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Two of the most used models of anxiety in zebrafish research, the novel tank-diving test (NTDT) and the black-white preference test (BWPT), are modifications of assays used in rodent research (open field test and light/dark test). There has been a thorough validation of these tests in rodents, but a similar level of knowledge is still missing in zebrafish. Adult zebrafish naturally live in shoals with conspecifics, and group housing is therefore assumed to be the optimal housing condition for zebrafish, as it allows for shoaling behavior. This study investigated how housing in social isolation affected whole-body cortisol and the behavioral responses in the NTDT and BWPT. We also examined the correlation between the behavioral responses in the two behavioral tests. We found that zebrafish housed in groups had significantly higher whole-body cortisol than individually housed zebrafish (F 1, 85 = 25.51, P < 0.0001). Regardless of treatment, all groups had a general preference for the lower compartment in the NTDT and the black compartment in the BWPT. Individually housed zebrafish had a higher total number of entries to the white compartment in BWPT compared to group housed zebrafish when their first test was BWPT (F 1, 48 = 5.79, P = 0.0201), but not when BWPT was their second behavioral test. Fish that had higher whole-body cortisol had a tendency toward fewer entries into the white compartment the first 3 min of the BWPT (F 1, 48 = 3.90, P = 0.0540). There was no effect of housing on the behaviors registered in the NTDT. There was a positive correlation (correlation coefficient 0.40; p = 0.003) between transitions from black to white compartment in BWPT and transitions from lower to upper compartment in NTDT, but we did not find any association between duration in white compartment in BWPT and upper compartment in NTDT. Considering this, we suggest that further model validation is needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Onarheim, Janczak and Nordgreen.)

Damaging behaviors (DB) such as tail and ear biting are prevalent in pig production and reduce welfare and performance. Anecdotal reports suggest that health challenges increase the risk of tail-biting. The prevalence of tail damage and health problems show high correlations across batches within and between farms. There are many common risk factors for tail-biting and health problems, notably respiratory, enteric and locomotory diseases. These include suboptimal thermal climate, hygiene, stocking density and feed quality. The prevalence of tail damage and health problems also show high correlations across batches within and between farms. However, limited evidence supports two likely causal mechanisms for a direct link between DB and health problems. The first is that generalized poor health (e.g., enzootic pneumonia) on farm poses an increased risk of pigs performing DB. Recent studies indicate a possible causal link between an experimental inflammation and an increase in DB, and suggest a link between cytokines and tail-biting. The negative effects of poor health on the ingestion and processing of nutrients means that immune-stimulated pigs may develop specific nutrient deficiencies, increasing DB. The second causal mechanism involves tail-biting causing poor health. Indirectly, pathogens enter the body via the tail lesion and once infected, systemic spread of infection may occur. This occurs mainly via the venous route targeting the lungs, and to a lesser extent via cerebrospinal fluid and the lymphatic system. In carcasses with tail lesions, there is an increase in lung lesions, abscessation, arthritis and osteomyelitis. There is also evidence for the direct spread of pathogens between biters and victims. In summary, the literature supports the association between poor health and DB, particularly tail-biting. However, there is insufficient evidence to confirm causality in either direction. Nevertheless, the limited evidence is compelling enough to suggest that improvements to management and housing to enhance pig health will reduce DB. In the same way, improvements to housing and management designed to address DB, are likely to result in benefits to pig health. While most of the available literature relates to tail-biting, we suggest that similar mechanisms are responsible for links between health and other DB., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors, SE., (Copyright © 2022 Boyle, Edwards, Bolhuis, Pol, Šemrov, Schütze, Nordgreen, Bozakova, Sossidou and Valros.)

