Individual variation in ranging behavior by free-range laying hens has been extensively reported [for example, Hartcher et al. (2016); Larsen et al. (2017); Campbell et al. (2018a) and Kolakshyapati et al. (2020)]. Yet, the reason for the variability remains largely unknown. To date, the most prevalent hypothesis to explain ranging variation is individual differences in fearfulness. This hypothesis has been tested in a variety of controlled and experimental settings using an assortment of methods to assess fearfulness with varied, and often contradictory, conclusions.

There is some evidence that range use may reduce hen fearfulness. For example, Grigor et al. (1995) reported that hens that spent more time on an outdoor range in commercial conditions were less fearful after range access, evident by shorter righting times in a tonic immobility (TI) test and a shorter latency to emerge from cover during an emergence test. Furthermore, both Campbell et al. (2016) and Hartcher et al. (2016) showed that hens that accessed the range more frequently were more fearful than hens that rarely or never accessed the outdoor range, evident by shorter TI righting times, fewer vocalizations and less activity in an Open Field Test (OFT). Of note, all of the aforementioned studies assessed fear after the hens had access to the outdoor range suggesting that range use may reduce fearfulness but did not provide evidence that fearfulness prevents hens from accessing the outdoor range. The positive effect of range use on fearfulness has also been reported in free-range meat chickens by assessing fearfulness (OFT and physiological stress responses to an acute stressor) both before and after range access (Taylor et al., 2020). A more recent study by Campbell et al. (2021) did assess fearfulness and exploration before range access - with a cohort of hens from the same research flock as the current study - using an OFT and maze test. The authors identified some inconsistent relationships between indicators of fearfulness (latency to vocalize in the OFT test and the time to leave start box when a maze contained a novel object) with subsequent ranging behavior (the number of visits and time spent on the range) but these relationships were present only with hens that were raised in standard conditions and not wtih hens that were reared in enriched environments. The Campbell et al. (2021) study does provide some evidence that fearfulness may be a predictor of range use in laying hens.

Assessments of range use and hen fearfulness on Australian commercial farms did not show similar results as those previously reported in controlled commercial conditions. Despite reporting significant variance in ranging behavior between individual layer hens in commercial flocks, Larsen et al. (2018) and Kolakshyapati et al. (2020) found no relationships between ranging behavior and fearfulness using TI, OFT and Human-Approach and Avoidance methods to assess fearfulness of hens on commercial egg farms. However, Kolakshyapati et al. (2020) did find that birds that accessed the range more frequently were more likely to approach a novel object during a novel object test (NOT) suggesting that inquisitive exploration may be associated with range use. It is possible that the reported discrepancy in research outcomes regarding fearfulness and range use by laying hens, could rather reflect relationships between range use and motivation to explore, which can be thwarted by fear. Additional evidence to suggest environmental exploration is an important factor to promote range use is provided in previous research work with free-range meat chickens. Ferreira et al. (2021) showed that meat chickens that contrafreeload (before and after range access was provided) were more likely to access an outdoor range when provided with the opportunity. It is therefore possible that range use reduces fearfulness in layer hens but accessing the range may be driven by motivations to explore.

The NOT method utilized by Kolakshyapati et al. (2020) likely reflects a hens’ motivation for inspective exploration and neophobia, such that hens that are motivated to obtain new information about a novel stimuli (inspective exploration) will approach the NO and faster than those less motivated. Of note, animals will show exploratory behavior even before new stimuli are presented, demonstrating that animals actively search for new information; referred to as inquisitive exploration (Wood-Gush and Vestergaard, 1991), curiosity (Špinka, 2019) or seeking (Wood-Gush and Vestergaard, 1991; Panksepp, 2004). Here we refer to curiosity as the motivator of inquisitive exploration, which is unrelated to survival (i.e. food or safety) (Hughes, 1997), and is in itself hedonistically rewarding (Panksepp, 2004) and competence building (Špinka, 2019). Curiosity may be the motivation for hens to access the outdoor range and may partially explain the heterogeneous ranging behavior of hen flocks.

Curiosity can be assessed with choice paradigms such as ‘willingness to sacrifice rewards’ or ‘willingness to wait for an answer’ (Wang and Hayden (2019). For example, male rhesus macaque monkeys have been shown willing to sacrifice a larger water reward to obtain information about a gambling outcome in advance of its delivery (Blanchard et al., 2015) and humans are willing to spend more scarce resources (limited tokens or waiting time) to obtain answers to trivia questions that they are more curious about (Kang et al., 2009) and even expose themselves to electrical shocks for access to the secret behind a magic trick (i.e. information) (Lau et al., 2018).

Franks et al. (2013) proposed a novel assessment of inquisitive exploration to reflect curiosity, using a trade-off paradigm providing animals with the opportunity to explore a novel area by forgoing a known reward and risking punishment. Franks et al. (2013) trained rats to expect reward in two arms and (mild) punishment in the opposite two arms of an eight-arm radial maze. Subsequently, environmental enrichment was removed from half of the population's home environment for two weeks before exposing the rats to the radial maze again. However, this time the radial maze test also provided rats with access to unfamiliar ambiguous arms for the final minute of the test. Both groups showed interest in the ambiguous arms but animals that were continuously housed with environmental enrichment were more likely to forgo reward and risk punishment to explore novel areas than rats where enrichment had been removed for two weeks prior to the test.

