Making the most of life: environmental choice during rearing enhances the ability of laying hens to take opportunities.

Introduction: The potential of aviary housing for improving laying hen (Gallus gallus domesticus) welfare will be constrained if rearing conditions limit the hens' behavioral ability to take opportunities. Incorporating theories on developmental plasticity and animal agency, this study aimed to determine: (1) whether a choice of litter and perch types during rearing would promote long-lasting changes in use of novel locations and resources, and (2) the influence of timing of choice provision. Methods: Laying hen chicks were assigned to either a "Single-choice" (one litter and perch type) or "Multi-choice" environment (four litter and perch types) during "Early" (day 1-week 4) and "Late" rearing (week 5-15). The environments were switched in half of the 16 pens in week 5, resulting in a 2 × 2 factorial design with four choice environment by period combinations. The allocation of perch and litter space was the same across all treatment combinations. In week 16, all groups were moved to standard aviary laying pens (Laying period, week 16-27). Results: When first moved to the laying pens, hens with Multi-choice in either or both rearing periods were quicker to spread out in their pen than hens with Single-choice throughout rearing. Multi-choice in Early rearing also reduced the latency to use novel elevated structures (perches and nests) in the laying pens. Multi-choice during Late rearing increased success in finding and consuming hidden mealworms (tested in weeks 9-17) and increased the proportion of eggs laid on elevated nesting trays. Numerically, hens switched from Multi-choice to Single-choice in week 5 used the outdoor range less than hens switched from Single-choice to Multi-choice. Discussion: These results support the hypothesis that offering multiple resource choices during rearing improves hens' ability to make the most of new opportunities by being more proactive in exploring and exploiting newly available resources. In different opportunity challenges, hens showed positive outcomes in response to choice during Early, Late or both stages of rearing, suggesting that best results can be obtained by offering environmental choice throughout rearing.

Resource choice during ontogeny enhances both the short- and longer-term welfare of laying hen pullets.

We hypothesised that resource choice during early life contributes to both current and longer-term beneficial effects on animal welfare. We investigated this hypothesis in a longitudinal cross-over experiment with laying hen pullets (Gallus gallus domesticus) reared in pens with one or four litter and perch types, respectively (n = 8 pens/treatment, all providing ample and identical litter and perch space). After 4 weeks (chick period), half the pens were modified to provide the opposite treatment (juvenile period). After 11 more weeks, all groups were moved to novel, identical laying pens (adult period; Week 16-27). In support of our hypothesis, the opportunity to choose between multiple litter and perch variants was associated with higher levels of positively-valenced behaviours, including play as chicks and dustbathing as juveniles and adults, and lower levels of negatively-valenced behaviours, including feather pecking as chicks and juveniles and aggressive pecking as adults. Resource choice in the juvenile period also led to better juvenile and adult plumage condition, and greater growth as adults. We conclude that the opportunity to choose among different litter and perch types, instead of having only one type of each, had both short- and longer-term positive effects on the birds' affective states and physical condition.

Oxytocin levels and self-reported anxiety during interactions between humans and cows.

Introduction: Positive social interactions with farm animals may have therapeutic benefits on humans by increasing brain oxytocin secretion, as inferred from circulating oxytocin levels. The aim of this observational study was to investigate acute changes in human plasma oxytocin levels and state anxiety associated with interactions with dairy cows. Methods: Data were collected from 18 healthy female nursing students who performed stroking and brushing of an unfamiliar cow for 15 min. Blood samples were drawn before entering the cowshed (T1, baseline), and after 5 (T2) and 15 (T3) min of interaction with a cow. At T1 and T3, the students filled out the Norwegian version of the Spielberger State-Trait Anxiety Inventory-State Subscale (STAI-SS). Results: Across participants, no significant changes in average plasma oxytocin concentration were detected between time points (p>0.05). There was, however, a modest decline in the STAI-SS scores between T1 and T3 (p=0.015) and a positive correlation between the change in individual level of state anxiety between T1 and T3 and the change in OT concentration of the same individual between T2 and T3 (p = 0.045). Discussion: The results suggest that friendly social interactions with cows are beneficial in lowering state anxiety, but any relationship with release of OT into the circulation was complex and variable across individuals. The acute reduction in state anxiety lends support to the value of interacting with farm animals in the context of Green Care for people with mental health challenges.

