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.     

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.     

Assessing Video Presentations as Environmental Enrichment for Laboratory Birds

The aim of the present study was to investigate the effects of video presentations of natural landscapes on European starlings'' (Sturnus vulgaris) stereotypic behaviours (SBs) and other abnormal repetitive behaviours (ARBs) and to evaluate the impact of past experience by comparing wild-caught and hand-reared starlings'' reactions. Ten wild-caught and five hand-reared starlings were presented 1-hour videos of landscapes twice a day for five successive days, while a control group of eight wild-caught and four hand-reared starlings was presented a grey screen for the same amount of time. The analysis of the starlings'' behaviour revealed that the video presentations of landscapes appeared to have a positive but limited and experience-dependent effect on starlings'' SBs and other ARBs compared to the controls. Indeed, whereas video presentations seemed to modulate high rates of SBs and ARBs, they did not appear to be enriching enough to prevent the emergence or the development of SBs and ARBs in an impoverished environment. They even appeared to promote a particular type of SB (somersaulting) that is thought to be linked to escape motivation. The fact that this effect was observed in hand-reared starlings suggests that videos of landscapes could elicit motivation to escape even in birds that never experienced outdoor life. These results highlight the importance of investigating stereotypic behaviour both quantitatively and qualitatively in order to provide crucial clues on animal welfare.     

Realized heritability of personalities in the great tit (Parus major)

Behaviour under conditions of mild stress shows consistent patterns in all vertebrates: exploratory behaviour, boldness, aggressiveness covary in the same way. The existence of highly consistent individual variation in these behavioural strategies, also referred to as personalities or coping styles, allows us to measure the behaviour under standardized conditions on birds bred in captivity, link the standardized measurements to the behaviour under natural conditions and measure natural selection in the field. We have bred the great tit (Parus major), a classical model species for the study of behaviour under natural conditions, in captivity. Here, we report a realized heritability of 54 +/- 5% for early exploratory behaviour, based on four generations of bi-directional artificial selection. In addition to this, we measured hand-reared juveniles and their wild-caught parents in the laboratory. The heritability found in the mid-offspring-mid-parent regression was significantly different from zero. We have thus established the presence of considerable amounts of genetic variation for personality types in a wild bird.     

Realized heritability and repeatability of risk-taking behaviour in relation to avian personalities

Personalities are general properties of humans and other animals. Different personality traits are phenotypically correlated, and heritabilities of personality traits have been reported in humans and various animals. In great tits, consistent heritable differences have been found in relation to exploration, which is correlated with various other personality traits. In this paper, we investigate whether or not risk-taking behaviour is part of these avian personalities. We found that (i) risk-taking behaviour is repeatable and correlated with exploratory behaviour in wild-caught hand-reared birds, (ii) in a bi-directional selection experiment on 'fast' and 'slow' early exploratory behaviour, bird lines tend to differ in risk-taking behaviour, and (iii) within-nest variation of risk-taking behaviour is smaller than between-nest variation. To show that risk-taking behaviour has a genetic component in a natural bird population, we bred great tits in the laboratory and artificially selected 'high' and 'low' risk-taking behaviour for two generations. Here, we report a realized heritability of 19.3 +/- 3.3% (s.e.m.) for risk-taking behaviour. With these results we show in several ways that risk-taking behaviour is linked to exploratory behaviour, and we therefore have evidence for the existence of avian personalities. Moreover, we prove that there is heritable variation in more than one correlated personality trait in a natural population, which demonstrates the potential for correlated evolution.     