Infectious and inflammatory conditions are common especially in growing pigs. Lipopolysaccharide (LPS) is an important antigenic structure of Gram-negative bacteria and can be used to induce inflammation experimentally. As pigs are usually group-housed in commercial conditions, it is difficult to detect sick individuals, particularly at an early stage of illness. Acute phase proteins such as haptoglobin (Hp) are known indicators of an activated innate immune system whereas adenosine deaminase (ADA) is a relatively novel inflammatory biomarker in pigs. Both parameters can be measured in saliva and could be used as indicators of inflammation. Compared with blood sampling, saliva sampling is a less stressful procedure that is rapid, non-invasive and easy to perform both at group and at individual level. In this blinded randomized clinical trial, 32 female pigs at their post-weaning phase were allocated to one of four treatments comprising two injections of the following substance combinations: saline-saline (SS), ketoprofen-saline (KS), saline-LPS (SL), and ketoprofen-LPS (KL). First, ketoprofen or saline was administered intramuscularly on average 1 h before either LPS or saline was given through an ear vein catheter. In all groups, saliva was collected prior to injections (baseline) and at 4, 24, 48, and 72 h post-injection for determination of ADA, Hp, and cortisol concentrations. A multivariate model was applied to describe the dynamics of each biomarker. Pairwise relationships between ADA, Hp, and cortisol responses from baseline to 4 h post-injection within the SL group were studied with Spearman correlations. A significant increase in the SL group was seen in all biomarkers 4 h post-injection compared to baseline and other time points (pairwise comparisons, p < 0.01 for all) and ketoprofen alleviated the LPS effect. We found a significant positive correlation between ADA and Hp within the SL group ( r = 0.86, p < 0.05). The primary and novel findings of the present study are the response of ADA to LPS, its time course and alleviation by ketoprofen. Our results support the evidence that ADA and Hp can be used as inflammatory biomarkers in pigs. We suggest further studies to be conducted in commercial settings with larger sample sizes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sali, Veit, Valros, Junnikkala, Heinonen and Nordgreen.)

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 × 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-γ, TNF-α, 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Veit, Janczak, Ranheim, Vas, Valros, Sandercock, Piepponen, Dulgheriu and Nordgreen.)

Sickness can change our mood for the worse, leaving us sad, lethargic, grumpy and less socially inclined. This mood change is part of a set of behavioral symptoms called sickness behavior and has features in common with core symptoms of depression. Therefore, the physiological changes induced by immune activation, for example following infection, are in the spotlight for explaining mechanisms behind mental health challenges such as depression. While humans may take a day off and isolate themselves until they feel better, farm animals housed in groups have only limited possibilities for social withdrawal. We suggest that immune activation could be a major factor influencing social interactions in pigs, with outbreaks of damaging behavior such as tail biting as a possible result. The hypothesis presented here is that the effects of several known risk factors for tail biting are mediated by pro-inflammatory cytokines, proteins produced by the immune system, and their effect on neurotransmitter systems. We describe the background for and implications of this hypothesis., (Copyright © 2020 Nordgreen, Edwards, Boyle, Bolhuis, Veit, Sayyari, Marin, Dimitrov, Janczak and Valros.)

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.

In adult chickens, the housing system influences hippocampal morphology and neurochemistry. However, no work has been done investigating the effects of the early life environment on chicken brain development. In the present study, we reared 67 commercial laying hens (Gallus gallus domesticus) in two environments that differed in the degree of complexity (aviary or cage system). These two groups were further divided into two age groups. At 20 weeks of age, 18 aviary-reared birds and 15 cage-reared birds were humanely euthanized and their brains dissected. At 24 weeks of age, a further 16 brains from aviary-reared birds and 18 brains from cage-reared birds were collected. These brains were prepared for immunohistochemical detection of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, in the hippocampus and the caudolateral nidopallium (NCL). There were no differences between the treatment groups in TH staining intensity in the hippocampus or the NCL. In the medial hippocampus, the right hemisphere had higher TH staining intensity compared to the left hemisphere. The opposite was true for the NCL, with the left hemisphere being more strongly stained compared to the right hemisphere. The present study supports the notion that the hippocampus is functionally lateralized, and our findings add to the body of knowledge on adult neural plasticity of the avian brain.

Spatial cognition in vertebrates 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. There are two main rearing systems for laying hens in the EU: aviaries and cages. These two systems differ from one another in environmental complexity. The aim of the present study was to test the hypothesis that rearing in a barren cage environment relative to a complex aviary environment causes long-lasting deficits in the ability to perform spatial tasks. For this purpose, 24 white Dekalb laying hens, half of which had been reared in an aviary system and the other half in a conventional cage system, were tested in a holeboard task. Birds from both treatment groups learnt the task; however, the cage-reared hens required more time to locate rewards and had poorer levels of working memory. The latter finding supports the hypothesis that rearing in a barren environment causes long-term impairment of short-term memory in chickens.