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The rearing environment of layer chicks can differ greatly in degree of complexity. With the industry moving towards cage-free housing systems, greater demands are placed on the birds’ cognitive abilities in order for them to find resources such as food, water and nest-boxes. Because early environmental complexity can influence cognition, we aimed at increasing our knowledge of how two different rearing environments affect the cognitive abilities of the hens. We habituated 64 hens to a spatial holeboard test, half of which were reared in cages and the other half in an aviary. Out of these 64 hens, 14 cage- and 14 aviary-reared White Leghorn hens were tested twice a day every workday in a holeboard test from 32 to 40 weeks of age. The test consisted of 4 consecutive phases, namely the uncued, cued, over-training and reversal phases, during which the hens had to find baits in a subset of cups in an arena. All cups were identical, so hens had to rely on spatial cues to find the baits which were always hidden in the same cups. During the cued phase, cues were added to the baited cups to give additional information to the hens. During the reversal phase, baits were hidden in a new subset of cups to study cognitive flexibility. The results show the birds were able to successfully complete the task. Aviary-reared hens had a higher reference memory score than cage-reared hens in the first block of the cued phase (F1,26= 4.21, p1,26= 5.26, p1,26= 6.32, p1,26= 6.29, p1,26= 6.19, p1,26= 5.87, p&nbsp

The immune system can influence social motivation with potentially dire consequences for group-housed production animals, such as pigs. The aim of this study was to test the effect of a controlled immune activation in group-housed pigs, through an injection with lipopolysaccharide (LPS) and an intervention with ketoprofen on centrality parameters at the individual level. In addition, we wanted to test the effect of time relative to the injection on general network parameters in order to get a better understanding of changes in social network structures at the group level. 52 female pigs (11-12 weeks) were allocated to four treatments, comprising two injections: ketoprofen-LPS (KL), ketoprofen-saline (KS), saline-LPS (SL) and saline-saline (SS). Social behaviour with a focus on damaging behaviour was observed continuously in 10 x 15 min bouts between 0800 am and 1700 pm 1 day before (baseline) and two subsequent days after injection. Activity was scan-sampled every 5 min for 6 h after the last injection in the pen. Saliva samples were taken for cortisol analysis at baseline and at 4, 24, 48, 72 h after the injections. A controlled immune activation affected centrality parameters for ear manipulation networks at the individual level. Lipopolysaccharide-injected pigs had a lower in-degree centrality, thus, received less interactions, 2 days after the challenge. Treatment effects on tail manipulation and fighting networks were not observed at the individual level. For networks of manipulation of other body parts, in-degree centrality was positively correlated with cortisol response at 4 h and lying behaviour in the first 6 h after the challenge in LPS-injected pigs. Thus, the stronger the pigs reacted to the LPS, the more interactions they received in the subsequent days. The time in relation to injection affected general network parameters for ear manipulation and fighting networks at the group level. For ear manipulation networks, in -degree centralisation was higher on the days following injection, thus, certain individuals in the pen received more interactions than the rest of the group compared to baseline. For fighting networks, betweenness decreased on the first day after injection compared to baseline, indicating that network connectivity increased after the challenge. Networks of tail manipulation and manipulation of other body parts did not change on the days after injection at the group level. Social network analysis is a method that can potentially provide important insights into the effects of sickness on social behaviour in group-housed pigs. (c) 2021 The Authors. Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Vocal expression of emotions has been observed across species and could provide a non-invasive and reliable means to assess animal emotions. We investigated if pig vocal indicators of emotions revealed in previous studies are valid across call types and contexts, and could potentially be used to develop an automated emotion monitoring tool. We performed an analysis of an extensive and unique dataset of low (LF) and high frequency (HF) calls emitted by pigs across numerous commercial contexts from birth to slaughter (7414 calls from 411 pigs). Our results revealed that the valence attributed to the contexts of production (positive versus negative) affected all investigated parameters in both LF and HF. Similarly, the context category affected all parameters. We then tested two different automated methods for call classification; a neural network revealed much higher classification accuracy compared to a permuted discriminant function analysis (pDFA), both for the valence (neural network: 91.5%; pDFA analysis weighted average across LF and HF (cross-classified): 61.7% with a chance level at 50.5%) and context (neural network: 81.5%; pDFA analysis weighted average across LF and HF (cross-classified): 19.4% with a chance level at 14.3%). These results suggest that an automated recognition system can be developed to monitor pig welfare on-farm.

Poor health is a risk factor for damaging behaviors, but the mechanisms behind this link are unknown. Injection of pigs with lipopolysaccharide (LPS) can be used to model aspects of poor health. Recent studies have shown that LPS-injected pigs perform more tail- and ear-directed behavior compared to saline-injected pigs and suggest that pro-inflammatory cytokines may play a role in these behaviors. The aims of this study were to test the effect of LPS on the social behavior of pigs and the neurotransmitters and modulators in their brains and to test the effect of a nonsteroidal anti-inflammatory drug on the effects of LPS. Fifty-two female pigs (11-12 weeks) were allocated to four treatments comprising two injections: saline-saline (SS), saline-LPS (SL), ketoprofen-saline (KS), and ketoprofen-LPS (KL). Activity was scan-sampled every 5 min for 6 h after the last injection in the pen. Social behavior was observed continuously in 10 x 15-min bouts between 8 a.m. and 5 p.m. 1 day before (baseline) and 1 and 2 days after the injection. Saliva was analyzed for cortisol and plasma for tryptophan and kynurenine. The frontal cortex, hippocampus, hypothalamus, and brain stem were sampled 72 h after the injection and analyzed for cytokines and monoamines. LPS activated the HPA axis and decreased the activity within 6 h after the injection. Ketoprofen lowered the effect of LPS on cortisol release and attenuated the behavioral signs of sickness in challenged pigs. SL pigs manipulated the ears of their pen mates significantly longer than SS pigs 2 days after the injection. LPS had no observed effect on IFN-gamma, TNF-alpha, and IL-18. At 72 h after the injection, plasma tryptophan was depleted in SL pigs, and tryptophan and kynurenine concentrations in the frontal cortex and brain stem of SL pigs were significantly lower compared to those in SS pigs. Dopamine concentrations in the hypothalamus of SL pigs were significantly lower compared to those in SS pigs. Serotonin concentrations in the hypothalamus and noradrenaline concentrations in the hippocampus of SL pigs were significantly lower compared to those in KL pigs. In conclusion, LPS influenced the different neurotransmitters and modulators in the brain that are hypothesized to play an important role in the regulation of mood and behavior.

