Maternal separation followed by isolation-housing differentially affects prepulse inhibition of the acoustic startle response in C57BL/6 mice.

Exposure to chronic stress is associated with an increased incidence of neuropsychiatric dysfunction. The current study evaluated two competing hypotheses, the cumulative stress and the match/mismatch hypothesis of neuropsychiatric dysfunction, using two paradigms relating to exposure to "stress": pre-weaning maternal separation and post-weaning isolation-housing. C57BL/6 offspring were reared under four conditions: typical animal facility rearing (AFR, control), early handling (EH, daily 15 min separation from dam), maternal separation (MS, daily 4 hr separation from dam), and maternal and peer separation (MPS, daily 4 hr separation from dam and from littermates). After weaning, mice were either housed socially (2-3/cage) or in isolation (1/cage) and then tested for prepulse inhibition in adulthood. Isolation-housed MPS subjects displayed greater deficits in prepulse inhibition relative to socially-housed MPS subjects while socially-housed AFR subjects displayed greater deficits in prepulse inhibition relative to isolation-housed AFR subjects. The results indicate that these treatment conditions represent a potentially valuable model for evaluating the match/mismatch hypothesis in regards to neuropsychiatric dysfunction.

Animals in flow - towards the scientific study of intrinsic reward in animals.

The concept of flow, a state of complete absorption in an intrinsically rewarding activity, has played a pivotal role in advancing notions of human well-being beyond minimising suffering towards promoting flourishing and thriving. While flow has played a fundamental role in human positive psychology, it has not yet been explored in non-human animals, leaving an enormous void in our understanding of intrinsic motivation in animals. As ethology and related fields keep progressing in uncovering complex cognitive and affective capacities of non-human animals, we propose the time is ripe to translate the concept of flow to animals. We start by embedding flow in the topic of intrinsic motivation and describe its impact on positive human psychology and potentially positive animal welfare. We then disambiguate flow from related concepts discussed in the animal literature. Next, we derive experimental approaches in animals from the canonical characteristics of flow in humans and provide guidelines for both inducing and assessing flow by focusing on two characteristics that do not necessarily depend on self-report, namely resistance to distraction and time distortion. Not all aspects of the human flow experience are (yet) translatable, but those that are may improve quality of life in captive non-human animals.

Using the Judgment Bias Task to Identify Behavioral Indicators of Affective State: Do Eye Wrinkles in Horses Reflect Mood?

Identifying and validating behavioral indicators of mood are important for the assessment of animal welfare. Here, we investigated whether horses' eye wrinkle expression in a presumably neutral situation is a measure of mood as assessed in a cognitive judgment bias task (JBT). To this end, we scored pictures of the left and right eyes of 16 stallions for different aspects of eye wrinkle expression and tested the same individuals on a spatial JBT with active trial initiation. Eye wrinkle expressions were assessed by a qualitative assessment, i.e., the overall assessment of how "worried" horses look, the number of wrinkles, and the angle measured at the intersection of lines drawn through the eyeball and the topmost wrinkle. Correlations between the three eye wrinkle measures and the optimism index as a measure of horses' decisions in the JBT were not statistically significant, but with increasing optimism index, horses tended to be scored as looking less worried (qualitative assessment). We discuss our findings from different perspectives and make suggestions for future research, e.g., by calling for experimental induction of mood and thus greater variation within and/or between individuals and by investigating the interplay between shorter-lasting emotional and longer-lasting mood states to further explore the potential use of the JBT to validate eye wrinkles and other facial or body expressions as indicators of mood.

Doing nothing and what it looks like: inactivity in fattening cattle.

