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.

Endocrine changes related to dog domestication: Comparing urinary cortisol and oxytocin in hand-raised, pack-living dogs and wolves.

Dogs are exceptionally well adapted to life close to humans, and alterations in their endocrine system during the domestication process may be an underlying mechanism. In particular, it has been suggested that low circulating cortisol concentrations in conjunction with simultaneously high oxytocin concentrations may have resulted in dogs' increased docility ('selection for tameness' hypothesis) and heightened propensity to interact and form relationships with humans ('hypersociability' hypothesis) compared to wolves. To investigate this, we analyzed cortisol and oxytocin metabolite concentrations from urine samples of hand-raised, pack-living domestic dogs and their non-domestic relatives, grey wolves. Based on the hypotheses outlined above, we predicted lower cortisol but higher oxytocin concentrations in dogs than wolves. In contrast to our prediction, we found higher cortisol concentrations in dogs than wolves. However, oxytocin concentrations were higher in dogs compared to wolves although the effect was relatively small. Indeed, male dogs had the highest oxytocin concentrations while female dogs' oxytocin concentrations were comparable to wolves'. Feeding status, reproductive phase, and conspecific social interactions also significantly affected cortisol and oxytocin concentrations. Furthermore, we compared two methods of correcting for variable water content of urine samples. We discuss our results in light of physiological and behavioral changes during domestication and highlight the importance of accounting for confounding variables in future studies.

Consistency and efficacy of two methods of intranasal oxytocin application in dogs.

Over the last few years, oxytocin (OT) administration to investigate the role of the oxytocinergic system in the social behavior of dogs has become of more and more interest. To date, the most common OT administration method for dogs is the intranasal spray commonly used for humans. Due to the different nasal conformation of dogs and the unpleasantness of the procedure, most dogs need to be restrained to allow administration. This has 2 main drawbacks-it may hinder reliable administration, which might lead to tremendous variance in the uptake of OT across individuals and it is likely to be stressful for the dogs. Alternatively, a vaporizer mask can be used to administer aerolized OT and dogs can be trained to voluntarily enter the mask, which might enable a more reliable administration without having to restrain the dogs. The aim of this study was to compare the efficacy of these 2 methods to identify a reliable non-invasive method for exogenous OT administration, thereby assisting future research on the role of OT in canines. We administered OT to pet dogs using either an intranasal spray bottle or a vaporizer mask and assessed urinary OT concentrations as a measure of OT uptake. We found that only when administered using a vaporizer mask, OT significantly increased in all subjects, while using a spray bottle led to considerable variance in OT uptake and an inconsistent increase in urinary OT concentrations across individuals. These results suggest that using a vaporizer mask should be preferred over using an intranasal spray bottle for OT administration in dogs. If not available, experimenters should at least monitor OT uptake after administration using spray bottles, to evaluate the success of the method.

Pet dogs' relationships vary rather individually than according to partner's species.

Most dogs worldwide are free-ranging animals that form relationships mainly with conspecifics, yet research has focused mainly on the dog-human bond, leading to the hypothesis that dogs evolved specific abilities to form a unique relationship with humans. Although widespread, this hypothesis has not, as yet, been tested. Here we compared the relationships pet dogs form with their owner and with other dogs living in the same household. Using a bottom-up approach, we analyzed dogs' behavior in a test battery with both dog and human partners. Results revealed that pet dogs' relationships are characterized by three components (i.e. reference, affiliation and stress). A comparison between dogs' intra- and inter-specific relationships found that overall dogs refer more to their owner, but also that some dogs form stronger affiliative bonds with conspecifics than with their owner. Moreover, we tested how different partners could help dogs cope with a stressful situation. We found that the type of relationship, rather than the partner species, predicts how dogs react to a social threat. Our results suggest that dogs can form relationships of comparable qualities with both humans and other dogs, and that these relationships vary along multiple components across different partners.

Critical issues in experimental studies of prosociality in non-human species.

