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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/).

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.

Objective To investigate motor and cardiovascular responses to dexmedetomidine or fentanyl in isoflurane-anaesthetized pigs. Study design Experimental, balanced, block randomized, two-group design. Animals A group of 16 crossbred pigs, 55 ± 8 days (mean ± standard deviation) old. Methods Deltoid electromyography (EMG) was recorded during isoflurane anaesthesia. Electrical stimulation using 5, 10, 20 and 40 mA of the distal right thoracic limb elicited a nociceptive withdrawal reflex (NWR), quantified by the area under the curve (AUC) for the simulation intensity versus EMG amplitude response curve. Latency to movement evoked by clamping a claw for maximum 60 seconds was noted. Arterial blood pressure and pulse rate were recorded. Data were sampled at baseline and during dexmedetomidine 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 μg kg–1 hour–1 or fentanyl 5, 10, 20, 40, 80 and 160 μg kg–1 hour–1 infusions. The influence of infusion rate on NWR AUC and spontaneous EMG was analysed using a mixed model, with p Results NWR AUC increased at fentanyl 5 μg kg–1 hour–1 but decreased at fentanyl 40, 80 and 160 μg kg–1 hour–1 and dexmedetomidine 4.0 and 8.0 μg kg–1 hour–1. All pigs at fentanyl 80 μg kg–1 hour–1, and three pigs at dexmedetomidine 8.0 μg kg–1 hour–1 had mechanical latencies greater than 60 seconds. Spontaneous EMG activity increased accompanied by visually evident ‘shivering’ at fentanyl 5, 10 and 20 μg kg–1 hour–1 but decreased at dexmedetomidine 2, 4 and 8 μg kg–1 hour–1. Clinically relevant effects of increasing infusion rates on blood pressure or pulse rate were not observed. Conclusion and clinical relevance If anaesthetic plane or antinociception is evaluated in pigs, response to claw clamping and NWR will not necessarily give uniform results when comparing drugs. If only one method is used, results should be interpreted cautiously.

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.

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

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.

Measuring glucocorticoid metabolites in faeces has proven a useful, non-invasive method to monitor adrenocortical activity in several farm and wild species. Unlike plasma cortisol, whose sampling requires restraint and blood draws, faecal cortisol metabolites (FCM) may be particularly suitable for farmed silver foxes as these animals are sensitive to handling by humans. Prior to using FCM as a potential indicator of stress in silver foxes, however, a proper physiological and/or biological validation is required. Here, we determined FCM concentrations in 30 silver foxes (10 adult vixens, 10 juvenile females and 10 juvenile males) every alternate hour for 24 h after 1) an increase in cortisol induced by injection with synthetic ACTH (hereafter ACTH), and 2) a 2 min period of handling and restraint. Baseline FCM values, recorded every fourth hour for 24 h before the ACTH and handling treatments, served as controls. FCM values increased significantly following ACTH injection (P = 0.0001) and handling (P

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]

The complexity of the rearing environment is important for behavioral development and fearfulness. The aim of this study was to test the hypothesis that laying hens reared in a complex aviary system with exposure to mild intermittent stressors would be less fearful, less sensitive to stress, and would use elevated areas of the pen more often as adults than hens reared in a barren cage environment. Laying hens (N = 160) were housed in the same rearing house; half of the birds (n = 80) in an aviary and the other half (n = 80) in cages. At 16 weeks of age, the birds were transported to the experimental facilities. Their behavior was recorded at 19 and 23 weeks of age and analyzed by analysis of variance on individual scores for a fearfulness-related principal component generated using principal component analysis. The results indicate that aviary-reared birds have lower levels of fearfulness compared with cage-reared birds both at 19 weeks and at 23 weeks of age. When comparing the response induced by initial exposure to a novel object at 19 and 23 weeks of age, more aviary-reared birds tended to fly up at 19 weeks compared to the cage-reared birds, indicating a tendency toward a more active behavioral response in the aviary-reared birds than in cage-reared birds. There was no difference between treatments in the flight response at 23 weeks. The groups did not differ in defecation frequency or the concentration of fecal corticosterone metabolites at either age. At 19 weeks, observation of the spatial distribution in the home pens indicated that more aviary-reared birds spent time on the low perch, the elevated platform, and the upper perch, compared to the cage-reared birds. However, at 23 weeks of age, these differences were no longer detected. The results of this study support the hypothesis that increased environmental complexity during rearing reduces fearfulness of adult laying hens.