In this study, we investigated the relationships between hen fearfulness and curiosity prior to range access and early ranging behavior. We predicted that hens reared with environmental enrichment would be more curious, less fearful and subsequently would range more frequently during the first three weeks of range access, compared to hens reared in more barren conditions. To assess the role of fearfulness and curiosity we assessed fearfulness and inspective exploration at 18 weeks of age with the well validated OFT and NOT tests. To determine whether range use is motivated by curiosity, we used the method developed by Franks et al. (2013) at 22 weeks of age to assess inquisitive exploration. Subsequently, range access was provided at 25 weeks of age and individual range visits were monitored by radio frequency identification (RFID) technology.

All animals and procedures used in this study were approved by the University of New England Animal Ethics Committee (Approval number 17-092).

We aimed to better understand the mechanisms behind the well-known variability in ranging behavior of free-range laying hens. Specifically, we hypothesized that hens that were curious and less fearful of novelty would access the outdoor range sooner after range access was first provided and would range more frequently during the first three weeks of range access relative to hens that were less curious and more fearful of novelty. However, we found few (weak) correlations between range use in the first three weeks of range access and behavioral indicators of inspective exploration and fearfulness. Yet, by utilizing a novel 8-arm radial test adapted from Franks et al. (2013), we found evidence that providing novelty during rearing increased a hen’s willingness to forgo reward and risk punishment to explore a novel area. Therefore, we provide evidence that a more novel and/or complex environment during rearing increases hen curiosity but is not related to ranging behavior.

Hens reared with novelty were quicker to enter the ambiguous arms of the 8-arm radial maze and entered the ambiguous arms more frequently than control hens, although there was no difference between hens provided with structures during rearing and hens provided with novelty. To our knowledge, this is the first time this choice paradigm has been reported in laying hens. Novelty in an environment (especially the rearing environment) has been shown to have an impact on various traits such as exploration and seeking novelty, learning and memory and anxiety and stress reactivity (De Jong et al., 2000; Altan et al., 2013). Although fearfulness has been shown to impact a hen’s ability to learn (De Haas et al., 2017), we found little evidence of this effect related to treatment in the 8-arm radial maze assessment and therefore we provide evidence that our novelty rearing treatment likely influenced inquisitive exploration. For example, it is unlikely that rearing treatment had an impact on learning as there was no difference between treatment groups on latency to enter a positive or negative arm (i.e. no differences in conditioning between treatment groups). Similarly, in a different group of hens from the same larger research flock, there were no effects of rearing treatment on learning during a maze test (Campbell et al., 2021). Additionally, although we saw habituation to the test arena [i.e. a reduction in the latency to step from habituation 1 to habituation 2, which is a validated indicator of fearfulness in chickens (Forkman et al., 2007)] this was not related to treatment suggesting that any differences in the motivation to explore the ambiguous arms were unlikely related to fearfulness or learning. It is possible that the differences observed may be related to reward expectation rather than curiosity per se.

Our results support those of Franks et al. (2013) that showed rodents forgo reward and risk punishment to explore a novel environment and therefore provide evidence that inquisitive exploration is valued. But the relationship between inquisitive exploration identified in the current study and hen welfare requires further investigation. Franks et al. (2013) removed environmental enrichment from rodents with the aim to reduce welfare (Bateson and Matheson, 2007; Latham and Mason, 2010) and subsequently showed that inquisitive exploration was reduced. Furthermore, Franks et al. (2013) showed that inquisitive exploration was positively related to sucrose consumption; sucrose was provided in the home pen as a reward to assess the rodent’s willingness to approach reward as well as an indicator of anhedonia and poor welfare. Sucrose consumption reduced when animals were exposed to chronic unpredictable stress and restored after treatment with an antidepressant (Willner et al., 1987). As such, Franks et al. (2013) suggest that the willingness to forgo reward and risk punishment to explore a novel environment may provide evidence as to an animal’s state of welfare. Further investigation is required to extrapolate these results to hens. However, much of the neurobiological research suggests that obtaining information that resolves uncertainty is rewarding (Panksepp, 2004; Bromberg-Martin and Hikosaka, 2009; Špinka, 2019). Therefore, enhancing curiosity in animals can help to ensure captive animals are permitted positive experiences throughout life, which is a critical component of animal welfare (Mellor, 2015a; Rault et al., 2020). It must also be noted that thwarting opportunities for inquisitive exploration for curious animals will compromise animal welfare [see Wood-Gush and Vestergaard (1989); Appleby et al. (2011); Mellor (2015b)]. Therefore, care must be taken to ensure that such curious animals are provided with an appropriate environment to seek information. Such opportunities may include problem solving tasks, as the success of completing such tasks itself is shown to be intrinsically rewarding in cattle (Hagen and Broom, 2004) and canines (Mcgowan et al., 2014). For example, physiological indicators of arousal were greater when cattle were rewarded with a food treat after completing a problem solving task compared to simply being provided with the food reward alone (Hagen and Broom, 2004).