Exploring dog ownership in the lives of people with substance use disorder: a qualitative study.

BACKGROUND: Recovery from substance use is commonly seen as a process of integrating social relationships and creating a sense of meaning in one's life. Dog owners describe a close relationship with their dog that impacts many aspects of their everyday life. Yet for individuals with substance use disorder (SUD), little is known about how dog ownership could affect their lives. The aim of this study was to explore how people living with SUD experience and describe their everyday life when owning a dog. METHOD: Eight semi-structured in-depth individual interviews were conducted with people having personal experience of living with SUD and owning a dog. Data were gathered and analysed using qualitative content analysis. RESULTS: The analysis yielded four categories, reflecting different aspects of dog ownership. Living with SUD and owning a dog was primarily something positive in their life. People increased their social connections personally and within society. They felt a belonging which gave a sense of agency and purpose, and they developed structure in their day and boundaries to their environment. Dog ownership, however, could hinder access to services which was found to be challenging for some participants. CONCLUSIONS: The owning of a dog can lead to changes that parallel those of a recovery process. This finding adds to the research on the connection that dogs can provide and shows how pertinent this can be particularly for vulnerable persons such as those with SUD.

The Aviary Transect-a practical welfare assessment tool to improve the management of cage-free laying hens.

The Aviary Transect (AT) is a method for assessing welfare in cage-free laying hen flocks, and comprises standardized walks along each aisle screening the flock for selected welfare indicators: feather loss (FL) on head, back, breast, and tail, wounds on head, back, tail, and feet, dirty plumage, enlarged crop, sickness, and birds found dead. The method is quick (20 min for a flock of 7,500 hens), has good interobserver agreement and shows positive correlations with individual bird sampling methods. However, it is less clear whether AT can be used to detect differences in flock health and welfare related to housing and management. The aim of this study was to evaluate how AT findings varied in relation to 23 selected housing, management, environmental, and production factors. The study was conducted on 33 commercial nonbeak-trimmed, white-feathered layer flocks of similar age (70-76 wk) kept in multitiered aviaries in Norway. The most prevalent findings across flocks were feather loss on the back (mean 0.97% of flock) and breast (0.94%) followed by feather loss on the head (0.45%) and tail (0.36%) with differences in feather pecking damage according to the hybrid used (P < 0.05). Better litter quality was associated with a lower prevalence of feather loss on the head and breast (P < 0.05), and addition of fresh litter during the production cycle resulted in fewer birds with feather loss on the head (P < 0.05) and tail (P < 0.001). Lower dust levels were linked to a lower prevalence of feather loss on the head, back, and breast (P < 0.05), and when access to the floor area underneath the aviary was provided at an earlier stage of production, fewer birds had wounds (P < 0.001), but more birds were observed with an enlarged crop (P < 0.05) and found dead (P < 0.05). In conclusion, findings from AT showed that results of the assessment varied according to housing conditions. These results support the validity of AT as a relevant welfare assessment tool for evaluating cage-free management practices.

Environmental change or choice during early rearing improves behavioural adaptability in laying hen chicks.

Laying hens are typically moved to a novel environment after rearing, requiring adaptability to cope with change. We hypothesized that the standard rearing of laying hen chicks, in non-changing environments with limited choices (a single variant of each resource), impairs their ability to learn new routines, use new equipment and exploit new resources. On the contrary, rearing in a changing environment that also offers a choice of resource variants could better prepare chicks for the unexpected. To explore this hypothesis, environmental change and choice were manipulated in a 2 × 2 factorial experiment. Compared to standard rearing, greater change during early rearing, through repeatedly swapping litter and perch types, reduced initial freezing when exposed to a novel environment suggesting a lower fear response. Greater choice during rearing, through simultaneous access to multiple litter and perch types, resulted in shorter latencies to solve a detour task, more movement in novel environments and less spatial clustering, suggesting improved spatial skills and higher exploration. However, combining both change and choice did not generally result in greater improvement relative to providing one or the other alone. We conclude that environmental change and choice during rearing have different positive but non-synergistic effects on later adaptability potential.

Developing a novel welfare assessment tool for loose-housed laying hens - the Aviary Transect method.

