Behavioural and endocrine fear responses in Japanese quail upon presentation of a novel object in the home cage

Most tests used to study fear in birds involve transferring them to a novel environment, which constitutes a bias in studies aiming at identifying the neural correlates of a specific fear-inducing situation. In order to investigate fear in birds with minimum interference by humans, behavioural and endocrine responses to the presentation of a novel object in the home cage were investigated in two lines of Japanese quail divergently selected for long or short duration of tonic immobility, a behavioural index of fear. Presentation of the novel object induced typical fear responses (avoidance of the object, increased pacing and increased plasma corticosterone levels) that were similar in the two lines of quail. Presentation of a novel object in the home cage thus appears to be a suitable stimulus to induce fear reactions in quail, with minimum interference from other motivational systems. The fact that quail of both lines reacted similarly in this test, while they are known to differ greatly in their behavioural responses to other fear-inducing tests, illustrates the multidimensional nature of fear.     

Selection for tonic immobility duration does not affect the response to novelty in quail

Genetic selection on a single fear test, the tonic immobility test, seems to result in selection on fearfulness, i.e. the propensity to exhibit fear responses, whatever the fear tests used. However, the conception of fear as a single variable has been challenged by the recognition that fear is multidimensional. This study was designed to test whether genetic selection on a classic index of fear in birds, tonic immobility duration, is accompanied by changes in the response to a single dimension of fear – novelty.Two lines of quail divergently selected for long (LTI) or short (STI) duration of tonic immobility were exposed to a novel object in their home cage. Quail of both lines showed typical fear reactions in response to novelty but there was no difference between lines. We conclude that genetic selection for tonic immobility duration does not affect all dimensions of fear, notably not novelty. Further studies are needed to investigate the dimensions of fear on which the two lines of quail could have been selected.     

Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats

Previous studies have indicated that neonatal handling influences development of hypothalamic-pituitary-adrenal (HPA) control of corticosterone. In addition, corticosterone influences memory consolidation processes in contextual fear conditioning. Therefore, neonatal handling may affect hippocampal-dependent memory processes present in contextual fear conditioning by influencing the development of HPA control of corticosterone. To investigate the effects of neonatal handling on early learning, rat pups were either handled (15-min removal from home cage) on the first 15 days after birth or left undisturbed in their home cage. Handled rats and nonhandled rats were fear conditioned at 18, 21, or 30 days of age and then tested at two time points--24 h following conditioning and at postnatal day 45. Subsequently, at approximately postnatal day 60, rats were exposed to restraint stress and corticosterone levels were assessed during restraint and recovery. Handled and nonhandled rats did not differ significantly in their freezing response immediately following footshock on the conditioning day. However, when tested for contextual fear conditioning at 24 h following conditioning and at postnatal day 45, handled rats showed more freezing behavior than nonhandled rats. When exposed to restraint stress, handled rats had a more rapid return of corticosterone to basal levels than nonhandled rats. These results indicate that neonatal handling enhances developmentally early memory processes involved in contextual fear conditioning and confirms previously reported effects of neonatal handling on HPA control of corticosterone.     

Landscapes of Coexistence for terrestrial carnivores: the ecological consequences of being downgraded from ultimate to penultimate predator by humans

Fear of predation can have major impacts on the behaviour of prey species. Recently the concept of the ecology of fear has been defined and formalised; yet there has been relatively little focus on how these ideas apply to large carnivore species which, although not prey sensu stricto, also experience fear as a result of threats from humans. Large carnivores are likely also subject to a Landscape of Fear similar to that described for prey species. We argue that although fear is generic, ‘human‐caused mortality’ represents a distinct and very important cause of fear for large carnivores, particularly terrestrial large carnivores as their activities overlap with those of humans to a greater degree. We introduce the idea of a ‘Landscape of Coexistence’ for large carnivores to denote a subset of the Landscape of Fear where sufficient areas of low human‐caused mortality risk are present in the landscape for long term coexistence of large carnivores and humans. We then explore aspects of terrestrial large carnivore behavioural ecology that may be best explained by risk of human‐caused mortality, and how the nature of a Landscape of Coexistence for these large carnivores is likely to be shaped by specific factors such as habitat structure, wild and domestic prey base, and human distribution and behaviour. The human characteristics of this Landscape of Coexistence may be as important in determining large carnivore distribution and behavioural ecology as the distribution of resources. Understanding the Landscape of Coexistence for terrestrial large carnivores is therefore important for their biology and conservation throughout large parts of their remaining ranges. Synthesis The Landscape of Fear concept describing the relationship between predator and prey also applies to the relationship between humans and top carnivores. We synthesise current research to introduce the Landscape of Coexistence concept, arguing that top predators respond to the risks of human‐caused mortality through spatiotemporal partitioning of activities to reduce contact with people. The character of the Landscape of Coexistence may be more important than the distribution of resources in determining large carnivore distribution and behavioural ecology in human dominated landscapes. Understanding their behavioural responses to human threats is crucial to successful conservation of large carnivores.     