Rearing treatment was related to the number of birds that entered the ambiguous arm and how quickly they entered the ambiguous arm. Hens reared with novelty were more likely to enter an ambiguous arm and were quicker to do so than hens raised in an industry standard environment. Although the difference was observed in the hen’s willingness to forgo reward but not risk punishment, hens from the novelty rearing group were more likely to enter ambiguous arms closer to the reward rather than the ambiguous arms located next to the punishment arms. This may suggest that hens that were reared with novelty were more curious than hens raised in more stable and barren conditions. Furthermore, the judgement of ambiguous cues has been shown to be related to the expectation of outcomes for human and non-human animals and is thought to reflect the underlying mood/state of the animal [see Roelofs et al. (2016) and Lagisz et al. (2020)]. For example, it has been shown that ambiguous cues are perceived more pessimistically if animals are anxious (evident by anxiogenic and anxiolytic pharmacological interventions) or more optimistically when animals are in a positive emotional state [see Neville et al. (2020)].

We defined our rearing treatment as a ‘novelty treatment’ as the environmental enrichment items were not biologically relevant and were changed every 7 days. However, we cannot determine if the effects here are reflective of a more complex rearing environment relative to the control group or the novelty provided every few days or a specific enrichment item(s) provided at one point in time during rearing, which may or may not have coincided with an important sensitive period for neurodevelopment (Jones and Waddington, 1993; Kempermann, 2019). Fox and Millam (2007) provide evidence that it is indeed novelty that is the important characteristic of environmental enrichment programs to reduce neophobia of orange-winged Amazon parrots. However, Abou-Ismail and Mendl (2016) found that the provision of novelty to rodents in the absence of diversity of objects at one time is less beneficial. A comparison of complexity and diversity compared to novelty in the rearing environment of laying hens is required to fully understand the relationships identified in the current study.

The lack of evidence that treatment affected a hen’s behavior during the novel arena and novel object tests suggests that the eight-arm radial maze is assessing a different component of the bird’s temperament, such as inquisitive exploration (rather than inspective exploration), reward expectation or even as Franks et al. (2013) suggests, a broader reflection of well-being. As evidence suggests that emotional reactivity impacts exploration but the propensity for novelty seeking is thought to be independent (Grossman et al., 2007), the 8-arm radial assessment utilized in the current study may be a useful tool to identify behavioral indicators specific to inquisitive exploration and may prove a useful tool to assess hen welfare that warrants further investigation.

We expected that a hen’s latency to first access the range would be related to fear or curiosity, such that fearful hens would take longer to access the range and curious hens would access the range shortly after access was provided. We did identify a few weak correlations between behavioral indicators in the novel arena and novel object tests and ranging behavior. Specifically, a shorter latency to first access the range and more range visits in the first three weeks of range access was associated with fewer vocalizations during the novel arena test and less time spent in the areas close to the novel object during the novel object test. These findings were unexpected and contrast findings from Kolakshyapati et al. (2020) who found that time spent interacting with a novel object was associated with more frequent range use of laying hens on a commercial farm. As Kolakshyapati et al. (2020) assessed hen behavior in the novel object test after range access in contrast to our findings which assessed hens prior to range access, the relationships identified with inspective exploration and range use in the Kolakshyapati et al. (2020) study may reflect an effect of ranging on laying hens as a consequence of the complex range environment rather than a hen’s motivation to range. Of note, Armstrong et al. (2020) found relationships between indicators of adult neurogenesis (proliferating cell nuclear antigen and doublecortin) in the rostral and caudal subregion of the hippocampus of laying hens that visited the range more frequently which further supports brain and behavioral changes associated with range use, perhaps from the more complex environment or simply an increase in activity associated with range use. However, Armstrong et al. (2020) did not find any differences in measures of TI between birds that accessed the range frequently and those that did not, suggesting that the associated brain changes were not associated with a bird’s response to unfamiliar stimuli. Pre- and post-ranging assessments of hen temperament may help unpack these complex relationships and explain some of the factors that influence ranging behavior of laying hens.

Whilst we identified some statistically significant relationships between ranging behavior during the first three weeks of access and behavioral responses during all three behavioral tests, results were very weak and inconsistent. This may indicate that no such relationships exist, or the presence of a type I error may be present due to the methodological constraints, i.e. small sample sizes (n = 30 hens/treatment each test) or the fact that treatments were only replicated across three pens during rearing and prior to testing. However, we did observe a willingness to forgo reward to explore a novel environment in hens that were reared with novel items. Further research utilizing this novel test is required to examine the effects of novelty during rearing on hen curiosity and ranging behavior.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://figshare.com/articles/dataset/Range_use_fearfulness_and_curiosity_test_data/21762485.

The animal study was reviewed and approved by University of New England Animal Ethics Committee.

PT wrote the manuscript. PT, PH, DC and CL developed the concepts and methodologies. PT, EJ and ND conducted all data collection and analysis. All authors contributed to the article and approved the submitted version.