This study compared welfare assessment results in aviary flocks using 3 approaches: 1) A novel Aviary Transect method, 2) AssureWel, and 3) the Norwegian farm advisors' NorWel method. The Aviary Transect time requirement, interobserver reliability, and within- and across-house sensitivity to detect welfare indicators were also evaluated. The study was conducted on 6 randomly chosen commercial white-strain layer flocks of similar age and flock size, kept in multitiered aviaries. The Aviary Transect method comprised standardized walks along each aisle while screening the whole flock for 12 welfare indicators: feather loss (FL) on head, back, breast, and tail, wounds on head, back, tail, and feet, dirty birds, enlarged crop, sick birds, and dead birds. AssureWel involved scoring FL on head and back, and dirtiness of 50 random birds, and flock-level evaluation of beak trimming, antagonistic behavior, flightiness, birds needing further care, and mortality. NorWel involved scoring 8 welfare indicators on 50 random birds: FL on head, back, breast, and tail, dirtiness, and wounds on head, back, and tail. The AssureWel detected flock differences in both minor and major FL on the back (P < 0.01) as well as somewhat dirty birds (P < 0.01). The NorWel method detected flock differences in both minor and major FL on the head (P < 0.01), back (P < 0.001), breast (P < 0.001), and tail (P < 0.001) and somewhat (score 1) dirty birds (P < 0.05). The Aviary Transect method detected flock differences in FL on head, back, breast, and tail (all P < 0.001), dirty birds (P < 0.05) and enlarged crop (P < 0.001). More birds with FL on breast, and more dirty birds, were found in wall vs. central transects (P < 0.05). There was good interobserver agreement, except for dirty birds (P < 0.01), and positive correlations (P < 0.05) were identified between the Aviary Transect method and the other sampling methods for FL on head and back, and dirtiness. The three methods took similar time to complete (about 20 min/flock). In conclusion, all 3 methods detected significant differences in welfare indicator prevalence between flocks. The new Aviary Transect method provides egg producers with an efficient and sensitive whole-flock assessment of hen welfare status in multitiered aviaries.

Prenatal and Early Postnatal Behavioural Programming in Laying Hens, With Possible Implications for the Development of Injurious Pecking.

Injurious pecking (IP) represents a serious concern for the welfare of laying hens (Gallus gallus domesticus). The risk of IP among hens with intact beaks in cage-free housing prompts a need for solutions based on an understanding of underlying mechanisms. In this review, we explore how behavioural programming via prenatal and early postnatal environmental conditions could influence the development of IP in laying hens. The possible roles of early life adversity and mismatch between early life programming and subsequent environmental conditions are considered. We review the role of maternal stress, egg conditions, incubation settings (temperature, light, sound, odour) and chick brooding conditions on behavioural programming that could be linked to IP. Brain and behavioural development can be programmed by prenatal and postnatal environmental conditions, which if suboptimal could lead to a tendency to develop IP later in life, as we illustrate with a Jenga tower that could fall over if not built solidly. If so, steps taken to optimise the environmental conditions of previous generations and incubation conditions, reduce stress around hatching, and guide the early learning of chicks will aid in prevention of IP in commercial laying hen flocks.

Effect of Environmental Complexity and Stocking Density on Fear and Anxiety in Broiler Chickens.

Barren housing and high stocking densities may contribute to negative affective states in broiler chickens, reducing their welfare. We investigated the effects of environmental complexity and stocking density on broilers' attention bias (measure of anxiety) and tonic immobility (measure of fear). In Experiment 1, individual birds were tested for attention bias (n = 60) and in Experiment 2, groups of three birds were tested (n = 144). Tonic immobility testing was performed on days 12 and 26 (n = 36) in Experiment 1, and on day 19 (n = 72) in Experiment 2. In Experiment 1, no differences were observed in the attention bias test. In Experiment 2, birds from high-complexity pens began feeding faster and more birds resumed feeding than from low-complexity pens following playback of an alarm call, suggesting that birds housed in the complex environment were less anxious. Furthermore, birds housed in high-density or high-complexity pens had shorter tonic immobility durations on day 12 compared to day 26 in Experiment 1. In Experiment 2, birds from high-density pens had shorter tonic immobility durations than birds housed in low-density pens, which is contrary to expectations. Our results suggest that birds at 3 weeks of age were less fearful under high stocking density conditions than low density conditions. In addition, results indicated that the complex environment improved welfare of broilers through reduced anxiety.