Poor health is associated with an increased risk of tail biting outbreaks in pigs. We propose that this is because illness changes social dynamics either by changing the behaviour of the sick pig towards its penmates, the behaviour of the healthy penmates towards the sick pig, or both. We tested the effect of immune stimulation (lipopolysaccharide (LPS) injection: O111:B4; 1.5 mu g kg(-1) IV) on social behaviour in gilts housed in triplets in a cross-over experiment. Each pen was subjected to the control treatment (all three pigs injected with saline) and then LPS treatment (one pig injected with LPS, two injected with saline), or vice versa. LPS injected pigs had a shift in social motivation and performed more tail- and ear- directed behaviour than saline pigs two days after injection. They seemed to fit the description of 'sick and grumpy'. This change was seen about 40 h after the signs of acute illness dissipated and was not accompanied by a similar increase in activity. We discuss possible mechanisms for this behavioural change in light of changes in neurotransmitter levels at three days after LPS injection described in a previous experiment.

Tail biting is detrimental to animal welfare and has negative consequences for producer economy. Poor health is one of the risk factors for tail biting. The first aim of this study was therefore to test for links between health status and behavior related to tail biting at the individual level. The second aim of this study was to test whether variation in cytokines was related to variation in social behavior. These small molecules produced upon immune activation are known to influence behavior both in the direction of withdrawal and increased aggression. This could potentially increase non-functional social behavior and thereby the risk of a tail biting outbreak. To investigate this, we collected behavioral data, health data, feeding data and blood samples from undocked boars at a test station farm in Norway. We compared groups with three different diagnoses: osteochondrosis diagnosed by computer tomography scanning (OCSAN), osteochondrosis diagnosed by clinical examination (OCCLIN) and respiratory tract disease (RESP), with healthy controls (CTR). We tested whether the diagnoses were associated with feeding and growth, social behavior and cytokine levels. We then tested whether there were correlations between cytokine levels and social behavior. We also provide raw data on cytokine levels in the extended sample (N = 305) as there are few publications on cytokine levels measured in pigs living under commercial conditions. OCCLIN pigs visited the feeder less, and fed longer compared to CTR pigs. Pigs diagnosed with RESP showed a large drop in growth the first week after filming, which corresponds to the week they were likely to have been diagnosed with illness, and a tendency to compensatory increase in the week after that. Social behavior differed between experimental groups with OCSCAN pigs receiving more social behavior (both aggressive and non aggressive) compared to CTR, and RESP pigs tending to perform more ear- and tail-biting than controls. There were no differences in absolute levels of cytokines between categories. However IL1-ra and IL-12 showed correlations with several behaviors that have been shown by others to be associated with current or future tail biting activity. To our knowledge, this is the first published study indicating a role for illness in non-functional social behavior in pigs and the first showing a correlation between cytokine levels and social behavior.

Emotions, unlike mood, are short-lived reactions associated with specific events. They can be characterized by two main dimensions, their arousal (bodily activation) and valence (negative versus positive). Knowledge of the valence of emotions experienced by domestic and captive animals is crucial for assessing and improving their welfare, as it enables us to minimize the negative emotions that they might experience and to promote positive ones. Emotions can affect vocalizations directly or indirectly through the brain, lungs, larynx or vocal tract. As a result, vocal expression of emotions has been observed across species, and could serve as a non-invasive and potentially very reliable tool to assess animal emotions. In pigs (Sus scrofa), vocal expression of emotions has been relatively well studied. However, it is not known if the vocal indicators revealed in previous studies are valid across call types and contexts. To find this out, we conducted a meta-analysis of the effects of emotional valence on pig vocalizations, including calls recorded in the most common emotional situations encountered by pigs throughout their lives, from birth to slaughter. Our analyses revealed that pigs produced calls characterized by a higher center of gravity, a shorter duration, less noise (lower Wiener entropy), lower formants (measured using the formant dispersion) and LPC coefficients in positive compared to negative contexts. Overall, these vocal parameters could be very useful for developing automated methods to monitor pig welfare on-farm.

Most of us have experienced deterioration of mood while ill. In humans, immune activation is associated with lethargy and social withdrawal, irritability and aggression; changes in social motivation could, in theory, lead to less functional interactions. This might also be the case for animals housed in close confinement. Tail biting in pigs is an example of damaging social behavior, and sickness is thought to be a risk factor for tail biting outbreaks. One possible mechanism whereby sickness may influence behavior is through cytokines. To identify possible mediators between immune activation and behavioral change, we injected 16 gilts with lipopolysaccharide (LPS; O111:B4; 1.5 mu g kg(-1) IV through a permanent catheter). In LPS-treated pigs, a significant increase in cortisol, TNF-alpha, IL-1 receptor antagonist, IL-6, and IL-8 was observed alongside decreased activity within the first 6 h after the injection. CRP was elevated at 12 and 24 h after injection, and food intake was reduced for the first 24 h after injection. Three days post-injection, LPS pigs had lower levels of noradrenaline in their hypothalamus, hippocampus and frontal cortex compared to saline-injected pigs. Pigs injected with LPS also had higher levels of the pro-inflammatory cytokine IFN-gamma in their frontal cortex compared to saline-injected pigs. Thus, a low dose of LPS can induce changes in brain cytokine levels and neurotransmitter levels that persist after inflammatory and stress markers in the periphery have returned to baseline levels.