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.

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

Stimulation of foraging behaviour during the first weeks of life by increasing environmental complexity such as scattering food items on the litter may be one way of preventing leg disorders, reducing fear and improving broiler welfare. However, studies on effects of access to various food based enrichment are inconclusive. Motivation of animals to search for resources in the litter may be driven by the motivational significance of the provided food resource. This study was conducted to test whether the motivational significance of food enrichment differentially affects activity, litter directed behaviours and fearfulness in broiler chickens. Chickens (120 chickens in three batches of 40 animals) were divided into four groups according to type of food-related enrichment: 1) mealworms (highly attractive), 2) whole wheat (less attractive), 3) wood shavings (no nutritional value), and 4) no enrichment. Enrichment materials were scattered on the litter daily for 12 days starting on day 6. On days 6, 9, 12 and 15 of age the chickens were video-recorded from 10 min before to 30 min after scattering. Duration and frequency of physical activity (time spent on legs), litter directed behaviour (litter pecking and scratching) and leg stretching were analysed from videos in 10-min intervals. All birds were subjected to a tonic immobility test (day 16) and a novel object test (day 17). Mealworm treatment induced a significant increase in activity, litter pecking and litter scratching (p < 0.001) during the first 10 min interval after the treatment. There were no effects on activity, litter directed behaviour, and on any of the parameters in the tonic immobility test and novel object test. In conclusion, motivational significance of the food items scattered on the litter affect litter directed behaviours in broiler chickens. More studies are needed to develop feasible attractive food enrichments and complex distribution methods that would increase time spent in activity.

Electric activity in the brain which is time-locked to a given stimulation of the somatosensory system can be recorded as a somatosensory evoked potential (SEP). We investigated whether a galvanic stimulation of the tail base in Atlantic salmon (Salmo salar) would elicit a SEP in the telencephalon. The telencephalon is central in learning and memory, and activity here may be a prerequisite for processing of external stimuli on a cognitive or emotional level. Anaesthetized salmon (n = 11) were subjected to craniotomy and a recording electrode was inserted into the telencephalon. The fish were given stimulations of four intensities, i.e., 2, 5, 10 and 20 mA. A SEP was elicited in the contralateral dorsal telencephalon for all intensities. This result agrees with findings in other fish species. Furthermore, there was a significant difference between the maximum peak amplitude and mean amplitude of the SEP elicited by putative non-noxious (2 mA) and putative noxious (20 mA) stimulation intensities (P

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.

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.

The pros and cons of using anaesthesia when handling fish in connection with experiments are debated. A widely adopted practice is to wait thirty minutes after anaesthesia before behavioural observations are initiated, but information about immediate effects of a treatment is then lost. This is pertinent for responses to acute stressors, such as acid injection in the acetic acid pain test. However, omission of anaesthetics in order to obtain data on immediate responses will compromise the welfare of fish and contribute to experimental noise due to stress. We therefore tested the effect of tricaine methanesulfonate on the behaviour of zebrafish. We predicted that tricaine (MS 222) would decrease swimming velocity and that the control fish would show an increased level of anxiety- and stress-related behaviours compared to the tricaine group. Following acclimatization to the test tank, baseline behaviour was recorded before immersion in either tricaine (168 mg l(-1), treatment group, N = 8) or tank water (control group, N = 7). Latencies to lose equilibrium and to lose response to touch were registered. The fish was then returned to the test tank, and the latency to regain equilibrium was registered in anaesthetized fish. When equilibrium was regained, and at five, thirty and sixty minutes after the fish had been returned to the test tank, behaviour was recorded. The tricaine fish showed the following responses (mean ± sd): latency to lose equilibrium 22.6 s±3.9; latency to lose response to touch 101.9 s±26.8; latency to regain equilibrium 92.0 s±54.4. Contrary to our predictions, neither treatment caused a change in any of the behaviours registered. This indicates that tricaine has no effect on several commonly used behavioural parameters, and that it may be unnecessary to postpone behavioural observations to 30 min after anaesthesia.