Is domestication driven by reduced fear of humans? Boldness,metabolism and serotonin levels in divergently selected red junglefowl (Gallus gallus)

Domesticated animals tend to develop a coherent set of phenotypic traits. Tameness could be a central underlying factor driving this, and we therefore selected red junglefowl, ancestors of all domestic chickens, for high or low fear of humans during six generations. We measured basal metabolic rate (BMR), feed efficiency, boldness in a novel object (NO) test, corticosterone reactivity and basal serotonin levels (related to fearfulness) in birds from the fifth and sixth generation of the high- and low-fear lines, respectively (44–48 individuals). Corticosterone response to physical restraint did not differ between selection lines. However, BMR was higher in low-fear birds, as was feed efficiency. Low-fear males had higher plasma levels of serotonin and both low-fear males and females were bolder in an NO test. The results show that many aspects of the domesticated phenotype may have developed as correlated responses to reduced fear of humans, an essential trait for successful domestication.     

Ease of capture in lines of Japanese quail (Coturnix japonica) subjected to contrasting selection for fear or sociability

Japanese quail (Coturnix japonica) of lines, which have been subjected to contrasting selection for duration of the tonic immobility (TI) reaction or social reinstatement (SR) behaviour over many generations show corresponding differences in underlying fearfulness and sociality. As fearfulness and sociality are particularly influential traits in domesticated species, the finding that such traits respond to artificial selection may have important implications for poultry welfare and performance. However, it is not known if or how such selection has influenced human-animal interactions. The present experiment investigated the influence of fearfulness and SR behaviour on the ease with which birds could be caught and handled. Birds of lines selected for duration of the TI response or SR behaviour were reared in mixed line groups (LTI and STI or HSR and LSR) of 491 and 346 birds, respectively, until 6 weeks of age. When the birds were 2, 4, and 6 weeks of age, they were caught one by one and their individual capture ranks noted. In the group of birds selected for duration of the TI response, birds selected of the line selected for short duration of TI were caught before those selected for long duration of the response. In the group of birds selected for SR motivation, birds of the high line were caught before their low lines counterparts. Coefficients of concordance between capture ranks were significant and capture ranks did not differ significantly across ages. These results imply that selection for low levels of fear or high levels of sociality produces animals that are less disturbed by human interventions than animals selected for the opposite traits. The greater ease of capture of low fear line birds than high fear line birds may be explained by reduced fear of humans. The fact that the birds selected for high levels of SR behaviour are easier to catch than birds selected for low levels of sociality is less readily explicable. One hypothesis is that HSR line chicks tend to be more strongly imprinted on each other and the human caretaker. However, SR behaviour is highly species specific in both lines, existing evidence for line differences in social discrimination is limited and birds of the two lines show similar duration of the TI response. Despite this, whatever their underlying causation, these results demonstrate that genetic selection can be used to reduce negative reactions to human beings and may be of value in the improvement of both animal welfare and productivity.     

Use and husbandry of captive European starlings (Sturnus vulgaris) in scientific research: a review of current practice

We reviewed the use of captive European starlings (Sturnus vulgaris) in scientific research published between 2000 and 2004. We estimated the numbers of birds used and documented their origin and the range of husbandry regimes employed with the aim of comparing current practice with the new European guidelines for husbandry of laboratory animals. Over the five-year period, 106 primary articles report the use of an estimated total of 2490 captive starlings. The majority of birds were caught from the wild either as adults or fledglings, and only 3% were hand-reared from chicks. There was considerable variation in husbandry. In the majority of cases, standards fell below those currently recommended as best practice in the UK and cited in new European guidelines. The median volume of home cages employed was 0.42 m3 (0.13-5.1 m3, interquartile range), whereas current recommendations suggest a minimum of 1.0 m3 for a singly-housed bird. The median volume of space allowed per bird was 0.13 m3/bird (0.08-1.05 m3/bird, Q1-Q3), whereas current recommendations suggest a minimum of 0.33 m3/bird. Only 27% of the articles mentioned providing any form of environmental enrichment for birds in their home cages. We recommend that more research be conducted into the welfare of starlings to inform legislation and guidelines, and thus maximize the welfare of captive animals.     

