1. Introduction

Zoos and aquariums aim to preserve wildlife through research, education, and conservation activities, while simultaneously providing good welfare conditions to the animals in their care [1]. Advances in husbandry practices and zoo animal welfare science are key to guaranteeing that zoological institutions work as scientific and ethical conservation centres [2].

According to Mellor and colleagues [3], an animal’s welfare state can be described as the balance between its negative and positive subjective experiences. Nonhuman animals (hereafter, referred to as animals) are deemed to reach a positive welfare state when they mostly experience positive affects (e.g., a sense of security, sometimes indicated by playfulness), when their physical and behavioural needs are met, and when they can exert choice and control over resources and the environment.

MZoos and aquaria aim to cony studies have trietribute to the preservation of wildlife through educational, research and conservation initiatives ^[1] and they must ensure goo evaluatd welfare for the animal s in their care to meet these goals ^[2]. Studies of their welfare byare important to verifying whether and how envguarantee high standards of animal husbandry and care in zoological institutions, and to enhance their role as conservation centers ^[3].

Animals experonmental and human stimuliience optimal welfare when their physical, behavioral and psychological needs are meet, when they have the opportunity to exert choice and control over the environment (e.g., ambient temperature andopportunity to decide where, when and with whom perform certain behaviors), and when they mainly experience positive affective states such as a sense of security and comfort ^[4].

Enclosure design, envisitor presence, respectively) affect animal behaviourronmental conditions, husbandry routine and exposure to animal care staff and visitors are among the most important features that can influence the welfare of animals within zoos and aquaria [4]^[5]. NeScholars havertheless, only a few researchers have exa usually tried to assess animal welfare by analyzing the effect of environmental and human-related factors on animals' behaviors separately ^[6]. A limited ned the combineumber of studies have recently focused on the integrated impacts of these stimuli factors on animals' well-being (see for example: ^[7], ^[8], ^[9], ^[10], ^[11]), often in dican attempt to obtain a clearer picture of animals’ experiencesting that visitor effect (i.e., the negative or positive influence exerted by visitors) has been over-estimated when other parameters (e.g., ambient temperature, time of day, weather conditions) were included in the analysis a^[7], ^[8], ^[9].

In this stud welfare statey, we aimed to examine this still un-researched topic, focusing on a pair of zoo-housed Asian small-clawed otters (Aonyx cinereus, hee for exreafter referred as ASCOs). A. cinereus is a good mple: [5-9]).odel species since it is the most Icommonteresti otter species housed in captivity ^[12], ^[13] and, despite its popularity and higlyh educational and conservational value ^[14], resome of thesearch on zoo-housed ASCOs' welfare is still scarce. Moreover, available studies detected an overestimation of the visitor effect (have reported conflicting results relating to time budgets, and have often detected the occurrence of abnormal repetitive behaviors (ARBs; ^[12], ^[15], ^[16], ^[17], ^[18], ^[19], ^[20], ^[21], ^[22]), i.e., thre positive or negative influenpetitive, unvarying and seemingly functionless behavioral patterns ^[23], which are exerted by visitor presence) wheusually interpreted as an indicator of poor welfare either currently or in the past ^[24].

In ohis study we investigated ther parameters, such as temperature and time of day, subjects’ behavioural time budgets, the occurrence and potential causes of ARBs, and applied multivariate research methods to evaluate whether and how the otters’ behaviours were included in thaffected by selected environmental and human-related parameters. To our knowledge, such a multivariate analysis [5, 6, 7]s has not previously been applied to the study of otter behaviour.

In this study, we aimed to assess the integrated effects of environmental conditions (i.e., ambient/water temperature, relative humidity, and background noise level) and caregiver/visitor presence on the behaviour of a pair of captive Asian small-clawed otters (hereafter, referred to as ASCOs) through the application of multivariate research methods. In particular, we investigated the subjects’ behavioural time budget, and the occurrence and potential causes of abnormal repetitive behaviour (ARBs), and evaluated whether and how the otters’ behaviours were affected by selected environmental and human-related parameters.