BACKGROUND: Animals kept in barren environments often show increased levels of inactivity and first studies indicate that inactive behaviour may reflect boredom or depression-like states. However, to date, knowledge of what inactivity looks like in different species is scarce and methods to precisely describe and analyse inactive behaviour are thus warranted. METHODS: We developed an Inactivity Ethogram including detailed information on the postures of different body parts (Standing/Lying, Head, Ears, Eyes, Tail) for fattening cattle, a farm animal category often kept in barren environments. The Inactivity Ethogram was applied to Austrian Fleckvieh heifers kept in intensive, semi-intensive and pasture-based husbandry systems to record inactive behaviour in a range of different contexts. Three farms per husbandry system were visited twice; once in the morning and once in the afternoon to cover most of the daylight hours. During each visit, 16 focal animals were continuously observed for 15 minutes each (96 heifers per husbandry system, 288 in total). Moreover, the focal animals' groups were video recorded to later determine inactivity on the group level. Since our study was explorative in nature, we refrained from statistical hypothesis testing, but analysed both the individual- and group-level data descriptively. Moreover, simultaneous occurrences of postures of different body parts (Standing/Lying, Head, Ears and Eyes) were analysed using the machine learning algorithm cspade to provide insight into co-occurring postures of inactivity. RESULTS: Inspection of graphs indicated that with increasing intensity of the husbandry system, more animals were inactive (group-level data) and the time the focal animals were inactive increased (individual-level data). Frequently co-occurring postures were generally similar between husbandry systems, but with subtle differences. The most frequently observed combination on farms with intensive and semi-intensive systems was lying with head up, ears backwards and eyes open whereas on pasture it was standing with head up, ears forwards and eyes open. CONCLUSION: Our study is the first to explore inactive behaviour in cattle by applying a detailed description of postures from an Inactivity Ethogram and by using the machine learning algorithm cspade to identify frequently co-occurring posture combinations. Both the ethogram created in this study and the cspade algorithm may be valuable tools in future studies aiming to better understand different forms of inactivity and how they are associated with different affective states.

Positive Welfare and the Like: Distinct Views and a Proposed Framework.

Positive welfare and related terms such as good welfare, happiness, and a good life are increasingly used in the animal welfare science literature. Overall, they highlight the welfare benefits of providing animals opportunities for positive experiences, beyond the alleviation of suffering. However, the various terms remain loosely defined and are sometimes used interchangeably, resulting in discrepancy. In this perspective article, we lay out the terms and concepts used in the literature. We identify two distinct views: "hedonic positive welfare," arising from likes and wants and their positive outcomes on welfare; and "positive welfare balance," as an overall positive welfare state based on positive experiences outweighing negative ones. Eudaimonia, satisfaction with one's life, may emerge as a third view. We propose a framework that is applicable across the different views. The "Vienna Framework" outlines different facets: frequency, duration, arousal, context, previous experience, individual differences, sense of agency, and long-term benefit. The framework aims to encourage researchers to consider the relevance of these facets for their own research, to indicate how the facets are affected by different interventions (e.g., greater sense of agency in enriched compared to non-enriched animals), or to compare different topics with respect to the different facets (e.g., high arousal of play behavior and low arousal of social affiliation). We encourage researchers to carefully consider and clearly state how their work falls along these views and facets, conceptually, and operationally. This should prevent dilution of the meaning of positive welfare and thereby preserve its potential to improve the welfare of animals.

Sex and Age Don't Matter, but Breed Type Does-Factors Influencing Eye Wrinkle Expression in Horses.

Identifying valid indicators to assess animals' emotional states is a critical objective of animal welfare science. In horses, eye wrinkles above the eyeball have been shown to be affected by pain and other emotional states. From other species we know that individual characteristics, e.g., age in humans, affect facial wrinkles, but it has not yet been investigated whether eye wrinkle expression in horses is systematically affected by such characteristics. Therefore, the aim of this study was to assess how age, sex, breed type, body condition, and coat colour affect the expression and/or the assessment of eye wrinkles in horses. To this end, we adapted the eye wrinkle assessment scale from Hintze et al. (1) and assessed eye wrinkle expression in pictures taken from the left and the right eye of 181 horses in a presumably neutral situation, using five outcome measures: a qualitative first impression reflecting how worried the horse is perceived by humans, the extent to which the brow is raised, the number of wrinkles, their markedness and the angle between a line through both corners of the eye and the topmost wrinkle. All measures could be assessed highly reliable with respect to intra- and inter-observer agreement. Breed type affected the width of the angle [F (2,114) = 8.20, p < 0.001], with thoroughbreds having the narrowest angle (M = 23.80, SD = 1.60), followed by warmbloods (M = 28.00, SD = 0.60), and coldbloods (M = 31.00, SD = 0.90). None of the other characteristics affected any of the outcome measures, and eye wrinkle expression did not differ between the left and the right eye area (all p-values > 0.05). In conclusion, horses' eye wrinkle expression and its assessment in neutral situations was not systematically affected by the investigated characteristics, except for "breed type", which accounted for some variation in "angle"; how much eye wrinkle expression is affected by emotion or perhaps mood needs further investigation and validation.

