Analytical and physiological validation of an enzyme immunoassay to measure oxytocin in dog, wolf, and human urine samples.

Oxytocin (OT) promotes pro-sociality, bonding, and cooperation in a variety of species. Measuring oxytocin metabolite (OTM) concentrations in urine or saliva provides intriguing opportunities to study human and animal behaviour with minimal disturbance. However, a thorough validation of analytical methods and an assessment of the physiological significance of these measures are essential. We conducted an analytical validation of a commercial Enzyme Immunoassay (EIA; Arbor OT assay kit) to measure OTM concentrations in dog, wolf, and human urine samples. To test the assay's ability to detect changes in OTM concentrations, we administered oxytocin intranasally to 14 dogs. Assay performance with regard to parallelism was acceptable. Assay accuracy and extraction efficiency for dog and wolf samples were comparable to a previously validated assay (Enzo OT assay kit) but variation was smaller for human samples. Binding sensitivity and antibody specificity were better in the Arbor assay. Average OTM concentrations were more than twice as high as in comparable samples measured with the Enzo assay, highlighting a lack of comparability of absolute values between different assays. Changes in OTM concentrations after intranasal treatment were detected reliably. The Arbor assay met requirements of a "fit-for-purpose" validation with improvement of several parameters compared to the Enzo assay.

Behavioral response of naïve and non-naïve deer to wolf urine.

Large carnivores are recolonizing many regions in Europe, where their ungulate prey have lived without them for >150 years. Whether the returning large carnivores will modify ungulate behavior and indirectly affect lower trophic levels, depends on the ability of ungulates to recognize risk based on past encounters and cues indicating carnivore presence. In two case studies, we tested, by means of camera trapping, the behavioral response of deer to wolf urine. The first case study was in the Netherlands where deer (still) live in absence of wolves, and the second in Poland with long-term wolf presence. As controls we used water (no scent) and all-purpose soap (unfamiliar scent). Deer vigilance level on control plots was 20% in both case studies indicating that wolf occupancy per se does not lead to a consistent difference in behavior. Placing wolf urine did not significantly affect deer behavior in either the wolf-absent or the wolf-present area. More intense cues, or a combination of cues, are likely needed to affect deer behavior. Moreover, we found an unexpected reaction of deer towards all-purpose soap of reduced foraging (and tendency for increased vigilance) in the wolf-present area, whereas it did not affect deer behavior in the wolf-absent area. We hypothesize that deer associate all-purpose soap with human presence, causing no response in human-dominated landscapes (the Netherlands), but triggering a behavioral reaction in more remote areas (Poland). This illustrates attention should be paid to controls used in scent experiments as they may be associated differently than intended.

Analytical validation of an Enzyme Immunoassay for the measurement of urinary oxytocin in dogs and wolves.

Assessing changes in oxytocin (OT) levels in response to a variety of social stimuli has become of major interest in the field of behavioral endocrinology. OT is involved in the regulation of various aspects of social behavior such as tolerance, and the formation and maintenance of social bonds but also the regulation of stress. All of these aspects have been identified as potential targets of selection during the domestication process. Therefore, comparing the role of the oxytocinergic system in various aspects of dog and wolf social behavior, might help to understand whether this system was involved in the domestication process. Studies assessing OT levels in dogs and wolves have used invasively collected plasma and serum samples and non-invasively collected urine samples. However, when using an assay system on a new species a careful and complete validation of the method is of crucial importance, and to date no proper validation, to assess urinary OT levels in dogs and wolves, has been reported. We therefore conducted an analytical validation of an Enzyme Immunoassay (EIA) for the measurement of OT in urine of dogs and wolves, using a commercially available EIA. Stability tests revealed that OT levels degrade over time when stored at 4 °C, but are little affected by repeated thawing. In addition, our results indicate that the variance in OT levels is slightly lower when phosphoric acid is added following collection to prevent OT degradation. Long term storage tests revealed that urinary OT levels are least variable when stored as extracts in ethanol at -20 °C, rather than as unextracted urine samples. Validation results were acceptable with regard to parallelism, but values for accuracy and extraction efficiency were not meeting the standard criteria usually applied to steroid EIAs, especially when assessed for the lower range of the assay. The results of this study highlight the importance of an analytical assay validation, since even if validation parameters are not optimal, if published, they allow readers to estimate the relevance of studies using the validated method.