Feather pecking is a multi-factorial behavioral disorder and a serious welfare issue in the poultry industry. Several studies report early life experience with litter to be a major determinant in the development of feather pecking. The current study aimed to test the large-scale on-farm efficiency of a simple and cheap husbandry procedure applied during the rearing period with the ultimate goal of reducing the incidence of feather pecking and plumage damage during the production stage in laying hens. Five laying hen-rearing farmers from across Norway participated in the study. These farmers were asked to create divisions within their hen rearing houses and to separate their chicks into 2 groups: one reared with access to a paper substrate from the first d of age, the other a control group without access to paper substrate during rearing. All flocks were visited at the production farms at 30 wk of age and observed for pecking behavior and feather damage. Birds in the control group had higher odds of having more feather damage compared to the birds from the treatment group. In addition, flocks provided with environmental enrichment at the production farms had a reduced incidence of feather pecking, irrespective of the treatment. These results indicate that husbandry procedures during both rearing and production stages have the potential to alleviate feather pecking and increase laying hen welfare.

A case control study with a questionnaire was carried out to compare feeding practices, diet composition, housing and management in 78 herds with or without a history of tail-biting in undocked pigs (Sus scrofa) in Finland. Tail-biting was measured as the mean annual prevalence score of tail-biting damage (TBD) for a farm. Logistic regression parameters were calculated separately for risk factors present in piglet (lactation), weaner, and finishing units. Risk factors found in piglet units for TBD were slatted floors and area of slats. In the weaner units, slatted floors, area of slats, use of whey or wheat in the diet, and use of purchased compound feeds were associated with a risk of TBD. In the finishing units, slatted floors, area of slats, increasing number of finisher pigs at the farm, absence of bedding, liquid feeding, several meals per day, specialised production type and a group size greater than nine pigs were found as risk factors for TBD. Increased farm size was connected to risk for TBD in the overall dataset. The nutritional risk factors seem to operate together with other risk factors, but with relatively lower odds. The risk factors of undocked herds in this study seem to be similar to the risk factors from earlier studies of docked pigs. This study provides information which can be used to refine decision-support tools for management of the potentially higher risk for tail-biting among long-tailed pigs, thus aiding compliance with EU law and enhancing pig welfare.

Feather pecking, toe pecking, cannibalism, smothering, social clumping, hens laying eggs outside the nest boxes, and reduced feather quality are examples of problem behaviors and consequences reported by egg producers. The aim of this study was to identify rearing- and production-related risk factors associated with producer-reported problem behaviors in Norwegian layer flocks. Questionnaires were distributed to 410 egg producers nationwide, and 120 producers responded to the survey (response rate 29%). After exclusion of data that did not comply with the instructions, the final dataset included 78 flocks (19%). The survey covered questions about the farm, the flock's production results, the housing environment, climate and management routines, and the behavior of the birds from 16 wk of age until the flock was euthanized at 70-80 wk of age. The individual problem behaviors were combined to generate a continuous index variable called "problem behavior", ranging from 0 (none) to 8 (all the listed problem behaviors) reported. Multilevel linear regression models were applied to evaluate associations between the index and selected risk factors during rearing and production. The primary predictor was housing system during egg production: producers with aviary flocks on average (± standard deviation) reported 1.6 (± 0.60) more problem behaviors compared to producers with furnished cages (P 0.001). Within aviaries (n = 40), producers, on average reported 1.7 (± 0.50) more problem behaviors in flocks that experienced problems with climatic conditions, compared to flocks without climatic problems (P = 0.001). For respondents with furnished cages (n = 30), on average 1.1 (± 0.50) fewer problem behaviors were reported in farms with ≥ 7,500 birds compared to farms with 7,500 birds (P = 0.027). In conclusion, this is the first study assessing management and housing factors during the rearing and laying phase associated with problem behaviors as reported by Norwegian egg producers. As this study relied on producer reported observations, future studies are needed to investigate whether objective measurements can verify these results.

Exaggerated fear-reactions are associated with injurious flying, smothering, feather pecking and other events that compromise animal welfare in laying hens. The aim of this study was to test the hypothesis that chicks with access to litter during the first five weeks of life would be less fearful as adult hens compared to birds reared without access to litter. The hypothesis was tested in a national on-farm study in commercial aviary flocks in Norway. Five rearing farmers divided the pullets into two groups within their rearing houses. While the chicks were enclosed inside the aviary rows during the first five weeks of life, paper substrate where food and other particles could accumulate, covered the wire mesh floor in the treatment group, whereas the control group was reared on bare wire mesh. At 30 weeks of age, 23 aviary flocks (11 control flocks reared without paper and 12 treatment flocks reared with paper) were visited. During the visit, the fearfulness of the adult birds was tested in a stationary person test and a novel object test. The data was analysed by ANOVA or logistic regression as appropriate. The access to litter during rearing did not influence the number of birds that approached within 25. cm of the stationary person (p = 0.51). All flocks, regardless of rearing treatment, had birds which came within 2. m of the stationary person. The latency to approach within 2. m of the stationary person tended to be influenced by provision of environmental enrichment as adults (p = 0.08) and by the interaction between treatment. ×. rearing farm (p = 0.08). The number of birds that approached within 2. m of the stationary person was influenced by the interaction between treatment during rearing and provision of enrichment as adults (p = 0.03), however, the post hoc test showed no pairwise differences. All flocks, regardless of rearing treatment, had birds that approached the novel object. The access to litter during rearing did not influence the birds' latency to approach the novel object. The number of birds approaching the novel object was affected by the interaction between access to substrate during rearing and provision of environmental enrichment as adults (p = 0.05). The results indicate that both adding paper substrate to chicks from the first day of life and environmental enrichment as adults, reduce fearfulness in laying hens.