Behavioural and physiological responses of birds to environmentally relevant concentrations of an antidepressant

Many wildlife species forage on sewage-contaminated food, for example, at wastewater treatment plants and on fields fertilized with sewage sludge. The resultant exposure to human pharmaceuticals remains poorly studied for terrestrial species. On the basis of predicted exposure levels in the wild, we administered the common antidepressant fluoxetine (FLUOX) or control treatment via prey to wild-caught starlings (Sturnus vulgaris) for 22 weeks over winter. To investigate responses to fluoxetine, birds were moved from their group aviaries into individual cages for 2 days. Boldness, exploration and activity levels showed no treatment effects but controls and FLUOX birds habituated differently to isolation in terms of the concentration of corticosterone (CORT) metabolites in faeces. The controls that excreted higher concentrations of CORT metabolites on day 1 lost more body mass by day 2 of isolation than those which excreted lower levels of CORT metabolites. CORT metabolites and mass loss were unrelated in FLUOX birds. When we investigated the movements of birds in their group aviaries, we found the controls made a higher frequency of visits to food trays than FLUOX birds around the important foraging periods of sunrise and sunset, as is optimal for wintering birds. Although individual variability makes interpreting the sub-lethal endpoints measured challenging, our data suggest that fluoxetine at environmentally relevant concentrations can significantly alter behaviour and physiology.     

Categorization and discrimination of "chick-a-dee" calls by wild-caught and hand-reared chickadees

Bloomfield and Sturdy [Bloomfield, L.L., Sturdy, C.B. All chick-a-dee calls are not created equally. Part I. Open-ended categorization by sympatric and allopatric chickadees. Behav. Proc., in press] previously reported that black-capped chickadees (Poecile atricapillus) discriminate conspecific from heterospecific (mountain chickadee, P. gambeli) 'chick-a-dee' calls, and their ability to accurately discriminate and classify the calls as belonging to separate species' defined categories was largely unaffected by their prior experience with mountain chickadees and their calls. To further examine the potential influence of experience on discrimination and categorization, we compare wild-caught black-capped chickadees, wild-caught mountain chickadees, and black-capped chickadees hand-reared among either adult laboratory-housed black-capped chickadees or adult laboratory-housed mountain chickadees on a true category/pseudo category chick-a-dee call discrimination task. Irrespective of group assignment, hand-reared birds performed as well as wild-caught birds and did not show a conspecific- or rearing-specific advantage in discrimination, categorization or memorization of chick-a-dee calls. While vocal learning is under the influence of ontogenetic experience, the results derived from the current methods suggest that experience (or a lack thereof) does not affect categorization and memorization abilities.     

Experience with adults shapes multisensory representation of social familiarity in the brain of a songbird

Social animals learn to perceive their social environment, and their social skills and preferences are thought to emerge from greater exposure to and hence familiarity with some social signals rather than others. Familiarity appears to be tightly linked to multisensory integration. The ability to differentiate and categorize familiar and unfamiliar individuals and to build a multisensory representation of known individuals emerges from successive social interactions, in particular with adult, experienced models. In different species, adults have been shown to shape the social behavior of young by promoting selective attention to multisensory cues. The question of what representation of known conspecifics adult-deprived animals may build therefore arises. Here we show that starlings raised with no experience with adults fail to develop a multisensory representation of familiar and unfamiliar starlings. Electrophysiological recordings of neuronal activity throughout the primary auditory area of these birds, while they were exposed to audio-only or audiovisual familiar and unfamiliar cues, showed that visual stimuli did, as in wild-caught starlings, modulate auditory responses but that, unlike what was observed in wild-caught birds, this modulation was not influenced by familiarity. Thus, adult-deprived starlings seem to fail to discriminate between familiar and unfamiliar individuals. This suggests that adults may shape multisensory representation of known individuals in the brain, possibly by focusing the young's attention on relevant, multisensory cues. Multisensory stimulation by experienced, adult models may thus be ubiquitously important for the development of social skills (and of the neural properties underlying such skills) in a variety of species.     

