Janicke Nordgreen

Behavioural Effects of the Commonly Used Fish Anaesthetic Tricaine Methanesulfonate (MS-222) on Zebrafish (Danio rerio) and Its Relevance for the Acetic Acid Pain Test

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.

Early life in a barren environment adversely affects spatial cognition in laying hens (Gallus gallus domesticus)

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.

Factors affecting mechanical (nociceptive) thresholds in piglets

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.

Exposure to Increased Environmental Complexity during Rearing Reduces Fearfulness and Increases Use of Three-Dimensional Space in Laying Hens (Gallus gallus domesticus)

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.

Ectoparasitic sea lice (Lepeophtheirus salmonis) affect behavior and brain serotonergic activity in Atlantic salmon (Salmo salar L.): Perspectives on animal welfare

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.

Anticipatory and foraging behaviors in response to palatable food reward in chickens: Effects of dopamine D2 receptor blockade and domestication

Behaviors associated with anticipation and search for palatable food may provide information about dopaminergic reward processes and positive motivational affect in animals. The overall aim was to investigate the involvement of dopamine signaling in the regulation of cue-induced anticipation and search for palatable food reward in chicken, and whether domestication has affected expression of reward-related behaviors. The specific aims were to describe effects of mealworms (palatable food for hens) and haloperidol (a dopamine D2 antagonist) on foraging behaviors and cue-induced anticipatory behaviors in Red Junglefowl (RJF; the wild ancestor of modern laying hens) and a white layer hybrid (LSL). RJF (n=26) and LSL (n=20) were initially trained on a conditioning schedule to anticipate mealworms (unconditioned stimulus; US) 25s after exposure to a red light (conditioned stimulus; CS). For the experiment, hens received haloperidol or saline injections 30 min before exposure to one CS+US combination. Behavior was registered 10 min before CS and 10 min after US (foraging behaviors), and during the CS-US interval (anticipatory behaviors). Higher frequencies of CS-induced anticipatory head movements, faster approach to rewards, and higher frequency of foraging behaviors were found in LSL compared to RJF. Haloperidol suppressed CS-induced head movements in both breeds, and the frequency of foraging behaviors after reward delivery. The results support a role of dopamine signaling in the regulation of reward processes in chickens, and suggest that domestication has changed the threshold for perceiving food incentives and/or for expressing reward-related behaviors that may be indicative of positive motivational affect in hens.

Effects of haloperidol, a dopamine D2-like receptor antagonist, on reward-related behaviors in laying hens.

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.

Pharmacokinetics of morphine after intramuscular injection in common goldfish Carassius auratus and Atlantic salmon Salmo salar

Teleost fish have a nociceptive system and likely perceive pain. This warrants the development 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 pharmacokinetics 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 substantial 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-glucuronide (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 gastrointestinal tract, while little tritium activity could be detected in the central nervous system.

DNA methylation profiles in red blood cells of adult hens correlate with their rearing conditions

ABSTRACT 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-term stress. 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. Summary: Differential DNA methylation occurs in red blood cells of adult hens reared under different conditions, i.e. in cages or open aviaries.

Does Early Environmental Complexity Influence Tyrosine Hydroxylase in the Chicken Hippocampus and "Prefrontal" Caudolateral Nidopallium?

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.