Studies using classical conditioning have shown that hens display high frequencies of dopamine-controlled cue-induced anticipatory behaviours in the cue-reward interval when signalling mealworm rewards. However, it is not known whether anticipatory behaviours are reward specific, and whether the opioid system is involved in their control. The purpose of the present study was to investigate (1) effect of incentive value of rewards, and (2) the involvement of μ-opioid receptor activation, on the expression of cue-induced anticipatory behaviours in laying hens. Incentive value was manipulated by reward type (mealworm and whole wheat) and by physiological state (sated and fasted hens). Hens ( n =14) were trained to associate a cue (green or red light) with a reward (whole wheat or mealworms). Blue light served as an unrewarded control stimulus. Cue-induced anticipatory head movements (latency to first head movement after cue presentation, and frequency of head movements in the cue-reward interval), steps (frequency), and pecking at reward (latency), were registered in sated and fasted hens during a 25s cue-reward interval. An involvement of the opioid system in mediating cue-induced anticipatory behaviours was tested by intraperitoneal injection of the μ-opioid receptor antagonist naloxone at 5.0mgkg −1 . Saline served as control. Injections were administered 30min before the light cues. Individual hens were tested on all treatment combinations: sated/saline, fasted/saline, sated/naloxone, and fasted/naloxone. Incentive value of signalled reward was differentially reflected by the frequency of cue-induced head movements ( P P P =0.0003, respectively), but there was no difference between the rewarded cues. Latency to initiate the first head movement was longer in response to the unrewarded cue than to cues signalling whole wheat and mealworms ( P =0.051 (tendency) and P =0.0017). Hunger amplified the frequency of head movements ( P P

International audience; In the laboratory, prenatal stress produces alterations in the structure and function of corticolimbic neurons. Here we report changes in gene expression and corticolimbic dendritic spine morphology in the offspring of pregnant ewes subjected to aversive interactions with human handlers during the last five weeks of pregnancy (AVS) compared to control dams that received gentle handling (GEN). AVS Lambs had higher spine density on pyramidal neurons in area CA1 of the hippocampus and in medial prefrontal cortex compared to GEN lambs, as well as a lower ratio of mushroom spines to stubby and thin spines in area CA1. Expression of genes involved in brain development and spine morphogenesis was decreased in hippocampus and prefrontal cortex in AVS compared to GEN lambs. This study is the first demonstration that an ecologically relevant aversive experience in a field setting alters neuronal structure similarly to previous reports from laboratory settings and that even for animals domesticated over 12,000 years ago, an apparently mild stressor, resulting from human-animal interactions, can have similarly profound impacts on corticolimbic morphology. (C) 2012 Elsevier Ltd. All rights reserved.

Granulomatous peritonitis is often induced by intraperitoneal vaccination in fish. Peritonitis is a very painful condition in mammals, but little is known about how fish experience this condition. In a previous experiment we found increased latency to eat and a tendency to decreased swimming during feeding in vaccinated salmon housed in groups of three. These changes in behaviour correlated with the severity of the peritonitis. However, dominance relationships may influence the degree of pain-related changes in behaviour and physiology shown by group-housed fish, and we therefore conducted the present study using singly-housed fish. The aim was to describe changes in latency to eat, hiding, swimming and bottom behaviour after vaccination, to test whether morphine would alleviate changes in behaviour, and finally to test whether vaccination and/or morphine would influence the response of the fish in the novel object test. In addition, we looked for microscopic changes of peritonitis two days after vaccination to test whether we would be able to detect inflammation at an early stage. Four treatment groups were used: VS (injected with vaccine intraperitoneally (ip) and saline intramuscularly (im)), VM (injected with vaccine ip and 300 mg kg −1 morphine im), SS (saline ip, saline im) and SM (saline ip, morphine 300 mg kg −1 im). Swimming during feeding decreased in the VS fish 2 days after treatment, both compared to baseline ( p = 0.031) and to the SS group ( p = 0.023). The latency to eat differed significantly between groups at 6.5 h after treatment ( p = 0.027), and showed a tendency towards a difference 2 days after treatment ( p = 0.092). The VS group showed significantly higher latency to eat compared with the saline group 6.5 h and 2 days after treatment ( p = 0.024 and 0.043, respectively). Contrary to predictions, we did not find an increase in bottom behaviour after vaccination. We could not detect any microscopic signs of peritonitis 48 h after vaccination. Morphine did not seem to have an analgesic effect in that there were no differences between the VM and VS groups. However, the SM fish spent significantly more time in the open during the novel object test compared with the SS fish ( χ 2 = 6.97, df = 1, p = 0.0083) one day after injection, indicating an anxiolytic effect of morphine at that time-point. It is important that the lack of analgesic effect is not taken to indicate lack of pain perception in salmon. Inter species differences in analgesic efficacy is well known in veterinary medicine, and the results from this paper indicate that we need to look at alternatives to morphine to find effective analgesics for Atlantic salmon.

In order to understand the impact of different experimental and husbandry procedures on fish, and ensure that the behavioural and physiological needs of fish used in research are met, it is necessary to have detailed knowledge of their behaviour. This knowledge is largely lacking for Atlantic salmon. Results from behavioural studies of rainbow trout are often extrapolated to Atlantic salmon even though this may not be correct. Pre-smolt Atlantic salmon show diurnal variation in behaviour in the wild, but there are no published descriptions of behaviour during the dark hours for salmon held in the laboratory. Thus, the first aim of this paper was to describe the time budget of singly-held salmon both during the light and dark hours on the first and seventh day after introduction to a new environment. Statistical differences in time budget between morning, afternoon and night are described. In addition, three environmental enrichments were provided, and the time spent using each resource was scored. In general, no behaviours differed in frequency between morning and afternoon registrations, but there were significant differences in the time-budget between day and night and also between day 1 and 7. Fish hid less and displayed more behaviours during the night than during the day, and also after 7 days in the tank compared to at the first day. The exceptions were bottom behaviours which were displayed with the same frequency during day and night and during day 1 and day 7, and hovering, which was unchanged at night from day 1 to day 7. Thus, different behaviours seem to be influenced by light and by the novelty of the surroundings to different degrees. Of the three enrichments included in the study, the hiding place and the inlet current were used most frequently. As far as the authors know, this is the first paper describing the daytime and night time behaviour of parr Atlantic salmon in a research facility in detail.