Long-term effects of different handling procedures on behavioural, physiological, and production-related parameters in silver foxes

Fifty-one silver fox vixens, subjected to three different handling treatments as cubs (no-handling, gentle or forced handling), were studied for long-term effects on behavioural, physiological and production-related parameters in a one and a half year period following the last handling session. As juveniles, the animals were exposed to three different behavioural tests at 18, 22, 28, and 32 weeks of age. Both forcibly and gently handled animals showed reduced fear responses compared with nonhandled controls in test situations involving close contact with humans (P < 0.05). The foxes were tested again as adults at 10, 13, 15, and 18 months of age with the same behavioural tests. It was revealed that forcibly handled animals persistently showed reduced fear responses compared with control animals, both in close contact with humans and when exposed to a novel object, whereas gently handled animals only differed from control animals in one of the tests involving some human contact and when exposed to a novel object (P < 0.05). Significant differences between adult gently handled and forcibly handled animals were found in the “confront’ test involving close human contact. In this test more flight responses were observed in the gently handled group than in the forcibly handled group (P < 0.01). Control animals had significantly larger adrenal weights compared with both forcibly handled animals and gently handled animals at 22 months of age (total mean adrenal weight: controls, 0.62 g; gentle, 0.54 g; forced, 0.54 g, P < 0.05, general linear models). Other physiological measures and production-related parameters such as body weight, body size, gastric ulceration and pelt qualities did not differ between treatments (P > 0.05). Early post-weaning handling made foxes less fearful towards humans. Forced handling seemed somewhat superior to gentle handling as a means to produce animals which, in the long term, adapted better to the farm environment both behaviourally and physiologically. Non-handled control animals suffered from long-term stress as reflected by high levels of fear responses and enlarged adrenals. Thus post-weaning handling may be beneficial in the long term for the wellbeing of farmed foxes. When applied as a management routine, handling had no significant influence on later production-related parameters and was not considered an economic risk.     

Estimation of fear in two lines of the domestic chick: Correlations between various methods

The assessment of fear is a controversial issue and low levels of correlation between different measures have been used to criticise the fear concept. The present study assessed fear levels in individual domestic chicks of each of two lines, flighty and docile, using four commonly employed methods of estimating fear. They were the hole-in-the-wall box, the open field, response to a bell and tonic immobility. On the basis of a wide variety of behavioural responses each chick was ranked for fearfulness in each of the four tests. The degrees of association or correlation between these ranking within lines were then calculated. The significant intra-individual correlations found in both lines provides some support for the use of these tests as methods of estimating fear, at least within the lines used. Differences in the degree or form of fear-responding between the lines are also discussed in terms of reactions to handling and to sudden auditory stimuli.     

Fear responses of individually-caged laying hens as a function of cage level and aisle

Chickens kept under intensive conditions are often housed in multi-deck battery cage systems. The effects on egg production of housing birds at different cage levels have been extensively studied, but little is known of the behavioural consequences of this. The present study examined the responses of individually-caged adult laying hens from each tier of a 3-tier battery system in a number of situations intended to elicit fear. Birds housed in the top tier showed (a) longer durations of tonic immobility, (b) greater avoidance of a novel rod placed at the front of the cage, and (c) lower levels of approach and greater inhibition when placed in a pen containing either a human being or one of two novel, inanimate objects, than did those from middle and bottom tiers. The latter birds behaved remarkably similarly. The consistently higher fear scores recorded by hens from the top tier are considered likely to reflect differences in general fearfulness as a function of cage level, and may be a consequence of long-term exposure to different degrees of environmental stimulation.     

Agonistic behaviour,responses to a novel object and some aspects of maintenance behaviour in feral-strain and domestic chickens

Feral-strain fowl, bred from birds captured on North-West Island (24°S, 150°E), were compared with domestic chickens hatched and raised under similar conditions. Aspects of behaviour considered included agonistic behaviour and responses to unfamiliar environments and objects. There were many similarities between the feral and domestic fowl in behaviour, but there were also many differences between the strains.Feral cockerels showed higher levels of agonistic behaviour than domestic cockerels, under some conditions. There were differences between young feral and Leghorn-cross chickens in the “freezing” response to handling and placement in an unfamiliar cage, with the feral chickens responding more rapidly.Feral cockerels showed a greater initial avoidance of a novel object than did Black Australorp bantam cockerels, but after a short interval they spent more time near the novel object than the bantams.These results are discussed in relation to selection pressures under conditions of domestication, and on North-West Island.     