Slow-growing broilers are healthier and express more behavioural indicators of positive welfare.

Broiler chicken welfare is under increasing scrutiny due to welfare concerns regarding growth rate and stocking density. This farm-based study explored broiler welfare in four conditions representing commercial systems varying in breed and planned maximum stocking density: (1) Breed A, 30 kg/m2; (2) Breed B, 30 kg/m2; (3) Breed B, 34 kg/m2; (4) Breed C, 34 kg/m2. Breeds A and B were 'slow-growing' breeds (< 50 g/day), and Breed C was a widely used 'fast-growing' breed. Indicators of negative welfare, behavioural indicators of positive welfare and environmental outcomes were assessed. Clear differences between conditions were detected. Birds in Condition 4 experienced the poorest health (highest mortality and post-mortem inspection rejections, poorest walking ability, most hock burn and pododermatitis) and litter quality. These birds also displayed lower levels of behaviours indicative of positive welfare (enrichment bale occupation, qualitative 'happy/active' scores, play, ground-scratching) than birds in Conditions 1-3. These findings provide farm-based evidence that significant welfare improvement can be achieved by utilising slow-growing breeds. There are suggested welfare benefits of a slightly lower planned maximum stocking density for Breed B and further health benefits of the slowest-growing breed, although these interventions do not offer the same magnitude of welfare improvement as moving away from fast-growing broilers.

On-farm broiler chicken welfare assessment using transect sampling reflects environmental inputs and production outcomes.

To evaluate the utility of transect sampling for assessing animal welfare in large chicken flocks, we quantified relationships between environmental inputs, welfare problems detected using transect sampling, and production outcomes. We hypothesised that environmental inputs including environmental complexity (i.e. number of environmental enrichment types provided), space allowance, underfloor heating (presence or absence), and photoperiod regimen (18 h continuous vs 16 h intermittent) would correspond to variations in welfare assessment findings, which would predict production outcomes. We conducted on-farm welfare assessment of Norwegian broiler flocks at approximately 28 days of age. We sampled four transects (rows between feeder and drinker lines) per flock to determine litter quality and the proportions of chickens with compromised welfare as indicated by visual signs of walking difficulties, illness, skin wounds and small bird size. Production outcome measures included mortality, reasons for carcass rejection at slaughter, footpad dermatitis, growth rate, feed conversion and an integrated production index. Greater environmental complexity was associated with a reduction in skin wounds and total welfare problems on the farm, lower mortality, fewer rejections due to wounds and underweight birds, and fewer rejections overall. Higher space allowances within levels of environmental complexity were associated with fewer walking difficulties and welfare problems overall, a reduction in rejections due to wounds, and a higher growth rate and production index. Underfloor heating was associated with a reduction in rejections due to leg deformity, and intermittent light was associated with lower illness and skin wound rates on the farm, and lower mortality. Furthermore, fewer welfare problems and better litter quality on the farm were associated with fewer carcass rejections at slaughter. Thus, data from transect sampling varied with environmental inputs and production outcomes, supporting the validity of transect sampling for practical, animal-based on-farm welfare assessment.

Effects of environmental enrichment on activity and lameness in commercial broiler production.

The aim of this pilot study was to investigate the effects of commercially applied environmental enrichments on behavior and lameness in broilers. Two consecutive flocks of broilers were observed at 16 days and 30 days of age to investigate differences between enriched (peat, bales of lucerne hay, and elevated platforms) and control birds with regards to behavioral activities and lameness. More running (p < .001), worm running (p = .006), play fighting (p = .015), dust bathing (p = .009), and ground pecking while standing (p < .001) were observed at 16 days than at 30 days. Across both ages, enriched birds showed more wing flapping (p = .016), wing stretching (p = .002), body shaking (p = .002), ground scratching (p < .001), and ground pecking while standing (p < .001) and lying (p = .003) compared with control birds. Even when no enrichments were close, enriched birds showed more body shaking (p = .008) and ground pecking while standing (p < 0.001) and lying (p = .010) than birds in comparable locations in control pens. There was a tendency for a lower gait score (i.e., reduced lameness) with the enriched treatment (p = .077). In conclusion, enriched birds showed higher levels of several activities compared with control birds, and demonstrated higher levels in areas where no enrichments were present.