Stressful conditions are common in the environment where production animals are reared. Stress in animals is usually determined by the levels of stress-related hormones. A big challenge, however, is in determining the history of exposure of an organism to stress, because the release of stress hormones can show an acute (and recent) but not a sustained exposure to stress. Epigenetic tools provide an alternative option to evaluate past exposure to long-termstress. Chickens provide a unique model to study stress effects in the epigenome of red blood cells (RBCs), a cell type of easy access and nucleated in birds. The present study investigated whether two different rearing conditions in chickens can be identified by looking at DNA methylation patterns in their RBCs later in life. These conditions were rearing in open aviaries versus in cages, which are likely to differ regarding the amount of stress they generate. Our comparison revealed 115 genomic windows with significant changes in RBC DNA methylation between experimental groups, which were located around 53 genes and within 22 intronic regions. Our results set the ground for future detection of long-term stress in live production animals by measuring DNA methylation in a cell type of easy accessibility. Funding Agencies|Coordenacao de Aperfeicamento de Pessoal de Nivel Superior (CAPES; Brazilian government); Fundaco de Amparo a esquisa do Estado de Sao Paulo (FAPESP) [2016/20440-3]; Foundation for Research Levy on Agricultural Products (FFL); Agricultural Agreement Research Fund; Animalia (Norwegian Meat and Poultry Research Centre) through the Norges Forskningsrad [207739]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq; Brazilian government); Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP; Sao Paulo State, Brazil) [2014/08704-0]; European Research Council [322206]

Objective To evaluate the stability and repeatability of measures of mechanical (nociceptive) thresholds in piglets and to examine potentially confounding factors when using a hand held algometer. Study design Descriptive, prospective cohort. Animals Forty-four piglets from four litters, weighing 4.6 ± 1.0 kg (mean ± SD) at 2 weeks of age. Methods Mechanical thresholds were measured twice on each of 2 days during the first and second week of life. Data were analyzed using a repeated measures design to test the effects of behavior prior to testing, sex, week, day within week, and repetition within day. The effect of body weight and the interaction between piglet weight and behaviour were also tested. Piglet was entered into the model as a random effect as an additional test of repeatability. The effect of repeated testing was used to test the stability of measures. Pearson correlations between repeated measures were used to test the repeatability of measures. Variance component analysis was used to describe the variability in the data. Results Variance component analysis indicated that piglet explained only 17% of the variance in the data. All variables in the model (behaviour prior to testing, sex, week, day within week, repetition within day, body weight, the interaction between body weight and behaviour, piglet identity) except sex had a significant effect (p < 0.04 for all). Correlations between repeated measures increased from the first to the second week. Conclusions and Clinical relevance Repeatability was acceptable only during the second week of testing and measures changed with repeated testing and increased with increasing piglet weight, indicating that time (age) and animal body weight should be taken into account when measuring mechanical (nociceptive) thresholds in piglets. Mechanical (nociceptive) thresholds can be used both for testing the efficacy of anaesthetics and analgesics, and for assessing hyperalgesia in chronic pain states in research and clinical settings.

We compared the effects of aversive and gentle handling in late pregnant ewes on fearfulness, heart rate variability and spatial learning in lambs. Twenty-four Norwegian-Dala ewes were studied. Ewes were subjected to gentle (i.e. soft talking and calm behavior) or aversive handling (i.e. swift movements and shouting) for 10 min twice a day during the last five weeks of pregnancy. Lambs from aversively (AVS) or gently (GEN) treated ewes were tested at 4 weeks of age. Lamb behavior was recorded during a) a human approach test, composed of 4 min of isolation and 4 min of exposure to an unfamiliar human, b) an umbrella startle test followed by 5-min recording, and c) two repetitions of a maze test. In addition, heart rate variability was recorded telemetrically before and after the human and startle tests. The baseline heart rate variability measures suggested a lower influence of vagal stimulation in AVS lambs. In the human approach test, AVS lambs vocalized and explored the environment less, and were slower to approach the human. They also tended to have higher flight distances during the startle test than the GEN lambs. The prenatal treatment had no significant effect in the maze test. In conclusion, we showed that aversive handling of pregnant ewes increased fearfulness and reduced vagal tone in their progeny compared to GEN lambs. These effects can have consequences for how lambs cope with rearing conditions.

Positive affective states may be as important for animal welfare as the absence of suffering. Thus, there is a need for knowledge of the mechanisms underlying positive affective states, including how they can be induced and assessed. Studies of behaviour during the anticipation of a signalled palatable food reward have been suggested as a promising tool for answering these questions for the following three reasons: Anticipatory behaviour is hypothesized to reflect an appetitive type of positive affective state associated with dopaminergic activity in the brain. Inducing anticipation is useful for inducing pleasure and reducing stress. Finally, anticipatory behaviour can be used to test a subject's sensitivity to reward. However, to test various hypotheses regarding anticipatory behaviour there is a need to develop standardized research protocols. The aim of the present study was to develop and test an automated method for studying anticipatory and consummatory behaviour in laying hens in their home environment based on trace conditioning. Experimental hens (EX) were first trained to acquire the association between CS (conditioned stimulus; a green light) and US (unconditioned stimulus; meal worm) using a trace interval of 3.5s. To test if EX hens had formed an association between CS and US, they were exposed to CS alone. Then, EX hens were trained to anticipate by gradually increasing the CS–US interval from the initial 3.5s trace interval with a 1s increment on every other CS+US presentation up to 22s after 3 days and 32s after 5 days of training. Finally, they were tested using a CS–US interval of 22 and 32s. Control hens (CO) were exposed to CS and US in a random order, and tested as described for the EX hens. The results demonstrated that the EX hens responded to the CS with an increased level of anticipatory behaviour compared to the CO hens, and that this difference between the groups was significant at a CS–US interval of 22s. No differences between treatments were found in any of the consummatory behaviours. In conclusion, trace classical conditioning can be used for inducing anticipatory behaviour in ad libitum fed laying hens freely moving in the home pen environment. This method will facilitate the testing of hypotheses about reward-related behaviour in laying hens.