Response to novel object in Wistar and wild-type (WWCPS) rats

Laboratory rats react to a novel object with a rapid redirection of exploratory behaviour towards the source of the novelty and a subsequent decline of this neotic preference with repeated object exposure. Studies with wild Rattus norvegicus have shown that a novel object in a familiar cage results in avoidance reactions (neophobia) in those animals, but it is also well established that the wild R. norvegicus show strong aversive reactivity to a variety of high intensity stimuli. In this study we aimed to create low-stress conditions enabling the comparison of spontaneous exploratory behaviour directed at a novel object in male (age = 80 days) “wild-type” WWCPS rats (n = 21; fourth generation bred in captivity) and Wistar (n = 24) rats. The study involved repeated placing of individual animals in the experimental chamber for 15 (6 min) trials on consecutive days. On the 11th day the novel object was introduced. Animals were tested in darkness and without human presence. Under these conditions neither WWCPS nor Wistar have shown behavioural signs of high emotional arousal, both lines have shown comparable general levels of experimental cage exploration and the positive new object exploratory reaction was observed only in Wistar rats.     

Reducing Mouse Anxiety during Handling: Effect of Experience with Handling Tunnels

Handling stress is a well-recognised source of variation in animal studies that can also compromise the welfare of research animals. To reduce background variation and maximise welfare, methods that minimise handling stress should be developed and used wherever possible. Recent evidence has shown that handling mice by a familiar tunnel that is present in their home cage can minimise anxiety compared with standard tail handling. As yet, it is unclear whether a tunnel is required in each home cage to improve response to handling. We investigated the influence of prior experience with home tunnels among two common strains of laboratory mice: ICR(CD-1) and C57BL/6. We compared willingness to approach the handler and anxiety in an elevated plus maze test among mice picked up by the tail, by a home cage tunnel or by an external tunnel shared between cages. Willingness to interact with the handler was much greater for mice handled by a tunnel, even when this was unfamiliar, compared to mice picked up by the tail. Once habituated to handling, C57BL/6 mice were most interactive towards a familiar home tunnel, whereas the ICR strain showed strong interaction with all tunnel handling regardless of any experience of a home cage tunnel. Mice handled by a home cage or external tunnel showed less anxiety in an elevated plus maze than those picked up by the tail. This study shows that using a tunnel for routine handling reduces anxiety among mice compared to tail handling regardless of prior familiarity with tunnels. However, as home cage tunnels can further improve response to handling in some mice, we recommend that mice are handled with a tunnel provided in their home cage where possible as a simple practical method to minimise handling stress.     

Hand-rearing reduces fear of humans in European starlings, Sturnus vulgaris

Pending changes in European legislation ban the use of wild-caught animals in research. This change is partly justified on the assumption that captive-breeding (or hand-rearing) increases welfare of captive animals because these practices result in animals with reduced fear of humans. However, there are few actual data on the long-term behavioural effects of captive-breeding in non-domestic species, and these are urgently needed in order to understand the welfare and scientific consequences of adopting this practice. We compared the response of hand-reared and wild-caught starlings to the presence of a human in the laboratory. During human presence, all birds increased their general locomotor activity but the wild-caught birds moved away from the human and were less active than the hand-reared birds. After the human departed, the wild-caught birds were slower to decrease their activity back towards baseline levels, and showed a dramatic increase in time at the periphery of the cage compared with the hand-reared birds. We interpret these data as showing evidence of a greater fear response in wild-caught birds with initial withdrawal followed by a subsequent rebound of prolonged attempts to escape the cage. We found no effects of environmental enrichment. However, birds in cages on low shelves were less active than birds on upper shelves, and showed a greater increase in the time spent at the periphery of their cages after the human departed, perhaps indicating that the lower cages were more stressful. In demonstrating reduced fear of humans in hand-reared birds, our results support one of the proposed welfare benefits of this practice, but without further data on the possible welfare costs of hand-rearing, it is not yet possible to reach a general conclusion about its net welfare impact. However, our results confirm a clear scientific impact of both hand-rearing and cage position at the behavioural level.     