In order to investigate the involvement of dopaminergic transmission in the regulation of reward-related behaviors in laying hens, the effects of systemic injections of dopamine D2-like receptor antagonist haloperidol (0.3, 0.5, 1.0 and 2.0 mg/kg s.c.) 30 min prior to a conditioned cue signaling a reward were tested and compared to the effects of a saline injection. Head movements and latency to initiate display of anticipatory behavior were significantly affected by 0.3 and 0.5 mg/kg haloperidol, respectively. More hens were found lying down resting prior to the cue at 0.5 mg/kg and higher doses, and increased latency to peck at reward and shorter duration of anticipatory behavior was significant at 2.0 mg/kg. The findings are consistent with the involvement of dopamine in control of reward-related behaviors in laying hens. It is suggested that the lowest dose of haloperidol (0.3 mg/kg) affects reward-related behaviors, whereas the effects of higher doses of haloperidol could be confounded by sedative effects. A high inter- and intra observer agreement in the assessment of head movements together with their dopamine dependency further suggest that this behavior in classical conditioning paradigm represents an indicator of the state of the reward system in laying hens that can be assessed with good reliability.

Teleost fish have a nociceptive system and likely perceive pain. This warrants the devel- opment of analgesic protocols both for experimental surgery and for various husbandry procedures. Morphine is the standard analgesic against which the efficacy of other analgesics is assessed, and is the analgesic that has been most used in fish. The aims of this study were to describe the pharmaco- kinetics of morphine after an intramuscular (i.m.) injection in common goldfish and Atlantic salmon, and to illustrate the whole-body distribution of morphine in salmon following i.m. injection of tritiated morphine. In the kinetic experiment, goldfish and salmon were respectively i.m. injected with 40 and 100 mg morphine kg -1 in the right dorsal epaxial musculature. Blood was drawn at predetermined time points. Plasma was analysed for morphine and metabolites using liquid chromatography-mass spectrometry (LC-MS/MS). Morphine had a Tmax (time at which the maximum plasma concentration was measured) of 0.5 h in both species. The Cmax (maximum plasma concentration) showed substan- tial inter-individual variation, with a mean (90% CI) of 187 (167 to 199) mg l -1 in salmon and 37 (29 to 43) mg l -1 in goldfish, as determined by bootstrap analysis. The mean elimination half-lives were 12.5 and 13.5 h in goldfish and in salmon, respectively. The degree of metabolism to morphine-3-glu- curonide (M3G) and morphine-6-glucuronide (M6G) was low, with levels of M3G exceeding those of M6G. The distribution study demonstrated that the levels of tritiated morphine in the anterior kidney surpassed those in the other organs. A substantial amount seemed to be excreted through the gas- trointestinal tract, while little tritium activity could be detected in the central nervous system.

Aviary and cage-rearing systems for laying hens differ from one another in environmental complexity. Spatial cognition, important for navigating the environment and finding resources, is adversely affected by a lack of environmental complexity during early life. However, to our knowledge, no previous studies have tested the effect of early exposure to varying degrees of environmental complexity on specific components of spatial cognition in chickens. The aim of the present study was to test the hypothesis that rearing in a cage environment relative to an aviary environment causes long-lasting deficits in the ability to perform spatial tasks. 52,000 white Dekalb laying hens were reared in separate corridors of a commercial rearing farm. Each corridor had either a cage- or an aviary-rearing system with densities of 25 and 12 birds/m2 respectively. The cage system consisted of aviary-row cages that remained closed throughout the rearing period. All housing conditions used are consistent with current Norwegian legislation. At 16 weeks of age, 24 hens, 12 from each rearing system, were transported to experimental facilities, housed in furnished cages, and trained and tested in a holeboard task. The holeboard is an open-field arena with nine cups, three of which are baited with a food reward. Over time of exposure to the arena, the birds are expected to remember the position of the baited cups. Trial duration and working memory performance for each trial were recorded and tested in a repeated-measures-ANOVA. Aviary and cage-reared hens did not differ in trial duration and working memory during training, indicating that birds from both treatments were physically capable of performing the task. However, during the final test phase, the cage-reared hens required more time to locate rewards (Aviary: 59.95±7.02sec. Cages: 161.21±14.51sec. F3,57=3.897; p=0.013; post hoc t=2.99; p=0.004) and had poorer levels of working memory (Aviary: 0.84±0.02. Cages: 0.65±0.04. F3,57=2.75; p=0.051; post hoc t=-2.88; p=0.005). The findings thus support the hypothesis that rearing in a cage environment causes long-term impairment of short-term memory in chickens.