full inhibition of thrombin generation triggered with 5 or 20 pM human recombinant tissue factor. To study the evolution of TGT parameters following difethialone intake, we adapted the test by complementing intoxicated rat samples with pooled normal rat plasma (3/1, v/v). Adapted TGT confirmed the known higher procoagulant basal level in females compared to males through higher endogenous thrombin potential (ETP) and peak height (PH) (p < 0.0001 and p = 0.0003, respectively). An exponential model fitted well the PH and ETP decay after intoxication. In contrast to PT, the decreases were observed immediately following VKA intake and had comparable time to halving values: 10.5 h (95% CI [8.2; 13.6]) for ETP and 10.4 h (95% CI [7.8; 14.1]) for PH. The decrease of FVII and FX preceded that of PH, ETP and FII while FIX decreased later on, contributing to the severe hypo-coagulability. We demonstrated that TGT performed in samples of intoxicated rats complemented with normal plasma is a reliable tool for evaluation of VKA rodenticide PD in rats.

Management of Rodent Populations by Anticoagulant Rodenticides: Toward Third-Generation Anticoagulant Rodenticides.

Second-generation anticoagulant rodenticides (SGARs) have been used since the 1980s for pest management. They are highly efficient even in warfarin-resistant rodents. Nevertheless, because of their tissue persistence, nontarget poisoning by SGARs is commonly described in wildlife. Due to this major problem, a new generation of anticoagulants must be developed to limit this risk. This study proposes a method of developing a new generation of anticoagulant rodenticides by revisiting the old SGARs based on the concept of stereochemistry. Each current SGAR is a mixture of diastereomers. Diastereomers of each compound were purified, and their biologic properties were compared by determining their ability to inhibit vitamin K epoxide reductase (VKOR) activity involved in the activation of vitamin K-dependent clotting factors and their toxicokinetic properties. Systematically, for each SGAR, both diastereomers are as effective in inhibiting VKOR activity. However, their toxicokinetic properties are very different, with one of the two diastereomers always more rapidly cleared than the other one. For all SGARs except flocoumafen, the less persistent diastereomer is always the less predominant isomer present in the current mixture. Therefore, the development of baits containing only the less persistent diastereomer would avoid the ecotoxicological risk associated with their use without decreasing their efficacy.

Synthesis and biological evaluation of C-3 aliphatic coumarins as vitamin K antagonists.

Since the discovery of Warfarin in the 1940s, the design of new warfarin-derived anticoagulants for rodent management has been challenging, with mainly structural modifications performed on the C3 position of the coumarin skeleton. In order to better understand the pharmacomodulation of such derivatives, we have synthesized a family of C3 (linear and branched) alkyl-4-hydroxycoumarins, which led to the identification of compounds 5e and 5f as potential short-term active anticoagulants.

Monitoring of antivitamin K-dependent anticoagulation in rodents - Towards an evolution of the methodology to detect resistance in rodents.

Vitamin K antagonists are used as rodenticides for pest control management. In rodents, prothrombin time is used to monitor their effect despite its limits and the emergence of many coagulation methods. The aim of this study is to explore different coagulation monitoring methods in order to propose the best method and the best parameter to monitor vitamin K antagonists effect in rodents. The coagulation function was thus monitored with global coagulation assays and specialty assays after difethialone administration in rats. Despite many parameters obtained by thromboelastometry, only clotting time and clot formation time obtained by ExTEM were modified. Their evolution was fast with doubling time respectively of 4.0h and 3.7h but their increases were delayed with a lag time higher than 8h. Conversely, prothrombin time evolution presented a lag time of only 2h, but a higher doubling time of 7.2h. The measurements of factor VII and X activities were the most sensitive assays to monitor vitamin K antagonists effect with almost no lag time and the fastest evolution. Nevertheless, factor X was shown to be the only key factor driving prothrombin time. Monitoring factor X activity enables to follow most effectively the anticoagulation status in rats after rodenticides administration.

Development of an Ecofriendly Anticoagulant Rodenticide Based on the Stereochemistry of Difenacoum.

Difenacoum, an antivitamin K anticoagulant, has been widely used as rodenticide to manage populations of rodents. Difenacoum belongs to the second generation of anticoagulant, and, as all the molecules belonging to the second generation of anticoagulant, difenacoum is often involved in primary poisonings of domestic animals and secondary poisonings of wildlife by feeding contaminated rodents. To develop a new and ecofriendly difenacoum, we explored in this study the differences in properties between diastereomers of difenacoum. Indeed, the currently commercial difenacoum is a mixture of 57% of cis-isomers and 43% of trans-isomers. Cis- and trans-isomers were thus purified on a C18 column, and their respective pharmacokinetic properties and their efficiency to inhibit the coagulation of rodents were explored. Tissue persistence of trans-isomers was shown to be shorter than that of cis-isomers with a half-life fivefold shorter. Efficiency to inhibit the vitamin K epoxide reductase activity involved in the coagulation process was shown to be similar between cis- and trans-isomers. The use of trans-isomers of difenacoum allowed to drastically reduce difenacoum residues in liver and other tissues of rodents when the rodent is moribund. Therefore, secondary poisonings of wildlife should be decreased by the use of difenacoum largely enriched in trans-isomers.