There are two main memory systems: declarative and procedural memory. Knowledge of these two systems in fish is scarce, and controlled laboratory studies are needed. Trace classical conditioning is an experimentally tractable model of declarative memory. We tested whether rainbow trout (Oncorhynchus mykiss) can learn by trace conditioning and form stimulus-stimulus, as opposed to stimulus-response, associations. We predicted that rainbow trout trained by trace conditioning would show appetitive behaviour (conditioned response; CR) towards the conditioned stimulus (CS; light), and that the CR would be sensitive to devaluation of the unconditioned stimulus (US; food). The learning group (L, N = 14) was trained on a CS + US contingency schedule with a trace interval of 3.4 s. The control group (CtrL, N = 4) was kept on a completely random schedule. The fish that learnt were further trained as either an experimental (L, N = 6) or a memory control (CtrM, N = 3) group. The L group had the US devalued. The CtrM group received only food. No fish in the CtrL group, but nine fish from the L group conditioned to the light. When tested, five L fish changed their CRs after US devaluation, indicating learning by stimulus-stimulus association of the light with the food. CtrM fish retained their original CRs. To the best of our knowledge, this experiment is the first to show that rainbow trout can learn by trace classical conditioning. The results indicate that the fish learnt by 'facts-learning' rather than by reflex acquisition in this study.

The question of whether fish can perceive pain is controversial, and pain is a potentially grave threat to fish welfare. To be able to study pain in a species, knowledge of its nociceptive system is necessary. There is therefore a need for standardised, repeatable and quantifiable measures of nociception and pain in fish. Sensitivity to noxious heat is readily quantifiable. We developed an apparatus to expose goldfish to controlled, localised heat stimulation, and tested the hypothesis that goldfish perceive heat as aversive. We predicted that they would respond to increasing heat with an escape response, that morphine would decrease their heat sensitivity and that the heat stimulation would affect post-test behaviour. A safety cut-off temperature of 50 8C was built into the test apparatus. All 16 fish responded to the heat with an escape response, with a mean baseline of 38 8C. However, morphine at 40 and 50 mg kg

Previous experiments indicate that artificial elevation of egg corticosterone content prior to incubation may increase fear of humans, reduce the willingness to compete with conspecifics and reduce the growth rate of chicks hatching from these eggs. It can also be hypothesized that corticosterone-treated chicks might be more sensitive to the effects of stress after hatching. Accordingly, we tested the effect of embryonic corticosterone exposure (5.5 ng/ml egg) and intermittent handling after hatching on the behaviour and growth of 88 vehicle-treated (40 males and 48 females) and 88 corticosterone-treated (40 males and 48 females) chicks. The injection treatment did not have any significant effects on tonic immobility ( P ≤ 0.23), but tended to increase the male's willingness to feed in a competitive environment ( P ≤ 0.10). However, post-hatch handling increased the duration of tonic immobility in corticosterone-treated birds but not in controls ( P ≤ 0.01), as reflected in the interaction between the injection and handling treatments ( P ≤ 0.01). Intermittent handling but not corticosterone treatment reduced the willingness of male birds to eat in a competitive situation, but neither treatment affected this behaviour in females. The present results suggest that the effect of embryonic corticosterone exposure may be dependent on the hormone dosage and the environment to which birds are exposed after hatching.

This experiment aimed at testing the hypothesis that exposing hens to stress increases their corticosterone secretion and leads to elevated corticosterone concentrations in their eggs. Furthermore, exposure to stress and the associated changes in egg hormone content was predicted to alter offspring behaviour. Parental hens were 40 White Lohmann Selected Leghorns, half of which were exposed to an unpredictable feed restriction treatment. After application of the treatment, birds in the treatment group had higher concentrations of faecal corticosterone metabolites than birds in the control group. The treatment had no effect on the weight of treated birds or on the concentration of corticosterone in the yolk or albumen of their eggs. Sixty progeny (30 per treatment) were tested for effects on growth and behaviour. No effects of the treatment on the hatchability of eggs or progeny body weight were registered, but treated birds had a higher duration of tonic immobility in response to manual restraint and spent less time eating, when competing with birds from the control group for access to feed in a novel environment. These results confirm that pre-hatch stress may influence behavioural development in chickens. The lack of an effect on the concentration of corticosterone in eggs suggests that other steroid hormones in addition to or rather than corticosterone may mediate the effects of pre-hatch stress in chickens.

Laying hens may face a number of welfare problems including: acute and chronic pain caused by beak trimming; exaggerated fearfulness that may cause stress and suffocation; difficulties in locating resources, resulting potentially in emaciation and dehydration; frustration and boredom, caused by an environment that is barren; feather pecking; cannibalism; foot lesions; and bone fractures. In Europe, a greater proportion of laying hens are housed in non-cage systems compared to the rest of the world. The extent of the different welfare problems may therefore vary between countries as the type of housing system influences the risk of suffering. More generally, many of these welfare problems are influenced by the rearing environment of the pullets. This article therefore focuses on welfare problems in laying hens that can be traced back to rearing. Factors that have been studied in relation to their effects on bird welfare include beak trimming, housing type, furnishing, enrichment, feeding, stocking density, flock size, sound and light levels, concentration of gasses, age at transfer from rearing to production facilities, similarity between rearing and production facilities, competence of staff, and interactions between bird strain and environment. The present review aims to summarize rearing-related risk factors of poor welfare in adult laying hens housed according to European Union legislation. It aims to identify gaps in current knowledge, and suggests strategies for improving bird welfare by improving rearing conditions. Two main conclusions of this work are that attempts should be made to use appropriate genetic material and that beak trimming should be limited where possible. In addition to this, the rearing system should provide constant access to appropriate substrates, perches, and mashed feed, and should be as similar as possible to the housing system used for the adult birds. Finally, young birds (pullets) should be moved to the production facilities before 16 weeks of age. The measures outlined in this review may be useful for improving the welfare of pullets and adult laying hens.