Pair housing makes calves more optimistic.

Individual housing of dairy calves is common farm practice, but has negative effects on calf welfare. A compromise between practice and welfare may be housing calves in pairs. We compared learning performances and affective states as assessed in a judgement bias task of individually housed and pair-housed calves. Twenty-two calves from each housing treatment were trained on a spatial Go/No-go task with active trial initiation to discriminate between the location of a teat-bucket signalling either reward (positive location) or non-reward (negative location). We compared the number of trials to learn the operant task (OT) for the trial initiation and to finish the subsequent discrimination task (DT). Ten pair-housed and ten individually housed calves were then tested for their responses to ambiguous stimuli positioned in-between the positive and negative locations. Housing did not affect learning speed (OT: F1,35 = 0.39, P = 0.54; DT: F1,19 = 0.15, P = 0.70), but pair-housed calves responded more positively to ambiguous cues than individually housed calves (χ21 = 6.79, P = 0.009), indicating more positive affective states. This is the first study to demonstrate that pair housing improves the affective aspect of calf welfare when compared to individual housing.

A cross-species judgement bias task: integrating active trial initiation into a spatial Go/No-go task.

Judgement bias tasks are promising tools to assess emotional valence in animals, however current designs are often time-consuming and lack aspects of validity. This study aimed to establish an improved design that addresses these issues and can be used across species. Horses, rats, and mice were trained on a spatial Go/No-go task where animals could initiate each trial. The location of an open goal-box, at either end of a row of five goal-boxes, signalled either reward (positive trial) or non-reward (negative trial). Animals first learned to approach the goal-box in positive trials (Go) and to re-initiate/not approach in negative trials (No-go). Animals were then tested for responses to ambiguous trials where goal-boxes at intermediate locations were opened. The Go:No-go response ratio was used as a measure of judgement bias. Most animals quickly learned the Go/No-go discrimination and performed trials at a high rate compared to previous studies. Subjects of all species reliably discriminated between reference cues and ambiguous cues, demonstrating a monotonic graded response across the different cue locations, with no evidence of learning about the outcome of ambiguous trials. This novel test protocol is an important step towards a practical task for comparative studies on judgement biases in animals.

Effects of Cage Enrichment on Behavior, Welfare and Outcome Variability in Female Mice.

The manner in which laboratory rodents are housed is driven by economics (minimal use of space and resources), ergonomics (ease of handling and visibility of animals), hygiene, and standardization (reduction of variation). This has resulted in housing conditions that lack sensory and motor stimulation and restrict the expression of species-typical behavior. In mice, such housing conditions have been associated with indicators of impaired welfare, including abnormal repetitive behavior (stereotypies, compulsive behavior), enhanced anxiety and stress reactivity, and thermal stress. However, due to concerns that more complex environmental conditions might increase variation in experimental results, there has been considerable resistance to the implementation of environmental enrichment beyond the provision of nesting material. Here, using 96 C57BL/6 and SWISS female mice, respectively, we systematically varied environmental enrichment across four levels spanning the range of common enrichment strategies: (1) bedding alone; (2) bedding + nesting material; (3) deeper bedding + nesting material + shelter + increased vertical space; and (4) semi-naturalistic conditions, including weekly changes of enrichment items. We studied how these different forms of environmental enrichment affected measures of animal welfare, including home-cage behavior (time-budget and stereotypic behavior), anxiety (open field behavior, elevated plus-maze behavior), growth (food and water intake, body mass), stress physiology (glucocorticoid metabolites in fecal boluses and adrenal mass), brain function (recurrent perseveration in a two-choice guessing task) and emotional valence (judgment bias). Our results highlight the difficulty in making general recommendations across common strains of mice and for selecting enrichment strategies within specific strains. Overall, the greatest benefit was observed in animals housed with the greatest degree of enrichment. Thus, in the super-enriched housing condition, stereotypic behavior, behavioral measures of anxiety, growth and stress physiology varied in a manner consistent with improved animal welfare compared to the other housing conditions with less enrichment. Similar to other studies, we found no evidence, in the measures assessed here, that environmental enrichment increased variation in experimental results.