2. Material and methodsthodology

This study fwas cocusnducted on a pair of adult ASCOs housed at the Giardino Zoologico di Pistoia (GZP), Pistoia, Italy. We collected data duover two-month pering od (14 sampling days selected within a two-month period (assigned randomly, from 19th Julyof July 2020 to 6th of September 2020). We videoed the otters to ensure comprehensive monitoring of their-recorded the otters' behaviourals patterns [10]. We conducted cross six one-hour observations dailysessions (S1: 9:15–10:15 am; S2: 10:45–11:45 am; S3: 12:15–1:15 pm; S4: 2:45–3:45 pm; S5: 4:15–5:15 pm; S6: 5:45–6:45 pm) to. In this way we could record data evenlyduring different time slots across the public opening hours (9:00 am–7:00 pm), and tocould include keeper-otter interactions (e.g., feeding sessions and enclosure cleaning) in the data collection periods.. During Eeach observation session was based on a, we recorded each individual for 30- minutes using a recording of eachcontinuous focal animal sampling technique otter^[25]. We conducted a total of 84 focal sessions for each subject, resulting in 40 hobtaining oursn and 18 minutes of observation material for thge male and40 39 hours and 25 minutes of observation matperial for the female. The order of the subjects was alternated to monitor them in both the first and second 30-mtter. Following relevant scienute portion of eachic one-hour observatlion session. Based on other similar studieserature  [5-7^[7], ^[8], ^[9], 11-12]^[12],^[22], we selected the following parameters to be included in the analyses: weaeather conditions, ambient and water temperature, wind speed, relative humidity, background noise level, number of visitor numbers, and caregiver presence. Weather conditions, wind speed, as parameters to be included in the analysis. We recorded humidity, and ambient and water temperature were record, weather conditions, and wind speed at the beginning of each observation session. We recordedgistered visitor numbers and background noise every five minutes and continuously recorded caregiver presence in and around the enclosure while re. We analyzed data with generalized linear mixed models ^[8], ^[9], ^[10] cording visitor numb the above-mentioned parameters and background noise every five minutes (seven times per focal observation). To tesanimal identity as fixed factors, date as a random factor, and behaviors as respondent variables.

3. Discoveries

Both the effect of the selected parameteotters were found to perform a rich behavioral repertoire ^[26]. Aggres onsive and avoidance behaviours, we ran generalised linear mixed modelrs were limited, suggesting that the subjects experienced mainly positive emotional states^[4]. (GLMMThe otters) [6-8].

3. Results

Both otters performed a wide array of species-specific behavioural patterns [13], with a limited occurrence of aggressive and avoidance behaviours, which are usually associated with negative emotional states, such as fear and anxiety [14]. Moreover, the presence of potential ARBs, which usually indicate poor welfare conditions, was limited. The otters could also exercise control and choice over the environment, choosing where to perform behaviours almost 24 hours a day.

Fcocusing in detail on the otters’ time budgets, tld also exercise control over their environment, choosing where to perform behaviours almost 24 hours a day. The occurrence of potential ARBs was also limited.

The dominant category within the otters' behavioral time budgets was out of sight,sight which accounting, on average,d for 41.59% of the subjects’ time budgets, on average. This was consistentconcurs with the results reported forof previous studies conducted on breeding pairs kepthoused with offspring in outdoor naturalistic outdoor enclosures [11 ^[12], ^[18], 15-16]^[19]. Conversely, pairs and triplets of siblings hosteld in indoor enclosures with limitedttle natural vegetation and a limited amount of shelters have been reporte were found to spend less time out of sight [12^[20], 17] ^[22]. Thuese findings, the results of this study seem to confirm that highlight that providing otters with the opportunity to choose among different and enriched hhiding options aris one of the key requirements in captive settings, in linaccordance with husbandry guidelines [18]^[27]. Nevertheless, a larger sample size is needed to exclude the influence of group composition on the results.

When visible, the subjottects mainrs mostly engaged in resting and , and performed vigilance behavioursr. The percentage of ttime allocated to resting was similar to that reported for breeding pairs held in family groups  [11^[12], ^[17], ^[19] 15-16]. VigilHowever, we found thance performt the level of vigilance was, instead, s slightly higher in comparison with the available literature, in which (16.70%) than found in other studies, wherein ASCOs were estimated to spendspent less than 15% of their time budget checking the surrounding environments performing vigilance behavior [12^[20], 17]^[22]. We also found that Tthe otters engaged less in land locomotion and affiliative interactions compared to the breeding pairs ofbelonging to two family groups studiobserved by Cuculescu-Santana et al.and co-authors [11]^[12] and Owen [15]^[18]. This discrepancy could be due to the absence of offspring in thisour study that could have stimulatedreduced the occurrence of affiliative interactions, and the overall increased activity level of the adults and could also explain the lower level of locomotory behaviors, since the subjects were not involved in providing parental care (i.e., carrying, following, and feeding cubs) [6]^[27].

NRegarding footably, thd-related behaviors, Gothard ^[19] found that a zoo-housed family group analysed by Gothard [16] alllocated 20% of its time budget to foraging and feeding, with a 13% increase when enriched withen crickets and mealworms were scattered in the enclosure. AConversely, in all the other available studies, including the present research,our study, the time dedicated by otters to food-related behavioural patterns reached a maximum of half of that percentage used for foraging and feeding. Since the species. Considering that ASCOs in the wild usually spends 40 to 60% of itstheir waking time foragingsearching for food in^[28], the wild [19], a greater usemplementation of an enrichment program with plenty of food-based enrichments [1](e.g. puzzle feeders) is highly recommended ^[1]. 

As hypothesised, we did not detect an inflOuence of visitor numbers, noise, or visitor interaction on behavioural responses,r results also confirminged the findings of recent studies indicating thatthat described how visitor presence tendis not to be the mainhe key factor affecting animals' behavioursr when other parameters are takeanalyzed ^[7], ^[8], ^[9]. Indeed, into consideration [5-7]. Our results mainly showewe did not find an influence of visitor numbers or noise on the subjects' behavioural responses. We did find an effect of time of day (i.e., observation session) on the individuals’ behaviours, as previously rereported for other species [5-7,in previous studies 9]^[7], ^[8], ^[9], ^[11]. IMoreover, individual identity was a significant predictor of the levevigilance, visitor–animal interactiofn, and juggling (i.e., fast, erratic movements that pass an object between the forepaws and sometimes the mouth), vconsigilance, and visitor–animal interactiondered as a feeding anticipatory behavior ^[21]), in accordalince with results reported byprevious research conducted on birds ^[10]. Since othper scholars studying bird spsonality influencies [18].es how An analysis of the otimals interact with humans and cope with the environment ^[29], ^[30], ^[31] assessing otters’' personality traits could be usefassist in planning enclosure design and daily husbandry routine.

4. Influences

To our knowl to pinpoint betteredge, this study was the first to investigate the individual specific traits that should be considered when applying changes to the enclosure and to husbandry and care procedures. Indeed, personality influences how animals cope wittegrated effect of environmental factors and human-related stimuli on the welfare of captive otters. It is worth noting that the small sample size does not allow us to generalize our results. Moreover, the limited number of video-recording devices might have led to an underestimation of affiliative and resting behaviors, since we could not record all the available shelters. In addition, the COVID-19 restrictions put in place by the host institution caused a reduction in visitor numbers ^[32]; hence we their environment and interacrecommend further evaluation of the visitor effect in relation to other parameters.

Despite with humans [20-22].

4. Conclusion

In conclusion, to our knowledge, this study was the first to investigate the integrated influence of environmental and human-related stimuli on the welfare of captive otters. Our results suggest that the otters involved in this study mainly experienced positive welfare states, indicating that they were likely provided with good welfare conditions. Enclosure design and husbandry practices were found to allow the subjects to perform a variety of species-specific behaviours, and to exert choice and control over their environment. Due to the small sample size, the results cannot be generalised, and the limited number of video recording devices might have led to an underestimation of resting and affiliative behaviours. The COVID-19 pandemic and associated restrictions caused a reduction in visitor numbers, meaning re-evaluation of the visitor effect in relation to other parameters taken into consideration in this study is warranted in the future. Moreover, further studies, aimed at investigating how the subjects’ behaviour varies during the day, across seasons, and with personality, are also recommended, to evaluate how otters’ needs change over time, and how this could alter visitor effects. This understanding could be used to optimise their husbandry routines and maximise positive experiences.

Despite the limitations acknowledged previously, this study increases the understanding of a scarcely researched topic and confirms the effectiveness of applying multivariate research methods to better analyse the array of lived experiences of captive animals. It also suggests suitable areas for further research and provides recommendations, such as the implementation of new enrichments, for improving the welfare of the studied subjects.

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experiences.