TYPE Original Research PUBLISHED 08 December 2022 DOI 10.3389/fvets.2022.1033821 Ackonc-AWA: A multi-species OPEN ACCESS animal welfare assessment protocol for wild animals under EDITED BY Rebecca E. Doyle, University of Edinburgh, United Kingdom REVIEWED BY human care to overcome the Barbara De Mori, Università degli Studi di Padova, Italy Eduardo J. Fernandez, use of generic welfare checklists University of Adelaide, Australia *CORRESPONDENCE Débora Silvia Racciatti1*† , Alejandra Feld1† , Laura Analía Rial1† , Débora Silvia Racciatti dracciatti@fvet.uba.ar Carlos Blanco2 and Oriol Tallo-Parra3,4 1 † These authors have contributed Cátedra de Bienestar Animal y Etología, Facultad de Ciencias Veterinarias, Universidad de Buenos equally to this work Aires (UBA), Buenos Aires, Argentina, 2 Cátedra de Sociología Rural, Facultad de Ciencias Veterinarias, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina, 3 Zoo Animal Welfare SPECIALTY SECTION Education Center (ZAWEC), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, This article was submitted to 4 Departamento de Ciencia Animal y Alimentaria, Facultad de Ciencias Veterinarias, Universitat Animal Behavior and Welfare, Autònoma de Barcelona (UAB), Barcelona, Spain a section of the journal Frontiers in Veterinary Science RECEIVED 01 September 2022 Introduction: Maintaining a high level of animal welfare is essential in ACCEPTED 22 November 2022 PUBLISHED 08 December 2022 zoos, sanctuaries and aquaria for ethical, legislative and functional reasons. CITATION Therefore, it is necessary to have welfare assessment protocols that can be Racciatti DS, Feld A, Rial LA, Blanco C incorporated into daily management programs. Currently, there are different and Tallo-Parra O (2022) Ackonc-AWA: approaches to assessing animal welfare in zoos. Those that can be applied A multi-species animal welfare assessment protocol for wild animals to multiple species consist of checklists or qualitative assessments, with under human care to overcome the limitations, especially regarding the lack of guidance in the selection and use of generic welfare checklists. Front. Vet. Sci. 9:1033821. interpretation of indicators. Validated protocols also exist, but they are for very doi: 10.3389/fvets.2022.1033821 few wild species. This study aimed to develop, test in the field, and describe an COPYRIGHT animal welfare assessment protocol for wild animals under human care, that © 2022 Racciatti, Feld, Rial, Blanco can be applied to multiple species, intended to overcome the use of generic and Tallo-Parra. This is an open-access article distributed under the terms of welfare checklists and offer an alternative to challenging and time consuming the Creative Commons Attribution species-specific tools. License (CC BY). The use, distribution or reproduction in other forums is Methods: The development process consisted of the elaboration of a permitted, provided the original protocol, substantiated by published literature on zoo animal welfare and author(s) and the copyright owner(s) are credited and that the original multidisciplinary focus group work, and its on-field feasibility test. This was publication in this journal is cited, in performed on 14 species of different taxa housed in an Argentinian zoo. accordance with accepted academic The protocol was structured in two forms: an initial form to serve as scan practice. No use, distribution or reproduction is permitted which does using various animal-based (ABM), resource-based (RBM), and management- not comply with these terms. based measurements (MBM), and a follow-up form using exclusively ABM. The protocol also included a user’s manual with information about preliminary preparation, equipment required, steps from arrival until completion, and details on how to assess each indicator. The scoring method consisted in rating each indicator on a 3-point scale. Results: 23 ABM, 19 RBM, and three MBM were tested and selected to integrate Ackonc-AWA, a multidimensional protocol covering the five animal welfare domains and applicable to multiple species. Discussion: This protocol was entirely developed in Spanish and can be applied noninvasively and at a low cost, which constitute features of high relevance for Latin America. Further applications of the described welfare Frontiers in Veterinary Science 01 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 assessment tool in other species and different institutional contexts will reinforce the validation of the proposed measurements and allow the systematic and routine evaluation of animal welfare in zoos. KEYWORDS animal-based measurements, animal welfare, assessment protocol, compassionate conservation, management-based measurements, resource-based measurements, zoo, animal welfare indicators Introduction Ensuring animal welfare requires knowledge, experience, and institutional commitment, as well as the deployment Individual animal welfare and species welfare are critical of comprehensive and robust animal welfare assessment obligations of zoos, sanctuaries, and aquaria (hereafter tools, which can be implemented at two levels: institutional simplified as “zoo(s)”). Even the most ambitious conservation (examining policies, resources, programs, and practices) or goals will not be adequate justification for keeping wild individual (providing an assessment of animals and their animals in captivity if zoos do not actively demonstrate high environments) (3, 16). As animal welfare is a multidimensional standards of animal welfare (1–4). The integration of animal field of study (17–19), welfare assessment should consider welfare and wildlife conservation has been reflected in the multiple criteria (20–22), with a holistic evidence-based emergence of new fields of study, such as compassionate approach (3). Therefore, most welfare assessments strategically conservation and conservation welfare. These multidisciplinary include animal-based measurements (ABM) that address aspects approaches attribute intrinsic value to some individual wild of the actual welfare state of the animals in terms of their animals and support our moral obligation to consider their behavior, mental state, health, and physical condition. They welfare, interacting with responsibilities to protect other also incorporate resource-based (RBM) and management-based aspects of nature, such as populations, species, ecosystems measurements (MBM) that can be correlated to ABM and used and biodiversity (5–8). Despite these similarities, there are to identify risks for animal welfare and causes of poor welfare, so differences in their ethical foundations, and pragmatism as to implement improvement strategies (23). that have been deeply discussed in the literature [e.g., The approach in the construction of protocols to assess (8, 9)]. animal welfare, their methods, and the way in which they should The past few decades have seen an increased interest in be evaluated or validated depend on the goals, which need animal welfare among researchers and zoo staff. Zookeepers to be clearly defined before starting the development process. identify training in this area as relevant and important to Botreau et al. (20) identify three main models for assessing their work (9) and the scientific community shows an increase animal welfare according to the intended goals: descriptive, in published research on animal welfare over time (10–12). normative, and prescriptive. The descriptive model is used to In addition, there is a growing public concern for animal depict a pre-existing situation that is stable and independent welfare and an ethical requirement to comply with international of any observation, thus providing the ability to characterize standards and national regulations on zoo animal welfare (4, 13). and compare observed situations. The normative model explains According to the Single Public Registry of Wildlife Operators how things should be or how people should act, and aims to (14), in Argentina there are 16 officially registered institutions provide evaluation procedures to verify the appropriateness of that house wild fauna, with numerous populations of diverse collected information in relation to predefined rules. Finally, native and exotic species, maintained under different conditions the prescriptive approach does not assume any pre-existing of animal welfare, and with dissimilar realities in terms of situation to be described; it aims to collect and organize relevant human and financial resources. In addition to the interest of information to facilitate the formulation of recommendations to researchers and zoo staff, the active demands of public opinion achieve a goal. and animal rights NGOs have led to official interventions to Currently, there are different tools to assess animal welfare initiate conversion processes in many zoos, with animal welfare in zoos. Those that can be applied to multiple species usually as the main driver. It has also led to an update of national and consist of extensive checklists with questions aimed at revealing territorial regulations, establishing animal welfare as a priority what the conditions of the physical and social environment are by applying the highest welfare standards for individuals, like and provide insight into the welfare of an individual animal through adequate facilities and management modalities in [e.g., (3, 16, 24)]. Some of them also consider and integrate zootechnical, ethological, sanitary, and genetic terms (15). life stages, in relation to species and individual differences Frontiers in Veterinary Science 02 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 [e.g., (25)]. Although these protocols can be useful to easily from January 2008 to July 2017, in English and Spanish, a improve animal welfare monitoring, they have some limitations, selection of scientifically supported indicators previously used especially regarding the lack of guidance in the selection and in welfare assessment protocols applied to farm, laboratory and interpretation of indicators, and thus, a non-tested reliability zoo animals was obtained. Some of these indicators and their on applicants’ criteria. Validated protocols also exist (21, 26– references were modified to adapt them to the characteristics 30), but they have been developed specifically for very few of of the zoo, to the variety and characteristics of species to be the enormous variety of wild animal species that could require evaluated, and taking into account previous experiences of the assessment (24). researchers on animal welfare assessment in zoos. Interviews and This study aimed to develop, test in the field, and meetings with personnel from different areas of the zoo were describe an animal welfare assessment protocol for wild animals conducted. During the interviews, questions related to animal under human care, which can be applied on a daily basis, welfare were asked (e.g., When you observe the animals