The young of precocial species such as the domestic chicken can, shortly after hatching, imprint on a visually conspicuous object. The chicks' filial behaviour is influenced by a predisposition, directing its attention towards conspecifics, and a learning process, allowing it to distinguish between individuals. The concentrations of maternally derived steroid hormones found in birds' eggs depend on environmental factors and may be how the hen influences her progeny's behavioural development. We tested the hypothesis that prenatal exposure to corticosterone affects filial behaviour in the chick. Eggs in the treatment group were injected with corticosterone before incubation; eggs in the control treatment were vehicle-injected. In experiment 1, coloured chicks were used as imprinting stimuli and subjects were tested in a simultaneous discrimination test. Control chicks showed a significant preference for the imprinting stimulus, but treatment chicks did not. There was no difference between groups in preference scores, which measured memory of the imprinting stimulus. However, colour affected preference score differently between groups. Treatment chicks spent more time with stimulus chicks, regardless of colour, than did control chicks, indicating that corticosterone increased the strength of the predisposition. In experiment 2, chicks were imprinted on objects and tested in a sequential discrimination test. The results indicated a tendency for discrimination in the control group but no discrimination in the treatment group. No difference in preference score between control and treatment chicks was found.

This experiment aims at validating the percentage of visible white in the eye as an emotional indicator, related to the frustration–contentedness axis, in cattle. Earlier experiments showed that the calculated percentage of white in the eyes was larger in frustrated or scared cows compared to cows that were offered a positive stimulus. This eye change was therefore suggested to be a dynamic indicator of emotions in dairy cattle, a high percentage of white in the eye indicating frustration, and a low percentage indicating satisfaction. In the present paper, the hypothesis was that treatment with the anti-anxiety drug diazepam should reduce the percentage of white in the eye in frustrated cows, but have no effect on the percentage of eye white in non-frustrated or rewarded cows. The test animals were 20 randomly selected commercial dairy cows (Norwegian Red Cattle). Two experiments were performed on hungry cows. The four different groups in the first experiment are described in the following. Ten cows were introduced to a frustrating situation, being thwarted from access to visible food (treatment I). Ten cows were introduced to a positive situation, access to food (treatment II). The 10 cows in group I were introduced to the same frustrating situation, but were now pretreated with diazepam (treatment III). The 10 cows in group II were introduced to the same positive situation after pretreatment with diazepam (treatment IV). The two groups in the second experiment were 20 untreated (treatment I) and 20 diazepam-treated (treatment II) cows introduced to a positive, conditioned stimulus, a stockman entering the stall at concentrate feeding time. All predictions were confirmed, supporting the hypothesis that increased percentage of eye white is an emotional indicator of frustration in cows.

We tested the hypothesis that exposure of chick embryos to corticosterone leads to increased fear, reduced competitive ability, reduced ability to cross a barrier and reduced growth in juvenile chicks. Behaviour was studied in birds subjected to three different egg injection treatments: a negative control (no treatment of eggs), a positive control (100 μl sesame oil vehicle) and a corticosterone treatment (0.6 μg corticosterone in 100 μl sesame oil). Eggs were injected prior to incubation and the behaviour of chicks was studied during the first 4 weeks of life. Corticosterone treatment increased fear in chicks, as indicated by greater avoidance of an observer in the home pen at 2 weeks of age (P

The coping styles hypothesis states that animals have consistent behavioural and physiological coping responses that can be characterised on a continuum from reactive to proactive. According to this hypothesis, animals can be characterised by consistency in responses across social and non-social situations as well as over time. We evaluated the validity of this hypothesis for predicting the coping responses of pigs by testing for predicted correlations between responses to a variety of challenges. Animals were exposed to an immobility test at 3 weeks of age. At 8 and 24 weeks, animals were exposed to an unknown pig in the test animal’s home cage, a human in the home cage, and a novel object (bucket) in a novel arena. None of the predicted correlations were found between the duration of immobility and behaviour in the later tests. Many behavioural measures had some repeatability between the ages of 8 and 24 weeks, despite significant changes in the observed level of responses, but there were few correlations between responses to social and non-social situations. Responses to a human and novel object were positively correlated when measured at 8 weeks but not at 24 weeks. The results indicate that the coping styles hypothesis has very limited value in predicting the coping responses of pigs representing the population as a whole. The existing correlations could more parsimoniously be described as reflecting responses to specific or closely related stimuli. It is suggested that future research on pig personality should investigate the possibility that specific coping behaviours—for example, those mediated by fear and anxiety—may have consequences for different elements of coping ability related to welfare and productivity.

The present experiment compared the anxiety- and activity-related behaviour of sub-adult females from a mouse strain selected for over 106 generations for high litter size with that of a randomly selected control strain, to illuminate possible differences in their ability to cope with exposure to a novel environment. Selected for large litter size, the H-strain has an average litter size of 21 pups, whereas the randomly bred C-strain has an average litter size of 10 pups. The elevated plus-maze was used to measure the behaviour of the mice expressed in response to novelty. The results are described and discussed in relation to the ability to cope with novel environmental challenges. In the elevated plus-maze, the H-strain was significantly more anxious (having a lower percentage of entries into and percentage of time spent on open arms) and less active (having a lower number of entries into closed arms, and a lower total number of arm entries) than the C-strain. Thus, there were clear anxiety- and activity-related differences between the strains, which may be related to selection for large litter size. A tentative hypothesis is presented whereby selection for large litter size may accelerate adaptation to the home environment, but decrease the ability of selected animals to cope with exposure to novelty.

The aim of the present experiment was to examine the effects of pre-test transport duration and animal age on the behaviour of 40 weaned pigs in a porcine elevated plus-maze (PEPM). The transport constituted of holding test animals together with pen mates in a transport trolley kept in motion by the experimenter. Multiple regression of behaviour on transport duration and animal age (between 33 and 54 days) indicated that longer duration of pre-test transport was associated with increases in the activity-related measure closed entries (CLE) in the PEPM, but was not associated with changes in the activity-related variable total entries (TE) or either of the anxiety-related behavioural elements percent open time (POT) or percent open entries (POE). Increasing animal age, on the other hand, was associated with decreases in both activity-related measures, but not with anxiety-related parameters. These results imply that animal age and the duration of transport prior to testing should be standardised across treatments in experiments in which evaluation of activity in this novel environment is important, but that measures related to PEPM anxiety are robust to the effects of variation in transport duration and animal age.