Pyrazine analogues are active components of wolf urine that induce avoidance and freezing behaviours in mice.

BACKGROUND: The common grey wolf (Canis lupus) is found throughout the entire Northern hemisphere and preys on many kinds of mammals. The urine of the wolf contains a number of volatile constituents that can potentially be used for predator-prey chemosignalling. Although wolf urine is put to practical use to keep rabbits, rodents, deer and so on at bay, we are unaware of any prior behavioural studies or chemical analyses regarding the fear-inducing impact of wolf urine on laboratory mice. METHODOLOGY/PRINCIPAL FINDINGS: Three wolf urine samples harvested at different times were used in this study. All of them induced stereotypical fear-associated behaviors (i.e., avoidance and freezing) in female mice. The levels of certain urinary volatiles varied widely among the samples. To identify the volatiles that provoked avoidance and freezing, behavioural, chemical, and immunohistochemical analyses were performed. One of the urine samples (sample C) had higher levels of 2,6-dimethylpyrazine (DMP), trimethylpyrazine (TMP), and 3-ethyl-2,5-dimethyl pyrazine (EDMP) compared with the other two urine samples (samples A and B). In addition, sample C induced avoidance and freezing behaviours more effectively than samples A and B. Moreover, only sample C led to pronounced expression of Fos-immunoreactive cells in the accessory olfactory bulb (AOB) of female mice. Freezing behaviour and Fos immunoreactivity were markedly enhanced when the mice were confronted with a mixture of purified DMP, TMP, and EDMP vs. any one pyrazine alone. CONCLUSIONS/SIGNIFICANCE: The current results suggest that wolf urinary volatiles can engender aversive and fear-related responses in mice. Pyrazine analogues were identified as the predominant active components among these volatiles to induce avoidance and freezing behaviours via stimulation of the murine AOB.

Predator and heterospecific stimuli alter behaviour in cattle.

Wild and domestic ungulates modify their behaviour in the presence of olfactory and visual cues of predators but investigations have not exposed a domestic species to a series of cues representing various predators and other ungulate herbivores. We used wolf (Canis lupus), mountain lion (Puma concolor), and mule deer (Odocoileus hemionus) stimuli (olfactory and visual), and a control (no stimuli) to experimentally test for differences in behaviour of cattle (Bos taurus) raised in Arizona. We measured (1) vigilance, (2) foraging rates, (3) giving up density (GUD) of high quality foods and (4) time spent in high quality forage locations in response to location of stimuli treatments. In general, we found a consistent pattern in that wolf and deer treatments caused disparate results in all 4 response variables. Wolf stimuli significantly increased cattle vigilance and decreased cattle foraging rates; conversely, deer stimuli significantly increased cattle foraging rate and increased cattle use of high quality forage areas containing stimuli. Mountain lion stimuli did not significantly impact any of the 4 response variables. Our findings suggest that domestic herbivores react to predatory stimuli, can differentiate between stimuli representing two predatory species, and suggest that cattle may reduce antipredatory behaviour when near heterospecifics.

Capillariidae eggs found in the urine of a free ranging maned wolf from Argentina.

The first finding of a Capillariid in the urinary tract of a free ranging maned wolf (Chrysocyon brachyurus) is described. The individual was an adult male attacked by dogs in the locality of Cayastacito (Santa Fe, Argentina, 31 degrees 05' S, 60 degrees 34' W). Eggs found in urine measured 64.6-66.9 micrometer (mean 65.4 micrometer) x 26.9-31 micrometer (mean 29 micrometer). Further studies are needed to determine whether this finding corresponds to a new Capillariid species, related to C. brachyurus, or it is an already described species that has been introduced by domestic dogs.