under noninvasively, and at a low cost, under the aforementioned your care, what do you look at? How do you notice if there is prescriptive model. That is, first the current welfare status of any discomfort, pain or something wrong with them?). Their the animal is assessed to understand the starting point and then responses were taken into account when selecting, eliminating, its evolution is monitored by collecting information that allows or adapting certain indicators in the protocol. the development of tailor-made recommendations and rapid The principle of feasibility was taken into account for the decision making. Hence, it was intended that the protocol would selection of the welfare indicators (32, 33). The researchers be able to provide two types of assessment: comprehensive also considered the need for the institution’s own staff to be (whether initial diagnosis or in the face of important events, able to collect the data easily, subject to adequate training such as changes in the environment, group structure, and/or and performance evaluation. Thus, all measurements involving management) and regular (frequent monitoring to detect early physical invasion or restraint of the animals, and indicators that deviations). Simultaneously, it aimed to obtain an intermediate require further laboratory analysis (e.g., metabolic profiling), solution between protocols that are easy to apply yet rely entirely were excluded. For this test, all the animal welfare assessments on the judgment of the assessors, and validated but species- were performed hands-off, by remote observations at a distance. specific protocols that are useful only for assessing the species In addition, two meetings were held with eleven for which it was developed. representatives of different areas of the zoo (Veterinary, Nutrition, Biology, Behavior, Animal Care, Animal Welfare Management and Planning) to submit their input to a multi- Materials and methods disciplinary discussion in a focus group in order to select agreed upon items for assessment, as a way to provide content Site validity (13). Once the first selection of the indicators to be assessed The protocol was tested at an Argentinean zoo, member of had been made, two types of forms were developed: an initial Asociación Latinoamericana de Parques Zoológicos y Acuarios form and a follow-up form. The initial form consisted on (ALPZA) and World Association of Zoos and Aquariums 45 indicators (23 ABM, 19 RBM, and three MBM) (Table 1), (WAZA), which was in the process of transformation and which was meant to be carried out the first time an animal restructuring. The protocol was applied and tested between is assessed, and then on a semi-annual basis, or in the face October and December 2017. of important changes in the environment, group structure and/or management of the animal under study. At this first step, the RBM and MBM were exhaustively considered together Elaboration of the protocol with ABM, to detect risk factors of poor welfare, even before the occurrence of identifiable manifestations by means of The protocol was given the name Ackonc-AWA, which ABM. The follow-up form consisted exclusively of ABM (23 combines the purpose of conducting animal welfare assessments indicators) to facilitate the data collection process and reduce (AWA) with the role of the individuals involved in the the time required to carry out the observations, and was observation and data collection process (hereafter, sentinels), intended to be applied daily or weekly. The frequency of use given that the phonetics of the name reflects the native Andean of the follow-up form can be adjusted according to need. As word “ackoncahua” which is translated as “sentinel”. a starting point, the researchers suggest a weekly application. The conceptual animal welfare framework adopted to create However, a higher frequency (i.e., daily) could be used for Ackonc-AWA protocol was the Five Domains Model (31), continuous monitoring of a newly moved animal or changes with a joint approach between the behavioral domain and the in group composition, management or enclosure characteristics mental domain. Based on a literature review through research to detect early alterations in ABMs that reflect a deterioration databases (PubMed and Google Scholar), with date restriction in welfare. Frontiers in Veterinary Science 03 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 TABLE 1 List of indicators selected to test on-field reliability and feasibility, and sentinels assigned according to their availability, area of daily performance and experience. Sentinels assigned to the on-field feasibility and reliability test of each indicator External Zoo staff (departments) Researchers V N Bi Be AC AWMP Nutrition domain Body condition score (ABM) Yes Yes Yes No No Yes Yes Food intake (ABM) Yes Yes Yes No No Yes Yes Food availability (RBM) Yes No Yes Yes No Yes Yes Nutritional quality and safety of food (RBM) Yes No Yes No No No Yes Macroscopic condition of food (RBM) Yes No Yes No No No Yes Food presentation (RBM) Yes No Yes No Yes Yes Yes Water intake (ABM) Yes No Yes No No Yes Yes Availability of water (RBM) Yes No Yes Yes No Yes Yes Macroscopic quality of water (RBM) Yes No Yes No No Yes Yes Presentation of water (RBM) Yes No Yes Yes Yes Yes Yes Environment domain Substrate (RBM) Yes No No Yes Yes Yes Yes Temperature/humidity/ventilation (RBM) Yes Yes No No No Yes Yes Lighting (RBM) Yes Yes No Yes No Yes Yes Enclosure maintenance (RBM) Yes No No No No Yes Yes Enclosure hygiene (RBM) Yes No No No No Yes Yes Enclosure dimensions (RBM) Yes No No Yes Yes Yes Yes Environmental complexity (RBM) Yes No No Yes Yes Yes Yes Surrounding enclosures (RBM) Yes No No No Yes Yes Yes Shelter availability (RBM) Yes No No No Yes Yes Yes Public (RBM) Yes No No No Yes Yes Yes Group composition (RBM) Yes No No Yes No No Yes Environmental choice and control opportunities (RBM) Yes No No No No Yes Yes Management choice and control opportunities (MBM) Yes No No No Yes Yes Yes Environmental enrichment (MBM) Yes No No No Yes Yes Yes Training procedures (MBM) Yes No No No Yes Yes Yes Health domain Defecation behavior (ABM) Yes Yes No No No Yes Yes Stool score (ABM) Yes Yes No No No Yes Yes Micturition behavior (ABM) Yes Yes No No No Yes Yes Urine appearence (ABM) Yes Yes No No No Yes Yes Coat/feathers/tegument (ABM) Yes Yes No No No Yes Yes Lesions/injuries (ABM) Yes Yes No No No Yes Yes Hooves/claws/teeth (ABM) Yes Yes No No No Yes Yes Locomotion (ABM) Yes Yes No No No Yes Yes Sleep/wakefulness (ABM) Yes Yes No Yes No Yes Yes Signs of illness (ABM) Yes Yes No No No Yes Yes Behavior and affective states domain Reaction to strangers (ABM) Yes No No No Yes Yes Yes Interaction with zookeepers (ABM) Yes No No No Yes Yes Yes Exploration (ABM) Yes No No No Yes Yes Yes Social, affiliative and maternal-filial behavior (ABM) Yes No No No Yes Yes Yes (Continued) Frontiers in Veterinary Science 04 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 TABLE 1 (Continued) Sentinels assigned to the on-field feasibility and reliability test of each indicator External Zoo staff (departments) Researchers V N Bi Be AC AWMP Reproductive behavior (ABM) Yes No No Yes Yes Yes Yes Agonistic behavior (ABM) Yes No No No Yes Yes Yes Use of environmental enrichment (ABM) Yes No No No Yes Yes Yes Stereotypic behavior (ABM) Yes No No No Yes Yes Yes Behavioral diversity (ABM) Yes No No Yes Yes Yes Yes Space use (ABM) Yes No No No Yes Yes Yes V, Veterinary; N, Nutrition; Bi, Biology; Be, Behavior; AC, Animal Care; AWMP, Animal Welfare Management and Planning; ABM, Animal-based measurement; RBM, Resources-based measurement; MBM, Management-based measurements. Additionally, a user’s manual was written with instructions sentinel provided additional information about this on the on the method used to assess and score each indicator. Both the “Notes” column. indicators selected and the instructions for their assessment were the same for the different species and individuals included in the pilot test, although changes and clarifications were made in the On-field feasibility test user’s manual to adapt them to the differential characteristics of each taxon. Animals Before beginning the assessment, sentinels had to be The selection of the species and individuals on which familiar with the following information about the species to be the protocol was tested was based on the following inclusion assessed: biological and behavioral features (including specie’s criteria: 1—prospective permanence of the animals in the ethogram); housing and handling requirements recommended zoo: longer than 2 years; 2—easy identification: phenotypic by international associations; nutritional information (diet characteristics or features that made it possible to individualize received by the animal or group being evaluated) and both the animals housed in groups; and 3—include species from routine and scheduled activities (e.g., feeding time, enclosure different taxonomic categories to test the ability of Ackonc- cleaning, training sessions, environmental enrichment, animal AWA protocol to be applied for different taxa. As a result, rotation and other interfering activities planned for the day of 14 individuals (ten mammals, two birds, and two reptiles) the assessment, such as capture for veterinary examination or from different orders and families were selected (Table 2). transfer to another enclosure). Likewise, sentinels should have a Ackonc-AWA was also tested on one group of 12 capybara layout/map and information about the location and dimensions (Hydrochoerus hydrochaeris), to explore the potential usefulness of the enclosure. of the protocol for group assessment, with proper modifications Every effort should be made to minimize the impact of the or adaptations. presence of the sentinels on the behavior of the animal under study. Sentinels should remain out of sight and avoid any kind of interaction with the observed animal during the data collection Sentinels to minimize the impact of his or her presence on the behavior Sentinels assigned to observe, record and score the of the animal under study (e.g., choosing an observation point indicators on site were selected from the different areas involved to allow the sentinel to be as hidden as possible or remaining in animal management and care, based on interviews, in as long as necessary without interacting with the animal until it search of those who met a combination of experience, training, withdrew its attention from the sentinel’s presence). predisposition and observation skills. Their election was also Indicators were rated on a 3-point scale (A—normal/no agreed with representatives of the institution in order to avoid observable welfare risk; B—mild deviation/welfare risk; C— hindering or disrupting daily activities. Hence, the team of severe deviation/welfare risk). For indicators that could be rated sentinels consisted of a group