The effects of cage volume and cage shape on the condition and behaviour of captive European starlings (Sturnus vulgaris)

Cage size is widely recognised as an important determinant of captive animal welfare, but in contrast, cage shape has received far less attention. Husbandry recommendations for flying birds state that cages should be long in shape because this allows greater potential for flight. However, so far no studies have investigated the impact of cage shape on the behaviour or welfare of captive flying birds. We measured the effects of cage size and shape on the condition and behaviour of captive wild-caught European starlings (Sturnus vulgaris) using a 2 × 3 factorial design in which birds were individually housed for 1 week in cages of one of two volumes (either medium at 0.3 m³ or large at 1 m³), and one of three shapes (long with an aspect ratio (i.e. length/height) of 3.43), standard with an aspect ratio of 1.72 or tall with an aspect ratio of 0.86). We found effects of cage size and the interaction of cage size and shape on the behaviour and condition of birds. In interpreting the welfare implications of our results we focused on stereotypic behaviour as measured by incidence of somersaulting and a novel statistic that quantifies sequential dependencies in the birds’ locations within the cage. The lowest measures of stereotypic behaviour were recorded in the large cages and the medium long cage. Cage shape was more important in determining the quantity of stereotypic behaviour in the medium cages than the large cages. Our findings support the recommendation that starlings be housed in long-shaped cages.     

Red Junglefowl (Gallus gallus) selected for low fear of humans are larger,more dominant and produce larger offspring

Many traits associated with domestication are suggested to have developed as correlated responses to reduced fear of humans. Tameness may have reduced the stress of living in human proximity and improved welfare in captivity. We selected Red Junglefowl (ancestors of all domestic chickens) for four generations on high or low fear towards humans, mimicking an important aspect of the earliest period of domestication, and tested birds from the third and fourth generation in three different social tests. Growth and plumage condition, as well as size of eggs and offspring were also recorded, as indicators of some aspects of welfare. Birds selected for low fear had higher weight, laid larger eggs and generated larger offspring, and had a better plumage condition. In a social dominance test they also performed more aggressive behaviour and received less of the same, regardless of whether the restricted resource was feed or not. Hence, dominance appeared to increase as a consequence of reduced fear of humans. Furthermore, egg size and the weight of the offspring were larger in the less fearful birds, and plumage condition better, which could be interpreted as the less fearful animals being better adapted to the environment in which they were selected.     

Longevity and migration distance differ between wild and hand-reared mallards Anas platyrhynchos in Northern Europe

The mallard Anas platyrhynchos is the world’s most widespread and numerous dabbling duck. It is also farmed and released to the wild by the millions each year, but the effects of this on wild populations remain little studied. By using historical national ringing–recovery data from Sweden and Finland, we here address three predictions based on previous studies: (1) longevity is higher in wild than in hand-reared mallards, (2) wild mallards migrate longer than hand-reared, and (3) migration distance in wild ducks surviving long enough to start fall migration has decreased over the last 50 years. Indeed, wild mallards lived longer than hand-reared (19 versus 9 months in Swedish birds and 13 versus 4 months in Finnish birds). Compared to wild mallards, a smaller proportion of hand-reared birds survived long enough to have the chance to enter the wild breeding population; less than 25 % of the Swedish birds and less than 10 % of the Finnish birds lived a year or longer. Wild birds migrated farther than hand-reared (mean distance in Swedish birds, 676 versus 523 km; in Finnish birds, 1,213 versus 157 km), a pattern caused by both shorter life span and lower migration speed in hand-reared birds. Mean migration distance in wild Swedish mallards was 787 km in 1947–1972 but 591 km in 1977–1993. This difference was not statistically significant, though, possibly due to the limited sample size and lack of data from the last two decades. In general, our study provides a conservative test of the predictions addressed, calling for more research about the consequences of restocking duck populations.     