The elevated plus-maze, a widely used and well-validated test for studying anxiety-related behaviour in rodents, has recently been suggested for studying anxiety- or fear-related behaviour in swine. There are, however, clear evolutionary, and thus behavioural differences, between these two species. The aim of the present experiment was therefore to compare the behaviour of mice and swine in the murine and porcine elevated plus-maze (PEPM), respectively. To analyse the behaviour of swine, a database including 84 naive weaned pigs was used, while the analysis of mouse behaviour was based on a database of 79 naive individuals. Plus-maze behaviour of the two species was compared on the basis of factor analysis. Based on the hypothesis that the elevated plus-maze induces behaviour related to anxiety in pigs, as well as in mice, it was predicted that factor analysis of pig behaviour should produce factor loadings similar to those found for mice. More specifically, elements predicted to be anxiety-related for pigs (percent open entries, open arm time, percent open arm time) should load highly and independently on a single factor. Furthermore, elements predicted to be activity-related (closed entries and total entries) should also load highly and independently on a separate factor. The orthogonal factor pattern confirmed these predictions. On the other hand, pigs did not express the unconditioned avoidance of open arms observed for mice. It is therefore concluded that the present data provide only equivocal support for the validity of the PEPM.

Scientific research and public debate on the welfare of animals in human custody is increasing at present. Fish are in this context mentioned with particular attention to the high numbers of individuals reared in aquaculture. Research on fish has also contributed to the understanding of individual variation in the ability to cope with stress and disease. One mediator of such variation is the brain serotonergic (5-hydroxytryptamine, 5-HT) system, which conveys physiological and behavioral responses to stress and sub-optimal rearing conditions. Here we study links between the 5-HT response, melanin-based skin pigmentation, and behavior in laboratory-reared Atlantic salmon (Salmo salar) experimentally infested with ectoparasitic sea lice (Lepeophtheirus salmonis). Lice numbers were more variable in less pigmented fish, while the neurochemical response to ectoparastic lice—increased levels of the main 5-HT catabolite 5-HIAA in the brain stem—did not differ between pigmentation groups. A strong depression of growth and locomotor activity was seen in all infested fish but less pigmented fish grew better than fish with more skin melanization regardless of infestation status. The observed combination of neurochemical and behavioral effects clearly suggest that animal welfare concerns can be added to the list of negative effects of ectoparasitic sea lice.

Models of anxiety and fear of novelty were evaluated using correlations and principal component analysis. A total of 84 pigs (Landrace×Yorkshire) from nine different litters were subjected to a tonic immobility (TI) test at the age of 2.5 weeks, an elevated plus-maze (EPM) at the age of 6 weeks, a light/dark (L/D) exploration test at the age of 7 weeks and an open-field (OF) test at the age of 8 weeks. The first component from the principal component analysis had the highest correlation with number of entries into open arms in the EPM but was also highly correlated to variables from the other three tests confirming a common aversion-related element in the four experimental tests. The second component was negatively correlated with percent entries into and time spent on open arms in the EPM, but positively correlated with the number of entries into closed arms in the same test, number of lines crossed in the OF and time spent in the lit compartment of the L/D test. The last point illustrates a negative relationship between “anxiety” and “activity” in the EPM and OF. To achieve purer measures of fear of novelty and activity in the tests, the components were rotated using the Varimax criterion. The rotated factor pattern demonstrated a simple structure where variables related to “anxiety” or “fear of novelty” (i.e., percent entries into open arms and time spent on open arms of the EPM) had the highest loading on factor 1, whereas variables related to activity (i.e., number of entries into the closed arms in the EPM, number of lines crossed in the OF and time spent in the lit compartment of the L/D test) had the highest loading on factor 2. TI duration loaded more strongly on factor 1 (“fear of novelty”) than on factor 2 (“activity”), but did not represent any pure measure of either fear of novelty or activity. In conclusion, all of the test variables were related to one another. Open-arm avoidance represented the purest measure of fear of novelty, whereas entries into closed arms and number of lines crossed in the OF were the purest measures of activity. The EPM appeared to provide the best way to separate the fear of novelty and activity-related elements, indicating that the EPM may be a useful behavioural model of fear of novelty or avoidance in pigs.

The present study examined the effects of diazepam (a widely used anxiolytic benzodiazepine) on the behavioural response of pigs to three novel experimental situations used to measure anxiety-related behaviour in rodents. Twelve weaned pigs (two pairs from each of the three litters) were tested in an elevated plus-maze at the age of 6 weeks, a light/dark test at the age of 7 weeks and an open-field test at the age of 8 weeks. Six of the pigs were pre-treated with diazepam (valium) and the other six with saline (control). In the elevated plus-maze, diazepam-treated pigs had a higher number of entries into open arms (P=0.04), spent more time on open arms (P=0.07), and had a higher number of total arm entries (P=0.05) than pigs from the control group. However, diazepam had no significant effects on behaviour in the light/dark test (i.e., latency to enter lit compartment, number of entries into lit compartment and the time spent in lit compartment) or the open-field test (i.e., number of lines crossed, number of entries into centre). In summary, the anxiolytic effects of diazepam on the pigs' behaviour were only demonstrated in the elevated plus-maze, where the time spent on open arms and the number of entries into open arms could be interpreted as measures of anxiety in pigs.

Feather damage due to severe feather pecking (SFP) in laying hens is most severe during the laying period. However, SFP can develop at an early age and is influenced by early rearing conditions. In this study we assessed the risk factors during the rearing and laying period for feather damage at 40 weeks of age, in ISA brown and Dekalb White laying hens. Variables related to housing conditions during the rearing and laying period, and variables related to fearfulness (response to novel object, stationary person, and social isolation) and feather pecking (SFP, feather damage and feather eating) were tested to affect feather damage at 40 weeks of age. Feather damage on the neck, back and belly region was assessed on 50 hens, resulting in a total body score, and averaged per flock (based on Welfare Quality ®, 2009). First, analysis was conducted by a two-way ANOVA to assess separate factors to influence feather damage at 40 weeks of age. Hereafter, the final GLM for predicting feather damage at 40 weeks of age included only variables which had P