of three external veterinarians in several contexts (e.g., animals that have access to different experienced in animal welfare assessments (the first three enclosures at different times), rating was made according to authors of this work, hereafter the researchers) and a group the context that represented a higher level of animal welfare of nine zoo staff members with no prior experience in animal compromise. When any indicator was rated “B” or “C”, the welfare assessments, belonging to different departments [one Frontiers in Veterinary Science 05 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 TABLE 2 Information about the animals on which the Ackonc-AWA protocol was tested. Family Species Gender Age (Years) Level of assessment Order mammals Primates Hominidae Pan Troglodytes Male 11 Individual Pongo spp. Female 31 Carnivora Canidae Chrysocyon brachyurus Female 16 Felidae Panthera tigris tigris Male 11 Otariidae Otaria flavescens Female 10 Pilosa Myrmecophagidae Myrmecophaga tridactyla Female 12 Proboscidea Elephantidae Elephas maximus Female 50 Perissodactyla Tapiridae Tapirus terrestris Male 10 Artiodactyla Camelidae Vicugna vicugna Male 13 Rodentia Caviidae Hydrochoerus hydrochaeris 6 females 5 males 1 Unknown 11 adults 1 young Individual and Group Order birds Cathartiformes Cathartidae Vultur gryphus Male 10 Individual Psittaciformes Psittacidae Anodorhynchus hyacinthinus Female 27 Order reptiles Testudines Chelidae Acanthochelys spixii Female 4 Individual Squamata Teiidae Salvator rufescens Male 8 from Veterinary, one from Nutrition, one from Biology, two Test-retest reliability from Behavior, two from Animal Care (zookeepers) and two Three sentinels were assigned the assessment of the same from Animal Welfare Management and Planning (AWMP)]. animal at two different time points. Test-retest agreement rate All inexperienced sentinels received a 4 h theoretical was corrected for chance by kappa statistics (34). Inter-observer and practical training on animal welfare assessment in reliability could not be assessed due to the limited availability of general and on the use of the protocol in particular, designed zoo staff involved in this pilot test. The statistical processing of and delivered by the researchers. A virtual library was the data was carried out using the software Infostat R (35) and also created with ethograms and information on each of VassarStats: Website for Statistical Computation (36). the 14 species’ nutritional, physiological, environmental and behavioral needs, selected by the researchers from books, husbandry manuals and peer-reviewed scientific Feasibility publications. All sentinels were given access to this virtual Completeness of the forms library and were instructed to read the documents selected For the animal welfare assessment to be comprehensive, for the corresponding species before beginning the on-field all indicators in Ackonc-AWA must be completed, except feasibility test. for those that do not apply to a given species, due to The researchers and the zoo staff from the AWMP its particular nature (e.g., water consumption in underwater Department were exclusively dedicated to this task, so species) or under specific situations (courtship behavior outside they evaluated the entire protocol (all indicators). On the the reproductive season). In such cases, sentinels were instructed other hand, the rest of the sentinels were assigned a to use the legend “does not apply” to differentiate them from different number of indicators to score, since they had those that could be left blank due to other reasons (e.g., different availability to collaborate with this research (Table 1). lack of time, impossibility of taking the measurement, not For the latter group, indicators would be scored during provided access/information). the zoo routine schedule and with minimum interference The average completeness of the forms was determined by to the daily management and procedures. Likewise, the averaging the degree of completeness achieved by all sentinels assignment of the indicators to be rated was made considering for all species. In addition, a ranking of the indicators most often their area of daily performance and previous experience. left blank was made by counting the number of times that each For instance, health-related indicators were assigned to the indicator was not evaluated when it should have, in relation to zoo veterinarian, and nutrition-related indicators to the the total of forms (both initial and follow-up) across species and nutrition expert. sentinel groups. Frontiers in Veterinary Science 06 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 TABLE 3 Test-retest reliability calculated for the on-field test of Ackonc-AWA protocol. Cohen’s kappa SE CI95% Proportion of agreement n Sentinels 1 0.7391 0.0798 (0.5827; 0.8955) 0.8421 57 2 0.7863 0.0821 (0.6255; 0.9471) 0.8846 52 3 0.7997 0.0623 (0.6777; 0.9217) 0.8696 69 Overall pondered 0.7763 0.8652 Indicators ABM 0.7574 0.0759 (0.6086; 0.9062) 0.8816 76 MBM 0.6774 0.2040 (0.2775; 1) 0.8000 10 RBM 0.7877 0.057 (0.6761; 0.8993) 0.8636 88 Overall pondered 0.7681 0.8678 Degree of difficulty represented by the observation and plant and/or human wellbeing. The participation of zoo staff in recording process this study was completely voluntary and under written informed Ackonc-AWA protocol was designed so that the consent. The survey responses were strictly confidential and observations and completion of the forms can be done by data from this research was reported only in the aggregate. The the zookeepers, combining this activity with their other information was coded and remains confidential. responsibilities. Therefore, it was important to determine the degree of difficulty perceived by the staff in applying the chosen indicators. For this purpose, after completing the Ackonc-AWA Results forms, each sentinel was asked to assign a degree of difficulty to fill out each form between 1 and 10, with 1 being the minimum On-field feasibility test and 10 the maximum. At the bottom of each form, the sentinels had to specify which indicator was found as the most difficult Test-retest reliability to evaluate. With these responses, the indicators were rated for The mean intra-observer proportion of agreement was their level of difficulty, from the most often reported to the 0.8652 among the sentinels and 0.8678 among indicators (ABM, least often reported. The results were analyzed by averaging RBM, and MBM). The mean observed Kappa was 0.7763 among the degree of difficulty assigned for all species and sentinels, the sentinels and 0.7681 among indicators, which on the Landis differentiating between initial and follow-up assessment forms. and Koch (34) scale is substantial agreement (Table 3). Although Cohen’s test ruled out a random component, more trials are needed to increase the statistical power of the test. Time required to complete the forms It was intended that the Ackonc-AWA protocol require <2 h per individual or group for data collection since long application protocols have more difficulties to be used regularly in zoological Feasibility institutions, especially in those lacking resources or exclusive Completeness of the forms personnel for this purpose, a very frequent situation in Latin The average completeness for the initial form was 86.21% America. The average time (in minutes) required to complete whereas for the follow-up form it was 79.07%. The top ten the two welfare assessment protocol forms was recorded for all indicators most often left blank were part of both assessment species and sentinels, differentiating between initial and follow- forms and were therefore analyzed together. No indicators were up assessment forms. left blank over 50% of the times. Only Water intake was left blank Ethical review of the project was requested to the over 40% of the times (56 times; 42.10%). Three indicators were Institutional Committee for the Care and Use of Laboratory left blank between 40 and 30% of times: Micturition behavior (44 Animals (CICUAL) of the Faculty of Veterinary Sciences of the times; 33.08%), Defecation behavior (42 times; 31.58%), and Use University of Buenos Aires, and a review exemption was granted of environmental enrichment (42 times; 31.58%); two indicators given the observational nature of the project.The study focused were left blank between 30 and 20% of times: Social behavior (37 on the non-invasive/intrusive assessment of animal welfare, so times; 27.82%) and Reproductive behavior (30 times; 22.56%); no interventions of any kind were carried out on the animals. and four indicators were left blank between 20 and 10% of times: There were no potential adverse effects, nor foreseeable risks Hooves/claws/teeth (24 times; 18.04%), Agonistic behavior (20 or hazards associated with this project, with regards to animal, times; 15.04%), Food intake (17 times; 12.78%) and Behavioral Frontiers in Veterinary Science 07 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 TABLE 4 Time in minutes (mean +/– SD) required to complete initial diversity” within the Behavioral and mental domain by and follow-up welfare assessment forms of Ackonc-AWA protocol. the introduction of activity budget sheets of 20 min each, Species Initial form Follow-up form on three (or four, when possible) different time slots (see Supplementary Table 1). After sentinels complete the activity Mean SD Mean SD budgets sheets, a trained analyst (external or personnel of the institution) should evaluate the data and assign the appropriate Pan Troglodytes 56.4286 28.9704 86.2500 22.5000 score (A, B or C) for the indicators “Behavioral diversity” Pongo spp. 46.0000 29.6648 40.0000 0.0000 and “Space use”. Chrysocyon brachyurus 47.8571 35.1019 31.2500 6.2915 No changes were made for the indicators included in the Panthera tigris tigris 60.0000 46.9042 40.0000 28.2843 Nutritional, Environmental and Health domains. As a result, a Otaria flavescens 66.6667 37.7712 37.5000 9.5743 total of 45 indicators (23 ABM, 19 RBM, and three MBM) were Myrmecophaga tridactyla 29.0000 20.7364 67.5000 74.2462 selected to integrate Ackonc-AWA, covering the five animal Elephas maximus 46.0000 25.8360 75.0000 32.7872 welfare domains. Tapirus terrestris 55.0000 27.3861 87.5000 12.5831 Vicugna vicugna 30.0000 7.0711 50.0000 29.4392 Hydrochoerus hydrochaeris 63.2500 21.1213 58.2222 15.8096 Nutritional domain Vultur gryphus 90.0000 42.4264 63.7500 25.6174 Three ABM (Body condition score, Food intake, and Anodorhynchus hyacinthinus 36.6667 23.5938 61.7500 13.3760 Water intake) and seven RBM (Food availability, Nutritional Acanthochelys spixii 41.6000 45.8290 43.7500 9.4648 quality and food safety, Macroscopic condition of food, Food Salvator rufescens 50.0000 18.7083 74.0000 43.6119 presentation, Availability of water, Macroscopic quality of water, Total 51.3192 29.3658 58.3194 23.1132 and Presentation of water) were selected to assess the