Relationship between the behaviour of sows at 6 months old and the behaviour and performance at farrowing

Piglet crushing remains a major problem in pig production. Reduced crushing might be obtained through genetic selection on sow behavioural traits. The aim of the study was to assess the relationship between behavioural responses at 6 months of age, around farrowing, and sows' reproductive performance including crushing levels. At 6 months of age, behavioural responses of 75 nulliparous sows were observed both during behavioural tests to human presence and to the presence of a novel object in their home pen, and their responses when placed in a weighing device. At first farrowing, nervousness of the sows was observed when placed in the farrowing crate 1 week before and the day of farrowing, as well as their fear responses when approached by a human from behind or at the front of the farrowing crate. At 6 months of age, escape from a human tended to be correlated with the reactivity in the weighing device (rs = 0.21, P = 0.09). Around first farrowing, the withdrawal reaction when a human approached at the front was correlated with the fear response when approached from behind and the nervousness of the sow in the crate (rs = 0.29, P < 0.05; rs = 0.37, P < 0.01). The fear response when approached from behind was correlated with nervousness in the crate and around farrowing (rs = 0.70, P < 0.001; rs = 0.25, P < 0.05), and nervousness in the crate was significantly correlated with the nervousness around farrowing (rs = 0.34, P < 0.01). The escape from a human at 6 months was correlated with withdrawal when approached from the front before farrowing (rs = 0.38, P < 0.01) and with nervousness of the sow in the crate (rs = 0.24, P < 0.05). The number of piglets crushed at first farrowing was correlated with the latency to approach a novel object at 6 months and nervousness around farrowing (rs = -0.27, P < 0.05; rs = 0.28, P < 0.05), and tended to be correlated with the escape behaviour from human at 6 months and withdrawal away from human presence before farrowing (rs = 0.21, P = 0.09; rs = 0.22, P = 0.08). These results suggest that behavioural responses to humans and during management practices of nulliparous sows at 6 months of age are, to some extent, related with their behaviour around farrowing and crushing levels of piglets at farrowing.     

Fear extinction in rodents: basic insight to clinical promise

Fear extinction, the reduction of fear by repeated exposure to the object of fear, is a crucial paradigm of inhibitory learning and the acknowledged preclinical model for behavior therapy of human anxiety. Recent insights have clarified roles for infralimbic prefrontal cortex, hippocampus and periaqueductal gray in extinction learning, while maintaining a central role for the basolateral amygdaloid nucleus in the acquisition and storage of this learning. Simultaneously, molecular insights have implicated several neurotransmitter and second messenger systems in extinction learning, and revealed that extinction is surprisingly easy to improve, yielding the promise of a novel approach to improved psychiatric treatments for a variety of human anxiety disorders.     

Responses to the change in the environment in pairs of male rats genetically selected for activity level

Laboratory Wistar strain rats were genetically selected for high (+A) and low (-A) activity level. In thirteen pairs of adult males of the 23rd filial generation reactions to changes in the external environment were studied. The animals were housed in breeding cages four each. Two parallel studies were conducted: in pairs simultaneously placed into a novel environment (NOV), empty cages of the same dimensions as the home cage (HC), in the second, behaviour of the second pair that remained in the HC, after removal of two cage-mates, was tested. Once a minute, for a period of one hour, the type of activity was recorded and noted whether it was an element effected in contact with the partner or without any contact. The animals +A and -A differed in the frequency of various types of activity and immobility, in the ratio between behavioural manifestations shown in or without contact as well as in the response to the type of modified environment. To changes in the situation, whether removed cage-mates from the HC or placed into NOV +A animals reacted with a high wave of environment exploration which gradually habituated. -A rats equally responded with exploration but on a lower level. In +rats we recorded more frequently exploration without contact with the partner in HC and NOV in comparison with -A, more frequent grooming, less immobility in contact and with no contact. Between +A partners there was a greater number of contacts in NOV than in HC whereas in the -A group the incidence of contact did not differ between HC and NOV. ANOVA revealed the influence of factors of genetics and environment and interaction in several behavioural categories. The simple and in time economical method demonstrated the possibility of use for the detection of differences between +A and -A lines even at relatively small changes in the external stimulatory situation.     

The effects of regular handling on fear responses in the domestic chick

Reduction of fear in the domestic chick by regular handling was assessed and an attempt was made to determine whether such an effect was due specifically to reduced fear of human beings or to a general reduction in fearfulness. Regular handling decreased the tonic immobility response, a fear-potentiated phenomenon, and increased approach to a human being, but had no effect on approach towards an inanimate object. These findings were common to three strains (two layer, one broiler), and suggest that handling does not depress general fearfulness, but specifically reduces fear of human beings, presumably through habituation.     