Tickling, a Technique for Inducing Positive Affect When Handling Rats.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals' playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats' fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

Modelling personality, plasticity and predictability in shelter dogs.

Behavioural assessments of shelter dogs (Canis lupus familiaris) typically comprise standardized test batteries conducted at one time point, but test batteries have shown inconsistent predictive validity. Longitudinal behavioural assessments offer an alternative. We modelled longitudinal observational data on shelter dog behaviour using the framework of behavioural reaction norms, partitioning variance into personality (i.e. inter-individual differences in behaviour), plasticity (i.e. inter-individual differences in average behaviour) and predictability (i.e. individual differences in residual intra-individual variation). We analysed data on interactions of 3263 dogs (n = 19 281) with unfamiliar people during their first month after arrival at the shelter. Accounting for personality, plasticity (linear and quadratic trends) and predictability improved the predictive accuracy of the analyses compared to models quantifying personality and/or plasticity only. While dogs were, on average, highly sociable with unfamiliar people and sociability increased over days since arrival, group averages were unrepresentative of all dogs and predictions made at the individual level entailed considerable uncertainty. Effects of demographic variables (e.g. age) on personality, plasticity and predictability were observed. Behavioural repeatability was higher one week after arrival compared to arrival day. Our results highlight the value of longitudinal assessments on shelter dogs and identify measures that could improve the predictive validity of behavioural assessments in shelters.

Aggressiveness as a latent personality trait of domestic dogs: Testing local independence and measurement invariance.

Studies of animal personality attempt to uncover underlying or "latent" personality traits that explain broad patterns of behaviour, often by applying latent variable statistical models (e.g., factor analysis) to multivariate data sets. Two integral, but infrequently confirmed, assumptions of latent variable models in animal personality are: i) behavioural variables are independent (i.e., uncorrelated) conditional on the latent personality traits they reflect (local independence), and ii) personality traits are associated with behavioural variables in the same way across individuals or groups of individuals (measurement invariance). We tested these assumptions using observations of aggression in four age classes (4-10 months, 10 months-3 years, 3-6 years, over 6 years) of male and female shelter dogs (N = 4,743) in 11 different contexts. A structural equation model supported the hypothesis of two positively correlated personality traits underlying aggression across contexts: aggressiveness towards people and aggressiveness towards dogs (comparative fit index: 0.96; Tucker-Lewis index: 0.95; root mean square error of approximation: 0.03). Aggression across contexts was moderately repeatable (towards people: intraclass correlation coefficient (ICC) = 0.479; towards dogs: ICC = 0.303). However, certain contexts related to aggressiveness towards people (but not dogs) shared significant residual relationships unaccounted for by latent levels of aggressiveness. Furthermore, aggressiveness towards people and dogs in different contexts interacted with sex and age. Thus, sex and age differences in displays of aggression were not simple functions of underlying aggressiveness. Our results illustrate that the robustness of traits in latent variable models must be critically assessed before making conclusions about the effects of, or factors influencing, animal personality. Our findings are of concern because inaccurate "aggressive personality" trait attributions can be costly to dogs, recipients of aggression and society in general.

Correction to 'Modelling personality, plasticity and predictability in shelter dogs'.

[This corrects the article DOI: 10.1098/rsos.170618.].

Using network analysis to study behavioural phenotypes: an example using domestic dogs.

Phenotypic integration describes the complex interrelationships between organismal traits, traditionally focusing on morphology. Recently, research has sought to represent behavioural phenotypes as composed of quasi-independent latent traits. Concurrently, psychologists have opposed latent variable interpretations of human behaviour, proposing instead a network perspective envisaging interrelationships between behaviours as emerging from causal dependencies. Network analysis could also be applied to understand integrated behavioural phenotypes in animals. Here, we assimilate this cross-disciplinary progression of ideas by demonstrating the use of network analysis on survey data collected on behavioural and motivational characteristics of police patrol and detection dogs (Canis lupus familiaris). Networks of conditional independence relationships illustrated a number of functional connections between descriptors, which varied between dog types. The most central descriptors denoted desirable characteristics in both patrol and detection dog networks, with 'Playful' being widely correlated and possessing mediating relationships between descriptors. Bootstrap analyses revealed the stability of network results. We discuss the results in relation to previous research on dog personality, and benefits of using network analysis to study behavioural phenotypes. We conclude that a network perspective offers widespread opportunities for advancing the understanding of phenotypic integration in animal behaviour.