nutritional domain. Table 5 summarizes the methods, references, and scoring system required for this purpose. diversity (16 times; 12.03%). The rest of the indicators included in the protocol were left blank <10% of the time. Environmental domain Degree of difficulty represented by the observation and Twelve RBM (Substrate, Temperature/humidity/ventilation, recording process Lighting, Enclosure maintenance, Enclosure hygiene, Enclosure The mean reported difficulty across species and sentinels dimensions, Environmental complexity, Surrounding was 4.79 +/– 1.13 for the initial form and 5.20 +/– 1.51 for enclosures, Shelter availability, Public, Group composition, the follow-up form. Analysis of sentinel responses showed that Environmental choice, and Control opportunities) and the the indicator most frequently reported as difficult to assess was three MBM (Management choice and control opportunities, Behavioral diversity (54 times; 40.60%) followed by Defecation Environmental enrichment, and Training procedures) were behavior (31 times; 23.31%), Micturition behavior (29 times; adopted. Table 6 summarizes the most relevant information 21.80%), Hooves/claws/teeth (13 times; 9.77%), Water intake (12 provided in the user’s manual for assessing environmental times; 9.02%) and Food intake (11 times; 8.27%). domain. To this end, sentinels had to be able to access and consider all areas destined to the animal (e.g., exhibitors, Time required to complete the forms sleeping quarters, pens, handling areas, etc) to rate each The average time across species and sentinels required indicator according to the sector(s) that imply a greater to complete the initial form was 51.32 min. +/– 29.36 min. compromise to the welfare of the animal (or group). Completion of the follow-up form took an average of 58.32 min +/– 23.11 min. Table 4 shows the amount of time (in minutes) required to complete initial and follow-up welfare assessment Health domain forms of Ackonc-AWA protocol for each of the species included Ten ABM (Defecation behavior, Stool score, Micturition in the study. Activity budget sheets were later added to the behavior, Urine appearance, Coat/feathers/tegument, protocol (see below). Lesions/injuries, Hooves/claws/teeth, Locomotion, Sleep/wakefulness, and Signs of illness) were selected to assess the health domain. Table 7 summarizes the most relevant Structure of Ackonc-AWA and application information provided in the user’s manual for assessing guidelines/criteria health domain. Given the multispecies purpose of the Ackonc-AWA In the face of on-field feasibility results, some changes were protocol, it is important to note that for some species (e.g., implemented for the assessment of the indicator “Behavioral reptiles, birds) it may be necessary to score the indicators Frontiers in Veterinary Science 08 frontiersin.org TABLE 5 Summary of the most relevant information that is provided in the user’s manual for assessing nutritional domain. Frontiers in Veterinary Science Racciatti et al. Indicator Method Reference Scoring Body condition score It shall be assessed visually. Only when there is no risk for humans or Does the animal have a body condition A: 3, B: 2 o 4, C: 1 o 5. In case B or C, clarify in “observations” to which BCS it (ABM) animal’s welfare, it may also be assessed by palpation. Use a appropriate to their species, age, sex and corresponds. standardized 5-point scale scientifically validated for the species physiological state? under study. Food intake (ABM) Observe the eating behavior and the daily amount of food consumed. Is the feed intake adequate for the animal A: normal appetite. B: hyporexia, pica, trichophagia, coprophagia (In some according to their age, sex, physiological state species coprophagia is not pathological). C: anorexia, polyphagia or any type of and health condition? disturbance that is not allowing adequate food intake (even if appetite is not affected). Food availability (RBM) Observe the time at which food is offered in the indoor and outdoor Is the food available and sufficient A: all the features are adequate. B: only one of the features is not adequate, but it enclosures (features to consider: number, competition for access, considering age, sex, physiological state and does not prevent access to the food. C: the food is not accessable and/or two or location and height, cleanliness and maintenance condition of health condition of the animal? more features are not adequate. feeders or feeding zone). Nutritional quality and Request information from the nutrition department. If possible, send Is the diet adequate in nutrients (according to A: the diet is adequate, safe and secure. C: either nutrient profile or food safety safety of food (RBM) food samples for analysis. Relevant literature should be used to the species, age, physiological and health criteria is not adequate. obtain information on the reference values and analyses required for status) and are the ingredients safe and the species under study. secure (free of contaminants and toxins, cold chain mantained)? Macroscopic condition Observe the food offered to the animal (alterations to consider: Is the food offered to the animal in good A: no alterations are observed. B: only one food or portion have only one of the 09 of food (RBM) bruises, insects, mold, rotting, fruit ripening, fecal matter mixed with condition? mentioned alterations. C: one or more foods or portions have two or more of the the food). mentioned alterations. Food presentation Observe and compare the way in which the food is presented in the Does the presentation of the food respect the A: all features to be considered are adequate. B: only one of the features is not (RBM) zoo with how it is found in the evolutionary environments of the way the species feeds in the wild? adequate, but it does not impede the ingestion of food. C: two or more features species (features to consider: frequency, portion size, timing, texture, are inadequate consistency, temperature and location). Water intake (ABM) Observe the drinking behavior and the daily amount of water Does water consumption match the animal’s A: normal intake. B: slight increase or decrease in water intake unrelated to consumed. requirements? weather conditions. C: significant increase or decrease in water intake unrelated to weather conditions and/or difficulty in swallowing or ingesting water. Availability of water Observe the water troughs and other water sources in the indoor and Is the animal provided with sufficient and A: all features to be considered are respected. B: only one of the features to be (RBM) outdoor enclosures (features to consider: number, competition for accessible water at all times? considered is not respected, but it does not prevent access to water. C: water is access, location and height, cleanliness and maintenance). not accessible and/or two or more features to be considered are not respected. Macroscopic quality of Observe the water offered to the animal (features to consider: color, Is the water offered to the animal in good A: all features to be considered are adequate. B: only one of the features to be 10.3389/fvets.2022.1033821 water (RBM) odor, presence of food debris and other visible particles, greenery) condition? considered is not adequate, but it does not prevent the ingestion of water. C: two or more features to be considered are not adequate. Presentation of water Observe and compare the way in which water is presented in the zoo Does the presentation of water respect the A: the presentation of water respects the way the species drinks in the wild. B: the (RBM) with how it is found in the evolutionary environments of the species way it is found in the wild and accordingly presentation of water partially respects the way the species drinks in the wild (if frontiersin.org and their drinking behavior. with the species drinking behavior? the species has more than one way of drinking water, its presentation does not allow to express at least one of them) C: the presentation of water does not respect the way the species drinks in the wild. ABM, Animal-based measurement; RBM, Resources-based measurement. Frontiers in Veterinary Science Racciatti et al. TABLE 6 Summary of the information provided in the user’s manual for assessing environmental domain. Indicator Method Reference Scoring Substrate (RBM) Observe the substrate of the enclosure and compare it with the Is the substrate suitable for the animal to rest A: the substrate is suitable for the animal to rest comfortably and deploy typical natural environment of the species (features to consider: level comfortably and exhibit species-specific species-specific behaviors. B: the substrate is inappropriate for the animal to rest of compaction, texture, hardness and temperature of the material, behaviors? comfortably or may prevent the manifestation of any species-specific behaviors. undulations and unevenness). If available, check the reference C: the substrate is inappropriate for the animal to rest comfortably and/or could substrate requirements for the species in the husbandry manual. prevent the manifestation of several species-specific behaviors. Temperature/humidity/ Observe the conditions offered in the enclosure and compare them Are the enclosure conditions adequate to A: the enclosure presents conditions that allow maintaining an adequate thermal ventilation (RBM) with the climatic characteristics of the ancestral environment of the allow the animal to maintain thermal comfort in all its aspects. B: one of the aspects of the enclosure is deficient to species (features to consider: sources of heat or cold, shade and sun, comfort? maintain adequate thermal comfort without threatening the life of the animal. C: and bathing facilities (e.g., water, mud or other). If the enclosure has two or more of the aspects of the enclosure are deficient to maintain adequate a controlled system for temperature, humidity and ventilation, or if thermal comfort, or only one aspect is deficient in a way that puts the animal’s life you have a device to measure these parameters, check and record the at risk. values. If available, check the reference temperature, humidity and ventilation requirements for the species in the husbandry manual. Lighting (RBM) Observe the lighting of the enclosure and compare it with the typical Does the lighting in the enclosure respect the A: natural and artificial lighting is suitable for the species. B: one of the natural environment of the species. If available, check the reference circadian cycle, the number of hours of components to be considered is not appropriate, without putting the animal’s life 10 lighting requirements for the species in the husbandry manual. light/darkness characteristic of the natural at risk. C: two or more of the components to be considered are deficient, or only environment of the species and does it not one is deficient but puts the animal’s life at risk. affect or hinder vision or generate somatic disorders? Is the amount of sunlight entering the enclosure adequate according to the characteristics of the natural environment of the species? Enclosure maintenance Observe the maintenance conditions of the enclosure (features to Does the condition in which the enclosure is A: the enclosure is in good maintenance conditions. B: there are some defects in (RBM) consider: defects in the structure of the enclosure that may cause maintained pose no risk to the health and the maintenance of the enclosure, which do not directly endanger the lives of damage to the animals, poisonous plants within reach, exposure to welfare of the animal or third parties? animals or people. C: there are many defects in the maintenance of the enclosure electrical appliances or poorly protected electrical outlets, vegetation and/or the defective feature(s) put the life of animals or people at direct risk. that could fall and cause damage, entry of disease-carrying animals 10.3389/fvets.2022.1033821 or pests such as rodents). (Continued) frontiersin.org Frontiers in Veterinary Science Racciatti et al. TABLE 6 (Continued) Indicator Method Reference Scoring Enclosure hygiene Observe the hygiene of the enclosure (features to consider: spoiled Is the enclosure maintained in adequate A: The enclosure is in good hygienic conditions and the cleaning routine is (RBM) food, stagnant water, accumulation of feces and urine and dead hygienic conditions? Are the chemicals used adequate for the species. B: there are some defects in the hygiene of the enclosure, animals). Consider that excess hygiene can also be detrimental adequate in type and concentration? Is the which do not put the health of animals or people at direct risk. C: there are many (inadequate for the species or in higher concentration than frequency of cleaning adequate? defects in the hygiene of the enclosure and/or the defective feature(s) puts the recommended or with higher frequency than recommended). If health of animals or people at direct risk. available, check the recommended chemical types, concentration and frequency for the species (husbandry manual). Enclosure dimensions Request the enclosure outline and verify that the declared Do the dimensions of the enclosure allow the A: the dimensions comply with existing recommendations and are adequate for (RBM) dimensions match the actual dimensions. Take the necessary animal to move freely? Do they comply with the animal to move freely and express the full locomotor repertoire of its species. measurements and record the dimensions of the enclosure. When the minimum space requirements stated in B: the dimensions allow the animal to move freely but hinder the expression of answering the reference question consider that the animal should be the husbandry manuals per individual? the full locomotor repertoire of its species and are below those recommended. C: able to express the full repertoire of locomotor movements of their dimensions do not allow the animal to move freely and/or impede the expression species, including running, climbing, flying or swimming at speed. If of the full locomotor repertoire of its species and are below those recommended. more than one individual is housed in the same enclosure, consider the number of animals per surface area. If available, check the reference requirements for the species (husbandry manual). 11 Environmental Observe the disposition of different areas and elements within the Does the design of the enclosure allow for A: the design of the enclosure allows for differential use of each part of the space complexity (RBM) enclosure. Consider feeding and elimination zones, characteristics of species-specific behaviors as well as as well as the occurrence of all species-specific behaviors. B: the design of the the environment, land/water/air space ratio, implements for the differential use of each part of the space? enclosure allows differential use of each part of the space as well as the vertical use of space. For an accurate evaluation of welfare it is occurrence of most species-specific behaviors. C: the enclosure design does not essential to distinguish it from environmental enrichment. allow differential use of each part of the space and/or prevents the occurrence of several of the species-specific behaviors. Surrounding enclosures Observe the surrounding enclosures (features to consider: presence Does the housing layout and design minimize A: the layout and design of the housing are adequate to minimize stressful (RBM) of visual barriers, pray, predators or competitors housed in adjacent stressful situations with animals in adjacent situations with animals in adjacent enclosures or loose animals. B: only one of the enclosures and distance between enclosures). enclosures or loose animals? features to be considered is deficient. C: two or more of the features to be considered are deficient. Shelter availability Observe the existence, availability and adequacy of shelters for Do the animals have shelters to protect them A: shelters provide full protection from inclement weather. B: shelters provide (RBM) various weather conditions. from adverse weather conditions? partial protection from inclement weather. C: shelters do not provide protection 10.3389/fvets.2022.1033821 from inclement weather or there is no shelter or repair. (Continued) frontiersin.org Frontiers in Veterinary Science Racciatti et al. TABLE 6 (Continued) Indicator Method Reference Scoring Public (RBM) Observe the possibility of hiding from the public (features to Does the housing layout and design minimize A: the layout and design of the housing are adequate to minimize stressful consider: visual barriers, impediments for direct contact; first, stressful situations for the animal generated situations with humans. B: only one of the features to be considered is deficient. second and third level barriers; free access to confinement areas). by the public? C: two or more of the features to be considered are deficient. Group composition Observe group composition, ALWAYS record in “observations”: Is the group composition representative of A: the composition of the group is representative of the species in all features. B: (RBM) number of adults (clarifying the sex of each one), juveniles (sex) and the species? the gregarious/solitary condition of the species is respected but one or more of young, number of species and individuals in the same enclosure. If the other features to be considered is deficient. C: the gregarious/solitary the enclosure is shared with another species, consider if this condition of the species is not respected and/or two or more of the other features association is adequate for the species you are working with. to be considered are deficient. (Features to consider:gregarious/solitary, number of individuals, proportion of males/females and offspring) Environmental choice Examine the enclosure and assess whether it offers the animals Does the enclosure design allow the animal to A: the design of the enclosure allows the animal to choose where to be or what to and control opportunities for control and choice. Consider: opportunities for choose where to be or what to do 24 h a day? do, in all its aspects, during 24 h of the day. B: the enclosure design allows the opportunities (RBM) choice of display or concealment, shade or sun, heat or cold, animal to choose where to be or what to do, in various aspects, during at least the companionship or solitude, need to alternate exit to the main exhibit, most active period of the day for the species. C: the enclosure design allows the access to the main exhibit during peak periods of the day—species animal to choose where to be or what to do in few or none of its aspects and/or with nocturnal or crepuscular habits, isolation from stressors derived opportunities for choice and control are present only during the period of the 12 from cleaning, maintenance and repair maneuvers. day of least activity for the species. Management choice Interview staff and assess whether the management offers animals Does management allow the animal to A: management allows the animal to choose where to be or what to do, in all its and control opportunities for control and choice. Consider all the aspects choose where to be or what to do 24 h a day? aspects, 24 h a day. B: management allows the animal to choose where to be or opportunities (MBM) mentioned in “Environmental choice and control opportunities” what to do, in several of its aspects, during at least the most active period of the day for the species. C: management allows the animal to choose where to be or what to do, in few or none of its aspects, and/or opportunities for choice and control are present only during the period of the day of least activity for the species. Environmental Interview staff, check documentary records and verify the Is there a formal, written EE program in place A: an EE plan/program is implemented according to a formal, written outline enrichment (MBM) implementation of an appropriate and comprehensive and implemented to promote species-specific that promotes behavioral opportunities and psychological well-being and all environmental enrichment (EE) program. Consider anything that is behavioral opportunities and psychological steps are followed, including analysis of the animal’s response to EE, as well as the not fixed or does not remain the same in the animal’s environment, well-being? Does it include nutritional, various types of EE. B: an EE plan/schedule is implemented but no observation 10.3389/fvets.2022.1033821 but can be placed and removed on a daily basis. For an accurate social, sensory, cognitive, and occupational or analysis of the animal’s response to EE is performed, or EEs does not go evaluation of welfare it is essential to distinguish it from environmental enrichment? through an approval process from all areas (veterinary, biology, behavior, environmental complexity. nutrition and keepers), or any of the types of EE mentioned in the question are not implemented. C: no EE is performed or it is only performed by the individual frontiersin.org will of the keeper or volunteers, without an official plan by the institution. (Continued) Racciatti et al. 10.3389/fvets.2022.1033821 “Defecation behavior” and “Stool score”, together with animal. B: only training for veterinary and handling maneuvers is carried out by and in the case of dangerous species, without direct contact between trainer and written plan. C: training is not carried out and/or is carried out by inadequately with direct contact between trainer and animal in the case of dangerous species duly trained personnel, using validated techniques, with a formal, written plan, strengthening of the human-animal bond and animal exercise is carried out by “Micturition behavior” and “Urine appearance” respectively, trained personnel and/or by means of techniques with doubtful results and/or between trainer and animal in the case of dangerous species, with a formal, duly trained personnel, using validated techniques, without direct contact A: training for veterinary and handling maneuvers, cognitive enrichment, due to their physiologic and anatomic features. Behavioral and mental domain Ten ABM (Reaction to strangers, Interaction with zookeepers, Exploration, Social, affiliative and maternal-filial behavior, Reproductive behavior, Agonistic behavior, Use of environmental enrichment, Stereotypic behavior, Behavioral and/or without a formal and written plan. diversity, and Space use) were selected to assess the Behavioral and mental domains. Table 8 summarizes the most relevant information provided in the user’s manual for assessing behavioral and mental domains, through ten ABM. Discussion Scoring This study introduced an innovative multi-species animal welfare assessment protocol for wild animals under human care, intended to overcome the use of generic welfare checklists Is there a formal, written animal training and offer an alternative to challenging and time consuming species-specific tools (24). Ackonc-AWA protocol has several features in common with those of Kagan et al. (16), Brando and plan in place for the animal? Buchanan-Smith [264], Sherwen et al. (3), and Ward et al. (24). They all cover the five domains of animal welfare (31), through indicators that provide information on physical, environmental, Reference behavioral, and social state, as well as husbandry practices, human-animal interactions and individual animal agency. These checklists can be applied to most wild species and, as Ackonc- AWA, fit the prescriptive model (20) since they are helpful in the development of action plans to improve welfare conditions implementation of a comprehensive and appropriate training plan. and to set priorities. However, one of the main challenges of working with wildlife is the great diversity of species, with Interview staff, check documentary records and verify the characteristics and needs that are very different from one another (38). Therefore, similar to the work of Asher et al. RBM, Resources-based measurement; MBM, Management-based measurements. (27), Clegg et al. (28), Salas et al. (22), Yon et al. (29), and Padalino and Menchetti (30), Ackonc-AWA provides specific indications and descriptions to assist sentinels in the assessment of each indicator. The distinctive feature of Ackonc-AWA is that, notwithstanding its multi-species applicability, it proposes a standardized and detailed guide on the method to adapt, assess and rate each indicator as required by each species. Therefore, in order to successfully implement the current Method protocol, prior preparation is a key stage when used on a species for the first time. This includes reviewing the most updated guidelines for the adequate maintenance of the species TABLE 6 (Continued) in captivity and its dietary, health, environmental, behavioral, Training procedures and affective needs (3, 38). Sometimes this information may not be available, and it becomes necessary to search for information Indicator on the species natural history, biology, ecology, diet, sensory (MBM) systems, natural habitat, social structure, ethogram, activity patterns, and most common health problems and signs of illness, Frontiers in Veterinary Science 13 frontiersin.org Frontiers in Veterinary Science Racciatti et al. TABLE 7 Summary of the information provided in the user’s manual for assessing health domain. Indicator Method Reference Scoring (ABM) Defecation behavior If the animal is observed during defecation, check body posture, facial Does the animal have difficulty or pain A: absence of difficulty or pain during defecation. B: slight difficulty or pain expressions and vocalizations. during defecation? during defecation. C: difficulty or moderate to severe pain during defecation. Stool score Observe the characteristics of stool with the aid of the approved fecal Is the stool adequate in terms of consistency, A: normal stool, without alterations in any of the aspects to be considered. B: condition scales for the species. shape, color, frequency of excretion and stool with some of the aspects to be considered slightly or incipiently altered. C: macroscopic composition (blood, mucus, stool with some of the aspects to be considered severely altered. undigested food, foreign matter)? Micturition If the animal is observed during urination, check body posture, facial Does the animal have difficulty or pain to A: absence of difficulty or pain on urination. B: slight difficulty or pain during behavior expressions and vocalizations. urinate? urination. C: difficulty or pain moderate to severe pain during urination. Urine appearance Observe the characteristics of urine such as stream fluidity, urine color, Are there any abnormalities in the urine? A: normal urine, without alterations in any of the aspects to be considered. B: frequency and quantity. urine with some of the aspects to be considered slightly or incipiently altered. C: urine with two or more of the aspects to be consider altered in a severe way or for several days. Coat/feathers/ Observe the characteristics of the skin and the phanerae (features to Is the plumage/fur/coat/ integument in good A: good condition of plumage/coat/integument. B: Slight alteration in the tegument consider: quantity, brightness and integrity). condition? quantity or condition of the condition of the coat/plumage/tegument without alteration of its integrity. C: severe alteration in the quantity or condition of the 14 coat/plumage/tegument. Lesions/injuries Note the presence of wounds (Pay attention to hair removal, abrasion, Does the animal appear free of lesions or A: absence of lesions and wounds. B: shallow wounds or lesions, small in size and redness,swelling, bleeding, abscesses, bruises, presence of flies). wounds? low in number, without infection, suppuration or flies, with mild and short-term effects on animal welfare. C: deep, medium or large wounds or lesions, several in number, with infection, suppuration or flies, with moderate to severe or long-term effects on animal welfare. Hooves/claws/teeth According to the species, observe the condition of hooves, claws and teeth as Is the animal free of overgrowth or lesions on A: hooves/claws/teeth are free of overgrowth and lesions. B: hooves/claws/teeth appropriate. Take advantage of situations where the animal is close enough to hooves, nails, claws, teeth? show mild to moderate overgrowth but are free of lesions. C: hooves/claws/teeth inspect them (e.g., in training sessions for clinical procedures, when show severe overgrowth and/or lesions. performed). Locomotion Observe how the animal moves around the enclosure (features to consider: Does the animal ambulate without difficulty? A: the animal moves without difficulty or evidence of pain. B: the animal presents lameness, reluctance to walk or jump, facial expressions of pain and/or mild lameness (grade 1 or 2). C: the animal presents moderate to severe lameness 10.3389/fvets.2022.1033821 vocalizations while moving) (grade 3 or 4) and/or is reluctance to move and/or experiences evident pain when walking. Sleep/wakefulness Observe sleep and activity behaviors at different times of the day. Does the animal show activity in accordance A: the animal’s activity is in accordance with the circadian rhythm of the with the circadian rhythm of its species in free-living species. C: the animal does not present an activity in accordance with frontiersin.org nature? the circadian rhythm of the free-living species. (Continued) Racciatti et al. 10.3389/fvets.2022.1033821 considering the different life stages (25). Some preparation is moderate or severe symptoms of disease, or mild but long-standing symptoms, A: the animal appears clinically healthy. B: mild and/or recent symptoms of also needed when assessing an individual for the first time, disease, with minimal effect on animal welfare and/or good prognosis. C: such as information on enclosure size and design, schematic with significant effects on animal welfare and/or unfavorable prognosis. segmentation of the enclosure according to the biological relevance of each sector, major life history events and medical records. Thus, before applying the Ackonc-AWA protocol for assessing the welfare of an individual, the sentinels should do a crucial (but guided) previous step: to adapt the protocol to the specific welfare-related characteristics and requirements of the target species. By completing a spreadsheet with the optimal conditions for the welfare of the specific species to be evaluated and by adapting the indicators included in the protocol, sentinels would be able to compare them with those observed for the assessed individual and identify potential welfare concerns or needs of improvement. The need for prior search for information on the species and, if not available, the realization of an ethogram, could take Scoring considerable time. This time may be longer or shorter depending on the species, since for some there are husbandry manuals and abundant bibliography, and for others information is very scarce Does the animal appear healthy and free of or absent. This prior preparation could be seen as a limitation in comparison to other tools. However, it is important to note that this procedure is done only once at the beginning of the assessment and then the sentinels use the protocol adapted to the species of interest, without the need to go back to the visible signs of disease? literature for each assessment. In the field trials, the average time used by the sentinels was 51.32 min +/– 29.36 min for the Reference initial form and 58.32 min +/– 23.11 min for the follow-up form. Even with the addition of activity budgets (60 min in total), an increase in the time required for assessment is not expected, as many indicators can be assessed during the same observation. structures where signs of disease may be evident, depending on the species). Look for any signs of disease (pay attention to ears, mouth, muzzle, perineal region, respiration, general condition and other anatomical regions or body Nevertheless, this should be evaluated in further studies. Consider signs of disease as identified in the available literature for the Ackonc-AWA implementation cost is low, it is non- invasive/intrusive and takes relatively little time. Although these are all desirable qualities for any animal welfare assessment protocol (39), they could become an essential prerequisite for a welfare evaluation tool intended to be applicable on a daily or weekly basis in institutions with such dissimilar realities, in terms of financial and human resources, as those found in Latin American zoos. It should be noted that the Ackonc-AWA protocol includes some indicators that can be assessed by close observation and even palpation (i.e., body condition