Heritability and genetic correlations of fear-related behaviour in Red Junglefowl--possible implications for early domestication

Domesticated species differ from their wild ancestors in a number of traits, generally referred to as the domesticated phenotype. Reduced fear of humans is assumed to have been an early prerequisite for the successful domestication of virtually all species. We hypothesized that fear of humans is linked to other domestication related traits. For three generations, we selected Red Junglefowl (ancestors of domestic chickens) solely on the reaction in a standardized Fear of Human-test. In this, the birds were exposed for a gradually approaching human, and their behaviour was continuously scored. This generated three groups of animals, high (H), low (L) and intermediate (I) fearful birds. The birds in each generation were additionally tested in a battery of behaviour tests, measuring aspects of fearfulness, exploration, and sociality. The results demonstrate that the variation in fear response of Red Junglefowl towards humans has a significant genetic component and is genetically correlated to behavioural responses in other contexts, of which some are associated with fearfulness and others with exploration. Hence, selection of Red Junglefowl on low fear for humans can be expected to lead to a correlated change of other behavioural traits over generations. It is therefore likely that domestication may have caused an initial suite of behavioural modifications, even without selection on anything besides tameness.     

Euthanasia of rats with carbon dioxide--animal welfare aspects

A method of inducing euthanasia by carbon dioxide (CO2) inhalation in the home cage of an animal is described and tested for distress by behavioural as well as by hormonal measures. The animals were maintained in their home cage while CO2 was induced at a flow of 6 l/min. The behaviour of the animals was measured continuously as were the serum concentrations of glucose, ACTH and corticosterone 30, 75 and 120 s after the CO2 was introduced into the cage. In order to test for distress, two groups of rats were pre-treated with acepromazine (orally) and pentobarbiturate (i.p. injection) respectively, in order to reduce possible distress caused by CO2 euthanasia, and were compared with control groups. There were no signs of distress by behavioural or by hormonal changes. All changes seen could be attributed to experimental effects and, especially as there was no difference between the pre-treated and the control groups of rats, it must be assumed that the described method of euthanasia is in concordance with animal welfare, it leads to rapid death without severe distress or pain, and it seems therefore to be 'humane'.     

Incremental Changes in Extracellular Noradrenaline Availability in the Frontal Cortex Induced by Naturalistic Environmental Stimuli: A Microdialysis Study in the Freely Moving Rat

Abstract: Changes in levels of extracellular noradrenaline (NA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the frontal cortex, induced by exposure of unrestrained, conscious rats to novel environments, were compared using in vivo microdialysis. NA efflux increased when rats were transferred to a novel cage, but this was not significant when compared with either basal efflux or with changes after equivalent handling in their home cage. Increasing the intensity of illumination of the novel cage by fivefold significantly increased NA efflux with respect to basal efflux but not handled controls. However, a sustained and significant increase in NA efflux (cf. basal efflux or handled controls) was found when an unfamiliar conspecific was also present in the novel cage. In all cases, basal efflux was restored within 1 h of returning rats to their home cage. Neither handling nor environmental stimuli described above affected DOPAC efflux. 5-HIAA efflux was increased (cf. basal) in the presence of an unfamiliar conspecific, but this increase was no greater than that in handled rats. It is concluded that different naturalistic stimuli cause incremental changes in the levels of extracellular NA in the frontal cortex; these changes affect both phasic and tonic components of the response.     

The assessment of fear in domestic hens exposed to a looming human stimulus

The behavioural and physiological responses of hens exposed to a slowly approaching human being were assessed using remote observation and radio telemetry of heart rate. Forty isolated hens of two strains and from two rearing environments were used. The sequence of behavioural changes included looking around, ceasing feeding, head shaking or complete withdrawal and finally crouching or escape behaviour. The heart rate rose from a mean level of 303 beats/min when the birds were at rest, to 465 beats/min when the cage was opened and the bird caught and held at the conclusion of the approach. The light hybrid strain showed more pronounced behavioural responses and a greater proportional rise in heart rate in the course of the approach than the medium hybrid birds, consistent with the induction of a higher level of fear. An environmental effect was also observed, pen-reared birds were more disturbed than caged ones. The similarity of the time course of the physiological and behavioural measures supports the concept of fear as an intervening variable which has simultaneous effects on heart rate and behaviour. Both may be effective in assessing fear, and they should be regarded as complementary rather than alternative measures.     

Expression of freezing and fear‐potentiated startle during sustained fear in mice

Fear‐potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear‐potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo‐randomized conditioned–unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano‐electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear.