1. Introduction

Zoos

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.

Many studies have tried to evaluate animal welfare by verifying whether and how environmental and human stimuli (e.g., ambient temperature and visitor presence, respectively) affect animal behaviour [4]. Nevertheless, only a few researchers have examined the combined impacts of these stimuli in an attempt to obtain a clearer picture of animals’ experiences and welfare state (see for example: [5-9]). Interestingly, some of these studies detected an overestimation of the visitor effect (i.e., the positive or negative influence exerted by visitor presence) when other parameters, such as temperature and time of day, were included in the analysis [5, 6, 7].

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 methods

This study focused on a pair of ASCOs housed at the Giardino Zoologico di Pistoia (GZP), Pistoia, Italy. We collected data during 14 sampling days selected within a two-month period (from 19th July to 6th September 2020). We videoed the otters to ensure comprehensive monitoring of their behavioural patterns [10]. We conducted six one-hour observations daily (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 record data evenly across the public opening hours (9:00 am–7:00 pm), and to include feeding sessions and enclosure cleaning in the data collection periods. Each observation session was based on a 30-minute recording of each otter. We conducted a total of 84 focal sessions for each subject, resulting in 40 hours and 18 minutes of observation material for the male and 39 hours and 25 minutes of observation material for the female. The order of the subjects was alternated to monitor them in both the first and second 30-minute portion of each one-hour observation session. Based on other similar studies [5-7, 11-12], we selected the following parameters to be included in the analyses: weather conditions, ambient and water temperature, wind speed, relative humidity, background noise level, number of visitors, and caregiver presence. Weather conditions, wind speed, humidity, and ambient and water temperature were recorded at the beginning of each observation session. We recorded caregiver presence in and around the enclosure while recording visitor numbers and background noise every five minutes (seven times per focal observation). To test the effect of the selected parameters on behaviours, we ran generalised linear mixed models (GLMMs) [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.

Focusing in detail on the otters’ time budgets, the dominant category was out of sight, accounting, on average, for 41.59% of the subjects’ time budgets. This was consistent with the results reported for breeding pairs kept with offspring in outdoor naturalistic enclosures [11, 15-16]. Conversely, pairs and triplets of siblings held in indoor enclosures with limited natural vegetation and shelters have been reported to spend less time out of sight [12, 17]. Thus, the results of this study seem to confirm that different and enriched hiding options are key requirements in captive settings, in line with husbandry guidelines [18]. Nevertheless, a larger sample size is needed to exclude the influence of group composition.

When visible, the subjects mainly engaged in resting and vigilance behaviours. The percentage of time allocated to resting was similar to that reported for breeding pairs in family groups [11, 15-16]. Vigilance performance was, instead, slightly higher in comparison with the available literature, in which ASCOs were estimated to spend less than 15% of their time budget checking the surrounding environment [12, 17]. The otters engaged less in land locomotion and affiliative interactions compared to the breeding pairs of two family groups studied by Cuculescu-Santana et al. [11] and Owen [15]. This discrepancy could be due to the absence of offspring in this study that could have stimulated affiliative interactions, and the overall increased activity level of the adults involved in providing parental care (i.e., carrying, following, and feeding cubs) [6].

Notably, the family group analysed by Gothard [16] allocated 20% of its time budget to foraging and feeding, with a 13% increase when enriched with crickets and mealworms scattered in the enclosure. All the other studies, including the present research, reached a maximum of half of that percentage used for foraging and feeding. Since the species spends 40 to 60% of its waking time foraging in the wild [19], a greater use of food-based enrichments [1] is highly recommended.

As hypothesised, we did not detect an influence of visitor numbers, noise, or visitor interaction on behavioural responses, confirming the finding of recent studies indicating that visitor presence tends not to be the main factor affecting behaviours when other parameters are taken into consideration [5-7]. Our results mainly showed an effect of time of day (i.e., observation session) on the individuals’ behaviours, as previously reported for other species [5-7, 9]. Individual identity was a significant predictor of the level of juggling (i.e., fast, erratic movements that pass an object between the forepaws and sometimes the mouth), vigilance, and visitor–animal interaction, in accordance with results reported by other scholars studying bird species [18]. An analysis of the otters’ personality traits could be useful to pinpoint better 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 with their environment and interact 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.

References

1. Hosey, G.; Melfi, V.; Pankhurst, S. Zoo Animals: Behavior, Management and Welfare, 2nd ed.; Oxford University Press: Oxford, UK, 2013.

2. Kagan, R.; Allard, S.; Carter, S. What is the future for zoos and aquariums? J. Appl. Anim. Welf. Sci. 2018, 21, 59–70.

3. Mellor, D.J.; Hunt, S.; Gusset, M. Caring for Wildlife: The World Zoo and Aquarium Animal Welfare Strategy; WAZA Executive Office: Gland, aquaria aim to contribute to the preservation of wildlife through educSwitzerland, 2015.

4. Gray, J. Zoo Ethics: The Challenges of Compassionate Conservation; Cornell University Press: Ithaca, NY, USA, 2017.

5. Goodenough, A.E.; McDonald, K.; Moody, K.; Wheeler, C. Are “visitor effects” overestimated? Behaviour in captive lemurs is mational, research and conservation initiatives ^[1] anly drivend they must ensure good welfare for the animals in their care to meet these goals ^[2]. Studies of their by co-variation welfare are important to guarantee high standards of animal husbandry and care in zoological institutions, and to enhance theirth time and weather. roleJ. Zoo Aquar. Res. 2019, as7, conservation centers ^[3]59–66.

Animals6. experience 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., opportunity 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].

EncRose, P.E.; Scales, J.; Brereton, J. Why the “visitor effect” is complicated. Unravellosure design, environmental conditions, husbandry routine and exposure to ng individual animal care staff and , visitors are among the most important features that can influence the welfare of animals within zoos and aquaria ^[5]. S number, and climaticholars have usually tried to assess animal welfare by analyzing the effect of environmental and human-related factors on animals' influences on behaviors separately, space ^[6]. A limited number of studies have recently focused on the integrated impact of these factors on animals' well-being (see for example:e and interactions with keepers—A ^{[7][8][9][10][11]}), often indicating that visitor effect (i.e., the negative or positive influence exerted by visitors) has been over-estimated when otherse study on captive hornbills. parametersFront. Vet. Sci. 2020,7, (e236.g

7., ambient temperature, time of day, weather conditions) were included in the analysisRiley, A.; Terry, ^[7][8][9]M.

In; this study, Fresearchers aimed to examine this still un-researched topic, focusing on a pair of zoo-housed Asian small-clawed otters (Aonyx cinereus, hereafter referred as ASCOs). A. cinereus is a good model species since it is the most common otter species housed in captivity ^[12][13] eman, H.; Alba, A.C.; Solts, J.; Leeds, A. Evand, despite its popularity and high educational and conservational value ^[14], researcuating th on zoo-housed ASCOs' welfare is still scarce. Moreover, available studies have reported conflicting results relating to time budgets, and have often detected the occurrence of abnormal repetitive effect of visitor presence on Nile crocodile (Crocodylus nilotcus) behaviors. (ARBs;J. Zoo Aqu. Res. 2021, ^{[12][15][16][17][18][19][20][21][22]})2, i115–129.e

8., repetitive, unvarying and seemingly functionless behavioral patternsRose, P.E.; Badman-King, A.; Hurn, ^[23],S.; whRich are usually interpreted as an indicator of poor welfare either currently or in the past ^[24].

Ie, T. Visitor presence an this study, researchers investigated the 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 affected by selected a changing soundscape, alongside environmental and human-related parameters. Such a multivariate analysis has not previously been applied to the study of otter behaviour.

2. Methodology

This , can predict enclosure ustudy was conducted on a pair of adult ASCOs housed at the Giardino Zoologico di Pistoiage in captive flamingos. (GZP)Zoo Biol. 2021, Pistoia40, Italy21615.

9. Researchers cGollected data over two-month period (14 sampling days assigned randomly, from 19th of July 2020 to 6th of September 2020). Researchers video-recorded the otters' beodenough, A.E.; Sewell, A.; McDonald, K. Behaviours across six one-hour observations sessioal patterns in zoo-housed Humboldt penguins (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 pmSpheniscus humboldti). In this way, researchers could record data during different time slots across the public opening hours (9:00 am–7:00 pm), and could include revealed using long-term keeper-otter interactions (e.g., feeding sessions and enclosure cleaning) in the data collection. During each observation session, researchers recorded each individual for 30 minutes using a continuous focal animal sampling technique ^[25]. Reseacollected data: Validation of approaches and imprchers conducted 84 focal sessions for each subject, obtainingved husbandry. onAppl. Anim. Behav. Sci. 2023,258, average 4105811.