score). This is so that future users of the protocol are able to collect the information in the most practical way for them, as many ABM can be assessed Method by training and conditioning or during a scheduled veterinary ABM, Animal-based measurement. species. capture. Given that zoos frequently train animals to cooperate TABLE 7 (Continued) in veterinary maneuvers without the need for physical or chemical restraint, and that there is abundant scientific evidence indicating that operant conditioning training is another strategy Signs of illness Indicator to improve animal welfare in zoos and the human-animal bond, and is even a form of environmental enrichment (40–42), close observation and hands-on assessment are not discarded. Frontiers in Veterinary Science 15 frontiersin.org TABLE 8 Summary of the most relevant information that is provided in the user’s manual for assessing behavior and mental domain indicators. Frontiers in Veterinary Science Racciatti et al. Indicator (ABM) Method Reference Scoring Reaction to strangers It should be assessed at any time when the public or zoo staff are Is the animal indifferent to the presence of A: indifferent or positive. C: fear, hidding, aggressiveness, freezing. unfamiliar to the animal. Assess whether the presence of strangers the public, unfamiliar staff, or observers (if modifies the occurrence or development of species-specific they are not people with whom it has daily behaviors, or if signs of fear (e.g., hiding), agonism (e.g., stalking), or contact)? habituation (e.g., begging for food or actively seeking interaction) are observed. Interaction with It should be evaluated any time the animal is in the presence of its Does the animal have a positive relationship A: alert, responds to call and commands. B: indifference. C: fear, agonistic zookeepers keepers. Assess whether this presence modifies the occurrence or with their keepers? behavior. development of species-specific behaviors, or if signs of fear (e.g., hiding), agonism (e.g., stalking) or social behaviors (e.g., asking for petting or actively seeking interaction) are observed. Exploration Observe the animal’s active exploration of its environment (consider Does the animal roam the enclosure and its A: exploration is observed. B: exploration is only observed in response to novel that in addition to wandering, the individual listens, sniffs, licks, or sorroundings directing their senses to stimuli (e.g., environmental enrichment). C: no exploration is observed. manifests any other component of species-typical exploratory relevant stimuli? behavior). Social, affiliative and Observe affiliative bonds, such as nurturing and maternal-filial Does the individual interact with others in a A: positive interaction with other animals. B: indifference or isolation. C: maternal-filial behavior relationship, grooming sessions, or any other component of positive way? aggressiveness, fear. 16 species-typical social behavior). If the animal is housed in solitary, observe if there are interactions with animals from adjacent enclosures. Reproductive behavior Observe the occurrence of reproductive behavior according to the Does the animal deploy species-specific A: appetitive and consummatory phases of reproductive behavior are observed in time of year (and species characteristics), proximity of individuals of reproductive behavior? animals housed in the same enclosure during the breeding season. B: incomplete the same species and different sex, presence of young, courtship repertoire of reproductive behavior (e.g., substitution behaviors or blank firing) behaviors (depending on species: sniffing, urination, marking spray, are observed in the breeding season. C: absence of reproductive behavior during vocalizations, sensory orientation, etc.). Consider these factors in the the breeding season. different possible contexts (e.g., animals housed in the same enclosure, animals housed in adjacent enclosures with different possibilities of direct contact, and animals housed in nearby enclosures but without direct contact). 10.3389/fvets.2022.1033821 Agonistic behavior Observe for agonistic interactions and weigh the results. If the Do animals interact with others of the same A: no more than 3 agonistic interactions marked on the time budget sheets animal is housed alone, observe for interactions with animals in or related species in a negative way? during the 60 min and NO obvious negative effects on animal welfare (e.g., adjacent enclosures. Specify in “observations” which individuals are moderate to severe injury or wounding). C: 4 or more agonistic interactions involved and the observed behavior. marked on the time budget sheets during the 60 min or <4 WITH obvious frontiersin.org negative effects on animal welfare. (Continued) TABLE 8 (Continued) Frontiers in Veterinary Science Racciatti et al. Indicator Method Reference Scoring Use of environmental Evaluate the animal’s response to environmental enrichment (EE) by Is there evidence of interaction with EE? A: 5 (DE) or 3 (IE), B: 2, 3 or 4 (DE) or 2 (IE), C: 1 (DE or IE). enrichment direct evidence (DE) (visualization of the animal interacting with EE, observing it at the time it is offered) or by indirect evidence (IE) (visualization of the EE or its remains after the animal interacted—or not—with it). Clarify in “observations” which type of EE was observed during assessment. Stereotypic behavior Observe for the presence of repetitive, unvarying behaviors with no Does the animal show any abnormal A: the animal does not deploy repetitive behavior. B: repetitive behavior occurs obvious functional goals. In case B or C, describe in “observations” repetitive behavior? but the pattern retains some variability (it does not always move the same body the behavior in question as detailed as possible. parts in the same way, it can do it with some variants) and low repeatability (no more than 5 repetitions in a row without stopping). C: the behavior has no variability (always moves the same body parts in the same way) or high repeatability (more than 5 repetitions in a row without stopping). Behavioral diversity Complete the “time and space budget sheets” provided by the Does the animal perform species-specific A: Time budget reflects 100% coverage of the functional categories, with no analyst, in different time slots (morning, noon, afternoon and behaviors at natural frequencies and deviations in their proportion as expected for the species. B: the time budget evening) as specified in the user’s manual. Attention! The observer appropriate diversity? reflects a coverage of between 70 and 100% of the functional categories, with should not assign a score for this indicator. The analyst will be the slight deviations in their proportion according to what is expected for the species. 17 one to assign the score in consideration of the richness of the C: Time budget reflects a coverage of <70% of the functional categories, with behavior (number of behaviors) as well as the uniformity (frequency marked deviations in their proportion according to what is expected for the of each behavior) following the Activity budget method (37). species. Space use Complete the “time and space budget sheets” provided by the Does the animal make full use of the available A: uses between 85 and 100% of the areas to which it has access. B: uses between analyst, in different time slots (morning, noon, afternoon and space? 50 and 84% of the sectors of the enclosure to which it has access. C: uses between evening) as specified in the user’s manual. Attention! The observer 0 and 49% of the sectors of the enclosure to which it has access. should not assign a score for this indicator. The analyst will be the one to assign the score. ABM, Animal-based measurement. 10.3389/fvets.2022.1033821 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 However, the protocol has been specifically designed so that low frequency of occurrence during brief observation periods. contact with the animal is not essential to perform the welfare However, we consider that they are indicators of great value assessment, and was tested hands-off. This flexibility reinforces for the welfare assessment of animals and their inclusion was the practicality and non-invasiveness attributes of the protocol. deemed necessary. Abnormalities in these two behaviors could In order to further increase its practicality, Ackonc-AWA be related to somatic conditions and pain or distress, arousal was designed in two forms: initial and follow-up. Although, and fear (55). In addition, Ackonc-AWA was intended to be as discussed above, preparation requires some time, once the applied by zookeepers, who routinely have the opportunity protocol has been adjusted to the species under study, the follow- and the skills to detect these subtle changes in the behavior up form can be applied as often as necessary, even on a daily of the animals in their care (13), which would overcome basis. Its practicality and low cost of implementation is partly this constraint. Although the addition of the activity budget based on the fact that, subject to prior training, it can be applied sheets could potentially increase the total time required for by the institution’s own personnel and done in the context of the assessment, it provides greater robustness in assessing the their daily duties. aforementioned indicators as well as greater flexibility to use the In this regard, a core component of developing and using protocol on crepuscular and nocturnal species, through direct animal welfare assessment tools in zoos is to leverage the or recorded observations. Moreover, the proposed behavioral experience and expertise of the staff (13, 43, 44). Zoos often budget form was designed to reduce time consumption and have keepers with years of experience working with a particular to be applied in institutions with time constraints, since its species, as well as the opportunity to observe individuals over interpretation is left to a trained person (analyst) other than long periods of time and in a variety of contexts. As such, the sentinels. they usually develop skills and abilities to detect and integrate The need for an analyst can also be discussed as a possible subtle changes in behavior, posture, attitude, expression, or disadvantage. Nevertheless, the analysis and interpretation of the movement (13). In addition, many of the indicators to be information obtained from behavioral budgets has been widely assessed are part of their daily tasks, so zookeepers do not need to used in zoos, and many of the institutions in Latin America have coordinate with another member of zoo staff the proper moment highly trained personnel within their staff to perform this task. to do it (e.g., to assess response to environmental enrichment). To assess affective states, Ackonc-AWA proposes a joint Furthermore, the inter-observer agreement of ratings performed approach of