Embedded lead shot in European starlings Sturnus vulgaris: an underestimated hazard for humans and birds of prey

The poisoning of wild animals by lead (Pb) ammunition fired by hunters has been known for many decades, especially in the case of waterbirds. More recently, it has been demonstrated that raptors are also exposed to the risk of plumbism when feeding on unretrieved quarry that was wounded or killed by hunters. Further studies reveal that even humans can be subject to a significant Pb dose while consuming game animals killed by traditional ammunition. Given the relevance of this issue, several pieces of research have been carried out to assess frequency, dimension, and the number of Pb fragments embedded in the carcasses of ungulates, partridges, ducks, and other birds to evaluate the risk related to the consumption of game meat. In spite of their great importance as quarry species across southern Europe, until now, no data have been available on small passerines. To assess the quantity and type of Pb embedded in songbirds, we x-rayed 196 starlings shot in Italy and found Pb pellets and/or visible fragments in 118 carcasses (60.2 %). We counted 128 shotgun pellets in 85 carcasses. In 28 birds, we detected both whole pellets and lead fragments; in 33, we found only small fragments. By excising and weighing a sample of 20 shotgun pellets (diameter 1.35–1.99 mm), we calculated a Pb load of 3.75 g in the whole sample of 196 starlings, corresponding to an average of 27.32 mg/100 g of body weight. This is a conservative estimation, because fragments were not considered. Compared to game birds of a larger size, the starlings in our study had a lower amount of embedded Pb, but the shot pellets and fragments embedded in their tissues were abundant and tiny. Given the results of previous studies, the quantity and level of fragmentation suggest that the risk of Pb poisoning cannot be ruled out for humans and birds of prey consuming the meat from songbirds killed with traditional ammunition.     

Starlings can categorize symmetry differences in dot displays

Fluctuating asymmetry is an estimate of developmental stability and, in some cases, the asymmetry of morphological traits can reflect aspects of individual fitness. As asymmetry can be a marker for fitness, it has been proposed that organisms could use morphological asymmetry as a direct visual cue during inter- and intraspecific encounters. Despite some experimental evidence to support this prediction, the perceptual abilities of animals to detect and respond to symmetry differences have been largely overlooked. Studying the ability of animals to perceive symmetry and factors that affect this ability are crucial to assessing whether fluctuating asymmetry could be used as a visual cue in nature. In this study, we investigated the ability of wild-caught European starlings Sturnus vulgaris to learn to discriminate symmetry from asymmetry in random dot patterns through operant learning experiments. The birds did not possess a spontaneous preference for either symmetry or asymmetry. The birds learned a symmetry preference, although the learning process took longer than that previously reported for pigeons Columba livia and was more error prone. After being trained to discriminate symmetry differences in random dot patterns, birds successfully transferred their symmetry discrimination abilities to a set of novel stimuli that they had not previously seen. This indicates that starlings can form a mental categorization of visual stimuli on the basis of a somewhat generalized symmetry phenomenon. We discuss these findings in relation to the probability that birds use fluctuating asymmetry as a visual cue.     

Linking fearfulness and coping styles in fish

Consistent individual differences in cognitive appraisal and emotional reactivity, including fearfulness, are important personality traits in humans, non-human mammals, and birds. Comparative studies on teleost fishes support the existence of coping styles and behavioral syndromes also in poikilothermic animals. The functionalist approach to emotions hold that emotions have evolved to ensure appropriate behavioral responses to dangerous or rewarding stimuli. Little information is however available on how evolutionary widespread these putative links between personality and the expression of emotional or affective states such as fear are. Here we disclose that individual variation in coping style predicts fear responses in Nile tilapia Oreochromis niloticus, using the principle of avoidance learning. Fish previously screened for coping style were given the possibility to escape a signalled aversive stimulus. Fearful individuals showed a range of typically reactive traits such as slow recovery of feed intake in a novel environment, neophobia, and high post-stress cortisol levels. Hence, emotional reactivity and appraisal would appear to be an essential component of animal personality in species distributed throughout the vertebrate subphylum.     