10 hours of observation per otter. Following relevant scientific literature  ^{[7][8][9][12][22]}riard, researchersO.; selected weather conditions, ambient and water temperature, wind speed, relative humidity, background noise level, visitor numbers, and caregiver presence as parameters to be included in the analysis. Researchers recorded humidity, ambient and water temperature, weather conditions, and wind speed at the beginning of each oGamba, M. BORIS: A free, versatile open-source event-logging software for video/audio coding and live observation sessions. ResearchersMethods Ecol. Evol. 2016, registered7, visitor1325–1330.

11. nCumbers and background noise every five minutes and continuously recorded caregiver presence in and around the enclosure. Researchers analyzed data with generalized linear mixed models ^[8][9][10] culescu-Santana, M.; Mason, J.; Purcoding the above-mentioned parameters and animal identity as fixed factors, date as a random factor, and behaviors as respondent variables.

3. Discoveries

Both ase, K.; McKie, R. Outdoor enclotters were found to perform a rich ure use and behavioral repertoireur of ^[26]. Aggressive and avoidance behaviors were limited, suggesting that the subjects experienced mainly positive emotional statesult and cub asian small clawed ^[4]. The otters couldAonyx cinereus also exercise control over their environment, choosing where to performn summer and winter. behavioursIUCN OSG Bull. 2021, almost38, 3–24 hours a day7.

12. The occurrence of potential ARBs was also limited.

The dominant category within the otterllis' behavioral time budgets was out of sight which accounted for 41.59% of the subjects’ time budgets, on average. This concurs with the results of previous studies conducted on breeding pairs housed with offspring in naturalistic outdoor enclosures ^[12][18][19]. Conn, M.L.; Reed, R.; Michels, E.; Boogert, N.J. The driversely, pairs and triplets of siblings hosted in indoor enclosures with little natural vegetation and a limited amount of shelter were found to spend less time out of sight ^[20][22]. These findinand functions of rock juggs highlight that providingng in otters. withR. SocOpen Sci. 2020, 7, the200141.

13. opportHunity to choose among different hiding options is one of the key requirements in captive settings, in accordance with husbandry guidelines ^[27]. Nessain, S.A.; Gupta, S.K.; de Silverthelessa, a larger sample size is needed to exclude the influence of group composition on the results.

When visibP.K. Biology and ecology of Asian small-clawe, the ottersd otter Aonyx cinereus most(Illy engaged in resting, and performed vigilance behavioriger, 1815): A review. TheIUCN OSG Bull. 2011, time28, allocated63–75.

14. to rMesting was similar to that reported for breeding pairs held in family groups  ^[12][17][19]. Hllor, D.J.; Beausowever, researchers found that the level of vigilance was slightly higher (16.70%) than found in other studies, wherein ASCOs spent less than 15% of their time budgets performing vigilance behavior ^[20][22]il, N.J.; Litlewood, K.E.; McLean, A.N.; McGreevy, P.D.; Researchers also found that the otters engaged less in locomotion and affiliative interactions compared to the breeding pairs belonging to two family groups observed by Cuculescu-Santana and co-authors ^[12] anJones, B.; Wilkins, C. The 2020 Five Domains Model: Includ Owen ^[18]. The absence of offspring in this study could have reduced the occurrence of affiliativeuman–animal interactions and could also explain the lower level of locomotory behaviors, since the subjects were not involved in providing parental care (i.e.,in assessments of animal welfare. carryingAnimals 2020, following10, and1870.

15. feOweding cubs) ^[27]n, C.

Regarding foDod-related behaviors, Gothard ^[19] visitors affoundect that a zoo-housed family group allocated 20% of its time budget to foraginge Asian short-clawed otter andAonyx cinerea feeding, with a 13% increase when crickets and mealworms were scattered in the enclosure. Conversely, in all the other available studies, including the r a captive environment? In Proceedings of the Sixth Annual Symposium on Zoo Research, the time dedicated by otters to food-related behavioural patterns reached a maximum of half of that percentage. Considering that ASCOs in the wild usually spend 40 to 60% of their waking time searching for food ^[28], the implementEdinburgh, UK, 8–9 July 2004; The Federation of an enrichment prZoogram with plenty of food-based enrichments (e.g. puzzle feeders) is highly recommended ^[1]. 

The resulogicalts also confirmed the findings of recent studies that described how visitor presence is not the key factor affecting animals' behavior when other parameters are analyzeGardens of Great Britain and ^[7][8][9]. Indeed, researchers did not find an influence of visitor numbers or noise on the subjects' behavioural responses. Researchers did find an effect of time of day (iland: London, UK, 2004; pp. 202–211.e

16., observation session) on behaviors, as reported for other species in previous studiesGothard, N. What is the ^{[7][8][9][11]}. Moreover, individual identity was a significant predictor of vigilance, visitor–animal interaction, and juggling (i.e., fast, erratic movements that pass an object between the forepaws and sometimes the mouth, considered as a feeding anticipatoryximate cause of begging behaviour ^[21]), in linea with previous research conducted on birds ^[10]. Since personality ingroup ofluences how animals interact with humans and cope with the environmentcaptive ^[29][30][31] aAssessing otters' personality traits could assist in planning enclosure design and daily husbandry routine.

4. Influences

This san short-clawed ottudy was the firstrs? toIUCN OSG Bull. 2007, investigate24, the14–35.

17. integrated effeCuct 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 researchers to generalize the results. Moreover, the limited number of video-recording devices might have led to an underestimation of affiliative and resting behaviors, since researchers 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 numbersulescu-Santana, M.; Horn, C.; Howe, C.; Briggs, R.N.; Bowe, C.; Geraughty, M.L. Seasonal changes in the behaviour ^[32]; hence researchers recommend further evaluation of the visitor effect in relation to other parameters.

Despite these limitations, this stenclosudy helped to increase the understanding of a scarcely researched topic. From an applied perspective, this project helped to identify potential welfare issues which could negatively impact the welfare of the studied use of captive Asian small clawed otters individualsAonyx cinereus. ConcurrentlyIUCN OSG Bull. 2017, it34, also29–50.

18. highlighted hBow enclosure design and husbandry routine, such as the presence ofylan, J.; Palmer, J. multipleBIAZA—Asian Small-Clawed Otter Husbandry Guidelines; hidingBIAZA: options and the 24 hour access to the outLondon, UK, 2018.

19. Heap, C.J.; Wright, L.; Andrews, L. Summary of Husbandry Guidelines for Asian Small-Clawed Otters in Captivity. IUCN Otter Specialist Group, Otters in Captivity Task Force 2008.

20. Stoinski, T.S.; Jaicks, H.F.; Drayton, L.A. Visitor effects on the behavior of captive western lowland gorillas: The importance of individual door section of the enclosure, played a key role in enhancing the subjects' fferences in examining welfare. FromZoo Biol. 2011, a methodological standpoint30, it all586–599.

21. Herrelko, E.S.; Vick, S.; Buchanan-Smith, H. Cognitive research in zoo-housed chimpanzees: Influence of personality and impact owed investigation of a wider range of parameters welfare. comparedAm. J. Primatol. 2012, to previous research ^{[7][8][9][10][11]}74, leading828–840.

22. to a better characterWization of the animals' experiences, and confirming the effectiveness of applying multivariate research methods to welfare assessment. Finally, it also provided meaningful recommendations, such as the implementation of new enrichments, that could further improve the welfare of the studied subjects.

5. Future perspectives

Tlliams, E.; Carter, A.; Hall, C.; Bremner-Harrison, S. Exploring the relationshe findings emphasize the importance of conducting further studies to investigate the effect of p between personality and environmental enrichments on otters' welfare. Researchers also recommend 24 hour monitoring to analyse whether and how the subjects’ behaviour and enclosure use changes, especially at night when the zoo is closed to the visiting public, and care staff are absent. Future research should also investigate how the otters' behaviour and space use changes between seasons, to identify any potential welfare issues related to low temperatures during winter. Such additional studies could be used to further optimise enclosure design and husbandry procedures, and hence to maximisesocial interactions in zoo-housed elephants: Incorporation of keeper expertise. positiveAppl. Anim. Behav. Sci. 2019, 221, experiences104876.