the behavioral domain with the mental domain. by zookeepers on zoo animals has been examined and high This is because some affective states are directly or indirectly levels of agreement have been reported (45–48). Therefore, assessed in this protocol using behavioral indicators. Due to the Ackonc-AWA protocol was conceived to benefit from a the type of institutions for which this protocol was designed, systematic collection of information by experienced zookeepers. the importance of assessing affective states in relation to Simplicity of implementation is also a key factor for the the human-animal bond is emphasized. The effects of the feasibility of animal welfare assessment protocols. Although the visitors and zookeepers over the animals’ experiences and their overall feasibility results were positive, adequate training and consequent welfare state are addressed in the protocol through coaching could be implemented to reduce some of the difficulties two ABM indicators: Reaction to strangers and Interaction with encountered by sentinels when filling out the forms in animal zookeepers. As stated by Mellor et al. (31), Domain 4 (Behavioral welfare assessments. As demonstrated by Rodríguez Ruiz and Interactions) is intended to capture behavioral outputs as indices Heredia Rico (49), training increases reliability of the results and of animals’ perceptions of their external circumstances. Hence, reduces the protocol application time, which becomes relevant the inclusion of Reaction to strangers aimed at evaluating since the accuracy of the measurement decreases as the observer the affective experiences that animals may have when they gets tired (50). In this study, although inexperienced sentinels direct their attention toward unfamiliar people. This could received a short training (4 h), the average difficulty values for be recognized as behaviors associated with negative states both forms were relatively low, suggesting that they could be (i.e., freezing, hypervigilance, fear, hiding, and aggressiveness). further improved with longer training. This could be explored Behaviors associated with positive states could also be found, as in future studies. animals actively seek interaction with such strangers. Regarding The indicators most frequently reported as difficult to the indicator Interaction with zookeepers, it is relevant to assess assess were “Behavioral diversity”, “Defecation behavior” and how the animals respond to the staff with whom they are “Micturition behavior”. In order to simplify the assessment of familiar: whether they respond to calls, remain indifferent or “Behavioral diversity”, the use of activity budget sheets through display behaviors associated with negative affective states such focal (individuals) and scan (group) sampling was incorporated, as those mentioned above. as it is an objective, quantitative and validated method for All of these responses tend to offer an approach to affective animal welfare assessment in zoos (37, 51, 52), as well as states in relation with the interactions that animals and humans for “Space use” (53, 54). Regarding “Defecation behavior” and have. In the future, further interventions on negative or positive “Micturition behavior”, the difficulty could reflect their relatively human attributes and attitudes toward animals could be useful to Frontiers in Veterinary Science 18 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 address this issue from another perspective, in order to acquire Conclusion a MBM that could operate as a welfare predictor. The qualitative nature of this protocol may be considered This study aimed to develop, test in the field, and describe controversial. Observer ratings are scores given to a variable an animal welfare assessment protocol for wild animals under using units of measurement defined by the researchers. Since human care, that can be applied on a daily basis, noninvasively, they involve subjective judgments, some researchers question and at a low cost, under the prescriptive model. Therefore, a whether they can be trusted to reflect reality in an unbiased protocol structured in two forms (one exhaustive and other for manner (56). However, several studies have shown that routine use) was tested on 14 species of different taxa housed observer ratings can be reliable and valid [e.g., (46, 57–60)]. in a zoo in Argentina. Representatives from different areas of They have been widely employed to assess physical traits the institution as well as 3 of the authors participated in the [e.g., (61, 62)], health-related variables [e.g., (63–65)], animal test. It was possible to demonstrate the feasibility and test-retest personality [e.g., (46, 59)], behavioral patterns [e.g., (45)], and reliability of the protocol. However, due to time limitations a number of variables relevant to animal welfare [e.g., (66)]. of the institution staff, its inter-observer reliability has yet to In addition to their practicality, non-invasive nature and low be tested. cost (56), observer ratings can be used to integrate multimodal As a result of this process, Ackonc-AWA, a information across time and situations, and for constructs that multidimensional protocol for welfare assessment in multiple would otherwise be very difficult to assess [e.g., pain: (65, animal species under human care, was obtained. This proposal 67)]. Furthermore, this method seems to be useful for most offers an intermediate solution between protocols that are easy species that have been tested so far (56). Biases are indeed a to apply yet rely entirely on the judgment of the assessors, and risk, especially when the ratings could reflect the observer’s or validated but species-specific protocols that are useful only for institution’s own care of the animals (68–70). Nevertheless, this assessing the species for which they were developed. risk can be minimized by careful wording of the questions to Further applications of the described welfare assessment tool be answered, development of appropriate scales, selection and in other species and different institutional contexts will reinforce training of observers, and field testing (56). the validation of the proposed measurements and allow the With regards to the final assessment results, Ackonc-AWA systematic and routine evaluation of animal welfare in zoos. provides a representation of an animal’s welfare and a temporal component that is easy to read and allows tracking changes over time, making it possible to differentiate between problems that Data availability statement affect animal welfare at the current time and those that pose a risk to animal welfare in the medium and long term. As a The original contributions presented in the study are protocol with a prescriptive approach, it does not give a final included in the article/Supplementary material, further inquiries numerical result, but looks at each indicator in order to identify can be directed to the corresponding author/s. potential welfare concerns, which prevents the institution from settling for an acceptable overall result that could be deceiving and could pose a severe threat to animal welfare. For example, a Ethics statement zoo that scores 8 out of 10 might be satisfied with the idea that it has a good overall score and not work on establishing a plan to Ethical review of the project was requested to the improve those indicators that were found to be compromised. Institutional Committee for the Care and Use of Laboratory The situation of these compromised indicators could become Animals (CICUAL) of the Faculty of Veterinary Sciences of chronic and begin to impact negatively on others that were the University of Buenos Aires, and a review exemption adequate. On the other hand, by letter-marking indicators it was granted given the observational nature of the project. is easy to identify those that require immediate resolution and The study focused on the non-invasive/intrusive assessment establish a prioritization plan. The proposed 3-point scale score of animal welfare, so no interventions of any kind were would facilitate a fast and practical prioritization of the identified carried out on the animals. There were no potential adverse welfare concerns, and to tag the more urgent correction actions. effects, nor foreseeable risks or hazards associated with The implementation of Ackonc-AWA in zoos could be this project, with regards to animal, plant and/or human very useful for decision-making within the ethical frameworks wellbeing. The participation of zoo staff in this study was of compassionate conservation and conservation welfare, by completely voluntary and written informed consent was evaluating the impact of different actions and situations, and obtained from all participants. The survey responses were guiding future decisions, so to ensure that ex situ conservation strictly confidential and data from this research was reported efforts do not harm (or do as little as possible) the welfare of only in the aggregate. The information was coded and individuals (8, 71). remains confidential. Frontiers in Veterinary Science 19 frontiersin.org Racciatti et al. 10.3389/fvets.2022.1033821 Author contributions authors are grateful to the institution managers for providing unlimited access to interviews with personnel, to reading daily DR, AF, and LR contributed to the elaboration of the reports and medical records, and entering the enclosures, first drafts of the protocol, interviewed the zoo staff, met chutes, and night houses of the animals included in this with the zoo staff/focus group to test validity, designed the study. Finally, we thank our reviewers, who gave us coherent, on-field test, trained the staff as sentinels, organized the consistent and precise feedback and helped us to deliver an scheduled observations for all the sentinels, did field work as improved manuscript. sentinels, analyzed the results of the observations including feasibility results, and elaborated the Ackonc-AWA protocol. DR contacted and coordinated actions with the institution where Conflict of interest the pilot test was conducted and wrote and edited the first draft of this manuscript. LR and AF wrote sections of the manuscript The authors declare that the research was conducted in the and edited the first draft and manuscript. CB was responsible absence of any commercial or financial relationships that could for the statistical analysis of the results for test-retest reliability be construed as a potential conflict of interest. and edited the manuscript. OT-P collaborated as the main counselor for methodological aspects of the study and edited the manuscript. All authors contributed to manuscript revision, and Publisher’s note read and approved the submitted version. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated Funding organizations, or those of the publisher, the editors and the reviewers. 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