VKORC1L1, an enzyme rescuing the vitamin K 2,3-epoxide reductase activity in some extrahepatic tissues during anticoagulation therapy.

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.

Structural investigation of vitamin K epoxide reductase domain-containing protein in Leptospira species: a potential target for the development of new leptospirosis treatments as an alternative to antibiotics.

Leptospirosis is a worldwide zoonosis caused by the motile bacterium Leptospira. This disease can cause hemorrhagic symptoms, multi-visceral and renal failures, resulting in one million cases and approximately 60,000 deaths each year. The motility of Leptospira is highly involved in its virulence and is ensured by the presence of two flagella in the periplasm. Several proteins that require the formation of disulfide bridges are essential for flagellar function. In Leptospira, these redox reactions are catalysed by the vitamin K epoxide reductase domain-containing protein (VKORdcp). The aim of the present work was to study the conservation of VKORdcp among Leptospira species and its interactions with putative substrates and inhibitor. Our results evidenced the presence of ten amino acids specific to either pathogenic or saprophytic species. Furthermore, structural studies revealed a higher affinity of the enzyme for vitamin K1 quinone, compared to ubiquinone. Finally, characterisation of the binding of a potential inhibitor revealed the involvement of some VKORdcp amino acids that have not been present in the human enzyme, in particular the polar residue D114. Our study thus paves the way for the future development of Leptospira VKORdcp inhibitors, capable of blocking bacterial motility. Such molecules could therefore offer a promising therapeutic alternative to antibiotics, especially in the event of the emergence of antibiotic-resistant strains.Communicated by Ramaswamy H. Sarma.

Stereoselective bioaccumulation of chiral anticoagulant rodenticides in the liver of predatory and scavenging raptors.

Second-generation anticoagulant rodenticides (SGARs) are persistent chiral pesticides used to control rodent populations. Raptors are protected species and may be exposed through the ingestion of rodents contaminated with SGARs. Commercial formulations of SGARs are a mixture of four stereoisomers (E1, E2, E3, E4): the cis- and trans-diastereoisomers are each a racemic mixture of two enantiomers. In this study, the residue levels of all SGARs (bromadiolone, difenacoum, brodifacoum, difethialone, flocoumafen) were evaluated in the liver of 529 raptor carcasses. All species (n = 18) and 75 % of individuals (n = 396) were SGAR positive and 29 % (n = 154) had summed hepatic concentrations above 100 ng/g ww. Concentrations were higher for predators with facultative scavenging behaviors than for predators and obligate scavengers. Bromadiolone, brodifacoum and difenacoum had equivalent hepatic prevalence (between 48.9 and 49.9 %), and difethialone was detected less frequently (31.7 %). Concentrations and enantiomeric fractions of the four stereoisomers of all SGARs are described in to demonstrate the biological enantioselectivity of these chiral pesticides in the food chain. A difference was observed between the proportions of SGARs diastereoisomers and stereoisomers in the liver of all raptor species and in commercial baits. The enantioselective bioaccumulation of E1-trans-bromadiolone, E3-cis-brodifacoum, E1-cis-difenacoum and E3-cis-difethialone was characterized and represented 96.8 % of total SGARs hepatic residues. While hepatic concentrations were heterogeneous, the proportions of stereoisomers and diastereoisomers were homogeneous with no inter-individual or inter-species differences (only E1-trans-bromadiolone is present in hepatic residues). However, proportions of brodifacoum stereoisomers and diastereoisomers were more scattered, probably due to their slower elimination. This could provide an opportunity to date the exposure of individuals to brodifacoum. We highlight the need to consider each SGAR as four molecular entities (four stereoisomers) rather than one. These findings suggest new commercial formulations with the less persistent stereoisomers could reduce secondary exposure of non-target species.

Interest of the faecal and plasma matrix for monitoring the exposure of wildlife or domestic animals to anticoagulant rodenticides.

Anticoagulant rodenticides (ARs), particularly second-generation compounds (SGAR), are known to be a potential threat to unintended species due to their tissue persistence. The liver is the storage tissue of ARs and is a matrix of choice in diagnosing exposure and intoxication of non-target fauna. However, it is only available on dead animals. Blood and faeces can be used on living animals. These two biological matrices were compared in terms of their relevance to exposure to ARs. In addressing this question, we compared the faecal, plasma and liver concentrations of bromadiolone, one of the SGAR frequently implicated in wildlife exposure. We studied this comparison at the individual level and at the population level, considering three influencing factors: dose, sex and time. Our findings demonstrate that faecal analyses are more valuable than plasma analyses for monitoring AR exposure of domestic and wild animals, even if faecal concentrations cannot be correlated with liver concentrations.