Prosociality and acts of altruism are defined as behaviours which benefit another with either no gain or some immediate cost to the self. To understand the evolutionary origins of these behaviours, in recent years, studies have extended to primate species; however, studies on non-primates are still scarce. In light of the fact that phylogenetic closeness to humans does not appear to correlate with prosocial tendencies, but rather differences in the propensity towards prosociality may be linked to allomaternal care or collaborative foraging, it appears that convergent selection pressures may be at work in the evolution of prosociality. It would hence seem particularly important to extend such studies to species outside the primate clade, to allow for comparative hypothesis testing of the factors affecting the evolution of prosocial behaviours. In the current review, we focus on the experimental paradigms which have been used so far (i.e. the prosocial choice task, helping paradigms and food-sharing tests) and highlight the strengths and weaknesses of each method. In line with the aim of encouraging a broader comparative approach to the topic of prosociality, particular emphasis is placed on the methodological issues that need to be taken into account. We conclude that although a number of the paradigms used so far may be successfully applied to non-primate species, there is a need to simplify the cognitive demands of the tasks and ensure task comprehension to allow for a 'fair' comparative approach of prosocial tendencies across species.

Social referencing and cat-human communication.

Cats' (Felis catus) communicative behaviour towards humans was explored using a social referencing paradigm in the presence of a potentially frightening object. One group of cats observed their owner delivering a positive emotional message, whereas another group received a negative emotional message. The aim was to evaluate whether cats use the emotional information provided by their owners about a novel/unfamiliar object to guide their own behaviour towards it. We assessed the presence of social referencing, in terms of referential looking towards the owner (defined as looking to the owner immediately before or after looking at the object), the behavioural regulation based on the owner's emotional (positive vs negative) message (vocal and facial), and the observational conditioning following the owner's actions towards the object. Most cats (79 %) exhibited referential looking between the owner and the object, and also to some extent changed their behaviour in line with the emotional message given by the owner. Results are discussed in relation to social referencing in other species (dogs in particular) and cats' social organization and domestication history.

Dogs' comprehension of referential emotional expressions: familiar people and familiar emotions are easier.

Dogs have been shown to discriminate between human facial expressions, and they seem to use human emotional communication to regulate their behaviour towards an external object/situation. However, it is still not clear (1) whether they just respond to the emotional message received with a corresponding increase/decrease in their level of activation or whether they perceive that the emotional message refers to a specific object, (2) which emotional message they use to modify their behaviour (i.e. whether they are following the positive message or avoiding the negative one) and (3) whether their familiarity with the informant has an effect on the dogs' behaviour. To address these issues, five groups of dogs were tested in two experiments. The first group observed the owner delivering two different emotional messages (happiness and fear) towards two identical objects hidden behind barriers, and the second group observed the owner delivering the same emotional messages but with no-objects present in the room. The third and the fourth groups observed the owner delivering a happy versus a neutral, and a negative versus a neutral emotional message towards the hidden objects. Finally, the fifth group observed a stranger acting like the owner of the first group. When the owner was acting as the informant, dogs seemed to be capable of distinguishing between a fearful and happy emotional expression and preferentially chose to investigate a box eliciting an expression of happiness rather than of fear or neutrality. Dogs, however, seemed to have greater difficulty in distinguishing between the fearful and neutral emotional messages delivered by the owner and between the happy and fearful expressions delivered by the stranger. Results suggest that dogs have learned to associate their owners' positive emotional messages to positive outcomes, and hence use their communicative messages to guide their actions. However, negative emotional messages and those delivered by strangers are not as clear to dogs.

The effect of preferential paw usage on dogs' (Canis familiaris) performance in a manipulative problem-solving task.

In a number of species, individuals showing lateralized hand/paw usage (i.e. the preferential use of either the right or left paw) compared to ambilateral individuals have been shown to be more proactive in novel situations. In the current study we used an established test to assess preferential paw usage in dogs (the Kong test) and then compared the performance of ambilateral and lateralized dogs as well as left- vs. right-pawed dogs in a novel manipulative problem solving task. Results showed an equal proportion of ambilateral and lateralized dogs but contrary to predictions non-lateralized dogs were faster at accessing the apparatus in test trials. No differences emerged between right- and left-pawed dogs. Results are discussed in relation to previous studies on lateralization.

Gaze alternation in dogs and toddlers in an unsolvable task: evidence of an audience effect.