Social Interaction with an "Unidentified Moving Object" Elicits A-Not-B Error in Domestic Dogs.

Mechanical "unidentified moving objects" (UMO's) are useful for controlled investigations into features of social interaction that generate cooperativeness and positive social affiliation in domestic dogs (Canis familiaris). We hypothesized that, if a UMO interacted socially with a dog, the UMO would become associated with certain social cues and would subsequently affect dog behaviour. We assigned dogs to a Human, Social UMO or Non-Social UMO partner. In the Human and Social UMO conditions, the partner interacted with the dog cooperatively whereas the Non-Social UMO partner was unresponsive to the dog's actions. We then tested dogs with their partner in a Piagetian A-not-B error paradigm, predicting that the Human and Social UMO partners would be more likely to elicit A-not-B errors in dogs than the Non-Social UMO partner. Five trials were conducted in which the dog watched its partner hide a ball behind one of two screens (A or B). As predicted, dogs in the Human and Social UMO conditions were more likely to search for the ball behind the A screen during B trials than dogs in the Non-Social UMO condition. These results reveal that the unfamiliar partner's social responsiveness leads rapidly to accepting information communicated by the partner. This study has generated a better understanding of crucial features of agents that promote dog social behaviour, which will facilitate the programming of robots for various cooperative tasks.

Positive Reinforcement Training for Blood Collection in Grizzly Bears (Ursus arctos horribilis) Results in Undetectable Elevations in Serum Cortisol Levels: A Preliminary Investigation.

Training nonhuman animals in captivity for participation in routine husbandry procedures is believed to produce a lower stress environment compared with undergoing a general anesthetic event for the same procedure. This hypothesis rests largely on anecdotal evidence that the captive subjects appear more relaxed with the trained event. Blood markers of physiological stress responses were evaluated in 4 captive grizzly bears (Ursus arctos horribilis) who were clicker-trained for blood collection versus 4 bears who were chemically immobilized for blood collection. Serum cortisol and immunoglobulin A (IgA) and plasma ß-endorphin were measured as indicators of responses to stress. Plasma ß-endorphin was not different between the groups. Serum IgA was undetectable in all bears. Serum cortisol was undetectable in all trained bears, whereas chemically immobilized bears had marked cortisol elevations (p < .05). The highest cortisol elevations were found in 2 bears with extensive recent immobilization experience. These findings support the use of positive reinforcement training for routine health procedures to minimize anxiety.

The social buffering effect of playful handling on responses to repeated intraperitoneal injections in laboratory rats.

Handling small animals for veterinary and experimental procedures can negatively affect animal wellbeing. We hypothesized that playful handling (tickling) would decrease stress associated with repeated injections in adult laboratory rats, especially those with prior tickling experience. We compared responses of 4 groups of male Sprague-Dawley rats to intraperitoneal injection of saline daily for 10 d. Rats either tickled or not tickled as juveniles (2 min/d for 21 d) were exposed as adults to either a passive hand or tickling for 2 min immediately before and after injections. Rates of vocalization (22- and 50-kHz ultrasonic vocalizations (USV), indicative of negative and positive affective states, respectively, and audible calls indicative of pain and discomfort) were quantified before, during, and after injection. Tickling before and after injection, especially when combined with juvenile tickling experience (ending 40 to 50 d earlier), increased 50-kHz USV rates before and after injection, reduced audible call rate during injection, and decreased the duration of the injection procedure. The treatments did not affect indicators of physiologic stress (body weight change; fecal corticosteroid levels). We conclude that playful handling performed in association with a mildly aversive procedure serves as a useful refinement by inducing a positive affective state that mitigates the aversiveness of the procedure and makes rats easier to handle, especially when they have been accustomed to tickling as juveniles.