Behavioural and hormonal effects of social isolation and neophobia in a gregarious bird species, the European starling (Sturnus vulgaris)

Separating gregarious individuals from their group members often results in behavioural and physiological changes, like increased levels of corticosterone. Testosterone and corticosterone, in particular, have been implicated in the response of mammals to novelty. Data in birds are, however, rare. The presence or absence of group members may also influence an individual's response to novel stimuli. We assessed the behaviour and hormonal response of European starlings (Sturnus vulgaris) to a novel object in two different situations and seasons: each starling was tested when separated and when in contact with its group members in May/June (breeding season) and again in September/October (non-breeding season). Starlings are gregarious throughout the year, but as foraging flocks are small during the breeding season and large during the non-breeding season, we assumed that non-breeding starlings would be more affected by social isolation. Overall, starlings had higher levels of corticosterone, lost more body mass, and were more active when they were separated from their group. Isolated individuals, however, did not show a greater neophobic response than individuals in the presence of their group members in either season. Circulating levels of testosterone and corticosterone were higher after a test with novel object than after a test with only the familiar feeding dish in both sexes and seasons. However, control tests for handling effects confirmed only the increase in testosterone. Our study shows that social isolation is stressful for unrelated and unpaired members of a wild flocking bird species and demonstrates that novelty can lead to a rise in testosterone in birds.     

Behavioural differences between two captive populations of red jungle fowl (Gallus gallus) with different genetic background,raised under identical conditions

Ex situ conservation of threatened species may lead to behavioural adaptation, which can affect success of reintroduction attempts. In previous studies, we investigated the effects of captivity on the behaviour of red jungle fowl (Gallus gallus) and found that captive populations differed behaviourally as well as genetically. The aim of the present study was to compare the behaviour of two of the previously studied populations, raised under identical conditions. Eggs were collected from birds at Copenhagen zoo (Cop) and Götala research station (Got) and were incubated and hatched together. Twenty-eight birds (16 Got and 12 Cop) were reared together and tested in eight different behavioural tests, measuring different aspects of fear-related behaviours as well as exploratory and social behaviours. The study revealed several differences in fear-related behaviours between the populations but none in exploratory or social behaviours. In general, one of the populations (Cop) showed more intense fear behaviours than the other (Got), which instead were less fearful in their behaviours. This indicates that breeding animals in captivity may lead to behavioural modifications, which can affect the outcome of reintroductions. The results further suggest that fear-related behaviours are dependent on the genetic background of the animals while social behaviours may be more influenced by the social environment. Since fear-related behaviours, such as predator avoidance and fear of humans, are essential for a life in the wild, these aspects are crucial for the breeding of animals in captivity for conservation purposes.     

Reaction of laying hens to humans in the home or a novel environment

Tests assessing the fear of humans by measuring avoidance or approach reactions of the test animals towards humans can be useful tools in welfare assessment schemes. In this study, we wanted to compare tests assessing the reactions of laying hens towards humans in the home environment to a test performed in a novel environment. As well we investigated inter-test correlations in the home environment. We performed several tests to assess the hens’ approach and avoidance reactions towards an unfamiliar human inside the home environment of laying hens in 14 caged flocks and 10 free-range flocks. We measured the reactions of hens to a stationary person and a moving person approaching individual hens in both systems, as well as reactions to a stationary person trying to touch individual hens in non-cage systems and reactions of caged hens to a person passing by. Additionally, a standardised arena test was performed outside the home environment to compare the birds’ avoidance reactions towards an approaching human in a novel environment. In caged hens no significant correlations of tests performed inside the home environment with the arena test could be found, but there were moderate to high correlations (r_s > 0.6) with one parameter of the arena test in non-caged hens. The different tests applied inside the home environment correlated significantly and moderately to highly with each other, supporting the validity of these tests to measure fear of humans. The caged laying hens in our study reacted differently in the novel environment from the non-caged birds, indicating that the birds’ reactions towards humans in an unfamiliar environment depend on the housing system. In summary, a comparison of the level of fear of humans between flocks in different housing systems by using an arena test outside the home environment seems difficult and was not possible in the present study.