Toward a Choice-Based Judgment Bias Task for Horses.

Judgment bias tasks for nonhuman animals are promising tools to assess emotional valence as a measure of animal welfare. In view of establishing a valid judgment bias task for horses, the present study aimed to evaluate 2 versions (go/no-go and active choice) of an auditory judgment bias task for horses in terms of acquisition learning and discrimination of ambiguous cues. Five mares and 5 stallions were randomly assigned to the 2 designs and trained for 10 trials per day to acquire different operant responses to a low-frequency tone and a high-frequency tone, respectively. Following acquisition learning, horses were tested on 4 days with 3 ambiguous-tone trials interspersed between the 10 high-tone and low-tone trials. All 5 go/no-go horses but only one active-choice horse successfully learned their task, indicating that it is more difficult to train horses on an active choice task than on a go/no-go task. During testing, however, go/no-go horses did not differentiate between the 3 different ambiguous cues, thereby making the validity of the test results questionable in terms of emotional valence.

Facial Indicators of Positive Emotions in Rats.

Until recently, research in animal welfare science has mainly focused on negative experiences like pain and suffering, often neglecting the importance of assessing and promoting positive experiences. In rodents, specific facial expressions have been found to occur in situations thought to induce negatively valenced emotional states (e.g., pain, aggression and fear), but none have yet been identified for positive states. Thus, this study aimed to investigate if facial expressions indicative of positive emotional state are exhibited in rats. Adolescent male Lister Hooded rats (Rattus norvegicus, N = 15) were individually subjected to a Positive and a mildly aversive Contrast Treatment over two consecutive days in order to induce contrasting emotional states and to detect differences in facial expression. The Positive Treatment consisted of playful manual tickling administered by the experimenter, while the Contrast Treatment consisted of exposure to a novel test room with intermittent bursts of white noise. The number of positive ultrasonic vocalisations was greater in the Positive Treatment compared to the Contrast Treatment, indicating the experience of differentially valenced states in the two treatments. The main findings were that Ear Colour became significantly pinker and Ear Angle was wider (ears more relaxed) in the Positive Treatment compared to the Contrast Treatment. All other quantitative and qualitative measures of facial expression, which included Eyeball height to width Ratio, Eyebrow height to width Ratio, Eyebrow Angle, visibility of the Nictitating Membrane, and the established Rat Grimace Scale, did not show differences between treatments. This study contributes to the exploration of positive emotional states, and thus good welfare, in rats as it identified the first facial indicators of positive emotions following a positive heterospecific play treatment. Furthermore, it provides improvements to the photography technique and image analysis for the detection of fine differences in facial expression, and also adds to the refinement of the tickling procedure.

Are Eyes a Mirror of the Soul? What Eye Wrinkles Reveal about a Horse's Emotional State.

Finding valid indicators of emotional states is one of the biggest challenges in animal welfare science. Here, we investigated in horses whether variation in the expression of eye wrinkles caused by contraction of the inner eyebrow raiser reflects emotional valence. By confronting horses with positive and negative conditions, we aimed to induce positive and negative emotional states, hypothesising that positive emotions would reduce whereas negative emotions would increase eye wrinkle expression. Sixteen horses were individually exposed in a balanced order to two positive (grooming, food anticipation) and two negative conditions (food competition, waving a plastic bag). Each condition lasted for 60 seconds and was preceded by a 60 second control phase. Throughout both phases, pictures of the eyes were taken, and for each horse four pictures per condition and phase were randomly selected. Pictures were scored in random order and by two experimenters blind to condition and phase for six outcome measures: qualitative impression, eyelid shape, markedness of the wrinkles, presence of eye white, number of wrinkles, and the angle between the line through the eyeball and the highest wrinkle. The angle decreased during grooming and increased during food competition compared to control phases, whereas the two phases did not differ during food anticipation and the plastic bag condition. No effects on the other outcome measures were detected. Taken together, we have defined a set of measures to assess eye wrinkle expression reliably, of which one measure was affected by the conditions the horses were exposed to. Variation in eye wrinkle expression might provide valuable information on horse welfare but further validation of specific measures across different conditions is needed.