Dogs have been shown to use human-directed gazing behaviour and gaze alternation in numerous contexts; however, it is still unclear whether this behaviour can be considered an intentional and referential communicative act. In the current study, adult dogs and preverbal toddlers were tested using the classic unsolvable task paradigm, but varying the attentional stance of the participating audience (the experimenter and the caregiver). The aims were to assess (1) whether dogs and toddlers would use gaze alternation behaviour in similar manners when the task became unsolvable, and (2) whether both dogs and toddlers would take into account the attentional stance of the audience when initiating a communicative interaction. Results indicated that both toddlers and dogs increased their gaze alternation behaviour between the apparatus and caregiver when the task became unsolvable, and toddlers also showed an increase in pointing behaviour. Furthermore, both species showed a capacity to take into account the attentional stance of the audience when manifesting gaze alternation behaviours towards them. Taken together, these results suggest that gaze alternation is both an intentional and referential communicative act and that both species can take into account the need for audience attention when communicating with them.

Social referencing in dog-owner dyads?

Social referencing is the seeking of information from another individual to form one's own understanding and guide action. In this study, adult dogs were tested in a social referencing paradigm involving their owner and a potentially scary object. Dogs received either a positive or negative message from the owner. The aim was to evaluate the presence of referential looking to the owner, behavioural regulation based on the owner's (vocal and facial) emotional message and observational conditioning following the owner's actions towards the object. Most dogs (83%) looked referentially to the owner after looking at the strange object, thus they appear to seek information about the environment from the human, but little differences were found between dogs in the positive and negative groups as regards behavioural regulation: possible explanations for this are discussed. Finally, a strong effect of observational conditioning was found with dogs in the positive group moving closer to the fan and dogs in the negative group moving away, both mirroring their owner's behaviour. Results are discussed in relation to studies on human-dog communication, attachment and social learning.

Are dogs (Canis familiaris) misled more by their owners than by strangers in a food choice task?

Dogs are highly skilled in understanding a large variety of human social cues and use them appropriately to solve a number of different cognitive tasks. They rely on human signals even when these are contradictory or misleading and ultimately prevent them from correctly solving a task. In the following two experiments, we investigated whether the owner and a stranger differently influenced dogs' choices in food discrimination tasks. In Experiment 1, 48 dogs were tested in 3 different conditions: (1) choice between a large and a small amount of dog pellets with no demonstration; (2) choice between a large and a small amount of dog pellets after having witnessed the owner/stranger favouring the small quantity; (3) choice between two single food pellets after observing the owner/stranger choosing one of them. In Experiment 2, 48 dogs could choose between two foods of different palatability: in Condition 1, dogs chose between a slice of sausage and a dry pellet with no demonstration. In Condition 2, the same choice was available but with a person (owner/stranger) showing a preference for the dry pellet. In Condition 3, dogs chose between a single dry pellet and 8 slices of sausage, with the person (owner/stranger) showing a preference for the pellet. In both experiments, dogs conformed to the human's indications even though these led to the selection of the less advantageous option (i.e. the smaller amount of food in Experiment 1 or the low quality food in Experiment 2). However, the owner and the stranger did not differently influence the dogs' behaviour. Results show that dogs are willing to follow a person's indication even when this is visibly (if perhaps only mildly) counterproductive to them and that they are socially prepared to rely equally on cues given by the owner and an unfamiliar friendly person.

Comment on "Differential sensitivity to human communication in dogs, wolves, and human infants".

Topál et al. (Reports, 4 September 2009, p. 1269) showed that dogs, like infants but unlike wolves, make perseverative search errors that can be explained by the use of ostensive cues from the experimenter. We suggest that a simpler learning process, local enhancement, can account for errors made by dogs.

Is your choice my choice? The owners' effect on pet dogs' (Canis lupus familiaris) performance in a food choice task.

This study investigates the influence of owners on their dogs' performance in a food choice task using either different or equal quantities of food. Fifty-four pet dogs were tested in three different conditions. In Condition 1 we evaluated their ability to choose between a large and small amount of food (quantity discrimination task). In Condition 2 dogs were again presented with a choice between the large and small food quantity, but only after having witnessed their owner favouring the small quantity. In Condition 3 dogs were given a choice between two equally small quantities of food having witnessed their owner favouring either one or the other. A strong effect of the owner on the dogs' performance was observed. In Condition 1 dogs as a group chose significantly more often the large food quantity, thus showing their ability to solve the quantity discrimination task. After observing their owner expressing a preference for the small food quantity they chose the large quantity of food significantly less than in the independent choice situation. The tendency to conform to the owner's choice was higher when the dogs had to choose between equally small quantities of food (Condition 3) rather than between a large and a small one (Condition 2). These results provide evidence that dogs can be influenced by their owners even when their indications are clearly in contrast with direct perceptual information, thus leading dogs to ultimately make counterproductive choices.